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Arndt, Martin (1)
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Characterization and Regulation of Virulence Factors in Staphylococcus aureus (2011)
Kusch, Kathrin
Staphylococcus aureus is a pathogenic bacterium infecting the human host. It's multifaced adaptation to various environmental conditions is mediated by a tight regulation of the virulence factors influencing the host's immune system. In this thesis two regulators of gene expression were analysed: (i) the global influence of the two-component system SaePQRS and (ii) the regulation of superantigen gene expression by the alternative sigma factor σB. At the outset of this thesis, single target genes induced by SaeRS were known (hla, hlb, cap5, fnbA, coa). In order to get a general idea of the Sae-regulon, the influence of SaePQRS on gene-expression was analysed in two strain backgrounds by proteomics and transcriptomics aproaches. Recapitulatory, expression of at least 18 secreted and two covalently cell-wall bound proteins was decreased following inactivation of the Sae-system. Sae-dependently expressed were, amongst others, well decribed virulence factors like the y-hemolysins HlgA, HlgB, HlgC, LukM and LukF, the innate immune system modulating proteins Efb, CHIPS and SCIN-B as well as the enterotoxin SEB. SaeR acts as an activator of its target genes. Some proteins were detected in increased amounts in the extracellular proteome of the Sae-deficient strain. However, these changes did not occur at the transcriptional level. The expression of virulence factors is determined by other global regulators. No influence of SaePQRS on the transcription of five substancial regulators, namely the Agr-system and its effector molecule RNAIII, the alternative sigma factor σB, the two-component system ArlRS and the DNA-binding protein SarA, could be shown. In the second part of this thesis the issue was broached to the regulation of gene-expression of a subgroup of virulence factors, the superantigens (SAgs) of S. aureus by SaePQRS and σB. In contrast to their well described molecule structure and function, the regulation of their gene expression was largely unknown. Six different S. aureus strains (two laboratory strains and four clinical isolates) encoding one to seven SAg-genes each, were used for analysis of a total of twelve SAgs regarding their transcription and mitogenic activity. The transcriptional units were characterized using Northern-Blotting. The expression of SAgs could be correlated to the respective growth phase. While egc-SAgs were expressed mainly at low optical densities, seb was induced during late growth phase. In contrast, the transcription of sea, seh, sek, tst and sep remained constant and growth-phase independent. The transcriptional dataset was verified using T-cell proliferation assays. The expression of seh, tst and the egc-operon was dependent on σB. A potential σB-dependent promotor could be identified preceeding seo, the first gene of the egc-operon. In contrast, the expression of seb was increased in sigB-deficient background. This might be due to indirect effects. Expression of seb required SaePQRS. Transcriptional datasets were verified by Immuno-Blotting and T-cell-proliferation assays. In conclusion, the same mutation in sigB but in different strain backgrounds could result in opposite phenotypes with respect to their mitogenic activity. Besides well characterized virulence factors, some secreted proteins with so far unknown function belong to the Sae-regulon. Given that the influence of SaePQRS was restricted to virulence factors and induced especially modulators of the innate immune system, it can be assumed, that these proteins potentially play a role in virulence of S. aureus. In the third part of this thesis, one of these potential new virulence factors, namely SACOL0908, was analysed in detail. In cooperation with the group of Prof. Stehle, Tübingen, the crystal structure was solved. The protein folding of SACOL0908 is new with only minor similarities to described protein structures. Recombinantly expressed SACOL0908 binds to granulocytes. These cells belong to the innate immune system, incorporate bacteria by phagocytosis and kill them. The receptor for SACOL0908 on the surface of granulocytes could not be identified using immunoprecipitation, antibody-blocking assays and functional assays in cooperation with the group of Prof. Peschel, Tübingen. The gene encoding SACOL0908 was deleted in two S. aureus strain backgrounds (COL and Newman). These mutants are currently in use to characterize their phenotype in mouse-infection studies.
Building Bridges in Biosphere Reserves – The role of natural and social values in integrative conservation and development concepts in Madagascar (2011)
Fritz-Vietta, Nadine Veronica Marion
The dissertation aims at developing means to integrate conservation and development in biosphere reserves in Madagascar. Despite a multitude of concepts such as UNESCO biosphere reserves, Integrated Conservation and Development Projects and community-based natural resource management, gaps between conservation and development remain to exist. In a qualitative case study in Mananara-Nord and Sahamalaza Iles-Radama Biosphere Reserves in Madagascar data was collected on biosphere reserve management, local use of natural resources and socio-cultural aspects that influence natural resource use. Furthermore, natural values local people associate with the forest were investigated. Analysis revealed that management capacities constitute a limiting factor in biosphere reserve management. Collaboration between management, local people and international organisations fosters the achievement of both conservation and development. However, collaboration is only possible if (i) clear rules are formulated and (ii) partners have a vision in common. Based on the theory of social capital, newly introduced and locally existent rules/institutions having an influence on the use of natural resource were categorized in bonding, linking and bridging social capital. Furthermore, the perception of natural values was classified in instrumental and non-instrumental values and assigned to ecosystem services identifying the importance of nature for human well-being. With the capabilities approach Amartya Sen defined human well-being as the achievement of those capabilities a person considers valuable. This includes aspects that assure livelihoods on the one hand and aspects that are conducive to well-being on the other, thus both being relevant for development. In the dissertation capabilities are based on both instrumental and non-instrumental natural values and consequently offer an opportunity to demonstrate and characterise the relationship between nature and human well-being. Social and natural values provide orientation for a biosphere reserve management. The category bonding social capital (social values) describes local socio-cultural aspects in communities and their importance for collaborative processes. Natural values provide the management with guiding principles to foster nature conservation and to integrate locally existent capabilities. Supporting and furthering these capabilities enables the development of new capabilities of all concerned persons. The dissertation demonstrates various possibilities to build bridges between (i) nature conservation and development, (ii) natural and social sciences, (iii) formal regulations and local socio-cultural aspects and (iv) diverse actors. Implementation of a social monitoring is recommended together with local stewards and Malagasy students to collect information about the perception of natural and social values and use them as guiding principles for biosphere reserves. Collaboration with national and international scientific institutions can foster this process.
