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A research of the temperature effect of the muon cosmic ray (CR) component on the MuSTAnG super telescope data (Greifswald, Germany) for the whole period of its work (from 2007) was carried out. The primary hourly telescope's data were corrected for the temperature effect, using vertical temperature atmospheric profile at the standard isobaric levels obtained from the GFS model. To estimate the model accuracy and applicability the air sounding data for some years were used.
Abstract
We formulate exact generalized nonequilibrium fluctuation relations for the quantum mechanical harmonic oscillator coupled to multiple harmonic baths. Each of the different baths is prepared in its own individual (in general nonthermal) state. Starting from the exact solution for the oscillator dynamics we study fluctuations of the oscillator position as well as of the energy current through the oscillator under general nonequilibrium conditions. In particular, we formulate a fluctuation–dissipation relation for the oscillator position autocorrelation function that generalizes the standard result for the case of a single bath at thermal equilibrium. Moreover, we show that the generating function for the position operator fulfils a generalized Gallavotti–Cohen-like relation. For the energy transfer through the oscillator, we determine the average energy current together with the current fluctuations. Finally, we discuss the generalization of the cumulant generating function for the energy transfer to nonthermal bath preparations.
Abstract
Many processes in nature are governed by the interaction of electro-magnetic radiation with matter. New tools such as femtosecond and free-electron lasers allow one to study the interaction in unprecedented detail with high temporal and spatial resolution. In addition, much work is devoted to the exploration of novel target systems that couple to radiation in an effective and controllable way or that could serve as efficient sources of energetic particles when being subjected to intense laser fields. The interaction between matter and radiation fields as well as their mutual modification via correlations constitutes a rich field of research that is impossible to cover exhaustively. The papers in this focus issue represent a selection that largely reflects the program of the international conference on ‘Correlation Effects in Radiation Fields’ held in 2011 in Rostock, Germany.
Abstract
Emperor penguins breed during the Antarctic winter and have to endure temperatures as low as −50 °C and wind speeds of up to 200 km h−1. To conserve energy, they form densely packed huddles with a triangular lattice structure. Video recordings from previous studies revealed coordinated movements in regular wave-like patterns within these huddles. It is thought that these waves are triggered by individual penguins that locally disturb the huddle structure, and that the traveling wave serves to remove the lattice defects and restore order. The mechanisms that govern wave propagation are currently unknown, however. Moreover, it is unknown if the waves are always triggered by the same penguin in a huddle. Here, we present a model in which the observed wave patterns emerge from simple rules involving only the interactions between directly neighboring individuals, similar to the interaction rules found in other jammed systems, e.g. between cars in a traffic jam. Our model predicts that a traveling wave can be triggered by a forward step of any individual penguin located within a densely packed huddle. This prediction is confirmed by optical flow velocimetry of the video recordings of emperor penguins in their natural habitat.
Abstract
The spectral properties of three-dimensional dust clusters confined in gaseous discharges are investigated using both a fluid mode description and the normal mode analysis (NMA). The modes are analysed for crystalline clusters as well as for laser-heated fluid-like clusters. It is shown that even for clusters with low particle numbers and under presence of damping fluid modes can be identified. Laser-heating leads to the excitation of several, mainly transverse, modes. The mode frequencies are found to be nearly independent of the coupling parameter and support the predictions of the underlying theory. The NMA and the fluid mode spectra demonstrate that the wakefield attraction is present for the experimentally observed Yukawa balls at low pressure. Both methods complement each other, since NMA is more suitable for crystalline clusters, whereas the fluid modes allow to explore even fluid-like dust clouds.
Abstract
The surface charge distribution deposited by the effluent of a dielectric barrier discharge driven atmospheric pressure plasma jet on a dielectric surface has been studied. For the first time, the deposition of charge was observed phase resolved. It takes place in either one or two events in each half cycle of the driving voltage. The charge transfer could also be detected in the electrode current of the jet. The periodic change of surface charge polarity has been found to correspond well with the appearance of ionized channels left behind by guided streamers (bullets) that have been identified in similar experimental situations. The distribution of negative surface charge turned out to be significantly broader than for positive charge. With increasing distance of the jet nozzle from the target surface, the charge transfer decreases until finally the effluent loses contact and the charge transfer stops.
Musicians use different kinds of imagery. This review focuses on kinesthetic imagery, which has been shown to be an effective complement to actively playing an instrument. However, experience in actual movement performance seems to be a requirement for a recruitment of those brain areas representing movement ideation during imagery. An internal model of movement performance might be more differentiated when training has been more intense or simply performed more often. Therefore, with respect to kinesthetic imagery, these strategies are predominantly found in professional musicians. There are a few possible reasons as to why kinesthetic imagery is used in addition to active training; one example is the need for mental rehearsal of the technically most difficult passages. Another reason for mental practice is that mental rehearsal of the piece helps to improve performance if the instrument is not available for actual training as is the case for professional musicians when they are traveling to various appearances. Overall, mental imagery in musicians is not necessarily specific to motor, somatosensory, auditory, or visual aspects of imagery, but integrates them all. In particular, the audiomotor loop is highly important, since auditory aspects are crucial for guiding motor performance. All these aspects result in a distinctive representation map for the mental imagery of musical performance. This review summarizes behavioral data, and findings from functional brain imaging studies of mental imagery of musical performance.
Background: Fetal growth failure has been associated with an increased risk of hypertension, cardiovascular disease and diabetes in adulthood. Exploring the mechanisms underlying this association should improve our understanding of these common adult diseases. Patients and Methods: We investigated 225 SNPs in 10 genes involved in growth and glucose metabolism (GH1, GHR, IGF1, IGF1R, STAT5A, STAT5B, MAPK1, MAPK3, PPARγ and INS) in 1,437 children from the multinational NESTEGG consortium: 345 patients born small for gestational age who remained short (SGA-S), 288 who showed catch-up growth (SGA-Cu), 410 idiopathic short stature (ISS) and 394 controls. We related genotype to pre- and/or postnatal growth parameters, response to growth hormone (if applicable) and blood pressure. Results: We found several clinical associations for GH1, GHR, IGF1, IGF1R, PPARγ and MAPK1. One SNP remained significant after Bonferroni's correction: IGF1R SNP rs4966035's minor allele A was significantly more prevalent among SGA and associated with smaller birth length (p = 0.000378) and birth weight (weaker association), independent of gestational age. Conclusion:IGF1R SNP rs4966035 is significantly associated with birth length, independent of gestational age. This and other associations suggest that polymorphisms in these genes might partly explain the phenotype of short children born SGA and children with ISS.
This study aims to analyze psychometric properties and validity of the Compulsive Internet Use Scale (CIUS) and the Internet Addiction Test (IAT) and, second, to determine a threshold for the CIUS which matches the IAT cut-off for detecting problematic Internet use. A total of 292 subjects with problematic or pathological gambling (237 men, 55 women) aged 14-63 years and with private Internet use for at least 1 h per working or weekend day were recruited via different recruitment channels. Results include that both scales were internally consistent (Cronbach's α = 0.9) and had satisfactory convergent validity (r = 0.75; 95% CI 0.70-0.80). The correlation with duration of private Internet use per week was significantly higher for the CIUS (r = 0.54) compared to the IAT (r = 0.40). Among all participants, 25.3% were classified as problematic Internet users based on the IAT with a cut-off ≥40. The highest proportion of congruent classified cases results from a CIUS cut-off ≥18 (sensitivity 79.7%, specificity 79.4%). However, a higher cut-off (≥21) seems to be more appropriate for prevalence estimation of problematic Internet use.
Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h.
Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1.
Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH.
Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.
Background: Patients with mucin-producing adenocarcinoma have an increased risk for venous and arterial thrombosis. When these patients present with thrombocytopenia, disseminated intravascular coagulopathy (DIC) is often the underlying cause. Case Report: We report 2 patients who were admitted due to bleeding symptoms of unknown cause, in whom further workup revealed adenocarcinoma-induced DIC. Conclusion: In elderly patients presenting with signs of DIC, such as reduced fibrinogen levels, elevated prothrombin time, elevated D-dimer, and thrombocytopenia, without any obvious reason (e.g., sepsis), adenocarcinoma-associated coagulopathy should be considered as the underlying cause. Paradoxically, in these patients bleeding symptoms improve when the patient is sufficiently anti-coagulated with low molecular weight heparin. Treatment of the underlying disease is of central importance in controlling acute or chronic DIC associated with malignant diseases and chemotherapy should be started as soon as possible.
Lung dendritic cells facilitate extrapulmonary bacterial dissemination during pneumococcal pneumonia
(2013)
Streptococcus pneumoniae is a leading cause of bacterial pneumonia worldwide. Given the critical role of dendritic cells (DCs) in regulating and modulating the immune response to pathogens, we investigated here the role of DCs in S. pneumoniae lung infections. Using a well-established transgenic mouse line which allows the conditional transient depletion of DCs, we showed that ablation of DCs resulted in enhanced resistance to intranasal challenge with S. pneumoniae. DCs-depleted mice exhibited delayed bacterial systemic dissemination, significantly reduced bacterial loads in the infected organs and lower levels of serum inflammatory mediators than non-depleted animals. The increased resistance of DCs-depleted mice to S. pneumoniae was associated with a better capacity to restrict pneumococci extrapulmonary dissemination. Furthermore, we demonstrated that S. pneumoniae disseminated from the lungs into the regional lymph nodes in a cell-independent manner and that this direct way of dissemination was much more efficient in the presence of DCs. We also provide evidence that S. pneumoniae induces expression and activation of matrix metalloproteinase-9 (MMP-9) in cultured bone marrow-derived DCs. MMP-9 is a protease involved in the breakdown of extracellular matrix proteins and is critical for DC trafficking across extracellular matrix and basement membranes during the migration from the periphery to the lymph nodes. MMP-9 was also significantly up-regulated in the lungs of mice after intranasal infection with S. pneumoniae. Notably, the expression levels of MMP-9 in the infected lungs were significantly decreased after depletion of DCs suggesting the involvement of DCs in MMP-9 production during pneumococcal pneumonia. Thus, we propose that S. pneumoniae can exploit the DC-derived proteolysis to open tissue barriers thereby facilitating its own dissemination from the local site of infection.
Background/Aims: Only a small percentage of pathological gamblers utilizes professional treatment for gambling problems. Little is known about which social and gambling-related factors are associated with treatment utilization. The aim of this study was to look for factors associated with treatment utilization for pathological gambling. Methods: The study followed a sampling design with 3 different recruitment channels, namely (1) a general population-based telephone sample, (2) a gambling location sample and (3) a project telephone hotline. Pathological gambling was diagnosed in a telephone interview. Participants with pathological gambling (n = 395) received an in-depth clinical interview concerning treatment utilization, comorbid psychiatric disorders and social characteristics. Results: Variables associated with treatment were higher age [odds ratio (OR) 1.05, 95% confidence interval (CI) 1.03-1.08], an increased number of DSM-IV criteria for pathological gambling (OR 1.34, 95% CI 1.06-1.70), more adverse consequences from gambling (OR 1.10, 95% CI 1.03-1.16) and more social pressure from significant others (OR 1.17, 95% CI 1.07-1.27). Affective disorders were associated with treatment utilization in the univariate analysis (OR 1.81, 95% CI 1.19-2.73), but multivariate analysis showed that comorbid psychiatric disorders were not independently associated. Conclusion: These results indicate that individuals with more severe gambling problems utilize treatment at an older age when more adverse consequences have occurred. Further research should focus on proactive early interventions.
Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food.
Background: Primary angiitis of the central nervous system (PACNS) is a rare but serious condition. A fraction of patients suffering from PACNS concurrently exhibit pronounced cerebral amyloid angiopathy (CAA) which is characterized by deposits of amyloid-β (Aβ) in and around the walls of small and medium-sized arteries of the brain. PACNS with CAA has been identified as a distinct disease entity, termed Aβ-related angiitis (ABRA). Evidence points to an immune reaction to vessel wall Aβ as the trigger of vasculitis. Objective: To investigate whether the inflammatory response to Aβ has (1) any effect on the status of immune activation in the brain parenchyma and (2) leads to clearance of Aβ from brain parenchyma. Methods: We studied immune activation and Aβ load by quantitative immunohistochemical analysis in brain parenchyma adjacent to affected vessels in 11 ABRA patients and 10 matched CAA controls. Results: ABRA patients showed significantly increased immune activation and decreased Aβ loads in the brain parenchyma adjacent to affected vessels. Conclusion: Our results are in line with the hypothesis of ABRA being the result of an excessive immune response to Aβ and show that this can lead to enhanced clearance of Aβ from the brain parenchyma by immune-mediated mechanisms.
Background: This study aims to assess the role of ductoscopy for detecting intraductal anomalies in patients with nipple discharge in comparison to conventional tests and to find an effective combination of both approaches. Materials and Methods: Prior to duct excision, ductoscopy was performed in 97 women. Histologic and all other diagnostic results were compared. Sensitivity, specificity, and efficiency were calculated for all methods. These parameters were also calculated for all possible test combinations in 12 patients who had completed all tests. Results: Breast sonography reached the highest sensitivity (64.1%) and efficiency (64%); mammography had the highest specificity (100%). The sensitivity of ductoscopy was 53.2%, its specificity 60%, and its efficiency 55.1%. Among combinations of all methods, the combination ductoscopy + galactography was the most sensitive (80%). Mammography, magnetic resonance imaging, and ductoscopy were each 100% specific. Ductoscopy was the most efficient (75%) single method. Conclusion: Ductoscopy is a valuable test for diagnosing intraductal lesions in patients with nipple discharge. It is more efficient than conventional tests in patients undergoing all tests.
