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Life-threatening fungal infections are becoming increasingly common for immunocompromised patients such as those with AIDS, or those undergoing organ transplantation or chemotheraphy, as well as for other health-vulnerable patients. Excellent targets for antifungal drugs are chitin synthases, which are essential for survival of the fungus and lacking in humans. To design new antifungal drugs, knowledge of the three-dimensional structure and mechanism of action of chitin synthases are crucial. Chitin synthases are members of an important family of enzymes that synthesize structural polysaccharides, such as cellulose, β(1,3)-glucan, β(1,4)-mannan and hyaluronan. Therefore, chitin synthases could be used as a model system to understand these more complex enzymes, which are also of major medical and commercial importance. Chitin synthase 2 from Saccharomyces cerevisiae (ScChS2), the protein under study, is an integral membrane protein that synthesizes the primary septum between mother and daughter cells in budding yeast. It is essential for proper cell separation and expected to be highly regulated. An important aspect is that ScChS2 shows 55% sequence identity and is functionally analogous to chitin synthase 1 from the human opportunistic pathogen Candida albicans, this enzyme is also essential for cell survival (Munro, Winter et al. 2001). ...
A mild synthetic method for N-formyl-Met-Leu-Phe-OH (1) is described. After Fmoc solid phase peptide synthesis, on-bead formylation and HPLC purification, more than 30 mg of the fully 13C/15N-labelled tripeptide 1 could be isolated in a typical batch. This peptide can be easily crystallised and is therefore well suited as a standard sample for setting up solid-state NMR experiments.
Self-inactivating gammaretroviral vectors for the gene therapy of chronic granulomatous disease
(2008)
Chronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency characterized by defective intracellular oxidative killing of ingested invading microbes by PMN and monocytes. It is caused by mutations in one of the four genes coding for the essential subunits of the NADPH oxidase (gp91phox, p47phox, p67phox and p22phox). Approximately 75% of the CGD cases are due to mutations in the gp91phox gene. If regular care and conventional therapy fail, the recommended therapy is allogeneic bone marrow transplantation (BMT), but only if a matched donor is available. A therapeutic option for patients lacking suitable donors is the genetic modification of autologous hematopoietic stem cells. The gene therapy offers an interesting alternative to BMT since it implies a less invasive treatment and represents a possibly unique curative option for patients with no suitable donor. Gammaretroviral vectors were already used in some gene therapy trials for CGD and other immunodeficiencies showing relevant clinical benefit. However, these trials uncovered an unexpected mutagenic side effect. If the retrovial integration ocurrs near to, or into proto-oncogenes this might lead to clonal dominance or even malignant transformation (Hacein-Bey-Abina et al., 2003a; Ott et al., 2006). Therefore, there was a need to further improve the safety of these vectors and to this end the self-inactivating gammaretroviral vectors were engineered. Non essential sequences for virus infectivity and integration, which might influence the surrounding gene expression, were deleted in these vectors. In the first set of experiments, a series of SIN gamma retroviral vectors was cloned driving the expression of the wild-type gp91phox cDNA under the control of a viral constitutive SFFV promoter. However initial studies with these vectors failed because the titers of the virus produced by transient transfection protocols were extremely low (<5x105 TU/ml). Therefore, a codon optimization of the gp91phox cDNA was considered as an alternative. The codon optimized synthetic gp91phox gene was used to construct a SIN gammaretroviral vector, again under the control of the SFFV promoter (Schambach et al., 2006c). With this vector an increase in titer was observed compared to the native gp91phox sequence, which was due to the improved transcription in 293T transfected cells. The enhancement of the synthetic gp91phox transcription led to a higher internal transcript production and protein expression. An enhanced superoxide production in transduced myelomonocytic X-CGD PLB-985 populations was also detected. All these data indicate that the synthetic gp91phox might represent an excellent alternative to those former constructs expressing the native gp91phox transgene. Since it was postulated that the SFFV promoter could still cause transactivation of neighboring genes due to its strength (Modlich et al., 2006), three different non-viral promoters were tested, one constitutive (the EFs promoter) and two myeloid-specific promoters (the c-fes and MRP8 promoter). The three SIN gammaretroviral vectors were able to generate high titers after transient transfection of 293T packaging cells, to efficiently transduce the X-CGD PLB-985 cell line and to reconstitute the NADPH oxidase activity to a high degree. In mouse transplantation experiments, the EFs promoter showed a high variable transgene expression in the different lineages analyzed, and