Immune Dysfunction in Stroke and Multiple Sclerosis: Analysis of T cell function (2011)
Vogelgesang, Antje
Multiple sclerosis (MS) and stroke share a number of mechanisms of neuronal damage. In both cases the balance between neurodestruction and neuroprotection appears modulated by the function of the adaptive immune system. MS is a chronic inflammatory disease of the central nervous system (CNS), leading to permanent disability. It seems certain that an autoimmune response directed against the CNS is central to the pathogenesis of the disease. While these CNS-specific T cells are activated in MS patients, they are inactive and naive in healthy. Therefore it is believed that an activation of autoreactive T cells by cross-reactivity with pathogens occurs outside of the CNS. In consequence T cells express adhesion molecules and proteinases which enable them to cross the blood-brain barrier. In stroke, however, the blood-brain barrier is disturbed in its integrity caused by the decreased blood flow. Cells can freely migrate from the periphery into the brain. CNS autoreactive cells from the periphery can be activated within the CNS and thus contribute to further tissue damage. While the local autoimmune response remains temporary in stroked brains, it is chronically destroyed in MS. The differences between the underlying mechanisms are not understood. This thesis investigated T cell responses in Multiple Sclerosis in response to the therapeutics Mitoxantrone and IFN-b. The induction of a TH1 to TH2 cytokine response appears to be a shared mechanism of action between both therapeutic agents. Primarily the post stroke immune response was investigated. Patients developed a stroke induced immune suppression characterized by monocytic dysfunction and lymphocytopenia explaining the high frequency of post stroke infections. Moreover early post stroke predictors of subsequent infections, like the CD4+ T cell count, were identified. The T cell response of stroke patients appeared primed to proinflammation and unsuppressed after mitogen stimulation. A detailed understanding of post stroke immune alterations may offer new avenues of intervention to improve the clinical fate of stroke victims. In addition, such knowledge could also further our understanding of Multiple Sclerosis, because, while increasing the infection risk, the dampening of the immune system could have an important protective function, if it limits autoimmune brain damage triggered by the massive release of brain antigens during stroke. If these two pathways could be modulated separately it would create the opportunity to develop distinct therapeutic approaches that inhibit autoimmunity and strengthen antibacterial defenses. To further delineate these mechanisms it is crucial to investigate the role of the innate immune system as compared to the adaptive immune system in stroke induced immune suppression.
Endocrine-metabolic markers and subclinical cardiovascular disorders (2011)
Lorbeer, Roberto
Background: Cardiovascular diseases are the leading cause of death worldwide. Subclinical alterations of the cardiovascular system, such as increased exercise blood pressure or an endothelial dysfunction confer a higher risk of manifest cardiovascular diseases and incident events. Detecting associations between circulating markers of the endocrine-metabolic system and the subclinical cardiovascular phenotypes could be useful to better understand cardiovascular disease progression and to improve risk prediction for manifest cardiovascular diseases. Methods: The associations between (a) serum thyroid-stimulating hormone and increased exercise blood pressure, (b) serum hemoglobin A1c and endothelial dysfunction as well as (c) serum insulin-like growth factor I and endothelial dysfunction were studied using cross-sectional data from around 1400 subjects aged 25 to 85 years collected during the 5-year follow-up of the population-based Study of Health in Pomerania (SHIP-1). Increased exercise blood pressure was defined as a value above the sex- and age-specific 80th percentile measured at the 100 W stage of a symptom-limited bicycle ergometry test. Endothelial dysfunction was defined as an impaired flow-mediated dilation measured as a continuous decrease or below the median of sex-specific distribution. Non-fasting blood samples were drawn from the cubital vein in the supine position. Results: The odds for increased systolic exercise blood pressure (odds ratio 1.24, 95% confidence interval 0.88; 1.76) and diastolic exercise blood pressure (odds ratio 0.98, 95% confidence interval 0.70; 1.39) as well as for exercise-induced increase of systolic and diastolic blood pressure were not significantly different between subjects with high and low serum thyroid-stimulating hormone levels within the reference range. In women without current use of antihypertensive medication, increasing serum hemoglobin A1c levels were associated with decreasing flow-mediated dilation levels (ß = -1.17, 95% confidence interval -2.03; -0.30). Such an association was not found in men. In men, logistic regression analysis revealed an odds ratio of 1.27 (95% confidence interval 1.07; 1.51) for decreased flow-mediated dilation for each decrement of serum insulin-like growth factor I standard deviation. In women, no significant association between serum insulin-like growth factor I levels and flow-mediated dilation was observed (odds ratio 0.88, 95% confidence interval 0.74; 1.05). Conclusions: Based on the presented results it is concluded that (a) serum thyroid-stimulating hormone levels are not associated with exercise blood pressure in the general population, (b) higher serum hemoglobin A1c levels in non-diabetic subjects are inversely associated with flow-mediated dilation in women without antihypertensive medication, but not in men, and (c) lower serum insulin-like growth factor I levels are associated with impaired endothelial function in men, but not in women. Therefore the metabolic marker hemoglobin A1c and the endocrine marker insulin-like growth factor I might be markers facilitating the identification of subjects at high risk of subclinical cardiovascular alterations.
Interactive Visualization for the Exploration of Aligned Biological Networks and Their Evolution (2011)
Brasch, Steffen
Network Visualization is a widely used tool in biology. The biological networks, as protein-interaction-networks are important for many aspects in life. Today biologists use the comparison of networks of different species (network alignment) to understand the networks in more detail and to understand the underlying evolution. The goal of this work is to develop a visualization software that is able to visualize network alignments and also their evolution. The presented software is the first software for such visualization tasks. It uses 3D graphics and also animations for the dynamic visualization of evolution. This work consists of a review of the Related Work, a chapter about our Graph-based Approach for Interactive Visualization of Evolving Network Alignments, an explanation of the Graph Layout Algorithm and some hints for the Software System.