The objective of the present investigation was to examine the residual antimicrobial activity after a topical exposure of reconstructed human epidermis (RHE) to equimolar solutions of either chlorhexidine digluconate (CHG, 0.144% w/v) or octenidine dihydrochloride (OCT, 0.1% w/v) for 15 min. RHE-associated antiseptic agents were more effective on Staphylococcus aureus than on Pseudomonas aeruginosa. S. aureus was not detected after 24 h of contact, which demonstrated a microbicidal efficacy of greater than 5-log<sub>10</sub> reduction. In contrast, P. aeruginosa was reduced by approximately 2 log<sub>10</sub> at the same incubation time, which parallels the growth of the initial inoculum. This result could be interpreted either as a microbiostatic effect or as an adherence of P. aeruginosa to a low positively charged surface. Small amounts of CHG and OCT can penetrate the stratum corneum. Using these antiseptic agents, the viability of keratinocytes was reduced to 65-75% of that of the untreated RHE control following 24 h incubation in the presence of test microorganisms. With consideration of antimicrobial activity and cytotoxic effect, OCT corresponds better to a biocompatible antiseptic agent than CHG.
The pollen record is a powerful proxy to reconstruct past terrestrial vegetation, but quantifying plant abundances is strongly limited because plants produce pollen in different amounts and pollen is dispersed differently. Further complications arise from the use of percentage data. Finally, a pollen grain deposited at a site may have arrived from proximate or distant sources, which implies that a single pollen sample may reflect very different vegetation scenarios. Present thesis suggests improving quantitative reconstructions of past vegetation by refined calibration of the pollen-vegetation relationship (paper I) and application of the downscaling approach (papers II-IV). Paper I primarily addresses the questions of pollen production and dispersal by calibrating the pollen-vegetation relationship. Data analysis employs the common extended R-value (ERV) approach and a new data-model comparison method, which appears more suitable than the ERV approach. For the first time PPEs have been calculated using three contrasting pollen dispersal options, including a Lagrangian stochastic (LS) model. The study proves that the underlying pollen dispersal model is a crucial parameter in PPE calculations and that the calculations with the LS model produce more reliable and realistic PPEs. Papers II to IV address quantitative reconstructions of past vegetation. Using the newly developed downscaling approach, the three studies explore fine scaled vegetation patterns in NE Germany during the Late Glacial and early Holocene. The main assumption of the downscaling approach is that the present day pattern of abiotic site conditions (e.g. the pattern of soil substrates) existed, at least to a large extend, also during the study periods. The basic principle of the approach is to test, whether pollen deposition in sites across a landscape is correlated to that site pattern. The first application of the approach (paper II) has shown a close correlation between PINUS pollen percentages and the distance weighted abundance of sandy soils and between BETULA pollen percentages and the distance weighted abundance of morainic till during the Allerød period, indicating that pine and birch formed rather separate stands on either substrate type. The cooling of the Younger Dryas induced significant changes in the vegetation of NE Germany. By combining pollen percentage and pollen accumulation rate data paper III identified a sharp vegetation boundary between the Mecklenburg and Brandenburg area at about 53 °N. The downscaling approach, here used with pollen accumulation rate data, suggests that in the North small tree stands could only exist in sheltered positions. The sharp vegetation boundary is possibly related to a climatic gradient and the southern permafrost limit, which itself may result from the formation of sea ice on the North Atlantic north of 53°N during winter. The warming of the Holocene again allowed the expansion of forests in the study area. Paper IV uses high resolution pollen (accumulation rate) data to study the successive forest formation, including the immigration of hazel, and explores vegetation patterns and composition during these successive stages using the extended downscaling approach. This approach addresses the problems related to differential pollen production, dispersal and the use of percentage data by applying simulations. It reveals that initially pine and birch established, as during the Allerød period, in largely separate stands with pine dominating on sandy soils and birch dominating on fine grained soils. Also open rich vegetation persisted, possibly due to seasonal drought, mainly on fine grained soils. Hazel later mainly spread on sites that received additional wetness from ground or surface water; it did not enter pine dominated forests on well drained sandy soils. Overall, the early Holocene vegetation of the study area was sharply differentiated by soil humidity and fertility. To conclude, present thesis has revealed vegetation patterns and species site preferences in NE Germany during three periods of the Lateglacial and early Holocene. The results improve our understanding of vegetation history in northern Central Europe, specifically for periods of rapid climate change. The approaches applied are flexible with respect to the type and quality of pollen data used and may be implemented using standard software packages.
Metabolomics is the scientific study of metabolites of an organism, cell, or tissue. Metabolomics makes use of different analytical approaches. In this thesis, an analytical platform consisting of proton nuclear magnetic resonance spectroscopy (1H-NMR), gas chromatography-mass spectrometry (GC-MS, EI/quadrupol) and liquid chromatography-mass spectrometry (LC-MS, ESI/TOF) was used for metabolite analysis. Due to the high physicochemical diversity of metabolites, the usage of different analytics is profitable. Focusing on metabolome analysis of microorganisms, the development of viable protocols was prerequisite. To ensure metabolome samples of best possible quality, particularly the sampling procedure has to be optimized for each microorganism to be analyzed individually. In microbial metabolomics, the energy charge value is a commonly used parameter to assure high sample quality (Atkinson 1968). The pathogenic bacterium Staphylococcus aureus and the biotechnical relevant bacterium Bacillus subtilis were main target of research. The sampling protocol development “A protocol for the investigation of the intracellular Staphylococcus aureus metabolome” (Meyer et al. 2010) and “Methodological approaches to help unravel the intracellular metabolome of Bacillus subtilis”s (Meyer et al. 2013) confirmed the need for development and verification of viable protocols. It was observed, that minor differences in the sampling procedure can cause major differences in sample quality. Using the validated analytical platform and the optimized protocols, we were able to investigate the metabolome of S. aureus and B. subtilis under different conditions. Investigations of the pathogenic bacterium S. aureus are of major interest due to its increasing resistance to antibiotics. Methicillin (multi)-resistant S. aureus (MRSA) strains are responsible for several difficult-to-treat infections. The cell wall of bacteria is the target of an array of antibiotics, like the beta-lactam antibiotics. Our study “A metabolomic view of Staphylococcus aureus and Its Ser/Thr kinase and phosphatase deletion mutants: Involvement in cell wall biosynthesis” (Liebeke et al. 2010) revealed the influence of the serine-threonine kinase on cell wall biosynthesis of S. aureus. LC-MS based metabolome data uncovered prevalent wall teichoic acid precursors in the serine-threonine kinase deletion mutant (ΔpknB), and predominantly peptidoglycan precursors in the phosphatase deletion mutant (Δstp), compared to the S. aureus wild type strain 8325. This uncovered a so far undescribed importance of the serine-threonine kinase on the cell wall metabolism and provides new insights into its regulation. The nasopharynx and the human skin are often the ecological niche of S. aureus. Furthermore, S. aureus exists outside its host, for example on catheters. Depending on its niche, S. aureus is exposed to several stress factors and limitation conditions, such as carbon source limitation and starvation. To cope with the latter, a number of regulatory cellular processes take place. In “Life and death of proteins: a case study of glucose-starved Staphylococcus aureus” (Michalik et al. 2012) protein degradation during glucose starvation was monitored. An intriguing observation was that proteins involved in branch chain amino acid biosynthesis and purine nucleotide biosynthesis were distinctly down-regulated in the clpP mutant. This lead to the assumption of a stronger repression of CodY-dependent genes in the clpP mutant. Intracellular metabolome data revealed higher GTP concentrations in the clpP mutant. This may explain the higher CodY activity and thereby stronger repression of CodY-dependent genes in the clpP mutant. Since different S. aureus strains are known to colonize different niches, global carbon source (glucose, glucose 6-phosphate, glycerol, lactate, lactose and a mixture of all) and carbon source limitation dependent exo-metabolome analyses were performed using three different S. aureus strains (HG001: laboratory strain, EN493: human endocarditis isolate and RF122: bovine mastitis strain). The most apparent observation was that RF122 can utilize lactose best, while EN493 and HG001 are better at utilizing glucose-6-phosphate compared to the bovine RF122 strain. Bacillus subtilis is an extensively studied Gram-positive and non-pathogenic bacterium. In the functional genomics approach “System-wide temporal proteomics profiling in glucose-starved Bacillus subtilis” (Otto et al. 2010) growth phase dependent changes in the proteome, transcriptome and extracellular metabolome were monitored. By mass spectrometric analysis of five different cellular subfractions, ~ 52% of the predicted proteins could be identified. To confirm and complete the proteomic data transcriptome and extracellular metabolome analyses were performed. The extracellular metabolome data ensured that cells were glucose-starved and revealed growth phase dependent metabolic footprints. In “A time resolved metabolomics study: The influence of different carbon sources during growth and starvation of Bacillus subtilis” ((Meyer et al. 2013) submitted) four different compounded cultivation media were investigated as only glucose, glucose and malate, glucose and fumarate and glucose and citrate as carbon source. It could be shown, that B. subtilis is able to maintain an intracellular metabolite homeostasis independent of the available carbon source. On the other hand, in the exo-metabolome, carbon source as well as growth phase dependent differences were detected. Furthermore, in this study the influence of ATP and GTP on the activation of the alternative RNA polymerase sigma factor B (σB) was discussed. The concentration of ATP and GTP decreased for all conditions, as cells entered the stationary growth phase. While cell growth on solely glucose and during growth on glucose and additional malate, the ATP and GTP concentrations increased slightly when the consumption of the second carbon source was initiated. Only under these conditions, a considerable σB activity increase during the transition from exponential to stationary growth phase was observed. Furthermore, the developed sampling protocol for metabolome analysis of B. subtilis enabled us to be part of a “multi omics” system biological approach to study the physiological adjustment of B. subtilis to cope with osmotic stress under chemostat conditions.
Gout was described by Hippocrates in the 5th century BC as a disease of rich people and linked with excess food and alcohol. It is caused by long-lasting hyperuricemia, which is a result of an imbalance between excretion and production of uric acid. The surplus of uric acid leads to deposition of monosodium urate crystals in the joints, which can initiate a painful inflammation called a gout attack. Despite various pharmacological treatments for this disease, a low purine diet remains the basis of all gout therapies. Since food is rich in purines, the aim of this project was to develop a novel enzyme system to decrease the purine content of food, what should result in reduced serum urate concentration in patients with hyperuricemia. The system consists of five degrading enzymes (adenine deaminase, guanine deaminase, xanthine oxidoreductase, urate oxidase and purine nucleoside phosphorylase) that combined in one product are able to hydrolyse all purines to a highly soluble allantoin, which can be easily removed from the body. This approach provides the patients a possibility to reduce the symptoms and frequency of gout attacks or even doses of prescribed drugs. In order to obtain necessary system components, yeast Arxula adeninivorans LS3 was screened for enzyme activities. A. adeninivorans is known to utilise various purines and this ability is a result of activity of desired enzymes, two of which, adenine deaminase and xanthine oxidoreductase, are in focus of this thesis. The analysis of growth of A. adeninivorans on various carbon and nitrogen sources gave the first insight into the cells’ nutrient preferences indicating the presence of purine degrading enzymes, such as adenine deaminase and xanthine oxidoreductase. Purines, such as adenine and hypoxanthine, could be utilised by this yeast as sole carbon and nitrogen sources and were shown to trigger the gene expression of the purine degradation pathway. Enzyme activity tests and quantitative real-time PCR method allowed for identification of the best inducers for adenine deaminase and xanthine oxidoreductase, as well as their concentration and time of induction. The adenine deaminase (AADA) and the xanthine oxidoreductase (AXOR) genes were isolated and subjected to homologous expression in A. adeninivorans cells using Xplor®2 transformation/expression platform. The selected transgenic strains accumulated the recombinant adenine deaminase in very high concentrations. The expression of AXOR gene posed difficulties and remained a challenge. Additional expression of both proteins in alternative E. coli system was undertaken but failed for AXOR gene. The recombinant adenine deaminase and wild-type xanthine oxidoreductase were purified and characterized biochemically. The characterization included determination of optimal pH and temperature, stability in different buffers and temperatures, molecular weight, substrate spectrum, enzyme activators and inhibitors, kinetics and intracellular localisation. The determination of these parameters was necessary to ensure optimal conditions for application of these enzymes in the industry. At the final stage, the enzymes were combined in one mix with provided guanine deaminase and urate oxidase and used to degrade purines in selected food constituents. The application was successful and demonstrated the potential of this approach for the production of food with lower purine concentration.
There is a growing interest in the application of non-thermal atmospheric pressure plasma for the treatment of wounds. Due to the generation of various ROS and RNS, UV radiation and electric fields plasma is a very promising tool which can stimulate skin and immune cells. However, not much is known about the mammalian cell responses after plasma treatments on a molecular level. The present work focusses on the impact of plasma on cell signaling in the human keratinocyte cell line HaCaT by using the methods DNA microarray, qPCR, ELISA and flow cytometry. Here, cell signaling mediators such as cytokines and growth factors which could promote wound healing by enhancing angiogenesis, reepithelization, migration and proliferation were of major interest. Additionally, the crosstalk between keratinocytes and monocytes was studied using a co-culture. For the first time extensive investigations on the impact of plasma on cell signaling in human keratinocytes were conducted. The most prominent cytokines and growth factors which were regulated by plasma at gene and protein level were VEGF-A, GM-CSF, HB-EGF, IL-8, and IL-6. The latter was not activated due to the JAK/STAT-pathway but probably by a combined activation of MAPK- and PI3K/Akt-pathways. By the use of conditioned medium it was found out that ROS and RNS generated directly after plasma treatment induced larger effects on cell signaling in keratinocytes than the subsequently secreted growth factors and cytokines. Furthermore, monocytes and keratinocytes hardly altered their secretion profiles in co-culture. From these results it is deduced that the plasma generated reactive species are the main actors during cell signaling. In order to differentiate the impact of ROS and RNS on the cellular response the ambience of the plasma effluent was controlled, varying the ambient gas composition from pure nitrogen to pure oxygen. Thereby a first step towards the attribution of the cellular response to specific plasma generated reactive species was achieved. While IL-6 expression correlated with ROS generated by the plasma source, the cell signaling mediators VEGF-A, GM-CSF and HB-EGF were significantly changed by RONS. Above all hydrogen peroxide was found to play a dominant role for observed cell responses. In summary, plasma activates wound healing related cell signaling mediators as cytokines and growth factors in keratinocytes. It was also shown that the generated reactive species mainly induced cell signaling. For the first time cell responses can be correlated to ROS and RONS in plasma treated cells. These results underline the potential of non-thermal atmospheric pressure plasma sources for their applications in wound treatment.
