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Severe trauma results in alterations in immune functions, correlated with a dysbalanced cytokine synthesis. This imbalance endangers severely injured patients for post-traumatic complications such as MODS, liver failure, renal dysfunction and ARDS. IL-10, a powerful immunosuppressive cytokine, plays a central role in the immune response after severe trauma. The relevance of IL-10 for single and multiple organ failure was studied in a prospective study at a level I trauma center. Blood was systematically obtained from a total of 118 severely injured [median (IQR) ISS=34 (27-34)] patients. IL-10 plasma levels were measured by ELISA. Patients showed elevated IL-10 levels throughout the whole observation period of 5 days. IL-10 plasma levels rose rapidly after trauma and gradually declined towards day 5. Patients who developed complications demonstrated significantly elevated IL-10 levels compared with patients who did not. The odds of developing MODS were 9.6 times greater in patients with IL-10 plasma levels higher than 124 pg/mL 6 hours after arrival at the ICU. Multivariate analysis showed that IL-10 plasma levels >124 pg/mL at time-point 6h, severe head injury and an arterial pH <7.34 were simultaneously significant predictors of the development of MODS in severely injured patients. The dynamic with rapid increase and gradual decline in IL-10 plasma levels indicated that IL-10 is a marker of the initial damage to the organism caused by trauma, rather than a marker of somatic dysregulations.
The present work consists of four parts, containing experimental data obtained from analysis of 'Bacillus subtilis' specific and general defense strategies against reactive oxygen species. In the first part, the peroxide and superoxide stress stimulons ob 'B. subtilis' were analyzed by means of transcriptomics and proteomics. Oxidative stress responsive genes were classified into two groups: the gene expression pattern was either similar after both stresses or the genes primarily responded to one stimulus. The high induction observed for members of the PerR-regulon after both stimuli supported the assumption that activation of the peroxide specific PerR-regulon represented the primary stress response after superoxide and peroxide stress. The second part focuses on protein carbonylation in 'B. subtilis' wild-type and 'sigB' mutant cells. The introduction of carbonyl groups into amino acid side chains of proteins represents one possible form of protein modification after attack by reactive oxygen species. Carbonyl groups are readily detectable and the observed amounts can thus serve as an indicator for the severity of protein damage. The resultsdemonstrate clearly that 'B. subtilis' proteins are susceptible to hydrogen peroxide (H2O2) mediated carbonylation damage. The application of low concentrations of H2O2 prior to the exposure to otherwise lethal levels of peroxide reduced markedly the degree of protein carbonylation, which also held true for glucose starved cells. Artificial preloading with general stress proteins resulted in a lower level of protein carbonylation when cells were subjected to oxidative stress, but no differences were detected between wild-type and 'sigB' mutant cells. In the third part, strains with mutations in genes encoding general stress proteins were screenedfor decreased resistance after H2O2 challenge. It was demonstrated that resistance to H2O2 challenge. It was demonstrated that resistance to H2O2 after transient heat treatment, likewise to conditions of glucose starvation, was at least partly mediated by the sB-dependent general stress response. The screening of mutants in sB-controlled genes revealed an important role for the deoxyribonucleic acid (DNA)-binding protein Dps in the context of sB-mediated resistance to oxidative stress underlining previous reports. Therefore, the experimental strategy opens a global view on the importance of DNA integrity in 'B. subtilis' under conditions of oxidative stress. The fourth part includes analysis of a 'B. subtilis' thioredoxin conditional mutant. The thiol-disulfide oxidoreductase TrxA is an essential protein in 'B. subtilis' that is suggested to be involved in maintaining the cytoplasmic thiol-disulfide state even under conditions of oxidative stress. To investigate the physiological role of TrxA, growth experiments and two-dimensional gel electrophoresis were carried out with exponentially growing cells that were depleted of TrxA. The observations indicate that TrxA essentially involved in the re-reduction of phosphoadenosyl phosphosulfate reductase CysH within the sulfate assimilation pathway of 'B. subtilis'.