Neural correlates of influencing factors on economic decision making (2011)
Linder, Nicolas
Decision making in everyday purchase situations requires mental processing of factors that are related to the items on display. These influencing factors – called persuasive information – can take various forms, like the price level, the design of the package or the display of certain product attributes. Despite the existence of persuasive information trying to influence our buying behavior, almost nothing is known about the underlying neural mechanisms responsible for processing this information. In this thesis functional magnetic resonance imaging was used to investigate neural activity correlated with product related persuasive information. As persuasive information organic, light and regular labeled food was chosen. The 1st experiment investigated the neural correlates of visually inspected organic and regular labeled food and the influence on willingness to pay (WTP) for the displayed items. It was hypothesized that organic compared to regular labeled food will be perceived as more rewarding which should be visible by an increased activity in the ventral striatum as a central area for reward processing and by a heightened WTP. As organic label information the national German eco emblem 'Bio-Siegel' was chosen (for stimuli details see 2.1). As there is no emblem indicating regular food, an artificially created logo was used for indicating a conventional product. 40 well- known food products (e.g. milk, bread, eggs etc.) were presented to the subjects. These products were marked with the organic emblem and the same 40 products with the regular label. We found that visual inspection of organic labeled food indeed led to an increase in neural activity in the ventral striatum and to a heightened WTP, suggesting a higher subjective value for these products. The 2nd experiment investigated the neural correlates of actually administered food stimuli labeled organic, light or regular and the influence on expected and experienced taste. For organic compared to regular labeled food we hypothesized an increase in expected and experienced taste pleasantness. Furthermore, light compared to regular labeled food should lead to a decrease in expected and perceived pleasantness and intensity ratings. During the active tasting process this should be accompanied by an increase in reward-related (e.g. organic vs. regular; regular vs. light) areas like the ventral striatum medial orbitofrontal cortex or aversion-related (e.g. regular vs. organic; light vs. regular) areas as the lateral orbitofrontal cortex (lOFC) and operculum/insula. As organic label information the national German eco emblem 'Bio-Siegel' was chosen. Light label information was issued in form of the internationally used 'Bewusst-Wählen®' ('Healthy Choice') label (for stimuli details see 2.2). However, inside the scanner the written forms 'Bio', 'Light' or 'Normal' (indicating regular food) were chosen. Subjects were randomly assigned in two groups and were either confronted with the organic or the regular label (organic group) or with the light or the regular label (light group) but otherwise identical milk drink. The results show that organic compared to regular labeling of identical food stimuli indeed led to an increase in expected and experienced taste pleasantness for organic labeled food. Light compared to regular labeling of identical food stimuli led to a decrease in expected and experienced taste pleasantness and intensity for light labeled food. Moreover, taste-related activity was found in aversion related areas like the operculum insula and the lOFC for food labeled regular compared to organic and in reward-related areas like the ventral striatum for food labeled regular compared to light. The results show that persuasive food-related information influences human cognition on the behavioral and neural level; the effects were shown during visual and gustatory evaluation of the stimuli. Taken together the results demonstrate that the same stimulus can vary dramatically in personal valuation depending on the applied information.
Synthesis of Novel Biaryl-Type P=C-N-Heterocyclic σ2P,N- and σ2P,σ3P-Hybrid Ligands (2011)
Niaz, Basit
Phosphines are highly versatile ligands for transition metal catalysts because of wide tuning abilites of their stereoelectronic properties. Bulky and basic phosphines, to a smaller extend also π-acidic phosphites were intensively studied whereas dicoordinated trivalent phosphorus compounds were comparatively little investigated in this respect. In part this may go back to the limited stability of many P=C compounds, in the case of the stable benzazaphosphole to low stabilityof complexes with non-zero-valent transition metals. With the availability of suitable chelate complexes this problems may be overcome. Because biaryl phosphines proved particularly useful as chelate ligands this work is focused on the development of convenient syntheses of new biaryl-type N-heterocyclic or functionally aryl substituted 1,3-benzazaphosphole P,N- P,P- and P,O-chelate ligands and the characterization of their structures. The pivotal point was to find an applicable synthetic route to the title ligands. Because currently transition metal catalyzed cross-coupling reactions are a hot field in catalytic research, the initial target of my work was the investigation of the applicability of suitable biaryl coupling reactions on 1,3-benzazaphospholes. There are several types of transition metal catalyzed biaryl couplings. One reaction, which is currently in the main focus by use of non-toxic and air stable coupling partners, often allowing water as environmental friendly solvent, is the Pd-catalyzed Suzuki-Miyaura coupling of an aryl halide with an arylboronic acid. To apply the Suzuki coupling to the synthesis of biaryl-type benzazaphospholes, the synthesis of either benzazaphosphol-2-boronic acids or reactive 2-halogen-benzazaphospholes have to be performed. Because of the successful introduction of functional groups in position 2 of benzazaphospholes via lithiation and reaction with electrophiles, the 2-lithiation of suitably available N-substituted benzazaphospholes and introduction of boryl groups or halogen by reaction with boronic acid esters or with a halogenating reagent like dibromoethane appeared as a realistic route and was chosen for closer study. N-Neopentyl-benzazaphosphole was selected by its relatively easy access and N-mesityl-benzazaphosphole as a N-aryl representative. From the two principal methods developed to synthesize 1,3-benzazaphospholes, only the synthesis and reduction of o-aniline phosphonic acid esters to o-phosphinoanilines and subsequent [4+1] cyclocondensation is promising to access N-substituted 2-CH benzazaphospholes. My first investigations targeted to improve the synthesis of the benzazaphosphole precursors. The invention of a Cu- instead of the earlier used Pd-catalyzed P-C coupling allows a more economical access to anilinophosphonates which were then transformed to 2H-1,3-benzazaphospholes by the established orthoformamide cyclocondensation. Several attempts of the coupling with careful control of dryness of all reagents and solvents were made in order to obtain pure 1,3-benzazaphosphole-2-boronic acid ester and, after mild hydrolysis, to isolate 1,3-benzazaphosphole-2-boronic acid. The coupling worked with