The thesis describes experimental results based on optical diagnostics of low- pressure discharges. The models, which are necessary for the interpretation of the experimental data, are developed and simulations are done. The contents can be categorized into the following topics: 1) the time-resolved tunable diode laser absorption spectroscopy of excited states of argon in pulsed magnetron discharge and modeling the plasma afterglow; 2) optical emission- and laser absorption spectroscopy of excited states of argon in radio-frequency (rf) discharge and calculation of the escape factor for self-absorption; 3) fast video recording of the oscillatory motion of a dust particle in rf discharge and analysis of the data.
Abstract: The Arctic has experienced a pronounced increase in air temperature over the last four decades, with an average increase of 0.4 °C per decade and thus an increase of almost the double rate than that of temperate regions. Remote sensing studies and repeat photography of historical images have shown large-scale increases of plant productivity in tundra ecosystems over the same time period. A pronounced size, abundance and biomass increase of shrubs has been observed. This so called shrub expansion has important repercussions for the vegetation, the animals, the soil, the energy and the carbon balance of the Arctic tundra and on regional and global climate. As the comparison of historical photographs with recent photographs has shown, this shrub expansion occurs on different temporal and spatial scales with areas of strong increase in shrub cover (expanding patches) and areas without noticeable changes in shrub vegetation (stable patches). While remote sensing approaches for the detection of changes in vegetation are limited in their temporal coverage and so far also in their resolution, historical photographs with high resolution are often not available. Experimental studies have shown that an increase in nutrients or temperature often resulted in increased shrub biomass, but findings were partly contradictory, referred to short term observations and usually confined to small areas. To bridge the gap between spatially limited plot-scale experiments and global large-scale assessment of plant productivity by satellite derived pictures, dendrochronology was used in this thesis to analyze the drivers for and the rate of shrub growth of different widespread evergreen and deciduous shrub species in alpine and arctic tundra and to reconstruct historic environmental conditions. In detail, this doctoral thesis was conducted to study shrub growth and to assess the applicability of traditional dendrochronological methods on shrubs that had been so far mainly applied to trees and to test whether shrubs differed morphologically from trees. Further, I was determined to look for evidence for a possible Scandinavian shrub range expansion and to assess which climatic factors – temperature, precipitation or snow – influenced shrub growth significantly. Moreover, we aimed to find the reason for the observed heterogeneity of the shrub expansion on the landscape and its relevance for the three most common shrubs on the Alaskan tundra. The methods applied followed the routines usually applied for dendrochronological analyses of treerings, with the exception that usually several stem discs of the main stem were analyzed and frequently had to be prepared with help of a microtome as thin-sections, that were stained and sealed on a coverglass before annual shrubrings were measured. The averaged shrubring widths were then compared with environmental factors through correlation and regression methods. This thesis gives first a general introduction to climate change in the Arctic, shrub expansion on the tundra, the scientific discipline of dendrochronology or -ecology on shrubs and its development, the main research questions and the thesis outline. Then seven research papers are presented and the main results and conclusions are synthesized and discussed and finally possible venues of future research are outlined. The most important insights gained from this thesis are the following: I) Dendroecological methods can be applied to shrubs. Insights into shrub morphology have been gained by detecting an interesting mechanism for coping with adverse environmental conditions of both, trees and shrubs that can save resources by confining the production of wood to the upper parts of the stem. II) Further, I found evidence for a shrub expansion in Scandinavia. III) I could establish the causal link between the current climate warming and increased radial and vertical shrub growth by identifying summer temperature as main driver for shrub growth. IV) Results from the Alaskan tundra indicate a strongly adverse role of snow for shrub growth in stable patches, refuting the popular snow-shrub-microbe hypothesis for this extensive area across species. The differing influence of snow is likely linked to the presence of permafrost and shallow active layers and the snow’s contribution to moist or even anoxic conditions in Alaska. V) Furthermore, we found that the different rates and the spatial heterogeneity of shrub expansion are accompanied by strong differences in the surrounding vegetation composition and the soil parameters of expanding (accustomed to more favorable conditions) and stable shrub patches. VI) These differences are predisposed by shrub patch position within the landscape, comprising different levels and rates of disturbance. VII) Additionally, shrub ring records were successfully used as natural archives to model past temperature dynamics respectively summer glacier mass balance with high accuracy. VIII) Finally, a synthesis of the climate-growth relationships of shrubs of more than 25 sites around the Arctic as joined effort together with other leading shrub researchers supports the presence of a circumpolar shrub expansion, gives recommendations for methods used in shrub dendroecology and lays out future research directions. The findings of my dissertation research show that the analysis of shrubs by dendroecological methods yields highly interesting results, and they greatly improved our understanding of factors that influence individual shrub growth, the reconstruction of earlier environmental conditions as well as the reconstruction and assessment of plant population dynamics.
Tourism is booming on global level since many decades and developing countries often capture tourism as a lever to push up their economy. Many regions in Vietnam invest lots of money in this development hoping to change the traditional economic structure. Located in the North Central Vietnam, about 170 km from Ha Noi capital to the south at the coastline, Sam Son is known as an ideal location for holiday makers. Currently the number of tourist has been counted to millions. Realizing its potential, the local authorities decided to enlarge and improve tourism to become the biggest tourism center in the North Central part of Vietnam. As many mass tourism destinations, Sam Son is facing several hidden drawbacks, which are generated by a (too) fast growth. Thats why unexpected problems may occur very soon. One path to mitigate weaknesses and to strengthen the positive impacts of tourism is to develop tourism in a more sustainable manner. As the very first step Sam Son needs to delineate a proper strategy and to establish a powerful "Destination Management System". Although several studies have been conducted already, the destination lacks reliable empirical data. This research intends to fill the gap by supplying concrete information with specific focus on “making tourism more sustainable”. In the study methodological triangulation has been applied to collect data and information. Besides literature review and secondary data analysis, two surveys - statistically well secured - have been conducted: A household survey with more than one thousand questionnaires to record the situation and opinion of local people and a visitor survey with 1139 face-to-face interviews to collect structural data about current guests in Sam Son as well as to explore their behaviour. Moreover expert and stakeholder interviews were included to supplement the information basis. As the research acquired the information directly from local people and from tourists by face-to-face interviews several weaknesses could be unveiled which official statistical data cannot show. There are three facets out of balance in terms of economic benefit: Compared with the political awareness and the financial support the tourism sector does not ensure the livelihood of an adequate number of people; the economic benefits from tourism are very unequally distributed and also the spatial dimension indicates a strong imbalance - only people living directly in the core area of tourism participate in tourism sector, the positive impact of tourism does not reach till the outskirts. In addition social and environmental problems are recognized by a majority of local people as consequences of tourism in the region. Regarding the touristic demand side a very low level of satisfaction has been detected: A small variety and low quality of touristic offers are the main reasons of dissatisfaction. The most exiting finding is that tourists treasure environment in the destination, whereas this aspect is underestimated in the locals view. In addition, tourists are really willing to pay for an improvement of environmental and touristic quality! Even if the amount of voluntary payments is very low, the total sum can enable the local government to develop the destination in a very proper way. Although this is a case study the results deliver valuable information and furthermore a concrete delineated roadmap for all destinations, which are in the same situation as Sam Son today. It is an example how to analyze and evaluate the present condition and how to continue in a proper way towards a more sustainable tourism development.
There is a wide variety of Alfvén waves in tokamak and stellarator plasmas. While most of them are damped, some of the global eigenmodes can be driven unstable when they interact with energetic particles. By coupling the MHD code CKA with the gyrokinetic code EUTERPE, a hybrid kinetic-MHD model is created to describe this wave–particle interaction in stellarator geometry. In this thesis, the CKA-EUTERPE code package is presented. This numerical tool can be used for linear perturbative stability analysis of Alfvén waves in the presence of energetic particles. The equations for the hybrid model are based on the gyrokinetic equations. The fast particles are described with linearized gyrokinetic equations. The reduced MHD equations are derived by taking velocity moments of the gyrokinetic equations. An equation for describing the Alfvén waves is derived by combining the reduced MHD equations. The Alfvén wave equation can retain kinetic corrections. Considering the energy transfer between the particles and the waves, the stability of the waves can be calculated. Numerically, the Alfvén waves are calculated using the CKA code. The equations are solved as an eigenvalue problem to determine the frequency spectrum and the mode structure of the waves. The results of the MHD model are in good agreement with other sophisticated MHD codes. CKA results are shown for a JET and a W7-AS example. The linear version of the EUTERPE code is used to study the motion of energetic particles in the wavefield with fixed spatial structure, and harmonic oscillations in time. In EUTERPE, the gyrokinetic equations are discretized with a PIC scheme using the delta-f method, and both full orbit width and finite Larmor radius effects are included. The code is modified to be able to use the wavefield calculated externally by CKA. Different slowing-down distribution functions are also implemented. The work done by the electric field on the particles is measured to calculate the energy transfer between the particles and the wave and from that the growth rate is determined. The advantage of this approach is that the full magnetic geometry is retained without any limiting assumptions on guiding center orbits. Extensive benchmarks have been performed to test the new CKA-EUTERPE code. Three tokamak benchmarks are presented, where the stability of TAE modes are studied as a function of fast particle energy, or in one case as a function of the fast particle charge. The benchmarks show good agreement with other codes. Stellarator calculations were performed for Wendelstein 7-AS and the results demonstrate that the finite orbit width effects tend to be strongly stabilizing.
The confinement of energy has always been a challenge in magnetic confinement fusion devices. Due to their toroidal shape there exist regions of high and low magnetic field, so that the particles are divided into two classes - trapped ones that are periodically reflected in regions of high magnetic field with a characteristic frequency, and passing particles, whose parallel velocity is high enough that they largely follow a magnetic field line around the torus without being reflected. The radial drift that a particle experiences due to the field inhomogeneity depends strongly on its position, and the net drift therefore depends on the path taken by the particle. While the radial drift is close to zero for passing particles, trapped particles experience a finite radial net drift and are therefore lost in classical stellarators. These losses are described by the so-called neoclassical transport theory. Recent optimised stellarator geometries, however, in which the trapped particles precess around the torus poloidally and do not experience any net drift, promise to reduce the neoclassical transport down to the level of tokamaks. In these optimised stellarators, the neoclassical transport becomes small enough so that turbulent transport may limit the confinement instead. The turbulence is driven by small-scale-instabilities, which tap the free energy of density or temperature gradients in the plasma. Some of these instabilities are driven by the trapped particles and therefore depend strongly on the magnetic geometry, so the question arises how the optimisation affects the stability. In this thesis, collisionless electrostatic microinstabilities are studied both analytically and numerically. Magnetic configurations where the action integral of trapped-particle bounce motion, J, only depends on the radial position in the plasma and where its maximum is in the plasma centre, so-called maximum-J configurations, are of special interest. This condition can be achieved approximately in quasi-isodynamic stellarators, for example Wendelstein 7-X. In such configurations the precessional drift of the trapped particles is in the opposite direction from the direction of propagation of drift waves. Instabilities that are driven by the trapped particles usually rely on a resonance between these two frequencies. Here it is shown analytically by analysing the electrostatic energy transfer between the particles and the instability that, thanks to the absence of the resonance, a particle species draws energy from the mode if the frequency of the mode is well below the charateristic bounce frequency. Due to the low electron mass and the fast bounce motion, electrons are almost always found to be stabilising. Most of the trapped-particle instabilities are therefore predicted to be absent in maximum- J configurations in large parts of parameter space. Analytical theory thus predicts enhanced linear stability of trapped-particle modes in quasi-isodynamic stellarators compared with tokamaks. Moreover, since the electrons are expected to be stabilising, or at least less destabilising, for all instabilities whose frequency lies below the trapped-electron bounce frequency, other modes might benefit from the enhanced stability as well. In reality, however, stellarators are never perfectly quasi-isodynamic, and the question thus arises whether they still benefit from enhanced stability. Here the stability properties of Wendelstein 7-X and a more quasi-isodynamic configuration, QIPC, are investigated numerically and compared with another, non-quasiisodynamic stellarator, the National Compact Stellarator Experiment (NCSX) and a typical tokamak. In gyrokinetic simulations, performed with the gyrokinetic code GENE in the electrostatic and collisionless approximation, several microinstabilities, driven by the density as well as both ion and electron temperature gradients, are studied. Wendelstein 7-X and QIPC exhibit significantly reduced growth rates for all simulations that include kinetic electrons, and the latter are indeed found to be stabilising when the electrostatic energy transfer is analysed. In contrast, if only the ions are treated kinetically but the electrons are taken to be in thermodynamic equilibrium, no such stabilising effect is observed. These results suggest that imperfectly optimised stellarators can retain most of the stabilising properties predicted for perfect maximum-J configurations. Quasi-isodynamic stellarators, in addition to having reduced neoclassical transport, might therefore also show reduced turbulent transport, at least in certain regions of parameter space.
