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A paradigm was developed to experimentally investigate the dysregulation of affective reactivity in clinical depression. The literature so far reported evidence for three directions of dysregulation - negative potentiation, positive attenuation, and emotion context insensitivity. Therefore a paradigm was designed to allow to test all three hypotheses simultaneously. Furthermore, to enable generalization across the specific stimuli used in the experiment, stimuli of two sensory modalities were used - pictures and sounds. Because it was hypothesized, that the specificity of affective reactivity of depressed patients will be especially prominent in long lasting affective situations, a categorically blocked presentation mode was chosen. Regarding the dependent variables, a multimethod approach was conducted. Besides self-report ratings of the feeling state, startle responses, skin conductance responses, heart rate, and the electromyogram of the corrugator and zygomatic muscle were recorded. In a separate session, BOLD-responses during picture viewing were collected by functional magnetic resonance imaging (fMRI). Both sessions were conducted with three samples: a healthy student sample, a depressed outpatient sample, and a healthy age and gender matched control sample. The results of the patient sample support an integration of the emotion context insensitivity and the negative potentiation hypothesis. Patients reported generally to feel more unpleasant and more aroused than healthy controls. Skin conductance and startle responses were modulated by valence to a smaller degree in the patients than in the controls. No group differences were found in the facial muscle activity. BOLD-responses were potentiated during unpleasant compared to neutral pictures in the patient but not in the control group in the amygdala, the insular cortex and the orbito frontal cortex. A model to integrate these results is developed. Its central assumption is, that the inability to respond to affective stimuli is an aversive experience and therefore leads to a negativity bias in attention and cognition. Direction of further research and implications for psychotherapies are discussed.
Degradation of branched chain aliphatic and aromatic petroleum hydrocarbons by microorganisms
(2008)
The overall aim of the work was to investigate the ability of several Gram-positive bacteria including Mycocbacterium neoaurum SBUG 109, Nocardia cyriacigeorgica SBUG 1472 and Rhodococcus ruber SBUG 82 and the yeast Trichosporon mucoides SBUG-Y 801 to degrade and transform branched chain hydrocarbons which occur in petroleum and its fraction products such as gasoline or gas oil and which are known as important and recalcitrant environmental pollutants. Pristane, iso-pentylbenzene and sec-octylbenzene were used in this work as model compounds. These compounds represent significant groups of petroleum constituents (branched chain alkanes and aromatic hydrocarbons). Three bacteria and the yeast T. mucoides SBUG-Y 801 were selected in a screen of 16 hydrocarbon-utilizing strains in the SBUG collection and from 21 isolated hydrocarbon-utilizing strains from oil-contaminated habitats of Saudi Arabian Desert and of Vietnam. The bacteria were identified in cooperation with DSZM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) as M. neoaurum SBUG 109, N. cyriacigeorgica SBUG 1472, R. ruber SBUG 82. These bacterial and yeast strains were shown to possess high potential for degrading and transforming pristane, iso-pentylbenzene and sec-octylbenzene. The intermediates produced by these bacteria during incubation with pristane were analyzed by GC and GC/MS. The products 4-methyl pentanoic acid; methyl butanedioic acid; 2-methyl pentadioic acid; methyl propanedioic acid; 4-methyl heptanedioic acid and 2,6,10,14–tetramethyl-pentadecan–3–one were detected in M. neoaurum cultures. In R. ruber, methyl butanedioic acid; 2-methyl pentadioic acid; 4,8-dimethylnonanoic acid, 4-methyl heptanedioic acid; 2,6,10–trimethylundecanoic acid; 3,7-dimethyl decanedioic acid and 2,6,10,14–tetramethyl–pentadecan–3-one were identified. In N. cyriacigeorgica, 2-methylpentanedioic acid; 4,8-dimethylnonanedioic acid; 2,6-dimethylheptanedioic acid and pristanic acid were found. The detection of 11 intermediates during pristane degradation by the three Gram-positive bacteria provided sufficient information to elucidate in detail three degradative pathways of pristane involving mono-, di- and sub-terminal oxidations. The sub-terminal oxidation by M. neoaurum and R. ruber was demonstrated for the first time. This occurence of a sub-terminal oxidation in these strains was strengthened by further results of aromatic compounds transformation (see below). During this pathway, ketone mono-oxygenation reactions seem to be involved. Because of this it will be of interest to look more closely at the catalytic processes involved and their possible extension to the bio-degradation of other branched chain hydrocarbons. Since in the present study 59 %, 51 % and 84 % of pristane were degraded in 3 weeks by M. neoaurum, R. ruber and N. cyriacigeorgica, this illustrated that the degradation rates of this isoprenoid alkane were high. The bacteria we studied were not only effective degraders of multiple branched chain alkane but also useful transformers of aromatic hydrocarbons. The intermediates produced were analyzed by comparing the retention times and UV/Vis spectra of the HPLC elution profile as well as the retention times and mass spectra of the GC/MS with those of available standards. Using iso-pentylbenzene as a substrate, 8 metabolites were generated by M. neoaurum transformation including product A (phenylacetic acid), B (acetophenone), D (iso-valerophenone), E (succinic acid), F (benzoic acid), G [(2-hydroxy-phenyl)-acetic acid] and H (2-methyl-4-phenyl-butyric acid). We additionally identified an alkyl hydroxylated iso-pentylbenzene derivative as 2-methyl-4-phenyl-butan-2-ol or 2-methyl-4-phenyl-butan-1-ol. Two metabolites (C and D) were detected by N. cyriacigeorgica transformation and three metabolites (A, D and F) were identified by R. ruber transformation which led to the complete biotransformation of this substance. iso-Pentylbenzene transformation by M. neoaurum was initiated by attack on the alkyl side chain followed by ring cleavage. The appearance of iso-valeorophenone confirmed the occurrence of a sub-terminal oxidation mechanism in M. neoaurum and R. ruber. In addition to products A, C, D and G, the identification X-(3–methyl–butyl)-phenol (X means that position of the hydroxy group on the aromatic ring system, such as 2, 3 or 4 remained unclear) in T. mucoides cultivation demonstrated for the first time the capacity of alkyl side chain attack by this organism which was hitherto known only for its ability of ring cleavage. The detection of 15 degradation products of sec-octylbenzene (including 2-phenylpropionic acid, 3-phenylbutyric acid, ß-methylcinnamic acid, 5-phenylhexanoic acid, acetophenone, 2-hydroxy-acetophenone, 2,3-dihydroxy-benzoic acid, succinic acid, 7-phenyloctan-2-one, benzoic acid, phenylacetic acid, 7-phenyl-octan-2-ol, hydroxy-phenylacetic acid and 2-hydroxybenzoic acid), in the studied bacteria pointed to an effective sec-octylbenzene degradation pathway in which dehydrogenation of 3-phenylbutyric acid to form ß-methylcinnamic acid is a newly described option. The identification of 2-phenylpropionic acid and 3-phenylbutyric acid in sec-octylbenzene transformation experiments by T. mucoides confirmed the possibility of alkyl side chain attack by this yeast. Summarizing the results, we describe for the first time in detail the biotransformation of sec-octylbenzene by M. neoaurum, N. cyriacigeorgica, R. ruber and T. mucoides. Our results suggest that these microorganisms may be useful as potential strains for hydrocarbon degradation and it may be of interest to investigate their suitability to solve specific environmental pollutant problems associated with branched chain aliphatic and alkyl-branched compounds which contribute to the persistence of hydrocarbon fractions in the environment.