the c-fes promoter showed also a ubiquitinous expression. In contrast, the MRP8 promoter showed a high myeloid specificity since gp91phox expression in mSca-1+ cells and lymphoid B cells from transplanted mice was extremly low and even absent. However, the lowest levels of transgene expression were observed in the myeloid populations both in bone marrow and peripheral blood with this vector. When the oxidase reconstitution ability of these promoters was tested, the numbers of superoxide producing cells obtained were similar than those observed in the clinical X-CGD trial conducted by the groups of Dr. M. Grez and Prof. R. A. Seger (over 35% in one patient and ~15% in the second), which led to the eradication of therapy refractory infections (Ott et al., 2006). Between the three constructs, the MRP8 promoter was less effective in restoring the NADPH oxidase activity than the EFs and c-fes promoters. The c-fes promoter reached the highest levels of DHR reactive cells in the highest number of mice. Overall, these data showed that between all constructs tested, the c-fes containing construct in combination with the codon optimized gp91phox sequence showed the best performance within the SIN gammaretroviral backbone. It generated the highest titers in combination with a better NADPH oxidase reconstituting ability. One main goal in the development of SIN gammaretroviral vectors is reducing the genotoxic effect due to random vector integration. An improved gene transfer and expression, and a constant performance are also highly desirable. The present study shows that the c-fes SIN vector in combination with the synthetic gp91phox may be considered as an effective gene therapy strategy for the restoration of the NADPH oxidase activity in CGD. It allows the use of a cellular promoter generating adequate physiological levels of the therapeutic protein and reduces the number of vector copies required for a therapeutic effect.
Development of chromium(VI)-free defect etching solutions for application on silicon substrates
(2008)
Determination of the distribution of halocarbons in the tropical upper troposphere and stratosphere
(2008)
The aim of this thesis was to investigate distributions of 32 volatile chlorinated and/or brominated halocarbons that are currently believed to be present in the tropical upper troposphere and stratosphere and to contribute to stratospheric ozone depletion and also to global warming. For this purpose an analytical system was established, which is capable to measure ultra-low concentrated atmospheric trace gases. A quadrupole Mass Spectrometric (MS) Detector was attached to an existing Gas Chromatograph with pre-concentration system and Electron Capture Detector (ECD). The characterisation of the chromatographic system was significantly enhanced by the subsequent identification of 48 additional volatile organic compounds. Furthermore a Gaussian fit algorithm, which was developed in the workgroup, was applied to the chromatographic signals. This algorithm was proven to reflect peaks quantitatively and to enhance the performance of the integration process – especially the reproducibilities for peaks with a low signal to noise ratio. As it is known that the Electron Capture Detector responds nonlinear the new MS detector was checked for such behaviour and found to respond linear. In logical consistency the complete quantification process including e.g. pre-concentration of trace gases and signal integration can be considered as linear responding within the investigated parameter ranges. Moreover, the long term stability of the targeted halocarbons was proven inside the calibration standard containers over a period of 25 months. Many substances were also found to be stable inside the containers used for storage of air samples but a number of substances showed significant concentration changes. These were mainly CH3Cl (methyl chloride), CH3Br (methyl bromide), CH2Cl2 (dichloromethane), CHCl3 (chloroform), CCl4 (tetrachloromethane), C2Cl4 (tetrachloroethene), CH3CCl3 (methyl chloroform), CH2ClCH2Cl (1,2-dichloroethane) und C2H5Cl (chloroethane). But the number of affected substances and also the corresponding concentration changes varied between the individual containers. A systematic investigation of the influence of possible causes (e.g. air sampling methods, container materials) is recommended. Results from both internal detectors were compared and revealed biases and disadvantages of the ECD caused by its lower selectivity and its nonlinear response behaviour. Consequently the MS detector was chosen for the quantification of atmospheric trace gases. The quantification process was performed relative to externally calibrated air standards. To assess the uncertainties connected with different absolute calibration scales cross-comparisons between calibration standards of three different laboratories were carried out. Most substances’ calibrations agreed within the measurement uncertainties but significant differences were observed for CF2ClBr (H1211), CH3Cl (methyl chloride), CH2Cl2 (dichloromethane), CHCl3 (chloroform), CCl4 (tetrachloromethane) and CH3CCl3 (methyl chloroform). As five of these substances were also observed to show concentration changes inside sample containers it is likely, that such changes are responsible for