The development of innovative coatings with multifunctional properties is an ambitious task in modification of material surfaces. A novel approach is a hybrid method combining the non-thermal plasma processing with nanotechnology for the development of multifunctional surface coatings. The conception of the hybrid coating process is based on three steps: the preparation of a suspension consisting of an organic liquid and functional nanoparticles, the deposition of the suspension as a thin liquid film on the material surface, and the plasma modification of the liquid organic film to achieve a thin solid composite film with embedded nanoparticles demonstrating multifunctional properties and good adherence on the substrate material. In this work the liquid polydimethylsiloxane (PDMS) was applied as a model system, and the experimental investigations were focused on the PDMS plasma modification. In particular, the specific role of the different plasma components and the influence of the plasma and processing parameters on the PDMS modification were studied. The applied capacitively coupled radio frequency (CCRF) plasma was analyzed by electric probe measurements and optical emission spectroscopy, whereas the molecular changes in PDMS due to plasma-induced chemical reactions were studied by the Fourier transform infrared reflection absorption spectroscopy. Additionally, the photocatalytic activity of thin composite films consisting of plasma cross-linked PDMS with embedded TiO2 nanoparticles was demonstrated. During the investigation it was found that the CCRF discharge modifies efficiently thin liquid PDMS films to solid coatings. The samples were positioned in the plasma bulk at floating potential. The penetration depth of particles like neutrals, ions, electrons and radicals in the film is strongly limited. The heating of samples in the CCRF discharge is weak to modify PDMS by itself and only the plasma radiation is able to transform the liquid bulk to solid one. It is known that the absorption onset of PDMS lies in the VUV region (below 200 nm). The energetic VUV radiation penetrates into the PDMS film on a thickness from several hundred nanometers to few micrometers and initiates photochemical reactions there. Thus, different gases like Ar, Xe, O2, H2O, air and H2 were tested to provide the strongest VUV emission intensity of the CCRF discharge. Discharge pressure and power were varied for all these gases and it was found that at all conditions the H2 plasma demonstrates drastically stronger emission. Thus, H2 gas was selected for the plasma treatment of liquid PDMS films. The IRRAS analysis revealed the transformation process of PDMS with the degradation of CH3 groups, the formation of new groups like SiOH, CH2 and SiH, the formation of the SiOx material and crosslinking. It was found that the modification effect is not uniform across the film thickness. The top region with an initial thickness up to 100 nm loses all CH3 groups, in the underlying region the CH3 concentration increases gradually from zero to the value for PDMS, if the film was thick enough. The methyl-free SiOx top layer contains also SiOH and SiH groups. Furthermore, the SiH groups are concentrated only in a very thin layer with a thickness below 10 nm. The presence of the unscreened polar SiOSi and SiOH groups on the surface causes the adsorption of H2O from the atmosphere, which was also observed by IRRAS. By means of the spectroscopic ellipsometry it was found out that all above described regions experience a shrinking. The reason is the crosslinking and loss of material. The most shrunken layer is the top SiOx layer with the shrinking ratio (final thickness/initial thickness) of 0.55 - 0.60. Further, this ratio gradually rise up to the value of 0.95 in the deeper region, which has the concentration of CH3 groups of about that for PDMS. After the analysis of all results the depth of effective modification was estimated at 300 400 nm for the most optimal conditions. The optimization of the plasma VUV intensity was realized by variation of discharge pressure and power. The strongest plasma emission at studied conditions provided the irradiance of the sample of ca. 13 mW/cm2. However, such strong radiation causes very strong production rate of the gases. These products leave the modifying film slower as they are produced, what causes their accumulation in there. Their pressure grows up leading to formation of bubbles, which later explode. Finally, the film becomes heavily damaged. To avoid this effect the pressure and the RF power were changed to reduce the irradiance to 6 - 7 mW/cm2. This resulted in the absence of any damages.
The investigated bacterial strain 64G3 was isolated from an offshore oil reservoir in Vung Tau, Vietnam. By means of 16S rDNA sequence alignment and DNA-DNA hybridization with Petrotoga mexicana DSM 14811, the isolate was identified as Petrotoga mexicana species. Morphologically, the 64G3 cells were rod-shaped and cell sizes varied widely from 1.0 µm up to 60 µm in length and from 0.6 to 1.2 µm in width. The cells appeared single, pairwise or in chains within a sheath-like structure (a typical characteristic of the order Thermotogales) that ballooned over the cell ends. Cells were immobile and no flagella were observed. Strain 64G3 grew anaerobically at temperatures ranging from 30 to 65°C and within the pH range of 5.0 to 8.5 with optimum growth at 55°C and the pH 7.0. Elemental sulfur and thiosulfate served as alternative electron acceptors whereas sulfate did not. Cellular extract of strain 64G3 grown in a basal medium containing soluble starch displayed hydrolytic activity towards soluble starch. The amylase system includes at least two individual enzymes. Amylase activity of the cell extract was detected in a wide temperature range (30-80°C), with optimal enzyme activity at 75°C. By using degenerate primer for PCR amplification of GH13 enzyme coding regions in combination with other molecular methods, a full amylase coding gene containing four conserved regions of α-amylase was obtained. The deduced sequence showed low identities (up to 40%) to other known amylases. This 1992 bp coding gene was heterologously expressed in E. coli and its product (amylase) was characterized. Under common expression conditions, the 77 kDa amylase (rAmyA) was predominantly produced as inclusion bodies (insoluble protein). The minor amount of soluble active amylase was used for purification and characterization of the enzyme. rAmyA was active on starch at temperatures between 30-55°C, with an optimum at 45oC. It is not thermostable because it was completely inactive after incubation at 65°C for 15 min. The enzyme was active over a pH range from 4.5-8.0, with an optimum at pH 6.5. Beside starch, rAmyA also hydrolysed glycogen, amylose, amylopectin and other oligosaccharides. Pullulan and cyclodextrins were not the substrates for this amylase. The enzyme hydrolyzed starch in an endo-acting manner, releasing maltose and maltotriose as major products and a lesser amount of glucose. On the basis of the primary structure, the substrate specificities and the hydrolysis pattern, rAmyA was classified as an endo-acting α-amylase (EC. 3.2.1.1). The cpn10/60 operon from psychrophilic O. antarctica was cloned and expressed in B. subtilis using a multi-copy plasmid. The amounts of soluble 60 kDa Cpn60 and 10 kDa Cpn10 produced at temperature ranging from 10 - 30°C were high and stable during cell growth. To investigate the impact of psychrophilic chaperonin on cold adaptation, cells with (cpn+) and without (cpn-) cpn10/60 operon were grown at 10 and 15°C. Growth comparison between two strains revealed that psychrophilic chaperonin did not support cold adaptation of B. subtilis at 10 and 15°C as it did in E. coli. A single copy of O. antarctica cpn10/60 operon was integrated into the amyE locus of the B. subtilis chromosome. The yeast α-glucosidase, a theoretic protein substrate for this chaperonin, was heterologously produced in B. subtilis at temperatures ranging from 15-30°C. Within this temperature range, the major amount of this protein appeared as inclusion bodies. Co-expression of O. antarctica cpn10/60 operon at 15°C, however, did not result in a higher activity of glucosidase. Moreover, SDS-PAGE analysis of cellular insoluble fractions revealed that the amount of insoluble enzyme produced in cpn+ cells did not decrease in comparison with that produced in cpn- cells, indicating that the recombinant chaperonin had no impact on recovery of active α-glucosidase from the inclusion bodies.
In many industrial sectors biotechnological production processes have replaced pure chemical methods and allowed new, ecologically friendly and enzyme-based processes. Microorganisms, such as modified Bacillus strains are used in particular for the industrial enzyme synthesis. The two organisms Bacillus licheniformis and Bacillus pumilus are of great industrial importance. B. licheniformis is able to secrete proteins in large amounts, while B. pumilus shows high resistance to oxidative stress. During production processes different conditions can occur that affect the physiology of the production hosts and may result in a quantitative, but also a qualitative impairment of the products. This influence is based on e.g. chemical processes, the setting of temperature, pH, or oxygen availability and can lead to various stress situations for the bacteria. Cells respond to changes in their environment by sensing stressors and initiate a response to the stress, which is usually implemented by an induction or derepression of various regulons. In order to conduct an optimal production process, the metabolism and stress responses of the utilized bacteria should be known exactly. The aim of this study was to analyze of the stress response of B. licheniformis to heat and salt stress, and the stress response of B. licheniformis and B. pumilus to oxidative stress. These analyses were performed at the level of transcriptomics using cDNA microarrays, which is the most direct and global method for the analysis of changes in the physiology of a cell. The identification of stress specific markers genes and their differentiation from the SigB regulated general stress response has been another purpose of this work. Knowledge of these marker genes enables a prompt analysis of the fermentation conditions and thus a possible optimization of the process. The transcriptome analyses of this work show that B. licheniformis responds to heat stress by the induction of heat shock genes belonging to different regulons. These include the htpG gene, the HrcA regulon or the CtsR regulon, encoding chaperones and proteases, which mainly contribute to the protein quality control. The heat stress response of B. licheniformis revealed no fundamental differences to the heat stress response of the Gram-positive model organism Bacillus subtilis. The general stress response (SigB regulon), which is activated by heat stress, could be analyzed in more detail by the study of a ΔsigB mutant of B. licheniformis. Salt stress also provokes a strong induction of the general stress response in B. licheniformis. Genes for the transport and synthesis of compatible solutes were strongly induced, as well as several genes for transport systems with more or less known functions. The synthesis of the osmoprotective metabolites proline and glycine betaine could be verified in more detail by a metabolomics approach. The response to oxidative stress showed differences between both B. licheniformis and B. pumilus, and also to the oxidative stress response of B. subtilis. In B. licheniformis, the genes of the glyoxylate cycle are induced during oxidative stress. An activation of the glyoxylate bypass under oxidative conditions could be confirmed by a metabolome analysis of B. licheniformis. In addition, the PerR regulon of B. licheniformis is extended to include another two genes compared to B. subtilis. In contrast, several genes of the PerR regulon lack in the genome of B. pumilus, such as katA (vegetative catalase) or ahpCF (alkyl hydroperoxide reductase). However, other genes were induced in B. pumilus that were upregulated under oxidative stress conditions neither in B. subtilis nor in B. licheniformis. In addition, known regulons, regulated by e.g. Spx, CtsR or SOS were induced in both organisms. In summary, this dissertation transcriptionally analyzes the stress responses of B. licheniformis to heat, salt and oxidative stress, and in addition the oxidative stress response of B. pumilus. Several stress-specific regulons were identified in both, B. pumilus and B. licheniformis, which also correspond to the stress response of B. subtilis. However, it was possible to additionally assign genes to the stress specific responses of both organisms and to find differences, such as the absence of parts of the PerR regulon of B. pumilus, or the activation of the glyoxylate pathway in B. licheniformis during oxidative stress.
The aim of this work is to further analyze the nature of the TiO2 passivation layer regarding structure, hydrophilicity and adsorption behavior, starting with the question how far metal and oxide properties are affected by the contact, regarding structural relaxation, atomic charges and work function. This determines how far the influence of metal has to be considered in simulations of TiO2 passivation layers. Mimicking the initial phases of implant contact with the biological environment, the adsorption of the inorganic ions on titanium oxides is to be investigated next, especially the influence of Ca2+ and HnPO4n-3 on the surface properties. Finally, biomolecule adsorption on TiO2 surfaces is investigated for understanding and improving their bioactivity.
Titanium and Titanium Dioxide
The properties of sharp interfaces formed between metallic titanium and a titanium dioxide layer with rutile or anatase structure and four different surface terminations were investigated. In all cases the work of separation is higher than the sum of surface energies, indicating the formation of an energetically very favorable interface region that glues the two phases together. The interface energy is negative, which means that for Ti and TiO2 bulk phases, mixing is energetically favorable.
The influence of the metal on the atomic and electronic structure of the oxide is limited to a few atomic layers. Depending on its modification, a passivation layer may give rise to up- (rutile) or downshift (anatase) of the work function of the underlying titanium metal.
Calcium and Phosphate
First principles molecular dynamics simulations in vacuum revealed stable bonds between Ca2+ and HnPO4n-3 ions and the investigated TiO2 surfaces. Ca2+ ions bind to 2–4 surface oxygen atoms, preferring peripheral positions as found on both rutile surfaces where adsorption energies reach 9 eV per ion. In solution the hydration energy drastically reduces these values.
Phosphate adsorbs to the TiO2 surface, but the adsorption energy is much lower than that of Ca2+ ions. The approach of phosphate is highly orientation dependent and hampered by the terminal oxygen atoms.
Both ab initio and force field simulations indicate enrichment of Ca2+ ions close to the surface, most of them directly bound to it, which results in a net positive charge. As the adsorption of phosphate takes longer and is strongly reinforced by adsorbed Ca2+ ions, it has become obvious that Ca2+ ions initiate the adsorption of calcium phosphate clusters to titania surfaces. However, the TiO2 surface does not necessarily act as a nucleation site for calcium phosphate crystallization, as adsorbed Ca2+ ions show reduced affinity towards phosphate compared to free ions in solution.
Collagen and Mechanical Stress
Coinciding force distance relations have been obtained for a variety of restraint force constants, expansion rates and environments. The resulting Young’s moduli are in the range of experimental values both at low and high strain ranges. For low strains the calculated Young’s modulus of about 2 GPa is comparable to experimental values between 3 and 5 GPa. For high strains it reaches 10 GPa. The Young’s moduli can be assigned to three different mechanisms of stretching, affecting the macroscopic linearity, the torsional angles and the bond lengths.