The insulin dependent type 1 diabetes mellitus (IDDM) and the metabolic syndrome are complex human diseases. Both diseases are heterogeneous, genetically inherited and do not follow a simple Mendelian single-locus pattern. The analysis of complex human diseases is complicated both by genetic heterogeneity and by environmental factors. One way to overcome the problem of genetic heterogeneity in humans may be to cluster patients by kinship. It was shown by analysis of maternal lines of type 1 diabetics using mitochondrial DNA that 89% of maternal lines are related to each other. Moreover, an alternative to the genetic differential analysis of complex mammalian diseases is the use of animal models. The availability of inbred animal models closely resembling the human disease is an essential component of genetic investigations in this field, as shown in the results of this work. These findings do not only underscore the utility of the congenic and subcongenic approach in differentially analyzing complex traits, but also show that candidate genes can be identified and that chromosomal exchange can variously influence the phenotype, leading to sub-phenotypes which may be representative for human beings. Furthermore, it will also be possible to locate the syntenic region in the human genome and congenic and subcongenic strains can also be used to study interactions between chromosomal regions and various selected environmental conditions. In this way, it may be possible to learn which region can be influenced by environmental factors and to which extent, an undertaking which will require prospective projects.
This work studies different alternatives for parallelization of ground-state DMRG, with a focus on shared memory multiprocessor systems. Exploiting the parallelism in the dominant part of a DMRG calculation (diagonalization of the superblock Hamiltonian), speedups of 5 to 6 on 8-CPU machines can be achieved. A performance analysis gives hints as to which machine is best siuted for the task. The parallelized DMRG code is then applied to current problems in theoretical solid state physics with electronics, bosonic and spin degrees of freedom. Stripe-like modulations of the hole density in the ground state of doped Hubbard with cylindrical boundary conditions are idenficied in the thermodynamic limit using extrapolation techniques. In the 1D Holstein model of spinless fermions at half filling, Luttinger parameters and the charge structure factor are determinde in order to derive the phase diagram that had previously been established only on small lattices. For the 1D half-filled Holstein-Hubbard model, a finite size analysisof spine and charge excitation gaps in the relevant sectors (Mott insulator, Peierls band insulator and bipolaronic Peierls insulator) is able to yield the phase diagram as well. Finally, is the Heisenberg spin chain with dynamical phonons is considered as a relevant model for a spin-Peierls transition in Copper Germanate. Using DMRG, the relation between singlet-triplet excitation gap and dynamical dimeriaztion is calculated for the first time.
The present work is a paleolimnological orientated approach to refine and improve the indicator ability of freshwater ostracods from Holocene and Late glacial deposits in northeast Germany. The thesis follows two different approaches, one utilizes quantitative paleoenvironmental analysis, while the other evaluates ecological investigations of living specimens to extend the potential indicator group. For the first time quantitative ostracod analysis are carried out for a lacustrine basin (lake Krakower See) and a near-shore locality (Pudagla lowland) in the study area. The ecological investigation of living ostracods comprises 96 localities. The evaluation focused on environmental variables, which explain significantly the species composition. A canonical correspondence analysis identified at least four environmental parameters - water temperature, conductivity, pH-value, and mean water depth – which have an effect on ostracod assemblages. An extended analysis, which included only a subset of lake sites, revealed also that the former three environmental parameters affect the ostracod lake fauna, whereas the water temperature is the dominant factor. A temperature-transfer function could be regressed and calculated from the given trainingset by a weighted average model. These estimates can now be use in future paleolimnological investigations in northeast Germany to quantify the paleotemperature.
