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Photochemische Abbauprozesse von polybromierten Diphenylethern in homogenen und heterogenen Systemen (2008)

Ana Maria Geller

Ziel dieser Arbeit war es, die photochemischen Abbauprozesse von technischen Produkten zu untersuchen, die als Flammschutzmittel verwendet werden: die polybromierten Diphenylether (PBDEs) Deca-, Octa- und PentaBDE. Die Bestrahlung der kommerziellen Mischungen wurde in einer Karussell-Bestrahlungsapparatur und/oder mittels Gesichtsbräuner durchgeführt. Für den photolytischen Abbau unter umweltrelevanten Bedingungen ist ein sonnenähnliches Spektrum notwendig. Dieses wurde durch die eingesetzte Xenon-Lampe (XOP-7, 500 W) mit einem Filter aus Duranglas (1,5 mm Wandstärken) und durch 4 oder 6 UV-Leuchtstoffröhren (TL29D16, 16 W) erreicht. Der Verlauf der Photolyse wurde mit HPLC-DAD und/oder mit GC-MS verfolgt. Von den PentaBDEs sind BDE 99 und BDE 100 am häufigsten in der Umweltprobe aufgetreten und haben zugleich den größten Gewichtsanteil in der kommerziellen Mischung DE-71. Während der Photolyse von BDE 100, 99 und 85 wird hauptsächlich TetraBDE 47 gebildet. Die Geschwindigkeitskonstanten für die Debromierung von BDE 85 und BDE 99 waren mit beiden Lampen deutlich größer als bei BDE 100. Die Quantenausbeuten der PentaBDEs 85, 99 und 100 in THF/Hexan (2/1 v/v) betrugen 0,22, 0,17 und 0,13. Die größere Quantenausbeute für BDE 85 verursacht den schnelleren Photoabbau dieses Penta-Kongeners im Vergleich zu BDE 99 und BDE 100. BDE 100 wurde als das photostabilste der drei Penta-Kongeneren der kommerziellen Mischung DE-71 identifiziert. Der Verlauf der Photolyse der kommerziellen Mischung DE-79 zeigt einen schnellen Abbau von DecaBDE 209 und NonaBDE 207+208. Nachdem DecaBDE 209 mehr als zur Hälfte abgebaut ist, beginnt die Debromierung des NonaBDE 206 mit einer Geschwindigkeit, die ca. der Hälfte der Geschwindigkeit der Summe BDE 207+208 entspricht. Auch beim Abbau der OctaBDEs 203, 197 und 196 und von HeptaBDE 180 in der kommerziellen Mischung DE-79 handelt es sich um Folgereaktionen mit fortschreitender Debromierung. Die Quantenausbeuten in THF der DecaBDE 209 und NonaBDE 207 waren 0,20 bzw. 0,14. Die Photodebromierung von HeptaBDE 183 als einzelnes Kongener verläuft über die Bildung von 7 Hexa-Kongeneren, hauptsächlich bei BDE 153. Eine Quantenausbeute von 0,11 wurde für den Abbau von BDE 183 in THF/Hexan (2/1 v/v) bestimmt. DecaBDE 209 wurde in THF-Lösung und auf anorganischem Trägermaterial photolysiert. Die Lebensdauer von DecaBDE 209 in Lösung ist ca. 1,7 Stunden. Als Octa-Produkte aus dem Abbau von DecaBDE 209 bzw. NonaBDEs traten (in Reihenfolge der Häufigkeit) BDE 196 > BDE 203 > BDE 201 > BDE 197 auf. Es wurden keine PBDEs mit Bromierungsgraden < 5 bei der Photolyse von DecaBDE auf den anorganischen Trägermaterialien Quarzglaspulver (Aerosil 380), Mikro-Glashohlkugeln und Kieselgel gefunden. Eine schnellere Debromierung von DecaBDE wurde auf Kieselgel und Aerosil 380 im Vergleich zu den Mikro-Glashohlkugeln beobachtet. Die unterschiedlichen Trägermaterialen haben einen Einfluss auf die Abbaugeschwindigkeit von DecaBDE, aber das Photolyseprodukt-Muster bleibt gleich. Bei der Photolyse der kommerziellen Mischung DE-79 und von DecaBDE 209 in Lösung wurden gleichzeitig mit der Debromierung der PBDEs verschiedene PBDFs und sogar das Muttermolekül Dibenzofuran gebildet. Die Bildung von polybromierten Dibenzofuranen spielt eine wichtige Rolle in dem Abbauweg bei der Photolyse von PBDEs mit höherem Bromierungsgrad. In der kommerziellen Mischung DE-79 nach 48 h Bestrahlung liegt der Gewichtsanteil von PBDFs bei. ca. 17 %. Während beim DecaBDE in Lösung nach 46 h Bestrahlung der Gewichtsanteil von PBDFs bei ca. 13 % liegt. Die PBDFs wurden bei der Photolyse von DecaBDE auf anorganischem Trägermaterial hauptsächlich bei Versuchen mit dem Kieselgel beobachtet. Bei DecaBDE in Lösung wurden polybromierte Dibenzofurane mit kleinerem Bromierungsgrad als bei der gesättigten Lösung mit Bodensatz und auf anorganischem Trägermaterial gebildet. Die Bildung der toxischen 2,3,7,8-bromierten Dibenzofurane konnte in allen Versuchen ausgeschlossen werden.