Summary Prostate cancer (PCa) is the most common type of cancer found in men from western countries and is the leading cancer death next to lung cancer and colorectal cancer. Proteomic studies on PCa identified a number of differentially expressed proteins and some of them were reported as potential markers, but clinical application of these markers is mostly missing. Most of the expression profiling studies have been carried out on radical prostatectomy specimens, formalin-fixed paraffin-embedded (FFPE) tissue sections, serum, urine and prostate fluids. To define the protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH) and PCa patients by two-dimensional gel electrophoresis (BPH n=11 and PCa n=12) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins identified. The important proteins identified included, Prohibitin and NDRG1 tumor suppressor proteins, HSPs, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin expression was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating the involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH, moderate staining in prostate intraepithelial neoplasia (PIN) and strong staining in PCa. From the list of differentially proteins compared to PCa, TPD52 is over expressed in prostate cancer and also mRNA estimation by real-time PCR confirmed over expression of TPD52 at transcriptional level in cancer. TPD52 is a protein over expressed in prostate and breast cancer due to gene amplification but its exact physiological function is not investigated in detail. In the present study, we explored the responsiveness of LNCaP cells after dysregulation of TPD52 expression. Transfection of LNCaP cells with specific shRNA giving efficient knockdown of TPD52 resulted in a significant cell death of the carcinoma LNCaP cells. As evidenced by the activation of caspases (caspase-3 and -9) and by the loss of mitochondrial membrane potential, cell death occurs due to apoptosis. The disruption of the mitochondrial membrane potential indicates that TPD52 acts upstream of the mitochondrial apoptotic reaction. To study the effect of TPD52 expression on cell proliferation, LNCaP cells were either transfected with EGFP-TPD52 or a specific shRNA. EGFP-TPD52 overexpressing cells showed an increased proliferation rate whereas TPD52-depleted cells showed a reverse effect. Additionally, we demonstrated that the exogenous expression of TPD52 promotes cell migration via ávâ3 integrin in prostate cancer cells through the activation of protein kinase B (PKB/Akt) pathway. In an attempt to identify new interacting proteins for TPD52, GST pulldown assays provided evidence for the physical interaction between TPD52 and Prx1 in LNCaP cells. Further, immunoprecipitation results confirmed this interaction. Our results demonstrates that protein profiling and mRNA studies can be performed on prostate biopsies. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCa and BPH. From the results for functional characterization of TPD52, we conclude that TPD52 plays an important role in various molecular events particularly in morphological diversification and dissemination of PCa. It may be a promising target to investigate further in detail to develop new therapeutic strategies to treat PCa patients. Caspases represent a family of cysteine proteases that are regarded as central executioners of apoptotic cell death. Activation of caspase cascade is an essential prerequisite in the induction of apoptosis in cellular systems. So far, in many tumors caspases were shown to be downregulated while anti-apoptotic Bcl-2 is up-regulated. To get insight in their putative role in PCa progression we determined the expression of caspase-1, uncleaved caspases 3 and 6, cleaved (activated) caspases 3 and 6, caspase-9 and antiapoptotic protein Bcl-2 in benign prostate epithelium (BPE) and prostate carcinoma. In the current study 20 prostates were obtained from patients undergoing radical prostatectomy due to PCa. Paraffin embedded prostate whole mounts were cut at (4 µm) and investigated immunohistochemically using anti-mouse monoclonal antibodies directed against caspases 1 and 9, uncleaved caspases 3 and 6, cleaved caspases 3 and 6, and Bcl-2. In BPE all caspases were localized in the cytoplasm of glandular cells. Comparing BPE to PCa, no differences were found for caspase-1, uncleaved caspases 3 and 6 as well as caspase-9. Immunostaining for cleaved caspases 3 and 6, however, revealed a statistically significant reduction in PCa compared to non-neoplastic tissue. Whereas in BPE Bcl-2 protein was detected in the basal compartment of epithelial gland cells no immunostaining was seen in PCa. As our results show a decreased amount of activated caspases may be due to the alterations of posttranslational cleavage rather than expression of caspases 3 and 6. This suggests that the modification in their activation pathway could play an important role during PCa progression.
The WEGA stellarator is used to confine low temperature, overdense (densities exceeding the cut-off density of the heating wave) plasmas by magnetic fields in the range of B=50-500 mT. Microwave heating systems are used to ignite gas discharges using hydrogen, helium, neon or argon as working gases. The produced plasmas have been analyzed using Langmuir and emissive probes, a single-channel interferometer and ultra-high resolution Doppler spectroscopy. For a typical argon discharge in the low field operation, B=56 mT, the maximum electron density is n_e~10^18m^{-3} with temperatures in the range of T=4-12 eV. The plasma parameters are determined by using Langmuir probes and are cross-checked with interferometry. It is demonstrated within this work that the joint use of emissive probes and ultra-high resolution Doppler spectroscopy allows a precise measurement of the radial electric field. Here the floating potential measurements using emissive probes have been compared to measurements of the poloidal rotation of the plasma which is also linked to the radial electric field. In order to alter the plasma parameters a biasing probe setup has been used during this work. The focus of this work is on demonstrating the ability to modify the existing radial electric field in a plasma by using the biasing probe. This technique is in principle not new, as it has been around for decades. Looking at details, it turns out that describing low field operation WEGA argon plasmas in connection with biasing is not covered by the present set of theoretical approaches and experimental cognition. This work will commence with a basic approach and first establishes the diagnostic tools in a well-known discharge. Then the perturbation caused by the biasing probe is assessed. Following the characterization of the unperturbed plasmas, plasma states altered by the operation of the energized biasing probe will be characterized. It is demonstrated that modifying the existing radial electric field can be achieved and reliably diagnosed using spectroscopy and probe measurements. In order to verify the different approaches for determining the radial electric field the diagnostics are cross-checked against another whenever possible. During biasing the plasma two different stable plasma states have been found. Stable here refers to the state existing much longer than the confinement time for WEGA. The presence of a calorimetric limiter placed in the scrape-off layer has an impact on the type of the plasma state. The two observed plasma states differ in plasma parameter profiles, such as density, temperature, electric field and confined energy. The results are compared to two simple models. One model relies on the relevant atomic processes and a second one is based on neoclassical theory. Both models can be used to derive the particle and power flux from the plasma. The losses predicted by the atomic models can be tested using bolometry. It can be shown that both models agree well in the description of the particle balance of the electrons for large regions of the plasma. By comparing the models the neoclassical heat flux turns out to be small compared to the energy fluxes caused by atomic processes. For the reference discharge taking the energy flux due to the atomic processes and balancing it by the input microwave power is satisfying the energy balance, without the need for transport. For the biased discharges it turns out that neoclassical transport can be neglected as well, but the additional biasing power has to be taken into account. A simple model for the biasing power is motivated and tested. An agreement in the energy balance can be reached in this way as far as the models are applicable. The models also allow drawing conclusions on the amount of absorbed microwave power.