Chondroitin Sulfate (CS) and Hyaluronic Acid (HA)
A force field model for CS and HA could be established that reproduced experimental torsion angles and showed the same free energy surface (FES) as an ab initio model. Hydration affects the overall FES, but does not alter the position of the energetic minima. Stabilization of the conformation via bridging water molecules as suggested by other works is not necessary.
Both glycosaminoglycans adsorb to a hydroxylated rutile (100) surfaces despite the negative net charge both on surface and adsorbate. The presence of Na + ions is enough to compensate for the negative surface charge and to allow for adsorption. Ca2+ ions form additional bridges between negative groups on the surface and in the adsorbate.
Background: Hepatitis E virus (HEV) is the etiological agent of an acute self-limiting hepatitis in humans worldwide. The main route of infection is by ingestion of food or water contaminated with the virus. In Germany, several hundred human cases are reported each year, while preliminary studies suggest a high infestation rate of herds of domestic pig (Sus scrofa domesticus) and sounders of wild boar (Sus scrofa). Autochthonous cases are originating mainly from zoonotic transmission from domestic pig and wild boar, but other animals may also be involved. Recently, a novel strain of HEV (ratHEV) had been found in Norway rats (Rattus norvegicus) in Germany, that could contribute to human epidemiology. Therefore, the aim of this study was to assess the seroprevalence of both HEV and the novel ratHEV in human, domestic pig and rat. For each of the three mammal species, an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was established, that based on an Escherichia coli-expressed carboxy-terminal segment (GT3-Ctr, amino acid (aa) 326–608) of the capsid protein of the autochthonous genotype 3 (GT3), derived from a wild boar from Germany. In parallel, a segment from ratHEV homologous to GT3-Ctr was also expressed in E. coli (ratHEV-Ctr, aa315–599) and was used in the ELISA. Hence, the established tests detect antibodies directed against HEV GT3 when using GT3-Ctr as antigen and ratHEV when using ratHEV-Ctr. Results: The GT3-based in-house human IgG test was validated using a commercial assay and showed high specificity and sensitivity. The average human population (represented by a panel of blood donors from Berlin and Brandenburg) reached a seroprevalence of 12.3% (37/301) with the in-house ELISA. A panel of forestry workers from Brandenburg had an even higher seroprevalence of 21.4% (119/555). Furthermore, ratHEV-specific antibodies could be detected in several sera of forestry workers. The novel ratHEV-based rat IgG ELISA could not be compared to similar tests, however, parallel testing with GT3-Ctr and statistical inference allowed conclusion of a seroprevalence. Rats trapped from several sites in Germany had an overall seroprevalence of 24.5% (36/147). The sera were reactive exclusively with ratHEV-Ctr. As with the in-house ELISA for human sera, the porcine IgG test was validated using a commercial assay, yielding high specificity and sensitivity. A panel of domestic pigs from ten federal states of Germany showed a seroprevalence of 42.7% (383/898) when tested with the in-house ELISA. Reactivity with ratHEV was present, but seemed to be caused mostly by cross-reactivity to GT3-Ctr. Conclusion: The HEV seroprevalence observed for human sera of the average population of Germany is among the highest in Europe and has been confirmed recently by other authors. The high seroprevalence found in forestry workers suggests that they should be counted as a risk group for HEV infection. Populations of rats have been shown to be infested heavily with ratHEV, as rats from all trapping sites situated within cities had a high prevalence for ratHEV exclusively and no serum reacted exclusively with GT3-Ctr. Seroprevalence in domestic pigs was demonstrated to be distributed evenly across federal states and districts. However, a vast difference of infestation could be detected in different herds, suggesting either differences in husbandry conditions, or an external source of infection that acts locally only. The rare but exclusive reactivity of human sera with ratHEV as well as the high cross-reactivity of swine sera with ratHEV suggests that viral strains other than the ones already known may contribute to cases of hepatitis E.
The LINC (Linker of Nucleo- and Cytoskeleton) complex is an evolutionarily conserved complex of nuclear envelope (NE) proteins that forms a direct connection between the nucleoskeleton and cytoskeleton. Primarily, members of two protein families form the complex: SUN and giant nesprin isoforms that reside in the inner and outer nuclear membrane, respectively, thus forming a “bridge” across the NE. Lamin A/C and emerin are additional LINC complex components. Mutations in the genes encoding the LINC complex components have been associated with at least a dozen diseases, the majority of them muscular diseases. Emery-Dreifuss muscular dystrophy (EDMD), an inherited neuromuscular disorder with variable clinical presentation, is one of these diseases. But only around 46% of all EDMD patients are linked to a disease allele. Except of SUN1 and SUN2 all known LINC complex components had been associated with EDMD. Following a functional candidate gene approach the SUN1 and SUN2 genes were sequenced in a cohort of pseudoanonymized 175 EDMD patients without a known mutation and 70 patients with known causative mutations in other LINC components. Based on these results the pathomechanism causing the phenotype in patients with SUN1 or SUN2 mutations was investigated. Autosomal recessive inheritance was observed in one patient with compound heterozygous SUN1 mutations. Patient myoblasts showed defective protein interactions within the LINC complex, altered mRNA expression levels of some LINC components, an enhanced differentiation rate and defects in myonuclear organization. This provides first insights into a new pathomechanism based on weakening of the LINC complex and resulting in disruption of myonuclear alignment. In six patients with known EMD or LMNA mutations additional heterozygous SUN1 or SUN2 mutations modifying the disease have been identified, causing a significantly more severe course. Thus the modifying effect of SUN mutations found in the present study helps to explain the clinical intra- and interfamilial variability observed in EDMD. Further evidence for the influence of mutations in LINC complex components on the molecular pathology of muscular dystrophies comes from a study on primary fibroblasts of a patient suffering Duchenne muscular dystrophy (DMD) and of a patient showing signs of EDMD and Charcot-Marie-Tooth syndrome (CMT). The DMD patient had apart from a mutation in the DMD gene two variants in genes encoding the LINC components nesprin 1α2 and SUN1. The EDMD/CMT patient carried two variants in nesprin 1α1 and SUN2. Fibroblasts of both patients showed changes in cell adhesion, cell migration, senescence and stress response as well as characteristics typical for laminopathies like changes in nuclear shape and NE composition. Mutations in genes encoding LINC complex proteins are also associated with a number of other diseases. Pleiotropic LMNA mutations have also been linked with progeroid syndromes – genetic diseases that mimic clinical and molecular features of aging including Hutchinson Gilford progeria syndrome (HGPS), mandibuloacral dysplasia (MAD), restrictive dermopathy (RD) and atypical Werner’s syndrome (aWS) as well as a couple of overlapping phenotypes. MAD and RD can also be caused by mutations in ZMPSTE24, a gene encoding the ZMPSTE24 metalloproteinase necessary for the processing of prelamin A to mature lamin A. It is expected that insights into the pathomechanism of this group of diseases might provide clues to normal aging process. Analyzing RD and MAD patients for mutations in the ZMPSTE24 gene, some novel mutations have been identified. Based on this results and a review of the literature regarding ZMPSTE24 mutations could be shown that all mutations involved in RD are null mutations, whereas all patients with MAD are compound heterozygotes carrying one loss-of-function mutation and one missense mutation. This shows a clear genotype-phenotype correlation. A further part of this work is the description of the molecular genetics and functional background of a rare, unclassified progeroid syndrome. The clinical course of the affected patient appeared as an accelerated HGPS finally ending up in a delayed RD with overlapping clinical features of MAD. Mutational analysis revealed a homozygous LMNA mutation caused by a partial uniparental disomy of chromosome 1. Immunohistological analyses of tissue samples taken at the beginning and the end of the disease course showed a decreasing amount of lamin A and increasing amounts of DNA double strand breaks. Functional analysis in transfected human normal fibroblasts showed an impaired ability of the mutant lamin A to recruit 53BP1, a component of the DNA repair pathway, to damaged DNA sites. This case provides the first evidence of human lamin A direct involvement in DNA repair and that increased DNA damage is a major pathophysiological factor in progeroid laminopathies.
The absolute density of the metastable N2(A,v=0) molecule was extensively studied in nitrogen barrier discharges at 500 mbar. For the detection of the metastables laser-induced fluorescence spectroscopy (LIF) was used, at which for the calibration of the absoute metastables density a comparison with Rayleigh scattering was performed. To get the ratio of the LIF signal to the Rayleigh signal it is shown that the LIF signal is the convolution of the Rayleigh signal with an exponential decay. Besides, the different cross sections are calculated and the ratio of the detection sensitivities at the laser and fluorescence wavelength is determined. As a first step on the way to atmospheric pressure barrier discharges, the laser-induced fluorescence spectroscopy was implemented in low pressure capacitively coupled radio-frequency discharges. The determined metastables density in the capacitively coupled radio-frequency discharge is somewhat below 10^12 cm^(-3) at 40 Pa and somewhat below 10^13 cm^(-3) at 1000 Pa. The axial density profiles show a nearly symmetric shape due to the long lifetime of the metastable state. At a pressure of 500 mbar the two discharge modes of the barrier discharge, the filamentary and the diffuse mode, were analysed. The filamentary mode was mainly investigated in an asymmetric discharge configuration. Typical densities in the detection volume are in the range of 10^13 cm^(-3), resulting in maximal densities of up to 10^15 cm^(-3) in the microdischarge channel. Such large densities are in agreement with the fast decay by the pooling reaction after the maximum of the metastables density in the afterglow of the discharge pulse. The time dependent measurements in the afterglow of single microdischarges offer a delay of the metastables production with respect to the discharge current. This delay indicates that the metastables production takes place mostly by cascades from higher triplet states, which are in turn excited by electron impact. The axial density profiles show a maximum in metastables density in front of the anode in agreement with optical emission spectroscopy, but which cannot be clearly identified because of the asymmetric discharge configuration. The measurements for the diffuse discharge mode were performed in a symmetric discharge configuration. The metastables density is in the range of 10^13 cm^(-3). It increases during the current pulse of the discharge and decays afterwards. The maximum of the metastables density is delayed with respect to the maximum of the discharge current. The depletion of metastables in the early discharge afterglow is dominated by the pooling reaction, afterwards quenching by nitrogen atoms becomes important assuming a nitrogen atom density in the order of 10^14 cm^(-3). As for the filamentary mode, the losses by diffusion are negligible for the measurement positions. The measured axial density profiles show an accumulation of metastables in front of the anode, whereas the density in front of the cathode is below the detection limit. To calculate the metastables current density to the dielectrics after the discharge pulse a simulation is developed including the dominant volume processes for the depletion of metastables and the axial diffusion. Starting point for the simulation is the axial metastables density distribution at the end of the discharge pulse. The calculated metastables current density at the dielectrics is in the range of 10^14 cm^(-2)s^(-1). With the use of recently calculated secondary electron emission coefficients a comparison of the secondary electron emission by metastables with the discharge current is done. It is figured out that the secondary electron emission current is large enough to be important during the discharge ignition. To expand the simulation to the whole voltage cycle, the excitation of metastables is assumed to be proportional to the discharge current and electron density. Using this model, the measured time dependences of the metastables density are well reproduced for the investigated parameter variations. This is not the case for the axial profiles, where a metastables loss process is missed to explain the formation of a density plateau in front of the anode during the discharge pulse. The intended calculation of the metastables current density shows that the delay of the metastables production with respect to the discharge current might be responsible for the ignition of microdischarges at the beginning of the discharge pulse.
In terms of climate change and climate change mitigation, the quantitative knowledge of global carbon pools is important information. On the one hand, knowledge on the amount of carbon cycling among – and stored in – global pools (i.e. Atmosphere, Biosphere, Cryosphere, Hydrosphere, and Lithosphere) may improve the reliability of models predicting atmospheric CO2 concentrations in terms of fossil fuel combustion. On the other hand, the carbon sequestration potential of specific ecosystems allows for estimating their feasibility regarding carbon trade mechanisms such as the Clean Development Mechanism or the Reducing Emissions from Deforestation and Degradation Program (REDD+). However, up to date, the majority of terrestrial carbon assessments have focused on forests and peatlands, leaving a data gap open regarding the remaining ecosystems. This data gap is likely to be explained by the relatively high carbon densities and/or productivities of forests and peatlands. Nevertheless, to get a precise as possible global picture, information on carbon pools and sequestration of other ecosystems is needed. Although desert ecosystems generally express low carbon densities, they may absolutely store a remarkable amount of carbon due to their large areal extent. In this context, Central Asian Deserts (in particular within the Turanian Deserts, i.e. Karakum, Kysylkum, Muyunkum) likely inhibit comparably high carbon pools as they express a sparse vegetation cover due to an exceptionally high annual precipitation if compared to the World’s deserts. In this dissertation, three important woody plant species – Populus euphratica and Haloxylon aphyllum and Haloxylon persicum – of Central Asian Deserts were investigated for their carbon pools and carbon sequestration potential. These species were chosen as they I) locally express high carbon densities, II) are dominant species, III) have a rather large spatial distribution, and IV) have experienced a strong degradation throughout the 20th century. Thus, they likely show a remarkable potential for carbon re-sequestration through restoration and thus for an application of carbon trade mechanisms (CHAPTER I). P. euphratica was investigated in the nature reserve Kabakly at the Amu Darya, Turkmenistan and in Iminqak at the Tarim He, Xinjiang, China. The assessment of Haloxylon species was restricted to the Turanian deserts west of the Tain Shan. To achieve a first scientific basis for large scale estimates, different methodologies, ranging from allometric formulas, over dendrochronology to remote sensing were combined (CHAPTERS II-V). In CHAPTER II allometric formulas were successfully developed for Haloxylon aphyllum and Haloxylon persicum and applied to six study sites distributed over the Turanian Deserts to represent the allometric variability of Haloxylon species in Central Asia. CHAPTER III derives another allometric formula (only based on canopy area) for H. aphyllum and combines it with a remote sensing analysis from the nature reserve Repetek. Thereby, a first large scale estimate covering the Northeastern Karakum Desert of carbon pools related to mono specific H. aphyllum stands is achieved. CHAPTER IV describes the wood structure of Populus euphratica forests in the nature reserve Kabakly (Turkmenistan) and in Iminqak (Xinjiang, China). In CHAPTER V a dendrochronological approach derives models for predicting the Net Primary Productivity (NPP) and the age of P. euphratica in the nature reserve Kabakly. Thereby, a first feasibility assessment regarding remote sensing analyses and the upscaling of the obtained NPP results is carried out. First estimates based on these local studies (CHAPTER VI), reveal carbon densities ranging from 0.1 – 26.3 t C ha 1 for the three investigated species. Highest maximum and median carbon densities were found for P. euphratica, but Haloxylon aphyllum expressed remarkable maximum carbon densities (13.1 t C ha-1), too. The total carbon pools were estimated at 6480 kt C for P. euphratica, 520 kt C for H. aphyllum stands and 6900 kt C for Haloxylon persicum shrubland. Accounting for the extent of degraded areas, the total re-sequestration potentials of the respective species were estimated at 4320 kt C, 1620 kt C and 21900 kt C, this highlighting the remarkable absolute re-sequestration potential of H. persicum shrubland despite its low average carbon densities. In the end, the main results were put into a broader context (CHAPTER VI), discussing the general feasibility of reforestations both in ecological terms as well as in terms of carbon trade mechanisms. A short example highlights the strong connection between the feasibility of reforestations and the global carbon market. Finally, open research questions are brought forth revealing the yet large research potential of Central Asian Desert ecosystems in general and in terms of carbon sequestration.