A highly stereoselective recombinant alcohol dehydrogenase aus 'Pseudomonas fluorescens' DSM50106
(2005)
The alcohol dehydrogenase was biochemically characterized. A broad range of arylaliphatic ketones is efficiently reduced to the corresponding optically active (R)-alcohols by a recombinant alcohol dehydrogenase (PF-ADH) produced by overexpression in 'Escherichia coli'. PF-ADH shows high activity and stereoselectivity in the reduction of acetophenone and various derivatives (45-99%), as well as in the reduction of 3-oxy-butyric acid methyl ester and 3-oxy-butyric acid methyl ester and 3-oxy-hexanoic acid ethyl ester (>99%). The highest activity was observed between 10 and 20°C. The copfactor NADH can be efficiently recycled by the addition of 10-20% of iso-propanol. A flow-through-polarimetry-based assay to determine oxidoreductase activity and stereoselectivity is described.
About 30 % of epileptic patients are non-responsive to multidrug antiepileptic therapy. One of non-responsiveness in epilepsy hypothesis claims that non-responsiveness occurs because of reduced access of antiepileptic drugs to their targets, as a result of increased efflux of antiepileptic drugs away from these targets. Transporters believed to be involved in non-responsiveness in epilepsy are mainly but not exclusively the members of the ABC superfamily including P-gp (MDR1, ABCB1), MRP1 (ABCC1), MRP2 (ABCC2) and others. These proteins are normally found in the blood-brain barrier and the blood-cerebrospinal fluid barrier where they function as protectors. There is emerging evidence that P-gp, MRP1 and MRP2 are up-regulated in epileptogenic brain tissue. The risk of non-responsiveness could be related also to the MDR1 or MRP2 gene polymorphisms. We hypothesised that changes in expression and function of multidrug transporters involved in non-responsiveness of epilepsy might be detectable not only in the brain but also in other tissues such as lymphocytes. Therefore we evaluated the expression of MDR1, MRP1 and MRP2 and function of P-gp in lymphocytes in patients with epilepsy and healthy subjects. Three groups of epileptic patients and 15 healthy subjects as a control group were included in the study. The patients’ group was defined as follows: Monotherapy – patients treated with carbamazepine monotherapy, without seizures - corresponded to group responders. Combined therapy – patients after monotherapy (two different medicines have been tried) and combined therapy (two trials of combined therapy), not free of seizures. Monotherapy and combined therapy groups each embraced 15 patients. Neurosurgery – patients who had undergone neurosurgery, afterwards were or were not additionally treated with carbamazepine, with or without seizures. This group comprised 24 patients. Combined therapy and neurosurgery groups composed the group of non-responders. The mRNA expression of MRP1, MRP2 and MDR1 by means of quantitative real-time PCR as well as MRP2 and P-gp protein content by Western blot in lymphocytes was measured. For P-gp functional analysis rhodamine efflux from lymphocytes and natural killer (NK) cells was performed. The influence of the polymorphisms C3435T, G2677T/A in the MDR1 gene and C24T, G1249A, C3972T in the MRP2 gene for the transporters expression, function and their association with non-responsive epilepsy phenotype was investigated. Our results showed that MRP1 expression in lymphocytes was significantly lower in epileptics than in healthy subjects. Non-responders had lower MRP1 mRNA content in lymphocytes than responders. We did not find any difference in MRP2 expression between epileptics and healthy volunteers. MRP2 mRNA levels in lymphocytes were higher in non-responders than in responders. However, at protein level epileptic patients had significantly lower MRP2 content in lymphocytes than controls. MRP2 protein content did not differ in responders and non-responders. There was no reliable correlation between MRP2 mRNA expression and MRP2 protein content in lymphocytes. Epileptics had significantly lower MDR1 expression in lymphocytes than healthy individuals. MDR1 expression was decreasing according to the consumption of antiepileptic drugs and seizures frequency: patients after neurosurgery had significantly lower MDR1 expression than patients after combined therapy and monotherapy. MDR1 expression was significantly lower in non-responders than in responders. At protein level epileptics had lower P-gp content than controls. Detected P-gp amount in lymphocytes did not differ between responders and non-responders. Rhodamine efflux from lymphocytes and NK cells did not differ significantly between epileptics and healthy subjects, but it was higher in patients after neurosurgery than in patients after monotherapy. Rhodamine efflux from NK cells, which are known to express the highest levels of P-gp, was significantly higher in non-responders than in responders. In this study, we showed that MRP1 mRNA expression in lymphocytes was significantly correlated to its expression in the brain. We detected also a significant co-correlation between MRP1 expression in the hippocampus and MDR1 expression in lymphocytes. We found no evidence regarding the impact of the MDR1 polymorphisms on mRNA expression, P-gp content and rhodamine efflux from lymphocytes. Our data showed lack of evidence regarding the impact of the MRP2 polymorphisms on mRNA expression and protein content. We did not detect any association between MDR1 or MRP2 polymorphisms and non-responsiveness in epilepsy or epilepsy in the main. In conclusion, our results suggest that lymphocytes are an appropriate surrogate for studies on changes of multidrug transporters expression in epilepsy. Lymphocytes as an easily accessible tissue might serve as a marker for responsiveness to antiepileptic drug therapy in epilepsy studies.