N-mesityl-1,3-benzazaphosphole 13e, but the benzazaphosphol-2-boronic acid could not be obtained in pure form because of easy B-C bond cleavage during crystallization, certainly by the two 'OH groups. For attempts with a reverted methodology, the synthesis of a 2-bromo-substituted benzazaphosphole was studied, which should be coupled with (hetero)arylboronic acids via Suzuki-Mijaura reaction. However, the 2-bromo-benzazaphosphole also could not be obtained in pure form, and a coupling experiment with phenyl boronic acid and catalysis with ligand free Pd/C failed. Therefore, other routes to biaryl-type benzazaphospholes were envisaged. Direct C-H functionalization has emerged over the past few years as an attractive strategy to enhance molecular complexity. This holds also for π-excess-type heterocycles like indoles, benzoxazoles or purines which allow direct CH-arylation in 2-position. These reactions generally involve palladium based catalysts and in some cases rhodium catalysts. In a series of experiments the catalytic arylation, heteroarylation and later also alkylation were studied with 1,3-benzazaphospholes 13a-e as precursors. The initial studies were carried out with iodobenzene, keeping similar reaction conditions as for 2-CH arylation of indoles. Then transition metal catalysts, bases and conditions were varied. The necessity and influence of a catalyst was established by blind experiments without transition metal catalyst which led to strong decrease of the reactivity. However, the transitional metal catalyzed reactions of N-substituted-1,3-benzazaphosphole with aryl- and heteroaryl halides did not give the desired 2-aryl-substituted 1,3-benzazaphosphole biaryl ligands but revealed a novel oxidative addition at the P=C double bond. In the presence of moisture benzazaphospholine-P-oxides are formed. Further exploration of the scope of this reaction showed that it is applicable to several functionally substituted aryl halides and heteroaryl halides. As besides PdX2 (X = Cl, OAc) also Pd(0)(PPh3)4 was found active as catalyst, it can be assumed, that the reaction occurs via a Pd(0) species and oxidative addition of the aryl halide at Pd(0). Because Pd(0) will coordinate stronger to the π-acidic benzazaphosphole than Pd(II) it is assumed that in the first step small equilibrium amounts of a Pd(0)benzazaphosphole complex will be formed which undergo the oxidative addition and then react to benzazaphospholium salt and furnish back a Pd(0) complex with 1,3-benzazaphosphole ligand. The benzazaphospholium salts are highly sensitive to moisture and react with traces of water to form benzazaphospholine-P-oxides 20 and acid, neutralized by the base. A cyclic species RR'P(OH)=CHR", where the halogen is replaced by OH, may be assumed as intermediate which undergoes a rearrangement to the more stable RR'P(=O)-CH2R" tautomer, driven by the high P=O bond energy. After various investigations of the optimum conditions for the reaction, a number of new functionally substituted P-aryl or P-heteroaryl benzazaphospholine P-oxides and 1,3-dineopentyl-benzazaphospholine-3-oxide were isolated and characterized by 1H, 31P, 13C and HRMS data and two by crystallography. The biaryl-type 2-phenyl-1,3-benzazaphosphole is known since the earliest reports of these heterocycles, synthesized by cyclocondensation of 2-phosphinoaniline with benziminoester hydrochloride or in low yield with benzaldehyde. The latter method was further developed because of the compatibility of the aldehyde group with various donor functions. 2-Phosphinoaniline (12a) and 2-phosphino-4-methylaniline (12b) were heated with pyridine-2-carboxaldehyde under varied conditions, and a crucial role of acid catalyst was observed in the investigation. The results showed that the dehydrogenating cyclocondensation, if catalyzed by a suitable type and amount of acid catalyst, works well for primary phosphinoanilines 12a,b and a variety of reactive aldehydes, including N-heterocyclic and o- or m-functionally substituted arylaldehydes. In an equimolar ratio, on heating usually hydrogen is eliminated, at least formally, to furnish the aromatically stabilized 1H-1,3-benzazaphosphole ring systems of 35 whereas in other cases reductive side reactions occur, e.g. the N-CH2R substitution to 36 in reactions with two equivalents of aldehyde. Thus the synthesis of 1,5-dimethyl-1,3-benzazaphosphole (36a) was achieved by double cyclocondensation of 12b and formaldehyde in a 1:2 molar ratio. This provides the so far shortest way to synthesize N-substituted 1,3-benzazaphospholes and suggests, that the reaction is generally applicable in reactions with two equivalents of monoaldehyde. This puts the question if N-secondary o-phosphinoanilines such as N-neopentyl-2-phosphinoaniline (12d) can be cyclocondensed with aldehydes to benzazaphospholes or if a primary amino group is required. The successful experiment shows that cyclocondensation of N-secondary o-phosphinoanilines with suitable aldehydes is possible. N-Neopentyl-2-pyrido-1,3-benzazaphosphole was obtained in high yield. An interesting extension of the above reaction are cyclocondensations with compounds bearing two aldehyde groups. Double condensation of 12b with o-phthaldialdehyde was performed. It proceeded fast and gave tetracyclic-1,3-benzazaphosphole in high yield. Based on the NMR monitored primary formation of organoammonium phosphino glycolates from amines, phosphines and glyoxylic acid, followed by conversion to phosphinoglycines, it is assumed that the reaction proceeds by initial attack of the primary phosphino group of 12b at the carbonyl carbon atom of R-CHO, polarized with the help of the acid catalyst. The resulting P-C bonded secondary phosphine, containing an α-hydroxy group, may release water after transfer of a proton to oxygen in equilibrium, followed by attack of amine. This leads to formation of the dihydro-intermediate 34, observed by NMR reaction monitoring in several cases. Possible ways are releasing of H2 during reflux, directly giving 2-substituted NH-1,3-benzazaphospholes 35, or hydrogen transfer, connected e.g. with N-substitution leading to 1,2-disubstituted 1,3-benzazaphospholes 36. The second path is observed mainly when excess or double molar quantities of aldehydes are used at the start of the reaction. The two hydrogen atoms at P and C2 are consumed during the second condensation and formation of the NCH2R group and generate the P=C double bond. Finally, cyclocondensation of o-phosphinoanilines with aldehydes has proven as a useful method for the synthesis of biaryl type benzazaphosphole ligands. After thorough investigations, N-primary and secondary phosphino anilines were found cyclisable with various heteroaryl aldehydes upon refluxing in toluene in the presence of a suitable acid catalyst, and 11 new compounds were synthesized following this procedure and characterized by 1H, 31P, 13C NMR and HRMS data. For two compounds crystal structures were also obtained. First attempts to synthesize chelate complexes with the 2-(hetero)aryl-1,3-benzazaphospholes were started. A soluble 2-(o-diphenylphosphinophenyl)-1,3-benzazaphoasphole-Cr(CO)4 chelate complex was detected by NMR spectroscopy, whereas most products of the new ligands with Rh(COD) or NiCp complexes were insoluble in usual NMR solvents and require further efforts for synthesis and full analytical and structural characterization.