Genetic heterogeneity and molecular genetic diagnostics in primary and secondary laminopathies
(2008)
Laminopathies are a group of rare genetic disorders caused by mutations in genes encoding proteins of the nuclear lamina. One can distinguish between primary and secondary laminopathies. Primary laminopathies representing at least fourteen disease phenotypes arise through pleiotropic mutations in LMNA - the gene that codes for the A-type lamins A and C, mutations in LMNB1 encoding lamin B1 and mutations in LMNB2 encoding lamin B2. Secondary laminopathies including disease phenotypes also observed in primary laminopathies are caused by genes encoding proteins related to the nuclear lamina like ZMPSTE24 (FACE1), LAP2, LBR and thus reflecting genetic heterogeneity in laminopathies. The goal of the present investigation was to study pleiotropy and genetic heterogeneity in primary and secondary laminopathies by analysis of genotype/phenotype correlations. Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy with conduction disturbances (CMD1A), familial partial lipodystrophy (FPLD), mandibuloacral dysplasia (MAD), progeroid syndrome, atypical Werner syndrome (aWRN), restrictive dermopathy (RD) and Hallermann-Streiff syndrome (HSS) were included as disease phenotypes to look for their association with LMNA (primary laminopathies) and ZMPSTE24 (secondary laminopathies). Additionally, EDMD patients without STA or LMNA mutations were tested for ZMPSTE24 mutations. A functional candidate gene approach was applied using NARF and SREBF1 in patients suffering secondary laminopathies including FPLD, MAD, HGPS and RD, who were excluded from having LMNA and ZMPSTE24 mutations. Finally, practical consequences of the present study have been considered in genetic counseling and prevention of primary and secondary laminopathies. Screening for mutations in LMNA, ZMPSTE24 (FACE1), NARF and SREBF1 was carried out by PCR using intronic primers flanking each of the exons of the genes tested. The PCR products were tested for changes by heteroduplex analysis and directly sequenced by a cycle-sequencing procedure. Each DNA variation found was checked for its frequency in 386 chromosomes of an ethnically matched control population. For primary laminolathies, 249 unrelated individuals suffering EDMD, CMD1A (DCM), FPLD, MAD, HGPS, aWRN, RD, Hallermann-Streiff syndrome or only partially showing clinical features of the afore mentioned disease phenotypes were tested for LMNA mutations. Eighteen independent LMNA mutations were found in 249 unrelated patients resulting in a general detection rate of 7.2% Summary Dissertation 83 Among the 79 unrelated Caucasian patients and seven families suffering EDMD or EDMD-like disease phenotypes, 14 were found with LMNA mutations, including p.E33G, p.R249Q, p.L263P, p.R377H, p.M348I, p.R249W, p.R453W, p.R527P, p.L530R and p.R644C have been found, resulting in a detection rate of 17.7%. Of the ten different mutations, the three mutations p.L263P, p.M348I and p.L530R are novel. The other seven mutations have been reported before to be pathogenic. There is strong evidence that indicates the pathogenicity of the three novel mutations, p.L263P, p.M348I and p.L530R. Firstly, the mutations exchanged evolutionary highly conserved amino acids as shown by orthologous gene comparisons. Secondly, they were not found in 386 alleles of a reference population. Moreover, the mutations are located in the α- helical rod or globular domains of lamins A and C that might lead to the disruption of their nuclear function causing in skeletal and cardiac muscular malfunction. The LMNA p.M348I mutation was found in a Belgian male patient (G-13730) who also carried a STA c.1A>G, p.0 mutation. The STA mutation leading to a loss of emerin has previously shown to be causative for X-linked recessive EDMD and would explain the lack of emerin and a pathogenic effect found in the affected male by itself. But co-segregation of LMNA p.M348I with cardiac conduction disturbances in female family members showed an additional cardiac effect of this mutation to the pathology. This observation is one of the very rare pieces of evidence for digenic (oligo-allelic) pathogenesis in a neuromuscular disease phenotype of laminopathies. It points to related pathogenic mechanisms in EDMD and CMD1A that are not associated with STA and LMNA but with other so far unknown genes functionally related to the nuclear envelope. The known mutation p.R453W of the LMNA gene represents a mutational hot spot. So, it was not unexpectedly found in four unrelated EDMD patients of this study. Other recurrent mutations p.R249Q and p.R377H were found in two patients each. Variable phenotypic expression of the LMNA p.R644C mutation, ranging from no clinical signs to fully expressed EDMD was observed in an Austrian family in the present study. This mutation has reportedly been associated with strikingly diverse phenotypes in unrelated patients including left ventricular hypertrophy, limb girdle muscle weakness, CMD1A, FPLD or atypical lipodystrophy, neuropathy and atypical progeria. But the mechanism of pathogenesis is unknown. The apparent non-penetrance in relatives raises questions about the clinical significance of this missense mutation. However, the observations Summary Dissertation 84 in the present family and in those previously published provide evidence that the risk to express a laminopathy in close relatives is likely to be low but reasonable. Of the 49 unrelated Caucasian patients suffering CMD1A four mutations, p.E161K, c.- 3_+12del, p.Y259C and p.R377H, were found resulting in a detection rate of 8.2%, which did not significantly differ from the 2.5% found in 197 dilated cardiomyopathy patients of an earlier study. This overall low detection rate reflects the wide genetic and environmental heterogeneity of the pathogenesis in dilated cardiomyopathy. Otherwise, LMNA mutations may cause dilated cardiomyopathy in about 5% of the cases. The wide overlapping phenotypic and genetic similarities between Hallermann-Streiff syndrome (HSS) and HGPS, made HSS a good candidate disease for a primary laminopathy caused by LMNA mutations. But there was no co-segregating disease causing mutation identified. Thus, this study excluded HSS for the first time to be associated to LMNA and adds to the molecular genetic differentiation by excluding HSS from primary laminopathies. Among 32 individuals of 12 families suffering restrictive dermopathy, 22 individuals have been found to carry the ZMPSTE24 mutations c.50delA, c.209_210delAT, c.1085 - 1086insT or c.1385T>G. The mutation c.1085 -1086insT is a recurrent mutation that occurred in the present sample with a frequency of 68% in all RD patients with a ZMPSTE24 mutation. Three mutations, c.50delA, c.209_210delAT and c.1385T>G, are novel mutations. Like the c.1085 -1086insT mutation, c.209_210delAT and c.50delA lead to a frame shift, which putatively results in a non-functional truncated peptide. As an additional indication for a pathogenic effect, the novel mutations c.50delA and c.209_210delAT were not found in 386 alleles of a normal reference population. The first ZMPSTE24 missense mutation c.1385T>G (p.L462R) changing a highly conserved amino acid was found in patient from Guinea suffering from a clinically unequivocal case of restrictive dermopathy. The mutation was heterozygous in the patient but also in the healthy mother. A second pathogenic mutation should be expected. This hypothesis could not be proven, as there was no sufficient test material available from the patient and other family members. Moreover, there was no appropriate African (Guinea) reference population available, which could have been used to estimate the frequency of p.L462R. Thus, it cannot be excluded that p.L462R might be a polymorphism or rare non-pathogenic variant in the ethnic group the patient belongs to. Genetic instability in ZMPSTE24 has interfered with the molecular genetic diagnosis of restrictive dermopathy leading to the inability to distinguish between homozygotes and heterozygotes for the ZMPSTE24 mutation c.1085-1086insT. The reason is a repeated Summary Dissertation 85 thymine (T)9 c.1076-1085 in ZMPSTE24 that can cause a slippage of DNA polymerases. By sequencing cDNAs obtained from homozygous wild-type [(T)9], heterozygous [(T)9/(T)10] and homozygous mutant [(T)10] individuals by using regular Taq polymerase (Fermentas) or high fidelity polymerase (Pfu) for the sequencing reaction the genetic instability was quantified. High error rates up to 23% were found if regular Taq polymerase (Fermentas) was used for sequencing while using high fidelity polymerase (Pfu) resulted in error rates of 6.2 % or lower. As a practical consequence, high fidelity polymerase should always be used to distinguish homozygous mutant [(T)10] individuals from heterozygous [(T)9//(T)10] by sequencing. A high percentage of EDMD patients was tested negative for mutations in STA or LMNA (Bonne et al., 2003). Therefore, other genes are supposed to be involved in the molecular pathology of EDMD. ZMPSTE24 was considered as a promising functional candidate gene in this study, as the gene product - the ZMPSTE24 peptide - takes part in the post-translational modification of lamin A. The negative result of the present study points to a rather unlikely association of EDMD with ZMPSTE24. Additionally, NARF can very likely be excluded by this study from being associated with FPLD, MAD, HGPS and RD, while SREBF1 has obviously no association with FPLD. By the present study, diagnostic tools have been established for molecular genetic diagnosis of several very rare primary and secondary laminopathies, which has a direct practical impact on disease management of laminopathies. Now, the molecular definition of the diseases by association with a specific mutation can be used for genotype/phenotype correlation, predictive diagnosis and prenatal diagnosis.