The biodiversity of marine microorganisms opens a promising potential for the discovery of new technical enzymes. During this study a characterization of marine microorganisms, isolated from Arctic or Antarctic ice, sea water or sediment from the ocean was performed based on a comprehensive strain collection at the Alfred-Wegener-Institut für Polar- und Meeresforschung. These marine psychrophilic bacteria indicated a wide spectrum of extracellular cold-active enzymes. 16S rRNA sequencing revealed that many of these psychrophilic bacteria represent new species. Characterization of selected isolates by means of transmission electron or raster electron microscopy showed remarkably pleomorphic cellular structures throughout their growth. The major part of this thesis focuses on a marine Antarctic, psychrophilic bacterium (strain ANT/505) isolated from sea ice covered surface water from the Southern Ocean, which was identified to express a very uncommon enzymatic activity for the marine environment, namely a pectinolytic activity. The sequencing of the 16S rRNA of isolate ANT/505 and biochemical tests indicated a taxonomical affiliation to the specie Pseudoalteromonas haloplanktis. The supernatant of this bacterial isolate showed after growth on citrus pectin three different pectinolytic activities. By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated. Subcloning and sequencing revealed two open reading frames of 1671 and 1968 nt corresponding to proteins of 68 and 75 kDa. The deduced amino acid sequence of the two orfs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans. The pectate lyases contain signal peptides of 17 and 26 amino acids length that were correctly processed after overexpression in E. coli BL21. Both enzymes were purified by anionic exchange chromatography. Maximal enzymatic activities for both pectate lyases were observed at a temperature of 30°C and a pH range of 9-10. The Km values of both lyases for pectate and citrus pectin were 1 g⋅l-1 and 5 g⋅l-1, respectively. Calcium was required for activity on pectic substrates, while the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes. These two cold-adapted enzymes represent the first pectate lyases isolated and characterized from a marine bacterium. Further cloning and sequence analyses revealed that PelA from P. haloplanktis is an exceptionally big bifunctional enzyme featuring pectate lyase and pectin methylesterase activity. The deduced amino acid sequence of the pectin methylesterase domain showed homology to group I pectin methylesterases from Erwinia chrysanthemi and Erwinia carotovora. The pectin methylesterase domain of PelA was found to show highest homology to a potential pectin methylesterase from Saccharophagus degradans strain MD2-40. Maximum pectin methylesterase activity of PelA was detected at a pH of 7.5 and a maximum temperature of 30°C. This cold-adapted enzyme revealed high remaining pectin methylesterase activity at low temperatures around 5°C and was quickly unstabilized at temperatures above 45°C. The analysis of the localization of the two pectinolytic genes on the genome of P. haloplanktis ANT/505 revelaed that these pectinase genes are expressed from independent cistrons, which are not clustered but located at distant positions on chromosome I of the P. haloplanktis genome. It was found that the transcription of both pectinase genes is induced by the presence of pectin. By means of primer extension the promoter regions of both cistrons were detected.