Aim: To evaluate the association of Insulin-like Growth Factor (IGF) I related variables with periodontitis in the population-based Study of Health in Pomerania (SHIP). Material and Methods: From the cross-sectional SHIP, 2293 subjects with clinical attachment loss (CAL) data and 2398 subjects with tooth count data aged 20-59 years were analysed. Serum IGF-I and IGF binding protein (BP)-3 levels were determined by chemiluminescence immunoassays. Linear and logistic regressions with fractional polynomials were used to study associations between IGF-related variables and mean CAL or high tooth loss. For non-linear relations between IGFBP-3 and mean CAL, graphical presentations of fractional polynomials were used to deduce knots for linear splines. Results: In fully adjusted models, for serum IGFBP-3 values ≤1200 ng/mL, mean CAL increased significantly for decreasing serum IGFBP-3 levels (B=-0.027 (95% CI, -0.049; -0.005), p=0.02). The odds for high tooth loss decreased significantly for high serum IGFBP-3 values (OR=0.97 (0.95; 0.99), p=0.02). Serum IGF-I levels and the IGF-I/IGFBP-3 ratio were not related to mean CAL or tooth loss after full adjustment. Conclusions: Low serum IGFBP-3 levels might be associated with higher levels of periodontal disease. Neither serum IGF-I nor IGF-I/IGFBP-3 ratios were associated with periodontitis.
This thesis aims at improving the current representation of adaptation in economic frameworks of climate change by a) accounting for the time-dependent evolution of the adaptive capacities of countries and b) quantifying unwelcome feedbacks of the adaptation process. In this context, it is proposed that economic assessments of climate change incorporate adaptation as a cyclic and phase-dependent process while devising their cost methodologies. A phase-dependent process acknowledges the existence of adaptation barriers while a cyclic process accounts for potential unwanted feedbacks of adaptation. By analyzing economic assessments against this framework, it is shown that dependencies between phases of adaptation and phases altogether are often disregarded. Furthermore, potential negative consequences associated with adaptation are rarely considered and adaptation is generally assumed to be unconstrained. The assumption of unconstrained adaptation is only acceptable in the context of high adaptive capacity. This concept was further investigated through a review of vulnerability assessments regarding their operation of the adaptive capacity component. It was found that adaptive capacity is mostly equated to proxies that reflect the knowledge, financial and livelihood capacities of the system under analysis. With this theoretical considerations in mind, a dynamic representation of adaptive capacity was elaborated at a country-level. The Human Development Index (HDI) was used as a proxy of the adaptive capacity of countries and its evolution in time extrapolated. The time required for countries to achieve developed world standards of human development was then estimated. The results indicate that between 2005 and 2020, half of the world population will live in countries with low adaptive capacity. This percentage is then progressively reduced to 15% in the year 2050, with marked regional differences. The time required for a country to achieve an appropriate level of development sets a clear constraint on when, and to what extent, the country can engage on climate change adaptation. This does not imply that adaptation will not take place before development occurs. Rather, it calls for adaptation options to be tailored in order to t the current and future adaptive capacities of countries. Obtaining higher levels of adaptive capacity is likely to be associated with negative consequences for the climatic system. The statistical relation between HDI and per-capita emissions of countries was established and future projections made. Between 2010 and 2050 approx. 300 Gt of CO2 are estimated to be associated with the increase of adaptive capacities of current developing countries. This value represents about 30% of the allowed CO2-budgets to restrict global temperatures to an increase of 2 degrees by 2100 compared to pre-industrial times - conditional to a 25% risk of failing to meet the target. For the case of sea-level rise, the modelling framework DIVA (Dynamic Interactive Vulnerability Assessment) was used in order to illustrate the drawbacks of a simplistic representation of adaptation. The results show that adaptation via the construction of protective infrastructure might be economically feasible for particular countries. For others, modeled results fail to provide a clear choice between adaptation or inaction. The assumption of unconstrained adaptation resulted in the valuation of costly protection options whose financial and knowledge requirements can be at odds with the capacities of some coastal countries - namely developing countries. Further, infrastructural protection as adaptive measure to prevent coastal damages can have the counter-productive effect of raising the amount and value of assets at risk. This is a direct result of DIVA disregarding the potential unwelcome feedbacks of adaptation itself. In conclusion, the full potential of economic assessments of climate adaptation is likely to remain unlocked as long as adaptation continues to be misrepresented. The methodologies discussed in this work provide a way forward to alleviate this deficiency in forthcoming assessments. For the case of sea-level rise, the modeling framework DIVA (Dynamic Interactive Vulnerability Assessment) was used in order to illustrate the drawbacks of a simplistic representation of adaptation. The results show that adaptation via the construction of protective infrastructure might be economically feasible for particular countries. For others, modeled results fail to provide a clear choice between adaptation or inaction. The assumption of unconstrained adaptation resulted in the valuation of costly protection options whose financial and knowledge requirements can be at odds with the capacities of some coastal countries - namely developing countries. Further, infrastructural protection as adaptive measure to prevent coastal damages can have the counter-productive effect of raising the amount and value of assets at risk. This is a direct result of DIVA disregarding the potential unwelcome feedbacks of adaptation itself. In conclusion, the full potential of economic assessments of climate adaptation is likely to remain unlocked as long as adaptation continues to be misrepresented. The methodologies discussed in this work provide a way forward to alleviate this deficiency in forthcoming assessments.
Objective: The purpose of this study was to determine the accuracy and reliability of Frankfort horizontal plane identification using displays of multi-planar reconstructed MRI images, and propose it as a sufficiently stable and standardized reference plane for craniofacial structures Materials and Methods: MRI images of 43 adolescent randomly selected subjects were obtained from the longitudinal population based cohort study SHIP-2 using a T1-weighted 3D sequence. Five examiners independently identified the three landmarks that form FH plane. Intra-examiner reproducibility and inter-examiner reliability, correlation coefficients (ICC), coefficient of variability and Bland-Altman plots were obtained for all landmarks coordinates to assess reproducibility. Intra-examiner reproducibility and inter-examiner reliability in terms of location and plane angulation were also assessed. Results: Intra- and inter-examiner reliabilities for X, Y and Z coordinates of all three landmarks were excellent with ICC values ranging from 0.914 to 0.998. Differences among examiners were more in X and Z than in Y dimensions. The Bland–Altman analysis demonstrated excellent intra- as well as inter-examiner agreement between examiners in all coordinates for all landmarks. Intra-examiner reproducibility and inter-examiner reliability of the three landmarks in terms of distance showed mean differences between 1.3 to 2.9 mm, Mean differences in plane angulation were between 1.0° to 1.5° among examiners. Conclusion: This study revealed excellent intra-examiner and inter-examiner reproducibility of Frankfort Horizontal plane through 3D landmark identification in MRI. Sufficiently stable landmark-based reference plane could be used for different treatments and studies.
Rich knowledge about global nutrient cycles and functional interactions can be gained from the perspective of complex microbial proteomes. In this thesis, the application of environmental proteomics allowed for a direct in situ analysis of habitat-specific proteomes expressed by respective microbial communities from two different marine ecosystems. In the first part of this thesis, unculturable symbiont populations from tubeworms that colonize hydrothermal vents of the Pacific deep sea became accessible by use of community proteomics. This branch of environmental proteomics is generally employed to ascertain simple microbial assemblages derived from in situ samples. The proteome study was aimed at analyzing adaptations of seemingly monospecific symbionts to different hosts, the tubeworms Tevnia jerichonana und Riftia pachyptila. A comparison of the newly sequenced genomes of symbiont populations from both hosts confirmed that both symbioses involve the same bacterial species. Also the proteome analysis by 2D-PAGE showed a high physiological homogeneity for symbionts from both worm species, although the hosts are exposed to different geochemical conditions. Thus, the hosts provide their symbionts with a relatively stable internal environment by attenuation of external influences. Only minor variations in the symbionts proteomes reflected the differential environmental conditions outside the worms. Hence, the symbionts were able to fine-tune major metabolic pathways and oxidative stress in response to only minor chemical changes within their hosts. Moreover, new components of important physiological processes of the bacterial symbionts, like the sulfide oxidation and carbon fixation, were identified by in-depth proteomics of the Riftia symbiosis model system. The in situ protein samples showed as well that, in contrast to an earlier hypothesis, nitrate is used as an alternative electron acceptor. In the second part of this thesis, another branch of environmental proteomics called metaproteomics was applied to investigate the response of a bacterioplankton community to a spring phytoplankton bloom in the North Sea. Recurrent plankton blooms are a common phenomen of coastal areas, which however has only been investigated with limited resolution in biodiversity. Based on large-scale proteomic data sets it was found that specialized populations of Bacteroidetes, Gammaproteobacteria and Alphaproteobacteria exhibited differential protein expression patterns. These involved oligomer transporters, glycoside hydrolases and phosphate acquisition proteins. A successive utilization of algal organic matter by microbes indicated a series of ecological niches occupied by the heterotrophic picoplankton. Key proteins, identified by metaproteomics, were further investigated by studying a model bacterium to define their specificities regarding the utilization of algal glycans. By isotope labeling of proteins, quantitative proteomics of the North Sea isolate Gramella forsetii KT0803, a Bacteroidetes representative could be conducted. The adaptation to the algal polysaccharides alginate and laminarin in comparison with glucose was analyzed. G. forsetii proved to be a specialist for the chosen algal polymers, in particular for glucans like laminarin. Primarily comprehensive clusters, the so-called polysaccharide utilization loci (PULs) were activated. The results of this model study complemented the basic concepts obtained by the metaproteomic approach about carbon cycling in coastal systems. The accessibility of numerous unculturable marine microbes by environmental proteomics allows to improve our understanding of interactions that drive symbioses or complex communities. Adaptations to environmental parameters, such as the abundance of substrates, can be analyzed and associated with respective populations. Thus statements can be made for functional groups of microorganisms, their ability for the creation of niches and their flexibility to respond to varying environmental impacts. The increasing number of marine model bacteria enables targeted analysis of specificities and adaptations and hence to support the environmental proteomics approach.
The concept of the electron surface layer introduced in this thesis provides a framework for the description of the microphysics of the surplus electrons immediately at the wall and thereby complements the modelling of the plasma sheath. In this work we have considered from a surface physics perspective the distribution and build-up of an electron adsorbate on the wall as well as the effect of the negative charge on the scattering of light by a spherical particle immersed in a plasma. In our electron surface layer model we treat the wall-bound electrons as a wall-thermalised electron distribution minimising the grand canonical potential and satisfying Poissons equation. The boundary between the electron surface layer and the plasma sheath is determined by a force balance between the attractive image potential and the repulsive sheath potential and lies in front of the crystallographic interface. Depending on the electron affinity x, that is the offset of the conduction band minimum to the potential in front of the surface, two scenarios for the wall-bound electrons are realised. For x<0 electrons do not penetrate into the solid but are trapped in the image states in front of the surface where they form a quasi two-dimensional electron gas. For x>0 electrons penetrate into the conduction band where they form an extended space charge. These different scenarios are also reflected in the electron kinetics at the wall which control the sticking coefficient and the desorption time. If x<0 electrons from the plasma cannot penetrate into the solid. They are trapped in the image states in front of the surface. The transitions between unbound and bound states are due to surface vibrations. Trapping of electrons is mediated by one-phonon transitions and takes place in the upper bound states. Owing to the large binding energy of the lowest bound state transitions from the upper bound states to the lowest bound state are due to multi-phonon processes. For low surface temperatures relaxation to the lowest bound state takes place while for higher temperature a relaxation bottleneck emerges. Desorption occurs in cascades for systems without relaxation bottleneck and as a one-way process in systems with a relaxation bottleneck. From the perspective of plasma physics the most important result is that the sticking coefficient for electrons is relatively small, typically on the order of 0.001. For x>0 electron physisorption takes place in the conduction band. For this case sticking coefficients and desorption times have not been calculated yet but in view of the more efficient scattering with bulk phonons, responsible for electron energy relaxation in this case, we expect them to be larger than for the case of x<0. Finally, we have studied the effects of surplus electrons on the scattering of light by a spherical particle. For x<0 the electrons form a spherical electron gas around the particle and their electrical conductivity modifies the boundary condition for the magnetic field. For x>0 the electrons in the bulk of the particle modify the refractive index through their bulk electrical conductivity. In both cases the conductivity is limited by scattering with surface or bulk phonons. Surplus electrons lead to an increase of absorption at low frequencies and, most notably, to a blue-shift of an extinction resonance in the infrared. This shift is proportional to the charge and is strongest for submicron-sized particles. The particle charge is also revealed in a blue-shift of the rapid variation of one of the two polarisation angles of the reflected light. From our work we conclude that the electron affinity is an important parameter of the surface which should affect the charge distribution as well as the charge-up. Therefore, we encourage experimentalists to study the charging of surfaces or dust particles as a function of x. Interesting in this respect is also if or under what conditions the electron affinity of a surface exposed to a plasma remains stable. Moreover, we suggest to use the charge signatures in Mie scattering to measure the particle charge optically. This would allow a charge measurement independent of the plasma parameters and could be applied to nano-dust where conventional methods cannot be applied.