Novel Cyclic α-Phosphino-α-amino Acids and α-Phosphonium Glycolates, P,O Ligands and Nickel Complexes (2011)
Basvani, Kaleswara Rao
Novel heterocyclic alpha-phosphinoamino acids, by structural relationship named 3-phosphaprolines, were obtained by cyclocondensation of 2-phenylphosphinoethylamines with glyoxylic, pyruvic or phenylglyoxylic acid at room temperature in diethylether. The reactions proceed via primary attack of the P-lone electron pair, as shown by the synthesis of phosphonium glycolates from tertiary phosphines and glyoxylic acid, and addition of PH at the carbonyl group. The ring closure proceeds by replacement of the hydroxy by the amino group and is kinetically controlled. NMR monitoring of the phosphaprolines in CD3OD over several days indicates changes of the diastereoisomer ratios leading to higher contents of the more stable trans-diastereoisomers. The zwitterionic compounds are soluble in part in CD3OD, DMF or DMSO, are somewhat sensitive to air in solution and may undergo hydrolysis with larger amounts of water. The structures are proved by multinuclear NMR spectra and two crystal structure analyses. Suitable phosphaprolines as well phosphonium glycolates and Ni(COD)2 allow to generate precatalysts, activated by NaH for the oligomerisation of ethylene to mainly linear products with methyl and vinyl end groups. Some additional investigations with phosphinophenolates, another type of P-C-C-O- ligands, were performed for comparison. Precatalysts prepared from 2-phosphinophenolesters and Ni(COD)2 at room temperature were characterized by multinuclear NMR but decomposed on heating to stable nickel cis-bis(P,O-chelate) complexes. Heating precatalysts generated from a phosphinophenolester or phosphinophenols and Ni(COD)2 in the presence of ethylene under pressure led to linear ethylene oligomers. These reactions are much faster than the above mentioned conversions with NaH activated P,O-Ni-catalysts. In the presence of 9-decenol with unprotected remote hydroxyl group incorporation of a small amount of isolated hydroxyoctyl side groups takes place, detected by 13C NMR spectroscopy. Finally it is stated that the development of a facile synthesis and the characterization of the properties of the phosphaprolines pave the way for derivatisation and further studies with these novel types of amino acids.
Proteolysis at a proteome-wide scale in low GC, Gram-positive bacteria (2011)
Michalik, Stephan
Proteolysis represents the final step in the life of a protein. It is one of the most important cellular processes assisted by chaperone systems and ensures an appropriate protein homeostasis. Protein degradation is essential for the removal of cytotoxic protein aggregates and mis-translated/mal-folded proteins, "unemployed" and regulatory proteins to enable rapid cell adaptation to altering environmental conditions (Gottesman, 2003; Wiegert & Schumann, 2001; Parker, 1981; Stansfield et al., 1998; Drummond & Wilke, 2008; Goldberg, 1972; Gerth et al., 2008). The bacterial Clp (caseinolytic proteins) protease complexes are analogous to the eukaryotic 26S proteasome and consist of Hsp100/Clp proteins of the AAA+ superfamily and an associated barrel-like proteolytic chamber (e.g. ClpP). The Clp proteases seem to be responsible for the major protein turnover in low GC, Gram+ bacteria. The main goal of this thesis was to develop new methods and tools to investigate global proteolysis more precisely and to get a detailed understanding of protein degradation during starvation conditions and it´s regulation in low GC, Gram-positive bacteria. To analyse protein degradation under starvation conditions the well established glucose starvation model was used. In Bacillus subtilis it could be shown that approximately 200 proteins are selectively degraded in a glucose depletion induced stationary phase. Furthermore radioactive pulse-chase labelling experiments coupled with 2D-PAGE analysis revealed that mainly the ClpCP protease complex is involved in the degradation of proteins in the stationary growth phase. To investigate proteolysis in the human pathogen Staphylococcus aureus in the same way, a newly developed chemically defined medium was established suitable for radioactive pulse-chase labelling experiments under stable glucose starvation conditions. The degradation kinetics of individual 2D spots was significantly better resolved using 14C-BSA as an internal marker protein for the sample normalisation. A rather huge overlap was found within the functional protein classes that were degraded in B. subtilis and S. aureus the stationary phase. Among others, especially proteins involved in amino acid, nucleotide and cell wall biosynthesis were rapidly degraded, whereby not always the same and sometimes another enzymes from a biosynthetic chain were targeted for proteolysis. Despite the resolution power of the 2D-PAGE method, there are some drawbacks such as a limited "protein window" with regard to the molecular weight and isoelectric point, loss of low abundance proteins and a rather low reproducibility for time course experiments. Therefore a mass spectrometry based approach for the simultaneous detection of protein synthesis, accumulation and degradation was developed. This pulse-chase SILAC approach provides a very good reliability with a broad spectrum of proteins that can be analysed. Through the combination with ultracentrifugation even non-soluble and aggregated proteins could be analysed. Several hundred proteins were degraded in S. aureus during glucose starvation. Among them was the functional cluster of ribosomal proteins which is degraded in the early stationary phase. Furthermore proteins belonging to complexes were degraded with the same kinetic (e.g. NrdE, NrdF). In addition selective protein degradation took place according to functional categories (e.g., ribosomal proteins, biosynthetic, glycolytic enzymes) and not to regulatory groups (e.g. CcpA, SigB regulon).The investigation of a clpP deletion mutant in S. aureus revealed a greater susceptibility to aggregation, where the cells try to counteract with the expression of chaperones like GroEL/ES, ClpB and DnaK. The renaturation process is very ATP consuming and only takes place in energy rich phases of growth (e.g. from exponential to transient growth phase). Protein aggregation was found enhanced in the stationary phase. Furthermore, a higher GTP level compared to the wild-type probably resulted in a stronger CodY mediated repression with a rather low level of amino acids in clpP mutant cell. In addition substances like glycerol, which thermodynamically stabilise proteins in refolding processes (Maeda et al., 1996; Feng & Yan, 2008), were found in higher levels compared to the wild-type. A strong response to reactive oxygen species was detected in the clpP mutant strain, which is probably due to ROS production during the early stages of protein aggregation. Altogether, different methods were used for investigation protein degradation at a proteome-wide scale. Hundreds of degradation candidates were identified by gel-based and gel-free approaches in S. aureus wild-type cells. "Unemployed" proteins (e.g. ribosomal proteins, biosynthetic enzymes) were degraded and proteins particularly required and synthesized in glucose-starved cells such as TCA cycle enzymes were stable in the stationary phase. Investigation of the clpP mutant strain supports a proposed model for the pleiotropic phenotype and provides a deeper insight in the fine-tuned protein quality control and the important role of ClpP during starving conditions.