Recent geochemical and mineralogical alteration processes in tropical coastal sediments of Vietnam
(2006)
The dissertation contains two main parts: (i) Syn-sedimentary hydrodynamic processes & relationship with elemental distribution, clay matter, (ii) Short-term mineral alteration during early diagenes. Samples were taken from the uppermost one metre (<50 year old in RRD, < 300 y old in south central VN). In part one, three principal hydrodynamic factors can be revealed based on End-Member Modelling Algorithm (EMMA), for the polymodal grain-size distribution patterns in coastline of VN (i) Accumulation factor: accumulation of terrigenous sediments linked with a grain size separation (distance dependence), (ii) Erosion factor: synsedimentary erosion by wave activities, (iii) Aeolian factor: deposition from neighbour sand bar by wind (typically only for low sedimentation rate, like in South Central Coast but not to detect in RRD). Distributions of clay matter and chemical elements in the coastal sediments in Vietnam are strongly influenced by hydrodynamic forces (distance from the coastline). In part two, particle-wise analysis by TEM allowed to determined four main mixed layer series: di-Vermiculite/Smectite-ml, Kaolinite/ Expandable-ml, Illite/Smectite-ml, Chlorite/Saponite-ml. Three principal mineral alteration processes can be detected in coastal sediments during early diagenesis: Dissolution, Smectitization, Kaolinitization, based on XRD (CSD, peak area) & TEM-EDX (particle morphology, chemical formula, polytype, particle frequency). Dissolution process of clay matter is typically in the coastal alkaline condition. It is a function of diagenetic time. Dissolution begins with a higher degree of dislocation and is followed by step-wise delamination & dissolution of the first stacks Smectitization (mirrored in diVS-ml series) alters mica-like structures (illite, dioctahedral vermiculite) to smectitic structures (Beidellite, Montmorillonite). Smectitization process occurs in group-wise layer by layer transforming mechanism (mica-like layer to smectitic layer). Each step is indicated by a gauss-like distribution of the octahedral layer charge with K as trigger. Kaolinitization of KE series alters smectitic structure (beidellite, smectitic KE-ml) to kaolinitic structure (kaolinitic KE-ml, kaolinite) "interlayer by interlayer" transformation of KE-ml series is comparable to kaolinitization mechanism discussed by Dudek et al. (2006).OH- groups in ambient could be the trigger. Mangroves biota influences the sediments in two main pathways: Nutrients supply & trapping function (clay matter, heavy metals)Interaction of active root-layer: intensification of dissolution, smectitization & kaolinitization: uptake of K, Na by mangrove root is possible trigger Human activities like shrimp cultivation have stopped the influences of the former active roots.Besides, erosion process related to water discharge gives rise to dispersion of clay species & heavy metals => more toxic for ecosystem.
The non-natural substances in commonly used UV protection creams such as TiO2, are known to have a photocatalytic side effect, which is very harmful to human skin. This study presents some properties of clays and clays minerals concerning UV protection potential, which can be very helpful for the development of new UV protection cream generation. Clays and clay minerals are demonstrated that they have potential to absorb UV-radiation. The structures of clay particles in cream were shown to be dependent on the layer charge of clay minerals. The total amount of Fe2C>3 in chemical composition of clay plays a key role in determining the UV-absorption ability of the clay matter. Moreover, the UV-absorption ability also depends on the expandable or non- expandable property of the clay. The studies were also performed on the mixtures of wool-wax-alcohol cream and nanosuspension obtained by the extraction of fungi mass Ganoderma pfeifferi by using plantacare together with clay. The combination of clays and nanosuspension increased its UV-absorption ability. The skin model test was performed in vivo in mouse ears with skin flora Escherichia coli and infectious bacteria Staphylococcus aureus in order to determine the effects of cream samples on skin under UV irradiation and skin infection. From the results of characterization of clays and clay minerals properties in UV protection cream, this study also brings some ideas about products designing.