Genome-wide responses and regulatory mechanisms to thiol-specific electrophiles in Bacillus subtilis
(2008)
The soil-dwelling bacterium Bacillus subtilis is regarded as model organism for functional genomic research of low GC Gram-positive bacteria. Recently, the group of Haike Antelmann has monitored the expression profile of B. subtilis after exposure to phenolic compounds. Interestingly, proteome and transcriptome analyses showed a strong overlap in the expression profile after exposure to catechol, MHQ that auto-oxidized to quinones and the thiol-reactive electrophile diamide. The response to electrophilic quinones and diamide is governed by a complex network of transcription factors, including Spx, CtsR, PerR, CymR and the novel MarR-type repressors MhqR (YkvE), YodB and YvaP. The regulatory mechanisms of these novel thiol-stress sensors YodB and YvaP are studied as part of this thesis in collaboration with the group of Peter Zuber (Oregon). YodB negatively regulates the expression of the nitroreductase YodC and the azoreductase YocJ (AzoR1) after exposure to electrophilic quinones and diamide. The azoreductase AzoR1 is a paralog of AzoR2 that is under control of MhqR. Both paralogous azoreductases (AzoR1 and AzoR2) have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry approaches suggested that YodB is regulated by a thiol-(S)-alkylation mechanism in response to quinones. Mutational analyses revealed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro. Recent studies further suggest that YodB is redox-regulated by intersubunit disulfide formation in vivo by diamide. In addition to the azoreductases, several thiol-dependent dioxygenases confer resistance to quinones. In collaboration with Kazuo Kobayashi (Nara), the YodB-paralogous MarR/DUF24-family regulator, YvaP was identified as repressor of the catechol-2,3-dioxygenase encoding yfiDE (catDE) operon. DNA binding activity of YvaP was also directly inhibited by quinones and diamide in vitro indicating that also YvaP is regulated via post-translational modifications. Mutational analyses showed that the conserved Cys7 is essential for YvaP regulation in vivo and serves as sensor for thiol-reactive compounds. In addition, also the basic amino acids K19, R20 are essential for YvaP repression in vivo as well as conserved basic arginine and lysine residues located in the DNA binding helix-turn-helix (HTH) motif. Non-reducing PAGE analysis suggests the formation of an intersubunit disulfide bond in a YvaP dimer upon treatment with quinones and diamide in vitro. Besides quinones, also aldehydes are electrophilic compounds which react via the thiol-(S)-alkylation reaction with thiols. Thus, we were also interested in the response of B. subtilis to the toxic electrophiles methylglyoxal (MG) and formaldehyde (FA). We analyzed the changes in the transcriptome and proteome of B. subtilis after exposure to MG and FA. Like quinone compounds, both MG and FA induce the thiol-specific stress response. Metabolomic approaches confirmed that these reactive aldehydes deplete the cellular thiol pool and thus act like quinones as another class of thiol-reactive electrophiles. Additionally, MG and FA also triggered responses to overcome DNA damage. Our studies further revealed the specific induction of two FA detoxification pathways regulated by the MarR/DUF24 family repressor HxlR, and the novel MerR/NmlR-type regulator YraB (AdhR). HxlR positively regulates the hxlAB operon encoding the ribulose monophosphate pathway. AdhR positively regulates an adhA-yraA operon that encodes the thiol-dependent formaldehyde dehydrogenase (AdhA) and the DJ1/PfpI-like cysteine proteinase (YraA), and the yraC gene that encodes a γ-carboxymuconolactone decarboxylase. Thus, the AdhR regulon is involved in the detoxification of FA to formate via the formaldehyde dehydrogenase AdhA which catalyzes the cleavage of S-hydroxymethylcysteine adducts. In addition, the cysteine proteinase YraA could be involved in the degradation of S-hydroxymethylcysteine-modified and damaged protein thiols. In collaboration with the group of John Helmann (Ithaca), it was shown that AdhR binds in vitro to a conserved inverted repeat between the -10 and -35 promoter elements upstream of adhA, yraB and yraC. In addition, we showed that the conserved Cys52 of AdhR is essential for aldehyde sensing and activation of adhA-yraA transcription in vivo. Thus, we speculate that redox regulation of AdhR involves thiol-(S)-alkylation of this Cys52 residue by aldehydes as another novel mechanism of bacterial physiology.