calibration differences. In addition to the detailed assessment of uncertainties connected with the analytical quantification process a set of air samples was available for measurements. These samples mainly originated from the upper troposphere and lower and middle stratosphere in the tropics and the determined halocarbon quantities were used to investigate their distributions in the respective atmospheric regions. In detail, the altitudinal distributions and interrelations of 17 long-lived halocarbons in the tropical stratosphere were determined and compared with those of other stratospheric regions. Tracer-tracer-correlations of these substances in the tropical stratosphere were found to differ from those in mid- and high-latitudes. Characteristic fit functions relative to CF2Cl2 (F12) which are valid for the tropical stratosphere in 2005 were derived as well as time-independent fit functions of fractional release factors (FRFs) relative to the mean age of air. Both sets of correlations could be used for the parameterisation and evaluation of models and also to reassess the Global Warming Potentials (GWPs) of the corresponding halocarbons which might affect future climate predictions. However, the data set on halocarbons in the tropical stratosphere is still insufficient to investigate the variability of tracer-tracer-correlations and FRFs caused by dynamical and photochemical processes. Therefore it is important for future research to perform additional measurements there and – if possible – to extend the measurements to the upper tropical stratosphere in order to characterise the sink of those halocarbons that are still present in these altitudes. In addition, the amount of chlorine and bromine present in the form of organic compounds inside and above the main stratospheric entrance region (the Tropical Tropopause Layer, TTL) was quantified in the frame of a case study. This was possible because of a cooperation with scientists from the University of East Anglia which carried out measurements of six additional halocarbons leading to a total of 28 quantified target substances. Ten of these substances have short atmospheric lifetimes compared with the mean transport times of tropospheric air to the stratosphere (i.e. lifetimes below 0.5 years) and show non-uniform distributions in the upper troposphere. The contribution of these substances to stratospheric ozone depletion is subject of an ongoing scientific debate. In the performed case study a fraction range of short-lived halocarbons of 6 – 8 % (0.98 – 1.25 ppt) relative to the sum of bromine from organic substances and of 1.1 – 1.4 % (36.6 – 47.1 ppt) for the corresponding sum of chlorine was calculated to enter the stratosphere above Brazil in June 2005. Moreover by combining the data with tropospheric reference data and age of air observations the abundances of inorganic chlorine and bromine (Cly and Bry) were derived. At an altitude of 34 km an amount of 3062 ppt of Cly and 17.5 ppt of Bry from organic source gases was calculated. The latter is significantly lower than Bry mixing ratios inferred from quasisimultaneous BrO measurements at 33 km altitude above Brazil (Dorf, 2005, Dorf et al., 2008). But at the University of East Anglia indications for the presence of unknown brominated organic substances in the TTL were found which might cause this difference. Finally, a major result of this thesis adds to the knowledge of the composition of the troposphere as three Chlorofluorocarbons (CFCs) were first observed. Trifluorochloroethene, 3-chloropentafluoropropene and 4,4-dichlorohexafluoro-1-butene were found in air samples collected at the Taunus Observatory near Frankfurt (Main) and the Jungfraujoch High Altitude Research Station in Switzerland (Laube and Engel, 2008). Identification was possible because of an air plume containing high concentrations of these substances. It is suggested that the abundances found on this occasion originated from a local source. The atmospheric lifetimes of these substances are expected to be rather short as they contain a double bond. A quantitative calibration could only be derived for trifluorochloroethene but not for the other species by now. Thus, a relative sensitivity method was derived to get a first indication of the observed atmospheric abundances. All three CFCs could also be detected in air masses representative of background conditions, though with much lower concentrations. These species and some of their degradation products are toxic and could also be relevant for stratospheric and tropospheric ozone depletion. It is important to find out more about their atmospheric distributions, lifetimes, sinks and sources and their ability to reach the stratosphere to assess their possible influence on the global atmosphere. This will be done in the frame of the project "CLEARFOGG – Checking Layers of the Earths AtmospheRe For halogenated Ozone-depleting and Greenhouse Gases". This research project aims to perform a systematic scan of the atmosphere because there are indications for the presence of a number of halogenated organic compounds which are unknown by now. It was recently decided to be funded by the British National Environmental Research Council and will be carried out at the University of East Anglia mainly by the author of this thesis.