In this work, various aspects of fundamental physics and chemistry of molecular gas discharges are presented with emphasis on the interaction between species, activated by low-pressure plasmas, and surfaces. As already known, synergistic effects of multiple plasma-generated species are responsible for surface modification. However, due to the large number of internal parameters of a discharge and the complex plasma processes the identification of correlations between plasma characteristics and their effects on surfaces are complicated. Therefore, the aim of this thesis is to improve the understanding of several phenomena associated with plasma–surface interactions by measuring or calculating fundamental kinetic, transport or spectroscopic data needed to interpret measurements and hereby, to support some future applications of plasmas.
Background: High resolution three-dimensional (3-D) magnetic resonance imaging (MRI) is widely used to predict the neurovascular anatomy within the cerebellopontine angle. Objective: To assess value of 3-D three-dimensional Steady-state free precession imaging (SSFP) and Time-of-flight magnetic resonance angiography (TOF MRA) and in detecting the offending vessels in hemifacial spasm by comparison to intraoperative endoscopic visualization. Methods: 42 patients underwent endoscope-assisted microvascular decompression (MVD). All available preoperative 3-D SSFP and TOF MRA images were checked. Intraoperative videos were captured by a high definition endoscopic camera attached to endoscopes while exploring the area of facial nerve root exit zone (REZ). Evaluation of the 3-D images was performed by two independent groups of observers and compared with the operative findings. Results: 3-D MRI had an average positive predictive value (PPV) of 89.1% in differentiating between simple and complex compression. Mean accuracy of the before mentioned images in detection of the offending vessels was 83.3% and 77% according to the first and second group of observers respectively. Averaged inter-observer agreement between the two groups of observers was substantial with an averaged Kappa coefficient (K) of 0.56. In simple compression group, mean accuracy was 97% and 89.4% according to the first and second group of observers respectively. Averaged K for agreement was substantial (K=0.65). Conclusion: According to endoscopic visualisation, 3-D SSFP and TOF MRA images are accurate in detecting the offending vessels in simple compression of the facial nerve, and in predicting presence of a complex compression with variable sensitivity in identifying all offending vessels.
Surface and electrode modifications allow the alteration of surface and electrode properties required for certain applications. In the first part of this thesis, a pH sensitive graphite/quinhydrone composite electrode for Flow-Injection-Analysis (FIA) systems was optimized by using polysiloxane as binder material. This allows an easier handling of the electrode. Furthermore, new applications of the FIA system in conjunction with the pH sensitive detection system were developed. The electrode used here in conjunction with a common reference electrode proved to be a very useful potentiometric detector for FIA acid-base titrations of aqueous solutions. Even acid-base titrations in buffered solutions were performed successfully with the FIA system allowing the determination of activities of enzymes, which catalyse reactions with increasing or decreasing proton concentrations. A FIA system was applied to measure calcium and magnesium ions in different water samples by measuring the hydronium ion release during the complexometric reaction between EDTA and calcium or magnesium ions. A method was established to determine sequentially the titratable acidity and the pH of different wine samples. The new FIA method fulfils the official requirements of the "Organisation Internationale de la Vigne et du Vin" with respect to reproducibility and repeatability and can be easily adjusted to the legal requirements in USA and Europe. In summary, the first part of this thesis shows that the FIA system in conjunction with the graphite/quinhydrone/polysiloxane composite electrode is very well suited for simple, rapid and automatic determinations of small sample volumes in the areas of water analysis, food analysis or even biochemical analysis, provided that hydronium ions are involved. For all applications, one and the same measuring device without changing the detection system is used. Only different carrier solutions are necessary, which can be provided by a proper stream selector. The second part of this thesis is focused on the modification of gold surfaces of medical devices by treatment with OH radicals. These investigations are based on previous studies of the impact of OH radicals on mechanically polished gold surfaces resulting in a smoothing of the surface by dissolution of highly reactive gold atoms. In this thesis, the effect of OH radicals, generated either ex vivo by Fenton solutions or in vivo by immune reactions, on gold implants was analysed using atomic force microscopy. It was found that there is an analogy between the exposure of gold to Fenton solutions and the exposure of gold to immune reactions. The pre-treatment of gold implants with OH radicals of Fenton solution prevents surface alterations of the gold implants in vivo. This indicates that the in vivo release of gold from implants can be reduced by exposing the gold implants to Fenton solution before implantation. Finally, the modification of gold surfaces by OH radicals was applied to a medical nanodetector, which is coated with a gold layer and functionalized with antibodies, for isolating circulating tumour cells (CTCs) from the blood stream of cancer patients. By treating the gold layer of the nanodetector with OH radicals generated by Fenton solution or by UV-photolysis of hydrogen peroxide, the cytotoxicity of the gold layer after gamma irradiation was reduced to almost zero. This modification of the gold surface with OH radicals allows applying the nanodetector for in vivo applications.
Sealing ability of ProRoot MTA when placed as an apical barrier using three different techniques
(2013)
A one-visit apexification protocol with Mineral Trioxide Aggregate (MTA) can be seen as an alternative to the traditional treatment practices with calcium hydroxide. The aim of this study was to investigate the sealing ability of ProRoot MTA when placed as an apical barrier using three different techniques. Sixty freshly extracted single-rooted human teeth were decoronated and standardized to a root length of 15mm. The root segments were prepared with Gates Glidden burs (size 1-6) to simulate the clinical situation of an open apex and randomly assigned into 3 experimental groups of 20 samples each. 5mm MTA was placed by pluggers (Group 1) paper points (Group 2) or ultrasonic tips (Group 3). Radiographs were taken to verify the placement of the apical barriers. After obturation of all samples with warm gutta-percha (Obtura) and AH26 sealer, the coronal portion of all samples was sealed with Cavit. The root segments were then double coated with nail varnish except for the open apex and were exposed to methylene blue dye for 48 hours at room temperature. Afterwards, the samples were sectioned longitudinally and the extent of dye penetration was measured with a stereomicroscope. The mean depth of dye leakage for Group 1 was 1.34mm (±0.5 SD) Group 2 1.25mm (±0.4 SD) and Group 3 1.14mm (± 0.4 SD). Statistical analysis showed that there were no significant differences among the 3 experimental groups (0.178<p<0.552). In conclusion, ProRoot MTA has a similar sealing ability when placed as an apical barrier with pluggers, paper points or ultrasonic tips.
This thesis describes the implementation and first on-line application of a multi-reflection time-of-flight (MR-ToF) mass analyzer for high-resolution mass separation at the ISOLTRAP mass spectrometer at ISOLDE/CERN. On the one hand, the major objective was to improve ISOLTRAPs mass-measurement capabilities with respect to the ratio of delivered contaminating ions to ions of interest. On the other hand, the time necessary to purify wanted from unwanted species should be reduced as much as possible to enable access to even more exotic nuclei. The device has been set up, optimized and tested at the University of Greifswald before its move to ISOLTRAP. The achieved performance comprises mass resolving powers of up to 200000 reached at observation times of 30ms and a contamination suppression of about four orders of magnitude by use of a Bradbury-Nielsen gate. With the characteristics, it outperforms clearly the so far state-of-the-art purification method of a gas-filled Penning trap. To improve the utilization of the MR-ToF mass analyzer, the in-trap lift method has been developed. It simplifies the application and optimization of the device, which is a crucial time factor in an on-line experiment. The device was the first of its kind successfully applied to radioactive ion beams for a mass analysis, for a mass separation (in combination with the Bradbury-Nielsen gate) as a preparatory step for a subsequent Penning-trap mass measurement and as a high-precision mass spectrometer of its own. The later was recently used for the first mass measurement of the neutron-rich calcium isotopes 53Ca and 54Ca. The so-far achieved mass-resolving power of 200000 belongs to the highest reported for time-of-flight mass analyzers at all. The first successful application of the MR-ToF system as the only mass separator at ISOLTRAP resulted in the mass measurement of 82Zn. The new mass value has been compared to mass extrapolations of the most recent Hartree-Fock-Bogoliubov (HFB) mass models, HFB-19 to HFB-21, of the BRUSLIB collaboration. The mass of the nuclide is of high interest for the compositions and depth profile of the outer crust of neutron stars. In the classical model of the outer crust of a cold, non-accrediting and non-rotating neutron star, the sequence of nuclides found within this parts is determined mainly by the binding energy of exotic nuclides. The crustal compositions determined with the three HFB mass models differed with respect to the appearance of a layer of 82Zn, originating from different mass extrapolations of this mass. With the new experimental data, the extrapolations could be evaluated. It was found that the HFB-21 mass value differs less from the experimental data than the ones from HFB-19 and 20. Therefore, in the classical model, 82Zn does not appear anymore in the outer crust. Due to its high resolution and very fast measurement time, the MR-ToF mass analyzer will be an important instruments for future activities at ISOLTRAP, at the ISOLDE facility in general, and at other radioactive ion-beam facilities.
Urban Green Areas – their functions under a changing lifestyle of local people, the example of Hanoi
(2013)
Hanoi is a rapidly developing city - in terms of area, population and economy. It also has to face social and environmental problems that often accompany the fast development of a city. Increasing environmental pollution leads to a decrease in living conditions, such as clean water, housing, social services, etc., for most of the city’s inhabitants. Other well-known social problems in cities worldwide also occur in Hanoi nowadays, for example inequality, food supply, and unemployment. In addition, the society is altering; lifestyle- change is a permanent process. Today, it seems that the changing process in Asian countries is rather leap-frogging than continuously. Although many research projects concerning to urban green areas in Hanoi have been completed recently, less is known about utilization of parks&gardens in a practical and systematic way. This study aims at contributing to fulfills the gaps with up-to-date facts and figures of parks’ utilization in the inner city of Hanoi. Based on “grounded theory”, triangular methodology was applied to collect empirical data in four main parks in the inner city of Hanoi namely: Thong Nhat, Bach Thao, Hoan Kiem, and Lenin. Scientific observations, visitor countings were done in 2010-2011; including 2143 face-to-face interviews to park users covering the course of the year and a small online-poll of 113 responses. Thirteen interviews with experts have been done in several phases and taken into account for discussing and testing hypotheses. Empirical results have shown that utilization of the parks is in abundance in terms of the use - numbers as well as activities. A comparison between the courses of the day for parks in Berlin and Hanoi shown that the time of using parks in Hanoi is earlier in the day. In details, there is 25% and 1.4% of total daily visitors went to parks in Hanoi and Berlin,, respectively. However, the biggest peak in park-visiting time for both cities is in the afternoon. The findings also clarified that students and retirees are the major users even though there is some different in structures of park user among the four parks. The length of stay depends on how large and interesting a park is. In this aspect, Hanoi parks are very poor in facilities showing by a long list of missing facilities in the parks. However, as there is no alternative, parks in Hanoi still attract a high number of visitors. In general, results from this study have shown a close relationship among socio-economic and political situations in Vietnam with UGAs and its utilization. Influencing factors on parks’ utilization were also indentified, which are social changes, lifestyle changes, economic situation, conflicts in land usage, and maintaining cost for UGAs. Three given hypotheses were confirmed: i) the demand of public UGAs in inner Hanoi will grow mid-term and increase strongly long-term; ii) Hanoi’s residents will claim strongly for more parks in the future; ii) The number of conflicts inside the area of UGAs, among the visitors will occur very soon and require a specific master plan for the development and the management of UGAs. Finally, based on the weaknesses of UGAs in Hanoi recently, two groups of recommendations were also suggested to improve UGAs. Firstly, in terms of quality – quantity: • Retain each square meter of green space as UGA – do not unblock any green space for construction; • The existing UGA have to be well maintained. Secondly, in terms of management – planning: • Elaborate a UGA-masterplan for (inner) Hanoi; • Keep UGA management in the responsibility of public bodies; • Implement participation of affected population defector in the process of planning and management (as it has already been written in planning guidelines); • Be aware of potential conflicts among park user groups; • Establish an UGA monitoring system – involving regular (or even permanent) countings based on up-to-date technology; Realize horizontal partnering structures.