Inter-and intra-annual variations in carbon fluxes in boreal peatlands (2011)
Gazovic, Michal
Northern peatlands are ecosystems with unique hydrological properties, storing about 400-500 Gt of carbon. As the production rate of organic material is higher than its decomposition, which is slowed down in the wet and cold environment, peatlands store a great amount of carbon. Carbon assimilated from the atmosphere during photosynthesis by plants is partly lost due to autotrophic and heterotrophic respiration as carbon dioxide (CO2), as methane (CH4) or/and as dissolved organic carbon. The proportion of each carbon component is strongly controlled by environmental conditions as temperature, radiation, precipitation and subsequent water table changes and active role of vegetation. With predicted changes in the global climate, changes in the influence of environmental parameters on peatland ecology are expected. Thus thorough research is essential for a better understanding of mechanisms which influence carbon cycling in peatlands. In this thesis, various components of the carbon cycle were studied at two boreal peatland sites (Ust Pojeg in Komi Republic in Russian Federation and Salmisuo in Eastern Finland) using the micrometeorological eddy covariance method. The focus was placed on the temporal changes of the controlling parameters, ranging from a few days during short snow thawing through the rest of the year. At the Salmisuo site, two measurement seasons allowed to address possible inter-annual variation. We observed that diurnal variations in methane emissions which are typically controlled by vegetation during the growing season, might appear during snow melt as a result of the influence of physical factors rather than biological factors. The diurnal pattern in methane emissions was caused by the interaction of the freeze-thaw cycle and near urface turbulence. During the night time, when surface temperatures fell below zero and caused formation of the ice layer, methane emissions were only around 0.8 mg m-2 h-1, however after the increase in temperature and melting of the ice layer they reached peak values of around 3 mg m-2 h-1. The near surface turbulence had a significant influence on methane emissions, however only after the thawing of the ice layer. The effect of changing environmental parameters over the year was further elaborated on a carbon dioxide time series from the Ust Pojeg site. The generally accepted effects of temperature on ecosystem respiration during the night are not stable throughout the year and can change rapidly during the growing season. Using moving window regression analysis I could show that the strength of the exponential relationship between ecosystem respiration and temperature is changing during the year. This was in correspondence with recent publications elaborating on sub-seasonal changes of the controlling parameters. In general, measurements from the Ust Pojeg site represent estimates of annual CO2 and CH4 fluxes with an annual carbon balance of -94.5 g C m-2 and a new contribution to the quantification of trace gases emissions from a Russian boreal peatland. The inter-annual comparison of net ecosystem exchange (NEE) measurements with previously published data on CH4 and DOC flux from the Salmisuo site showed that the NEE of CO2 is the most important component of the carbon balance at this site. However, primary production was not responsible for the inter-annual changes in NEE. Rather, the effects of water table position during the year had a strong influence on ecosystem respiration, which was probably due to the influence on soil respiration, and higher NEE was observed during the year with smaller primary production, but higher water table levels. The effects of higher precipitation and higher water table during the wet year were shown to increase CH4 flux and the export of DOC, but their effects could not compensate for changes in ecosystem respiration. In the presented thesis intra- and inter- annual changes in carbon flux components and their controls, in our case attributed mostly to hydrological conditions in combination with other environmental parameters as temperature and the role of peatland vegetation, are discussed.


Mercedes Homes, LLC, a Florida limited liability company, and its subsidiaries and affiliates (collectively "Mercedes Homes") have initiated a plan of liquidation ("Plan") to wind down and conclude business operations. The sale of homes by Mercedes Homes is made pursuant to the Plan. Since Mercedes Homes will be concluding its business operations, Mercedes Homes extends no warranties, either express or implied with respect to any home sale. The only warranty to be provided is a third party warranty issued by Bonded Builder, an unaffiliated third party home warranty provider.
The website address is: www.mercedeshomes.com.


text:
Miss peregrine's home for peculiar children – Ransom Riggs
11 junio, 2015 Ransom Riggs Inglés Miss Peregrine's Peculiar Children (4)
A mysterious island. An abandoned orphanage. And a strange collection of very curious photographs. It all waits to be discovered in Miss Peregrine's Home for Peculiar Children, an unforgettable novel that mixes fiction and photography in a thrilling reading experience. As our story opens, a horrific family tragedy sets sixteen-year-old Jacob journeying to a remote island off the coast of Wales, where he discovers the crumbling ruins of Miss Peregrine's Home for Peculiar Children. As Jacob explores its abandoned bedrooms and hallways, it becomes clear that the children who once lived here—one of whom was his own grandfather—were more than just peculiar. They may have been dangerous. They may have been quarantined on a desolate island for good reason. And somehow—impossible though it seems—they may still be alive.
A spine-tingling fantasy illustrated with haunting vintage photography, Miss Peregrine's Home for Peculiar Children will delight adults, teens, and anyone who relishes an adventure in the shadows.
MRI: The Basics – William G. Bradley Jr.
Karl Marx – Isaiah Berlin
The Beach of Dreams – H. De Vere Stacpoole
Auberon – James S. A. Corey
How to talk dirty and influence people: an autobiography – Lenny Bruce
Force Collector – Kevin Shinick

redpajama_set_name: RedPajamaCommonCrawl


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Bipolarity is all around us. It is what creates balance in our environment and in our bodies. This law can be found in biological processes such as acid/base, nervous pole/metabolic pole, and the sympathetic/parasympathetic nervous systems.