Proteorhodopsin (PR) originally isolated from uncultivated γ-Proteobacterium as a result of biodiversity screens, is highly abundant ocean wide. PR, a Type I retinal binding protein with 26% sequence identity, is a bacterial homologue of Bacteriorhodopsin (BR). The members within this family share about 78% of sequence identity and display a 40 nm difference in the absorption spectra. This property of the PR family members provides an excellent model system for understanding the mechanism of spectral tuning. Functionally PR is a photoactive proton pump and is suggested to exhibit a pH dependent vectorality of proton transfer. This raises questions about its potential role as pH dependent regulator. The abundance of PR in huge numbers within the cell, its widespread distribution ocean wide at different depths hints towards the involvement of PR in utilization of solar energy, energy metabolism and carbon recycling in the Sea. Contrary to BR, which is known to be a natural 2D crystal, no such information is available for PR til date. Neither its functional mechanism nor its 3D structure has been resolved so far. This PhD project is an attempt to gain a deeper insight so as to understand structural and functional characterization of PR. The approach combines the potentials of 2D crystallography, Atomic Force Microscopy and Solid State NMR techniques for characterization of this protein. Wide range of crystalline conditions was obtained as a result of 2D crystallization screens. This hints towards dominant protein protein interactions. Considering the high number of PR molecules reported per cell, it is likely that driven by such interactions, the protein has a native dense packing in the environment. The projection map represented low resolution of these crystals but suggested a donut shape oligomeric arrangement of protein in a hexagonal lattice with unit cell size of 87Å*87Å. Preliminary FTIR measurements indicated that the crystalline environment does not obstruct the photocycle of PR and K as well as M intermediate states could be identified. Single molecule force spectroscopy and atomic force microscopy on these 2D crystals was used to probe further information about the oligomeric state and nature of unfolding. The data revealed that protein predominantly exists as hexamers in crystalline as well as densely reconstituted regions but a small percentage of pentamers is also observed. The unfolding mechanism was similar to the other relatively well-characterized members of rhodopsin family. A good correlation of the atomic force microscopy and the electron microscopy data was achieved. Solid State NMR of the isotopically labeled 2D crystalline preparations using uniformly and selectively labeling schemes, allowed to obtain high quality SSNMR spectra with typical 15N line width in the range of 0.6-1.2 ppm. The measured 15N chemical shift value of the Schiff base in the 2D crystalline form was observed to be similar to the Schiff base chemical shift values for the functionally active reconstituted samples. This provides an indirect evidence for the active functionality of the protein and hence the folding. The first 15N assignment has been achieved for the Tryptophan with the help of Rotational Echo Double Resonance experiments. The 2D Cross Polarization Lee Goldberg measurements reflect the dynamic state of the protein inspite of restricted mobility in the crystalline state. The behavior of lipids as measured by 31P from the lipid head group showed that the lipids are not tightly bound to the protein but behave more like the lipid bilayer. The 13C-13C homonulear correlation experiments with optimized mixing time based on build up curve analysis, suggest that it is possible to observe individual resonances as seen in case of glutamic acid. The signal to noise was good enough to record a decent spectrum in a feasible period. The selective unlabeling is an efficient method for reduction in the spectral overlap. However, more efficient labeling schemes are required for further characterization. The present spectral resolution is good for individual amino acid investigation but for uniformly labeled samples, further improvement is required.