The primary objective of this study is to practically apply geostatistical tools that can help to improve an evaluation of groundwater quality for a particular area. The Nam Dinh area, an area of 70 x 70 km2, located in the Southern part of the Red River Delta, was selected as a source for different data sets to be used as case studies. A set of geostatistical tools has thus been applied to the different real data sets which were collected from the coastal Quaternary aquifers in the different campaigns. This gives us a yardstick by which the success of a specific approach can be measured. Throughout the thesis a series of the case studies are, in turn, represented in order to get insight into and an understanding of what various geostatistical tools can do and, more importantly, what their shortcomings are. There are nine different methods of data analyses use in this thesis, which include: (1) Major Ion Comparison, (2) Graphic Plots, (3) Exploratory Statistical Data Analysis, (4) Variogram Analysis, (5) Spatial Estimation Using Kriging, (6) Cluster Analysis, (7) Principle Component Analysis, (8) Multivariate Regionalization Analysis, and (9) Contamination Risk Mapping Using Indicator Kriging. First, major ion comparison and graphic analysis are performed in order to get a general hydrogeochemical view of the collected datasets, before stepping into a further geostatistical approach. By these analyses, various groundwater types are observed and a general hydrochemical trend is visualized using Stiff- and Piper diagrams as well as site maps. The major ion ratios in relation to TDS concentration are compared to investigate the origin of water. Second, a statistical exploratory data analysis is applied to describe the important features of the data by which the character of a specific hydrogeochemical variable might be recognized. Deviations from the Gaussian probability model are detected and appropriate transformations for a formal analysis in geostatistics are selected. Since the Theory of Regionalized Variables (Mathéron 1971; short term: Geostatistics) assumes Gaussian distribution the hydrochemical variables used here are checked for normality. These analyses show that although the data are facing some problems such as outliers and they are very positively skewed at the linear scale, this can effectively be minimized by transforming the data to log-scale. Third, both variogram analyses and Kriging techniques are used to spatially estimate a rectangular 36x36 estimation grid within an area of 70 x 70 km based on the sampled locations (85, 45 and 74 and 38 visited locations for the Pleistocene RS, the Pleistocene DS, the Holocene RS, the Holocene DS, respectively). These estimated values are then used to map the spatiotemporal variability of groundwater quality. In practice, estimation of unknown values and mapping of concentrations of a specific variable can, of course, easily be created by many available software programs. However, error variances are always present in any estimation due to a level of uncertainty, so the reliability of how these estimates could be yielded has also been evaluated in this case study. A critical assessment of all possible variations, tightly related to the seasonal change, directional influence, spatial distribution and prediction error is conducted and concluded. Fourth, Cluster Analysis (CA), Principle Component Analysis (PCA) and Multivariate Regionalization Analysis (RA) are applied to three main datasets of all Quaternary aquifers in the Nam Dinh area to discover the relationships among measured hydrochemical parameters by which we can detect and regionalize major factors which have an impact upon groundwater quality. These approaches are also to overcome the plethora of data that is usually a common problem for any one who has already tackled groundwater data. In this case study both clustering and R mode principal component analyses are thus performed based on the following parameters: The log-transformed concentrations of all measured major ions and of NO3-, NO2-, NH4+, PO42-, i.e. 11 variables from three different datasets of the main aquifers. By cluster analysis three classes of water types, ranging from freshwater to brackish-saltwater types, are typically grouped. Finally, Indicator Kriging (IK) is performed to evaluate the risks of arsenic contamination. The focus of this approach is to assess contamination risk expressed as probability of exceeding threshold- values. The region may thus be subdivided into “safe” and “unsafe” zones on the basis of probability maps which mark contaminated all places where the risk of arsenic contamination exceeds a given threshold for drinking water purpose. By this case study, it is shown that Indicator Kriging is a useful method which has some advantages for many contamination studies. Firstly, it is well known as the non-parametric technique which can be appreciably used when a dataset does not reach normal shape or nearly normal shape as in this situation. Secondly, the outlier problem that often exists in any analysis can be overcome when applying this method. Thirdly, it can be applied in practice to delimit a study area into “safe zone” or “unsafe zone” from which decision-making on the water supply can be decided for the remediation of a contaminated water source or selecting an appreciative source for exploitation. The combined use of spatial (Geostatistics) and multivariate statistical measures have proven to be of major assistance in questions of assessing groundwater quality especially in less sampled regions. A major advantage lies in the possibility of simultaneously creating spatial estimates as well as estimation confidence limits.
Interaction of injected dust particles with metastable neon atoms in a radio frequency plasma
(2008)
Spatial density and temperature profiles of neon metastables produced in a radio frequency (rf) discharge were investigated by means of tunable diode laser absorption spectroscopy. The experiments were performed in the PULVA1 reactor, which is designed for the study of complex (dusty) plasmas. The line averaged measured density is about 1.5×1015 m−3 in the bulk and drops almost linearly in the plasma sheath. The gas temperature is in the range of 370–390 K. The flow of metastable atoms in the plasma sheath deduced from the spatial density distribution is dominated by the flow towards the rf electrode. The sheath length is supposed as the effective diffusion length in the plasma sheath region. This approximation was used to investigate the interaction of injected particles with the plasma. The observations and estimation provide evidence for a significant interaction between metastable atoms and powder particles which is important for energy transfer from the plasma to the particles. The power per unit area absorbed by dust particles due to the collision of metastable atoms with the dust particle surface is in the range of a few tens of mW m−2.
The properties of the ion feature of the Thomson scattering signal are investigated. Firstly, the description of the atomic form factor by hydrogen-like wave functions is reviewed and better screening charges are obtained. Then the ionic structure in systems with several ion species is calculated from the HNC integral equation.
Mind the gap: Information gaps and bridging options in assessing in-situ conservation achievements
(2008)
The biodiversity crisis has gained political attention on a global level. The “2010 Target” of the Convention on Biological Diversity (CBD) aims to significantly reduce the loss of biodiversity by 2010. In order to achieve this, a network of representative and effectively managed protected areas is to be established. The effectiveness of protected areas thus represents one indicator for progress towards the CBD’s 2010 Target. However, indicators require information. The present study, in a first step, reviews the availability of open access long-term ecological data for assessing protected area effectiveness. This review shows two parallel – though contradictory – phenomena: data overkill and data scarcity. While the number of online databases providing open access data on biodiversity has grown tremendously, no long-term ecological data for a larger set of protected areas can be openly accessed. Reasons for this data scarcity are discussed. Based on this lack of information, in a second step, a method to bridge information gaps through social science research is aspired. An innovative Conservation Success Framework is developed, which defines and relates conservation needs, conservation capacity and conservation actions, its three main components. The basic assumption is that conservation can only be successful where the conservation capacity exists that is required to implement the conservation actions determined by the conservation needs. The framework was used to develop open and closed questionnaires for application in two Mexican biosphere reserves, the Sierra Gorda and the Sierra de Manantlán. As "conservation success" is often immeasurable in protected areas in practice due to unspecific conservation objectives the term is for the case studies substituted by “conservation achievements”, i.e. clearly noticeable effects from conservation actions. Overall, almost 60 interviews were conducted with different stakeholder groups. The gained information is validated through social science research techniques, such as triangulation of perspectives and active and passive observation. Based on this, conservation needs are identified and conservation capacities summarised and discussed for both case study sites. Implemented conservation actions addressing identified conservation needs and conservation capacity constraints are then analysed. In addition, noticeable effects from conservation actions on the state of biodiversity at case study sites, i.e. the conservation achievements, are described. Where locally available, non-open access data (as opposing open access data) are used to verify the findings from the social science research. Identified conservation achievements at both case study sites are evident both from quantitative information (for example forest cover increase according to non-open access data) and qualitative information (for example perceived change in the occurrence of illegal activities according to interviews). In addition, rather “intangible” indicators that can only be revealed through qualitative surveys are identified for both sites. This study thus highlights the crucial importance of integrating different types of data, ecological and socio-economic, as well as quantitative and qualitative ones. The present study concludes with a series of recommendations 1) to local practitioners at the two case study sites, and 2) to the international conservation community. Local practitioners may benefit from the present study because its results provide for each site a) an overview of existing conservation needs and implemented conservation actions; b) an easy way to identify action gaps; c) a baseline to identify progress indicators; and d) an overview of diverse perspectives on the current effectiveness of the biosphere reserves. These benefits are considered of particular importance as they can be influential in the revision of the site’s management plans, which both are now approximately ten years old and will soon be revised. The international conservation community will not be able to make a clear statement in the year 2010 about the effectiveness of protected areas on a global level due to a lack of information and transparency. However, the year 2010 should not be considered an end point for measuring progress in in-situ conservation; instead protected area quality standards must be created, effectiveness evaluations institutionalised and efforts to foster regular reporting must continue. Consequently, a scheme of consolidated actions from local to national and international level is proposed that could help to sustainably bridge existing information gaps and close them on the long run. In the end, progress reporting on the effectiveness of protected areas, and other indicators, can only improve if different governance levels “mind the information gaps” in cooperation, until continued information gathering and sharing hopefully closes these gaps one day.