Genomics is the field of modern biology that studies the genome as the sum of all genes of a given organism. Genomics includes the analysis of genomic variations in order to identify genetic susceptibility loci for various human diseases. Besides genomics, there are related fields summarized by the term "Omics" such as transcriptomics and proteomics, studying the sum of all transcripts and proteins in a defined biological system, respectively. Genetic variants, namely single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) are used to identify genomic loci associated with human traits and diseases. Genome-wide association studies (GWASs) based on SNP data have been performed for a wide range of human traits and diseases. In the population-based Study of Health in Pomerania (SHIP) and the independent SHIP-TREND study, whole-genome genotyping data were available for 4081 and 986 individuals, respectively. In contrast to the widely used GWAS based on SNPs, association studies using CNV data are difficult to implement and thus less common. Therefore, one aim of this work was to detect CNVs using the whole-genome genotyping data available for 4081 individuals from SHIP. Another aim was to develop an efficient workflow for the analysis of these CNVs. As most common genetic variants exhibit only relatively small effects on phenotypic variability, large sample sizes are needed to maximize the statistical power to detect such effects. Therefore, the integration of data from multiple collaborating studies is indispensable. In this context, several CNV studies with the SHIP data have been performed and published, for example on body mass index (BMI) phenotypes where the SHIP cohort was used as a population-based control. Trait-associated genetic markers identified through GWASs are often intergenic or synonymous coding, and those loci identified through whole-genome CNV analyses often contain multiple genes, making it difficult to identify the causal variants. In this context, the functional analysis of identified loci aids in determining causal variant(s). One possibility to conduct functional analysis is the expression quantitative trait loci (eQTL) analysis, defined as the association of genome-wide genotyping data with genome-wide gene expression data based on measured transcriptomes. This allows the identification of genetic variants influencing the expression levels of defined genes. A further example are transcriptome-wide association analysis (TWAS), defined as the association of phenotype data with whole-genome expression data. Thus, another aim of this work was to establish an analysis pipeline for processing such expression data, which were available for about 1000 individuals from the SHIP-TREND study. Here, array-based gene expression data were generated using RNA prepared from whole-blood. Interpretation of TWAS results is often difficult, because of possible reverse causation on gene expression data. Furthermore, technical errors of measurement may bias the results. In a comprehensive work, biological and technical factors influencing measured gene expression data have been identified and were subsequently taken into account to improve the association analyses. To further elucidate the molecular mechanisms underlying the relationship of gene expression levels with human traits or diseases, pathway analyses using the Ingenuity Pathway Analysis (IPA) tool have been performed in connection with the TWAS. As for GWASs, the associations identified in TWAS usually exhibit only small effect sizes, highlighting the need for larger studies or meta-analysis to identify all susceptibility variants. In this context several eQTL- and TWAS meta-analyses using the SHIP-TREND data have been performed, for example on the phenotypes age, sex, BMI, smoking status and serum lipid traits. The results of these analyses are in preparation for publication and the most advanced example, the correlation of expression data with BMI, is presented here. The integration of whole-genome genotyping and expression data provides new functional information of the underlying biological mechanisms of complex human traits and diseases. Within the frame of this work, this could be demonstrated for the example of susceptibility to Helicobacter pylori infection.
The electron and negative ion densities in an asymmetric capacitively coupled low-pressure RF plasma in oxygen were systematically studied and compared to the electropositive argon RF plasma during continuous and pulsed power input. This work presents the careful design and realization of a non-invasive 160.28 GHz Gaussian beam microwave interferometry (MWI) as an innovative diagnostic tool. MWI directly provides the line integrated electron density without any model assumption. The high microwave frequency enables one to accurately describe the microwave free space propagation by means of Gaussian beam theory. The microwave interferometer is simultaneously coupled with laser photodetachment to experimentally determine the negative ion density in the CCRF oxygen discharge. This is the first time that both diagnostics were combined in low-pressure capacitively coupled RF oxygen plasmas. This thesis first presents comprehensive measurements of the steady state line integrated electron density in dependence on RF power and pressure for an argon and oxygen plasma. For both gases the electron density increases with RF power. However, the line integrated electron density in oxygen is about a factor 3 to 10 smaller than in argon. The reduced electron density is accompanied by a high number of negative ions, which exceeded the electron density and resulted in a high electronegative mode. With increasing RF power, the plasma switches into a low electronegative mode. Consequently, the discharge operates in two different modes, which are distinguished by their degree of electronegativity. The transition between the high and low electronegative modes is step-like and it was concluded that one can here directly see the discharge switches from the &alpha-mode to the &gamma-mode. The &gamma-mode (low electronegative mode, high RF power) is characterized by a strong increase of the electron density and a simultaneous decrease of the negative ion density. The increase may be connected to the production of secondary electrons by collision detachment of negative ions within the RF sheath (“pseudo-secondary electron”), in addition to the classical &gamma process due to positive ion bombardment of the powered electrode. In comparison to the &gamma-mode the &alpha-mode (high electronegative mode, low RF power) reveals more negative ions than electrons. Furthermore, a simple 0d attachment-detachment model was applied to calculate the effective rate coefficients for dissociative electron attachment and collisional detachment from the experimentally determined values of steady state electron and negative ion density, as well as the detachment decay time constant. Hence, the attachment rate coefficient of the molecular ground and the excited metastable state in dependence on RF power were determined. Moreover, the density of metastable molecular oxygen was estimated to 10% of the molecular ground state oxygen. The influence of each electronegative mode to the entire temporal behavior of the oxygen discharge was intensively investigated by pulsing the discharge. Here it was shown that for the low electronegative mode the afterglow behavior is similar to that of an electropositive argon plasma. In the high electronegative mode an electron density peak in the early afterglow was observed. It was concluded that the electron production originates from the collisional detachment of negative ions. The negative ion loss and the electron production in the early afterglow were modeled numerically with a 0d rate equation system. The model accurately describes the afterglow behavior of both electronegative modes and the additional electron density peak in the early of the high electronegative mode. For the high electronegative mode the molecular oxygen plays an important role as a detachment partner for the production of electrons in the early afterglow. Furthermore, the presence of the negative ions causes fluctuations of plasma parameters. 2d spatial and temporal fluctuations of the ion saturation current are measured during the instability. The temporal and phase resolved optical emission spectroscopy shows a strong change in emission pattern during the instability, which becomes more obvious for one RF cycle at characteristic instability phases. Here, the excitation patterns reveal significant changes in the electron heating mechanisms.
Bacteria are an integral part of modern biotechnology. They are used to make a variety of products, such as foods, drugs, as well as a multitude of chemicals. In order to increase their production rates molecular biotechnology offers many tuning points, starting from the selection of an applicable host, over its geno- and phenotypical characterization, followed by genetic manipulations for an optimized metabolism and stabilisation of production processes. This work comprises the optimization of Bacillus subtilis as an expression system. It describes the steps taken for selection and genomic characterization of the B. subtilis wild type strain ATCC 6051, the subsequent optimizations of the strain in respect to growth and productivity, as well as the characterization of its behaviour in a variety of cultivation conditions. The B. subtilis strain most commonly found in laboratories around the world is the first sequenced Gram-positive organism B. subtilis 168. Zeigler et al. showed that strain 168 is not a real wild type. Instead it was created through random mutagenesis with X-rays and selected for transformability. This strain has been used as the basis for popular B. subtilis strains in heterologous gene expression such as the extracellular protease deficient WB strains. Growth experiments showed the real wild type strain ATCC 6051 to be superior to its mutated ancestor 168, making it a solid basis for the construction of an optimized B. subtilis expression system. In order to gain a full understanding of the genomic and corresponding physiological differences between the two systems, B. subtilis ATCC 6051 was sequenced and compared to the genome of B. Subtilis 168. Several variations on geno- and phenotypic level could be revealed, that resulted in particular from genes involved in natural competency, the metabolism of amino acids and chemotaxis. This genomically well characterized B. subtilis ATCC 6051 was improved in respect to its application as an expression host. Improvements were achieved through the inactivation of both sporulation and reduction of autolysis, leading to a more robust behaviour during the overproduction and secretion of a reporter enzyme. A positive effect on the activity of an acetoin induced promoter by the addition of second copies for its transcription factors SigmaL and AcoR could be observed. Anaerobic zones and areas with excess glucose caused by insufficient mixing are common conditions in large scale bioprocesses and lead to oscillating conditions for the cells. In turn, this oscillation provokes an excretion of so called overflow metabolites, which can negatively affect the bacterial productivity. Detailed scientific characterizations of industrial scale processes under such oscillating conditions are scarce due to the high costs and logistics involved. A B. Subtilis sporulation mutant was thus examined in respect to its extra- and intracellular metabolites in a scale-down, two-compartment reactor giving hints about conditions the host is exposed to and how it reacts. To improve tolerance thresholds and utilization capacity for such metabolites in B. subtilis, the glyoxylate cycle was transferred from its close relative Bacillus licheniformis into the genome of B. subtilis. This feature enabled our B. subtilis ACE mutant to grow on acetate. The improved strain showed higher tolerance towards excess glucose in a fed-batch as well as higher productivity during the expression of a reporter enzyme in comparison to the wild type. The ACE strain and B. licheniformis showed an increased formation of glycolate during growth with the glyoxylate cycle. This with regard to bacteria undescribed metabolite seems to play a role as a by-product of the glyoxylate cycle. Summarizing, this thesis deals with the characterization and optimization of B. subtilis for growth on overflow metabolites, enhancements of the acoA-expression system and the influence of sporulation and lysis mutants on its activity. Complementary, the host was begun to be characterized in respect to its behaviour in industrial scale processes.
The focus of the first two articles was the engineering and application of enzymes for the conversion of the bio-based resources glycerol and its oxidation product glyceraldehyde for the production of the value added product glyceric acid. Article III focuses on the cloning, exploration and engineering of a polyol dehydrogenase, which later on was used as cofactor recycling system in order to produce ε-caprolactone from cyclohexanol as presented in arti-cle IV. The following paragraphs will give a short outline of each article. ARTICLE I: ASYMMETRIC SYNTHESIS OF D-GLYCERIC ACID BY AN ALDITOL OXIDASE AND DIRECTED EVOLUTION FOR ENHANCED OXIDATIVE ACTIVITY TOWARDS GLYCEROL. GERSTENBRUCH, S., WULF, H., MUßMANN, N., O’CONNELL, T., MAURER, K.-H. & BORNSCHEUER, U. T. (2012). Appl. Microbiol. Biotechnol. 96, 1243-1252. The alditol oxidase of Streptomyces coelicolor A3(2) (AldO) was used to catalyze the oxida-tion of glycerol to glyceraldehyde and glyceric acid. The enantioselectivity for the FAD-de-pendent glycerol oxidation was elucidated and different strategies were used to enhance the substrate specificity towards glycerol. Directed evolution by error-prone PCR led to an AldO double mutant with 1.5-fold improved activity for glycerol. Further improvement of activity was achieved by combination of mutations, leading to a quadruple mutant with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. In small-scale biotransformation concentrations up to 2.0 g•l-1 D-glyceric acid could be reached using whole cells. Investi¬gation of the effects of the introduced mutations led to a further identification of es¬sential amino acids with respect to enzyme functionality and structural stability. ARTICLE II: KINETIC RESOLUTION OF GLYCERALDEHYDE USING AN ALDEHYDE DEHYDROGENASE FROM DEINOCOCCUS GEOTHERMALIS DSM 11300 COMBINED WITH ELECTROCHEMICAL COFACTOR RECYCLING. WULF, H., PERZBORN, M., SIEVERS, G., SCHOLZ, F. & BORNSCHEUER, U. T. (2012). J. Mol. Catal. B Enzym. 74, 144-150. Two aldehyde dehydrogenases (ALDH) from Escherichia coli BL21 and Deinococcus geother-malis were cloned, characterized and evaluated according to their applicability for a bio-catalysis setup with electrolytic cofactor recycling. Both ALDHs turned out to have a sim¬ilar substrate scope and favor short to medium chain aldehydes and both oxidize glyceralde¬hyde to D-glyceric acid. The ALDH variant of D. geothermalis shows higher specific activity towards glyceraldehyde and has an elevated optimum temperature compared to the BL21 enzyme. Due to the higher specific activity of the ALDH of D. geothermalis, this enzyme was used to conduct a kinetic resolution of glyceraldehyde with electrolytic NAD+ recycling at a glassy carbon foam electrode with ABTS as redox mediator yielding in 1.8 g•l-1 glyceric acid. ARTICLE III: PROTEIN ENGINEERING OF A THERMOSTABLE POLYOL DEHYDROGENASE. WULF, H.*, MALLIN, H.*, BORNSCHEUER U.T. (2012). Enzyme Microb. Technol. 51, 217-224 (*equally contributed). The new enzyme polyol dehydrogenase PDH-11300 from D. geothermalis was extensively characterized regarding its temperature optimum and thermostability. A peptide stretch responsible for substrate recognition from the PDH-11300 was substituted by this particular stretch of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scopes were deter-mined and basically the chimeric enzyme represented the average of both wild-type en-zymes. A rather unexpected finding was the notably increased T5060, by 7°C to 55.3°C, and an increased specific activity against cyclohexanol. Finally, the cofactor specificity was suc¬cess-fully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD+ binding cleft, without influencing the catalytic properties of the dehydrogenase. ARTICLE IV: A SELF-SUFFICIENT BAEYER-VILLIGER BIOCATALYSIS SYSTEM FOR THE SYNTHESIS OF Ɛ-CAPROLACTONE FROM CYCLOHEXANOL. MALLIN, H. *, WULF, H. *, BORNSCHEUER U.T. (2013). Enzyme Microb. Technol., online, DOI: 10.1016/j.enzmictec.2013.01.007 (*equally contributed). The application of the engineered PDH-loopN mutant [1] (Article III) for the production of ε-caprolactone from cyclohexanol was investigated in a co-immobilization approach with the cyclohexanone monooxygenase from Acinetobacter calcoaceticus. Biotransformation with solubilized enzymes led to an isolated yield of 55% pure ε-caprolactone with no residual cy-clohexanol to be detected. During the immobilization experiments a higher enzyme ratio in favor of the CHMO led to higher reaction velocities. Similarly, the addition of soluble fresh CHMO during reuse of co-immobilization batches significantly increased the activity identi-fying the CHMO as the bottleneck in this reaction setup.