Bipolarity is all around us.
It is what creates balance in our environment and in our bodies. This law can be found in biological processes such as acid/base, nervous pole/metabolic pole, and the sympathetic/parasympathetic nervous systems.
During spring, the cerebral pole or the cerebral cortex is paired with the metabolic pole or the liver in conjunction with the gallbladder. The conception/governor meridian ensures communication between the lower and upper body.
If there are other functions/meridians that you haven't fully restored then they can also become animated during spring. A deep disturbance in any of the meridians will eventually disturb the liver/gallbladder and you may feel it in spring. Therefore, in spring, people can exhibit all kinds of symptoms from a lungs/colon cold to feelings of depression and loneliness.
Remember, "issues" come up in the spring! They can be emotional or physical. Pay close attention to how you are feeling and learn what to do for your health.
Be careful of the way to talk to you! If you are feeling a bit disturbed and grumpy then chances are you are also more self-critical. This may cause you to feel like you can't accomplish anything, that you are not good enough, or that everything is going wrong.
First of all, take the nutriments for the season and then stop telling yourself that everything is going wrong! Instead, encourage yourself with positive messages. You will feel a lot better!


Anny Waysun is a mystic singer, a researcher of human potential. The voice and inspirer of Demarsimo music project. Performs magical, spiritual and classical music, mostly in the style of Classical crossover, recognized by the world's music experts as beneficially influencing a person. Angelic vocal vibrations of Anny Waysun are inspiring, awaken the meaning of life and the desire to create and create. Music promotes the opening of potential and rejuvenates the human body.


Music is the best way to celebrate this magical season! There are lots of FREE concerts at the St. George Tabernacle to enjoy. St. George Tabernacle is located at 18 S Main Street, St. George, UT 84770. Here's the list!
December 8th at 7:00 PM: Geoffrey Myers and Paul Solum - "The Music of Christmas"
December 22nd at Noon: Ben Alder & Friends - "Come Let us Adore Him"


In the news yesterday, a girl accidentally killed her sister carelessly while drunk driving. She sounded apathetic when she talked. I felt sharp anger towards her. I thought she didn't deserve this world nor this life anymore. She was a disgrace to society; a murderer of her own innocent sister. She seemed to sound crazy when she talked. She looked emotionless; it was as if she was possessed. She's the type that no one will ever understand. She's probably hurt. This world is full of hurt. Full of walking souls; miserable, in pain. In the end, I wanted to give her a hug. I imagined the faraway and numb look on her face.
I wanted to go home and hug my little sister. I'd give up a limb for her.
I got confronted at the bus stop several weeks back. It wasn't a big deal but I still recall how I chose to remain silent and composed while the bus driver laughed.
I got yelled by the same guy for a mistake he made while he was inattentive. I'm sure he realized his mistake and felt bad afterwards, but I still got hurt.
When my mom yells at me due to frustrations in her own life, I know she's not the perpetrator and nor am I the victim. I just know that sometimes we get treated as people's punching bags. Sometimes it hurts being passive; people really may not know how sensitive I really am inside. It hurts me, and my eyes moisten in the silence amid darkness. But the sounds of crickets at night whisper to me that they really do know. My sensitivity is as clear as daylight; it really isn't something that I could ever hide very well. Yet I understand why we get treated as punching bags. The perpetrators are hurting as much as the victims themselves. Everybody's swimming in a sea of hurt. A dead man killed on the cross is a hurtful sight that's inspired an entire religion. We're all just walking around carrying our own stories and burdens. It's a quiet world if we just let our minds hush. It's a loving world if we just look into each other's teary eyes.
The voice tells me to get into the elevator and to go to a certain floor. I do so as told. Instinct tells me to walk down the hallway. There's promise of a grand prize; the greatest thing, the highest goal. There's supposed to be something that I've always wanted at the finish. I'm told to open the door. I do so.
wearing a black and white tuxedo. Your chiseled face looking even sharper against the shadows. Your creamy skin and shiny hair, contrasting each other. Your glass-like eyes, contracting in the spotlight directed at you.
but with a look of worry. Wordlessly, you give a notion that you want me back, that you're begging for me now. That you're willing to disregard everything for me and that I could too, for you.
But there's something unkind, untrustworthy, and cold about this whole new setup. There's doubt brewing in my gut.
There's an uneven tune playing in my ears.
that I wished it were true.
In my childhood, I saw something on TV that stuck with me. This guy entered an empty house full of… birds? They flew around in slow motion. They shined. They were women. It was a house full of beautiful women. They smiled and they greeted each other silently. They walked around in slow motion. He observed them as he walked between them and around then. A woman, with a sweater on, looked at his direction and smiled. He smiled idly and waved back at her. She walked towards him and his happiness lingered with a sigh of relief. She walked past him and greeted her friend. His smiled dropped. It was as if he was completely nonexistent.
I'd say hi to him. I wouldn't ignore him. I'd comfort him. I'd lead him through this oddly foreign territory. I'd smile next to him. I'd put my head on his shoulder and link my arm to his. We'd sit on the sofa underneath the ray of sunlight penetrating through the window. I'd look at him and he'd look at me, and we'd be lost in each other's eyes in a vast blue sea of wonder. We'd form a pyramid with our finger tips. I would whisper of love and my dreams and fill the void in his empty heart. It's true. That's what I'd do, if I were a magical bird.


Is a cheaper Pixel 3 incoming as Pixel 2 disappears?
"Apex Legends" is available on Xbox One, PlayStation 4, and Origin for PC. A Kotaku report suggests that the game was "well into development" as of 2017, and was set to launch previous year, but based on McCoy's comments it seems that work on that Titanfall 3 has essentially morphed into Apex Legends. If you'd rather play a mainline Titanfall game with a single-player campaign, Titanfall 2 is still on Amazon. Each game holds up to 60 players with three players to a team. Rod Breslau was the first to report this, stating that streamers took part in an event last week to check the game out ahead of launch.
Apex Legends promises to deliver a squad-based battle royale experience with a uniquely Respawn flavor. Players can opt for characters like Lifeline, the battlefield medic who can shield teammates from harm as well as revive them, or tracker Bloodhound, who can see recent enemy movement via the map. The decision to forego specific abilities or items ultimately came down to a difference in goals between the core Titanfall games and the new battle royale.