Cellular metabolism can be envisaged by fluorescence lifetime imaging of fluorophores sensitive to specific intracellular factors such as [H+], [Ca2+], [O2], membrane potential, temperature, polarity of the probe environment, and alterations in the conformation and interactions of macromolecules. Lifetime measurements of the probes allow the quantitative determination of the intracellular factors. Fluorescence microscopy taking advantage of time-correlated single photon counting is a novel method that outperforms all other techniques with its single photon sensitivity and picoseconds time resolution. In this work, a time- and space-correlated single photon counting system was established to investigate the behavior of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) in living cells. DASPMI is known to selectively stain mitochondria in living cells. The uptake and fluorescence intensity of DASPMI in mitochondria is a dynamic measure of membrane potential. Hence, an endeavour was made to elucidate the mechanism of DASPMI fluorescence by obtaining spectrally-resolved fluorescence decays in different solvents. A bi-exponential decay model was sufficient to globally describe the wavelength dependent fluorescence in ethanol and chloroform. While in glycerol, a three-exponential decay model was necessary for global analysis. In the polar low-viscous solvent water, a mono-exponential decay model fitted the decay data. The sensitivity of DASPMI fluorescence to solvent viscosity was analysed using various proportions of glycerol/ethanol mixtures. The lifetimes were found to increase with increasing solvent viscosity. The negative amplitudes of the short lifetime component found in chloroform and glycerol at the longer wavelengths validated the formation of new excited state species from the initially excited state. Time-resolved emission spectra in chloroform and glycerol showed a biphasic increase of spectral width and emission maxima. The spectral width had an initial fast increase within 150 ps and a near constant thereafter. A two-state model based on solvation of the initially excited state and further formation of TICT state has been proposed to explain the excited state kinetics and has been substantiated by the de-composition of time-resolved spectra. The knowledge of DASPMI photophysics in a variety of solvents now provides the means of deducing complex physiological parameters of mitochondria from its behavior in living cells. Spatially-resolved fluorescence decays from single mitochondria or only very few organelles of XTH2 cells signified distinctive three-exponential decay kinetics of viscous environment. Based on DASPMI photophysics in a variety of solvents, these lifetimes have been attributed to the fluorescence from locally excited state (LE), intramolecular charge transfer state (ICT) and twisted intramolecular charge transfer (TICT) state. A considerable variation in lifetime among mitochondria of different morphology and within single cell was evident corresponding to the high physiological variations within single cells. Considerable shortening of the short lifetime component (τ1) under high membrane potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and dramatic decrease of lifetime in polar solvents. Under these conditions τ2 and τ3 increased with decreasing contribution. Upon treatment with ionophore nigericin, hyperpolarization of mitochondria resulted in remarkable shortening of τ1 from 159 ps to 38 ps. Inhibiting respiration by cyanide resulted in notable increase of mean lifetime and decrease of mitochondrial fluorescence. Increase of DASPMI fluorescence on conditions elevating mitochondrial membrane potential has been attributed to uptake according Nernst distributions, to de-localisation of π electrons, quenching processes of the methyl pyridinium moiety and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI stained mitochondria in living XTH2 cells, revealed dependence of anisotropy on membrane potential. Such changes in anisotropy attributed to restriction of the torsional dynamics about the flexible single bonds neighboring the olefinic double bond revealed the previously known sub-mitochondrial zones with higher membrane potential along its length. Membrane-potential-dependent changes in anisotropy have further been demonstrated in senescent chick embryo fibroblasts. In conclusion, spectroscopic observations of excited-state kinetics of DASPMI in solvents and its behavior in living cells had revealed for the first time its localisation, mechanism of voltage sensitive fluorescence and its membrane-potential-dependent anisotropy in living cells. The simultaneous dependence of DASPMI photophysics on mitochondrial inner membrane viscosity and transmembrane potential has been highlighted.
In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.
A high-precision pressure probe is described which allows non-invasive online-monitoring of the water relations of intact leaves. Real-time recording of the leaf water status occurred by data transfer to an Internet server. The leaf patch clamp pressure probe measures the attenuated pressure, Pp, of a leaf patch in response to a constant clamp pressure, Pclamp. Pp is sensed by a miniaturized silicone pressure sensor integrated into the device. The magnitude of Pp is dictated by the transfer function of the leaf, Tf, which is a function of leaf patch volume and ultimately of cell turgor pressure, Pc, as shown theoretically. The power function Tf=f(Pc) theoretically derived was experimentally confirmed by concomitant Pp and Pc measurements on intact leaflets of the liana Tetrastigma voinierianum under greenhouse conditions. Simultaneous Pp recordings on leaflets up to 10 m height above ground demonstrated that changes in Tf induced by Pc changes due to changes of microclimate and/or of the irrigation regime were sensitively reflected in corresponding changes of Pp. Analysis of the data show that transpirational water loss during the morning hours was associated with a transient rise in turgor pressure gradients within the leaflets. Subsequent recovery of turgescence during the afternoon was much faster than the preceding transpiration-induced water loss if the plants were well irrigated. Our data show the enormous potential of the leaf patch clamp pressure probe for leaf water studies including unravelling of the hydraulic communication between neighbouring leaves and over long distances within tall plants (trees).
C2-symmetric bisamidines : chiral Brønsted bases catalysing the Diels-Alder reaction of anthrones
(2008)
C2-symmetric bisamidines 8 have been tested as chiral Brønsted bases in the Diels- Alder reaction of anthrones and N-substituted maleimides. High yields of cycloadducts and significant asymmetric inductions up to 76% ee are accessible. The proposed mechanism involves proton transfer between anthrone and bisamidine, association of the resulting ions and finally a cycloaddition step stereoselectively controlled by the chiral ion pair.