Multiply negatively charged aluminium clusters and fullerenes were generated in a Penning trap using the "electron-bath" technique. Aluminium monoanions were generated using a laser vaporisation source. After this, two-, three- and four-times negatively charged aluminium clusters were generated for the first time. This research marks the first observation of tetra-anionic metal clusters in the gas phase. Additionally, doubly-negatively charged fullerenes were generated. The smallest fullerene dianion observed contained 70 atoms.
In this work we will analyse the capabilities of several numerical techniques for the description of different physical systems. Thereby, the considered systems range from quantum over semiclassical to classical and from few- to many-particle systems. For each case we address an interesting, partly unsolved question. Despite the different topics we address in the individual chapters, the problems under study are somehow related because we focus on the time evolution of the system. In chapter 1 we investigate the behaviour of a single quantum particle in the presence of an external disordered background (static potentials). Starting from the quantum percolation problem, we address the fundamental question of a disorder induced (Anderson-) transition from extended to localised single-particle eigenstates. Distinguishing isolating from conducting states by applying a local distribution approach for the local density of states (LDOS), we detect the quantum percolation threshold in two- and three-dimensions. Extending the quantum percolation model to a quantum random resistor model, we comment on the possible relevance of our results to the influence of disorder on the conductivity in graphene sheets. Furthermore, we confirm the localisation properties of the 2D percolation model by calculating the full quantum time evolution of a given initial state. For the calculation of the LDOS as well as for the Chebyshev expansion of the time evolution operator, the kernel polynomial method (KPM) is the key numerical technique. In chapter 2 we examine how a single quantum particle is influenced by retarded bosonic fields that are inherent to the system. Within the Holstein model, these bosonic degrees of freedom (phonons) give rise to an infinite dimensional Hilbert space, posing a true many-particle problem. Constituting a minimal model for polaron formation, the Holstein model allows us to study the optical absorption and activated transport in polaronic systems. Using a two-dimensional variant of the KPM, we calculate for the first time quasi-exactly the optical absorption and dc-conductivity as a function of temperature. Concerning the numerical technique, the close relation to the time evolution in the other chapters get clear if we identify temperature with an imaginary time. In chapter 3 we come back to the time evolution of a quantum particle in an external, static potential and investigate the capability of semiclassical approximations to it. Considering various one-dimensional geometries, we address basic quantum effects as tunneling, interference and anharmonicity. The question is, to which extend and at which numerical costs, several semiclassical methods can reproduce the exact result for the quantum dynamics, calculated by Chebyshev expansion. To this end we consider the linearised semiclassical propagator method, the Wigner-Moyal approach and the recently proposed quantum tomography. A conceptually very interesting aspect of the compared semiclassical methods is their relation to different representations of quantum mechanics (wave function/density matrix, Wigner function, quantum tomogram). Finally, in chapter 4 we calculate the dynamics of a classical many-particle system under the influence of external fields. Considering a low-temperature rf-plasma, we investigate the interplay of the plasma dynamics and the motion of dust particles, immersed into the plasma for diagnostic reasons. In addition to the huge number of involved particles, the numerical description of this systems faces the challenge of a large range of involved time and length scales. Exploiting the mass differences of plasma constituents and dust particles allows for separating the PIC description of the plasma from the MD simulation of the dust particles in the effective surrounding plasma.
The term atomic cluster relates to compounds of at least two or three atoms. Thereby the physical properties are size dependent and the property transitions between single atoms and bulk material are not always smooth. Ion traps allow it to observe internal cluster properties independent from the influence of external forces. In this work the electron induced decay of singly negatively charged atomic clusters was observed. The dissociation cross section of the clusters is dominated by detachment of the only weakly bound outer electrons. For simple atoms at low electron energies a simple scaling law can be obtained that includes only the binding energies of the valence electrons. Nevertheless for larger sizes theoretical calculations predict so called "giant resonances" as dominant decay process in metal clusters. Due to mass limitations in storage rings exist so far only cross section measurements for simple anions and small negative molecules. In this work the electron detachment cross sections of small negatively charged carbon (Cn- n=2-12), aluminium (Aln- n=2-7) and silver clusters (Agn- n=1-11) were measured in an electrostatic ion beam trap. The classical scaling law, including only the binding energies of the valence electrons, turned out to be not sufficient, especially for larger clusters. In order to improve the correlation between measured and predicted values it was proposed to involve the influence of the cluster volume and the specific polarisability induced by long range coulomb interaction. For silver clusters the best agreement was obtained using a combination of the projected area reduced by the polarisability. The existence of "giant resonances" could not be confirmed. According to theory for clusters with a broad internal energy distribution, a power-law decay close to 1/time is expected. For some clusters the lifetime behaviour would be strongly quenched by photon emission. The thermionic evaporative decay of anionic aluminium and silver clusters in a size range from one to ten constituents was tested but a correlation could be only found incidentally for a few cluster sizes.