Liverpool defender Virgil van Dijk has urged patience as the pressure increases in the title run-in. Klopp said he was not aware of the tweet, but when told about it, said: "I can not help that".
The rationale, that source said, was that the game's underlying technology was starting to feel dated and that Titanfall 3 might not feel or look as good if it came out too much later. When a teammate is killed, they'll have a second chance with Respawn Beacons. "In other words, a third Titanfall is not now in development, despite the critical acclaim for the first two". One way Apex Legends will remain fresh for players is through different seasons. They made a decision to use the same engine they used for Titanfall and Titanfall 2, but found that the game looked rather dated.
The game will feature a seasonal content roadmap, with new Legends and weaponry coming on a quarterly basis.
We don't know because this man (Maduro) doesn't want to leave and is very defiant". Washington has imposed sweeping sanctions on state-owned oil firm PDVSA.
Samsung and Huawei will be introducing their first generation foldable smartphones at their respective events later this month. What's odd is when the phone is unfolded, the external display was still running in the video .
The executive has blamed his legal troubles on a "plot" by Nissan executives to block his planned merger of Renault and Nissan. He said the payment to this businessman, Khaled Juffali, had been signed by "four officers". "There are no exceptions".
The yearbook images were first published on Friday afternoon by the conservative news outlet Big League Politics. Northam issued a statement apologizing for being in that racist costume , though he didn't specify which one.
However, Levine told Entertainment Tonight that "no one thought about it more than I did". Nor did Adam Levine adhere to the advice of Roger Waters and take a knee .
That won't be easy, and getting anything less than seven points could put them in a super tough spot midway through the month. When a lot of teams play with a very deep block, it's because they need to play like that and can also have chances to win.
Seven EU states had given Maduro a Sunday midnight deadline to call presidential elections or they would recognise Guaido. The EU powers held off in joining the US pressure campaign to see whether Venezuela would agree to new elections.
The intelligence leaders reached the same assessment the United Nations atomic watchdog agency concluded after 13 inspections. Trump said his aversion to letting his son play football is because "it's a unsafe sport and I think it's really tough".
Corn syrup is an affordable alternative to sugar that has been associated with obesity, diabetes and other ailments. Representatives for Bud Light's parent company, Anheuser-Busch, did not respond Monday to a request for comment.
The company notes in a statement that all users voluntarily signed up for the Research app , and were free to opt out at any time. Most users got $20 monthly gift cards from Facebook in exchange for using the app, and some were also given paid referral fees.
Amazon has been expanding grocery delivery from Whole Foods stores, and the service is now active in more than 60 US cities. Revenue in its Amazon Web Services cloud business surged 45.3 percent to $7.43 billion to beat estimate of $7.26 billion.
He is one those lads who want to improve, who sees his game and wants to see what could have done better". He said, "I think he can look back on these games and say he is on his way.
The Sunderland plant, which has been active since 1986, employs nearly 7,000 people and produces around 2,000 cars a day. In 2016 the carmaker said it would build the new vehicle in the United Kingdom after "assurances" from the government.
Saturday, Punxsutawney (puhnk-suh-TAW'-nee) Phil emerged from his burrow in Pennsylvania at sunrise and didn't see his shadow. Whether or not he returns to his hole determines an early spring or prolonged winter.
Dwarf spheroidal galaxies are defined by their small size, low-luminosity, lack of dust and old stellar populations . The team published their discovery January 31, in the Monthly Notices of the Royal Astronomical Society: Letters .


text:
Ve dnech 7. a 8. října 2016 se v městysu Protivanov konalo místní referendum, které proběhlo současně s krajskými volbami. Předmětnou otázkou referenda bylo, zda obyvatelé chtějí podpořit rozšíření stávajícího větrného parku o dalších pět větrných elektráren.
Referendum obyvatelé Protivanova schválili, pro ANO se vyjádřilo 56,82 % obyvatel a pro NE 39,14 %.
Celková účast byla 48,9 % oprávněných voličů obce. Aby referendum bylo platné bylo potřeba splnit podmínku účasti alespoň 35 % oprávněných voličů, což se podařilo.
Celkem se referenda zúčastnilo 396 obyvatel z 810 oprávněných voličů, kteří mají trvalé bydliště v Protivanově. Kladně se ohledně případné stavby dalších větrných elektráren vyjádřilo 225 voličů a proti jich naopak bylo 155. Zbylých 16 hlasů bylo neplatných. Voliči, kteří se rozhodli volit v referendu odpovídali na otázku ve znění: "Souhlasíte s tím, aby zastupitelstvo městyse Protivanov podporovalo rozšíření stávajícího větrného parku nacházejícího se v katastrálním území Protivanov o dalších maximálně 5 větrných elektráren?".

Příprava referenda
Již v roce 2015 se v zastupitelstvu začalo projednávat rozšíření větrného parku, který do té doby byl tvořen pouze dvěma větrnými elektrárnami. Referendu předcházelo vypsání ankety a až na základě jejího výsledku se mělo zastupitelstvo rozhodnout o vypsání či nevypsání referenda.
V březnu roku 2016 byla vytvořena anketa, jejímž cílem bylo zjisti postoj obyvatel Protivanova k větrným elektrárnám a jejich případnému rozšíření. Anketa ukázala, že obyvatelstvo nemá k elektrárnám negativní postoj, nevadí jim a neobávají se jejich případného negativního vlivu na své zdraví. Celkem se této ankety zúčastnilo 53 % občanů obce starších 18 let, z nichž 47 % souhlasila s rozšířením větrného parku. Ná základě těchto výsledků dotazníku začalo pracovat na vypsání referenda.
Město z provozu elektráren získává 1 % z ročního obratu společnosti Wind invest, což představuje pro obci roční příjem kolem 200 tisíc korun. Roční rozpočet obce Protivanov se pohybuje kolem 12 milionů korun a svůj příjem z provozu větrných elektráren zpravidla využívá na rozvoj obce.

Reference

Protivanov
Místní referenda v Česku
Volby v Česku 2016
Politika v Olomouckém kraji
Události v Olomouckém kraji

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