The New Zealand coal covering the complete maturity range from peat to high volatile bituminous, thus from early diagenetic to catagenesis coalification levels, has been studied in order to bring out new insights into molecular alterations, macromolecular structural evolution, elemental-compositional changes as function of maturation and to propose which processes cause these changes. As particular note from the previous observations that many immature coals from around the world often have rather high extraction yields. It is uncertain whether or not bitumen affecting on petroleum potential and structural evolution of coals. My purposes were therefore to find out the possible interaction between kerogen and bitumen during pyrolysis, and to elucidate the role of bitumen in defining petroleum potential and structural evolution of coals. Furthermore, it is assumed that low rank coals appear to be well suited for feeding the deep subsurface microbes. The products are released as either CO2 or CO that could be substrates for microbial activity. Thus, in this dissertation, I have calculated the loss of CO2 during diagenesis to give the quantitative feeding potential link to deep biosphere, using a mass balance model. To achieve these purposes, at the primary step, the facies variability as well as the molecular compositional changes within the coal band sequence in regard to distinguish the influences of organofacies and maturity need to be clear. Hence, the first aim was to gather information about depositional environment and insights into the plant communities that have contributed to New Zealand coals. Numerous organic-geochemical techniques were used to analyse the free lipids and macromolecular organic matter. Total organic carbon determination (TOC), bulk δ13Corg isotope analysis, the Rock-Eval pyrolysis, pyrolysis- gas chromatography and infrared spectrometry were performed on the original samples and the residue after solvent extraction. The crude lipid extract was separated into fractions that were then analysed by gas chromatography and gas chromatography-mass spectrometry. The obtained data shows that organic matter of New Zealand coals contains mainly terrestrial higher plant material, with a more or less constant background supply of bacterial biomass, deposited in oxidising environment. Angiosperms contributed as the main proportion of the organic matters. Gymnosperms, particularly the Podocarpaceae, Cupressaceae, Taxodiaceae, Pinanceae and Araucariaceae conifer species, still dominated during the Cretaceous. New Zealand coal is classified as mixed gas- and oil-prone. Hydrogen index values increase from 120 to 280 (mg/g TOC) with increasing maturity, which has been explained by the loss of oxygen during diagenesis. A Transformation Ratio of CO2 (TRCO2) has been formulated here in order to quantify the loss of CO2 for any given coal type. It obviously shows CO2 generation is one of the major features of diagenesis that might feed the deep biosphere. In case of study, about 10 to 105 mg CO2 per gram of total organic carbon have been released during maturation from peat to high volatile bituminous. This is equivalent to 0.23 to 2.4 millimoles CO2 per g TOC. For methanogenesis via CO2 reduction, between 0.92 and 9.6 millimoles hydrogen would be required for complete CO2 reduction during diagenesis. Future work must determine if this is feasible or not. The important role of bitumen in defining the petroleum generation potential was elucidated. The presence of hydrogen transfer agents in bitumen helps to stabilize free radicals hence prevents recombination/ repolymerization processes thus preserving the petroleum generating potential in original coals. Specially, second-order reactions between kerogen and bitumen occurred during pyrolysis that reduce the primary gas yield, but increase the potential secondary gas as well as oil yields. Therefore, it is proposed that pre-extraction of source rocks before pyrolysis, especially coals where extraction yields are particularly high, is not recommended. The comparative investigation with previously studied higher rank Carboniferous German coals showed an excellent fit for both pyrolysis and infrared spectrometry data, suggesting that the New Zealand coals can be considered as natural precursors of the German coals. The structural evolution of coals during maturation is firstly characterized by the enrichment of the aliphatic structures in low rank, peat to high volatile bituminous, then decreases with further maturation. This enrichment of aliphatic carbon content in low rank is accompanied by an increase in the average aliphatic chain length. A slightly enrichment of CH3 group is observed in maturity range 0.9- 2.1% vitrinite reflectance. Secondly, during coalification the content of protonated aromatic carbons increases until R0 ~ 1.6%, then decreases with further maturation.
The rapid neutron-capture or the r-process is responsible for the origin of about half of the neutron-rich atomic nuclei in the universe heavier than iron. For the calculation of the abundances of those nuclei, atomic masses are required as one of the input parameters with very high precision. In the present work, the masses of the neutron-rich Zn isotopes (A=71 to 81) lying in the r-process path have been measured in the ISOLTRAP experiment at ISOLDE/CERN. The mass of 81Zn has been measured directly for the first time. The half-lives of the nuclides ranged from 46.5 h (72Zn) down to 290 ms (81Zn). In case of all the nuclides, the relative mass uncertainty (∆m/m) achieved was in the order of 1E-8 corresponding to a 100-fold improvement in precision over previous measurements.
The study of sow reproduction traits is important in livestock science and production to increase animal survival and economic efficiency. This work deals with the detection of different effects on within-litter variance of birth weight by applying different statistical models with different distributional assumptions. The piglets within one litter were separated by sex. The trait of sow was formed from the sample variances of birth weights within litter separated by sex to consider the sex effect on mean birth weight. A linear mixed model (LMM) approach was fitted to the logarithmized sample variance and the sample standard deviation. A generalized linear mixed model with gamma distributed residuals and log-link function was applied to the untransformed sample variance. Appropriate weights were constructed to account for individual litter sizes. Models were compared by analysing data from Landrace and Large White. The estimates of heritability for the different traits ranged from 6-14%. The LMM for the weighted standard deviation of birth weights was identified as most suitable in terms of residual normality. Furthermore, the impact of piglets´ sex on birth weight variability was tested, but it was only proved for one practical dataset. Additionally, we analysed the influence of including or not including birth weights of stillborn piglets on the estimates of variance components of birth weight variability. With omitted stillborns the estimates of heritability resulted in about 2% higher values than in investigations of total born piglets. We were interested in the presence of the random boar effect on birth weight variability. The corresponding variance component was tested via restricted likelihood ratio test. Among others, the null distribution of the test statistic was approximated by parametric bootstrap simulations which were computational intensive. We picked up a two-parametric approach from literature and proposed a three-parametric approach to approximate the null distribution of the test statistic. We have analysed correlated data in balanced (simulated data) and unbalanced (empirical data) designs. The two-parametric approach using a scaled mixture of chisquare-distributions as well as a three-parametric approach, that uses a mixture of the point mass at zero and a gamma distribution, behaved most solid in all investigations and were most powerful in the simulation study.
Understanding of the regulatory mechanisms controlling stress gene expression of S.aureus in response to environmental stress is very essential in studying its fitness and virulence. In this work, the changes in protein expression profiles as well as the gene transcription of S.aureus after heat exposure, osmotic stress and in response to the antibiotic puromycin were studied in order to provide detailed insights into the response of S.aureus to various kinds of environmental stress under in vitro conditions, namely: (1) to investigate the global response of S.aureus to heat stress conditions using transcriptomic and proteomic analyses. (2) to study the transcriptome and proteome of S.aureus in response to antibiotic substance puromycin. (3) to define the proteome signatures of S.aureus under NaCl stress condition. (4) to complete the proteome map of cytoplasmic proteins of S.aureus by identifying proteins exclusively synthesized during the exposure to stress. Firstly, the high resolution 2-D protein gel electrophoresis technique combined with MALDI-TOF-MS and a DNA array approach were used to investigate the cellular response of S.aureus to heat stress. A switch from normal growth temperature to high temperature condition revealed complex changes in the protein expression pattern as well as the genes expression profile. The effect of puromycin stress on S.aureus cells was analyzed, using a gel-based proteomic approach and transcriptomic analyses with DNA microarrays. We compared the protein synthesis pattern as well as the transcription data of S.aureus in response to puromycin stress with that in response to heat shock. The results demonstrated that both stress conditions induced specific, overlapping and general responses. Finally, the protein expression profile of S.aureus in response to NaCl stress was analyzed with 2D gel based proteomic approach. Our proteome analyses revealed the repression of the synthesis of many enzymes belong to different metabolism pathways . In summary, the signatures for stress or starvation stimuli can be used as diagnostic tools for the prediction of the mode of action of new antibiotics or for studying the physiological state of cells grown. Expression of the respective genes under in vivo conditions could provide some ideas on the environmental signals that specifically influence the survival of S.aureus within and outside the host.
The human antibody response to experimental colonization with Staphylococcus aureus NCTC8325-4
(2008)
The four main work packages and their most important results are briefly described as following. 1. Characterization of the extracellular proteome of S. aureus NCTC8325-4 Reference maps of the extracellular proteins of S. aureus NCTC8325-4 were produced at pH ranges 6-11 and 4-7. In total, 119 (pH 6-11) and 177 (pH 4-7) protein spots were identified, corresponding to 48 and 114 proteins, respectively. Among them were many well-known virulence factors such as alpha-hemolysin (Hla), beta-hemolysin Hlb, gamma-hemolysin subunits (HlgA-C), hyaluronate lyase (HysA) and staphylococcal superantigen-like protein 11 (Ssl11). We also detected various extracellular enzymes, which can cause tissue degradation and are involved in nutrient acquisition, for example, autolysin (Atl), glycerol ester hydrolase (Geh), lipase (Lip), thermonuclease (Nuc), several serine proteases SplA-F (SplA-F), V8 protease (SspA), cysteine protease (SspB), staphopain thiol proteinase (88195808, SspP). Many of these proteins probably also contribute to the virulence of S. aureus. 2. Optimization of a 2-D immunoblot (IB) method for the comprehensive investigation of IgG binding to S. aureus extracellular proteins (strain NCTC8325-4) The immune proteome of S. aureus NCTC8325-4 was revealed by probing 2-D blots of S. aureus extracellular proteins at the two pH ranges 6-11 and 4-7 with a pool of sera from 16 volunteers. IgG binding was detected with high sensitivity using a peroxidase-coupled secondary Ab in combination with an ECL-substrate. With application of the software package Delta2D, we could clearly define 66 immune reactive spots on the immunoblots (IBs) of pH range 6-11 and 38 spots on IBs of pH range 4-7. 72 of these 104 immune reactive spots could be identified by matching the IBs with the protein reference maps. These spots represented 36 identified proteins, many of which are known virulence factors, or they are involved in bacterial cell wall biosynthesis and degradation. Generally, the most abundant proteins were also highly immune reactive, but there was no strict correlation between protein abundance and immune reactivity. Some low abundance proteins, especially basic proteins, showed high immune reactivity on 2-D IBs, for example, Atl, 88195808 (SspP) and iron-regulated surface determinant protein A (IsdA). On the other hand, we observed proteins, which were present in large amounts but did not bind IgG such as peptidoglycan hydrolase (LytM) and a hypothetical protein 88193909 (SAOUHSC_00094). 3. Determination of the anti-staphylococcal Ab profiles of S. aureus carriers and noncarriers Comparing the serum IgG binding patterns of sera from the 16 individual volunteers, we observed pronounced heterogeneity in total IgG binding, spot patterns and spot intensities. Five spots were stronger in carriers than in noncarriers (P< 0.05, Mann-Whitney U test). These spots represent IgG binding to SspA, SspB, IsaA, and two hypothetical proteins. A principal component analysis based on differential IgG binding to these spots showed that the carriers were more closely related to each other than the noncarriers, but that they could not be clearly separated from the noncarriers. 4. Does experimental colonization induce changes of the anti-staphylococcal Ab profiles? Finally, we tested whether symptom-free experimental colonization of the 16 volunteers with S. aureus NCTC8325-4 elicited an IgG response. When we compared sera obtained before colonization with those taken 4 weeks after the inoculation with the laboratory S. aureus strain, we did not observe major changes in the Ab patterns. We conclude that short- term colonization with a strain of low virulence does not suffice to induce an Ab production, which is comparable to that present already before the colonization. Thus, either long term high density colonization is required, or as we consider most likely, the adaptive immune response is primarily triggered by (minor) S. aureus infections. Taken together, in this work we have separated the soluble proteins from complex extracellular S. aureus protein extracts with good reproducibility, large coverage (pH 6-11 and 4-7) and high resolution. With application of an ECL substrate, our 2-D immunoblotting procedure resulted in the highly sensitive detection of IgG binding over a wide range of signal intensities. The most important finding with this technique was the pronounced variability of anti-staphylococcal Ab profiles in healthy adults. This could well explain differences in susceptibility to S. aureus infection and its complications. The Ab responses are presumably triggered by long-term colonization or, more likely, by minor infections with S. aureus, since experimental nasal colonization of healthy volunteers with a bacterial strain of low virulence did not induce impressive changes in the Ab profiles.
The present work is the first work dealing with turbulence in the WEGA stellarator. The main object of this work is to provide a detailed characterisation of electrostatic turbulence in WEGA and to identify the underlying instability mechanism driving turbulence. The spatio-temporal structure of turbulence is studied using multiple Langmuir probes providing a sufficiently high spatial and temporal resolution. Turbulence in WEGA is dominated by drift wave dynamics. Evidence for this finding is given by several individual indicators which are typical features of drift waves. The phase shift between density and potential fluctuations is close to zero, fluctuations are mainly driven by the density gradient, and the phase velocity of turbulent structures points in the direction of the electron diamagnetic drift. The structure of turbulence is studied mainly in the plasma edge region inside the last closed flux surface. WEGA can be operated in two regimes differing in the magnetic field strength by almost one order of magnitude (57mT and 500mT, respectively). The two regimes turned out to show a strong difference in the turbulence dynamics. At 57mT large structures with a poloidal extent comparable to the machine dimensions are observed, whereas at 500mT turbulent structures are much smaller. The poloidal structure size scales nearly linearly with the inverse magnetic field strength. This scaling may be argued to be related to the drift wave dispersion scale. However, the structure size remains unchanged when the ion mass is changed by using different discharge gases. Inside the last closed flux surface the poloidal ExB drift in WEGA is negligible. The observed phase velocity is in good agreement with the electron diamagnetic drift velocity. The energy in the wavenumber-frequency spectrum is distributed in the vicinity of the drift wave dispersion relation. The three-dimensional structure is studied in detail using probes which are toroidally separated but aligned along connecting magnetic field lines. As expected for drift waves a small but finite parallel wavenumber is found. The ratio between the average parallel and perpendicular wavenumber is in the order of 10^-2. The parallel phase velocity of turbulent structures is in-between the ion sound velocity and the Alfvènvelocity. In the parallel dynamics a fundamental difference between the two operational regimes at different magnetic field strength is found. At 500mT turbulent structures can be described as an interaction of wave contributions with parallel wavefronts. At 57mT the energy in the parallel wavenumber spectrum is distributed among wavenumber components pointing both parallel and antiparallel to the magnetic field vector. In both cases turbulent structures arise preferable on the low field side of the torus. Some results on a novel field in plasma turbulence are given, i.e. the study of turbulence as a function of resonant magnetic field perturbations leading to the formation of magnetic islands. Magnetic islands in WEGA can be manipulated by external perturbation coils. A significant influence of field perturbations on the turbulence dynamics is found. A distinct local increase of the fluctuation amplitude and the associated turbulent particle flux is found in the region of magnetic islands.
1. The study confirms an association between transverse arch dimensions and severity of ankyloglossia, which reflects relationship between molar difference and free tongue. 2. Molar difference in group A (complete and severe ankyloglossia cases) has high negative values (average -4.38) which was much smaller than the data from the literature concerning many different types of occlusion. 99 3. The exact relation between transverse arch dimension and severity of ankyloglossia needs further research.