Elasticity measurements at extreme conditions: application to FeO and FeNi-alloy (2008)

Anastasia Kantor

The picture of Earth’s deep interior is rapidly improving from the seismic tomography data and indicates more complexity than previously thought. The presence of Earth’s seismic anisotropy requires the knowledge of fully anisotropic elasticity data for mineral phases. The single-crystal elastic constants of minerals, Cij, are elements of the fourth-rank elasticity tensor, which relates stress to strain. The fact that elastic strain also defines seismic wave propagation, the elastic tensor of minerals can be applied to interpret the bulk mineralogy of the interior from seismological observation. Knowledge of the elasticity of crystalline materials as a function of pressures and temperature is also of primary interest for solid state physics because elastic tensors reveal the nature of interatomic interactions. In order to determine the full elastic tensor of minerals under high pressure and temperature, several techniques are available, including ultrasonic interferometry and inelastic x-ray scattering methods. One of the most accurate techniques is high-frequency acoustic interferometry, which is capable for measuring sound wave velocities in very small samples under high pressures. The ultrasonic interferometry system operating at 0.5-2.0 gigahertz (GHz) frequencies was developed in the Bavarian Geoinstitut of the University of Bayreuth for in situ high pressure and temperature experiments. Here, GHz-ultrasonic interferometry has been used to study the elastic properties of monoxide minerals such as FeO, liquids and nanocrystalline samples, each with particular importance to Earth or material sciences. FexO, wüstite, is the end-member phase of the (Mg,Fe)O solid solution, thought to be the most abundant non-silicate oxide in the mantle. The full elastic tensor of wüstite is determined by three elastic constants (C11, C12, C44), which have been probed at high-pressures. At about 17-20 GPa, FeO is known to undergo a displacive cubic-to-rhomobhedral phase transformation. Prior to this transformation, we observe a pressure-induced mode softening of the C44 elastic constant. In addition, previously undetected discontinuities in the pressure derivatives of C11 and C12 at 4.7  0.2 GPa were observed. This pressure is consistent with that of the magnetic ordering commencement, as was observed by high-pressure Mössbauer spectroscopy in a 57Fe-enriched sample of FeO. The results indicate that an intermediate, partially magnetic but still cubic phase of FeO probably exists at room temperature and in pressure range from ~5 GPa to ~17 GPa. In order to provide deeper knowledge of the magneto-elastic coupling in the material, neutron diffraction experiments were performed under ambient pressure and low temperatures. The results indicate that the magnetically ordered cubic phase of FexO that was observed at high pressures also exists at ambient pressure at temperatures between 160 and 201 K. Combined inelastic x-ray scattering and x-ray diffraction studies on a single crystal of Fe0.95O were performed up to 20 GPa at room temperature. The results show strong anelastic behaviour of wüstite, which should be accounted for at high pressure. Transition-metal oxides, non-stoichiometric compounds, and materials with complex mesostructure have some internal degree of freedom, and could therefore experience internal relaxation and show deviations from normal elastic behaviour. A methodology to measure inelastic x-ray scattering in externally heated diamond anvil cells have also been developed. This technique was used to study polycrystalline fcc-Fe0.78Ni0.22 alloy at high pressures (up to 72 GPa) and temperature (up to 715 K). The bulk elasticity and its P and T derivatives were obtained for the material. No significant deviation of the elastic properties from those of pure iron was observed and furthermore no deviation from Birch’s law. Although the bulk elasticity of fcc Fe-Ni alloy and Fe seem to be very similar, the elastic anisotropy of hexagonal and cubic phases should be quite different. If the metal phase in the inner core is not hexagonal, but cubic (or a mixture of the two phases exists), seismic anisotropy may provide a better way to discriminate between them two.

Assessment of the environmental acceptability of refrigerants by discrete mathematics: cluster analysis and Hasse diagram technique (2008)

Guillermo Restrepo

The recognition of the adverse environmental impact of chlorofluorocarbons (CFCs), mainly used as refrigerants, has lead to look for environmentally acceptable CFC replacements. Main environmental concern CFCs face is their ability to deplete the stratospheric ozone layer, quantified by the ozone depletion potential (ODP). Some of the first replacements mooted were hydrochlorofluorocarbons (HCFCs) and hydrofluorocarbons (HFCs), which contribute to the global warming, quantified by the global warming potential (GWP). ODP and GWP are related to the atmospheric lifetime (ALT), a third indicator. Hence, the environmental impact of a refrigerant may be characterised by a triple of ODP, GWP and ALT values. In this respect, an acceptable refrigerant is a chemical with low ODP, GWP and ALT values. One of the first steps to assess the environmental acceptability of refrigerants is to classify them to find substances with common features. Hence, a supervised and unsupervised classification was performed over 40 refrigerants. First one was a classification based upon elemental composition and functional groups present in refrigerant molecules which leads to: CFCs, HCFCs, HFCs, hydrocarbons (HCs), hydrofluoro ethers (HFEs), chloromethanes (CMs), carbon dioxide, trifluoroiodomethane, dimethyl ether and ammonia. The unsupervised classification was performed using hierarchical cluster analysis. In this case, refrigerants were characterised by three kinds of descriptors: Environmental properties (ODP, GWP, ALT), thermodynamic features related to their refrigeration performance and molecular descriptors derived from their molecular structure. Eight clustering methodologies were applied to each kind of refrigerant descriptors. To assess the stability of these classifications, the cluster index, a method for quantifying classification similarities was developed and further applied to refrigerant results. It was found that environmental descriptors are the only case in which refrigerant classes are stable when varying the classification method. The chemotopological procedure, a method for studying similarity relationships, was applied to the environmental classification of refrigerants. It was found that CFCs are similar to themselves and also to 1,1,1,3,3,3-hexafluoropropane, a HFC. The most similar substances to all CFCs considered were trichlorofluoromethane and 1,1,2-trichloro-1,2,2-trifluoroethane. The other refrigerant families were found to be similar to many other substances, therefore there is no clear affiliation of refrigerants of one family to one certain class. It was found a disagreement between the supervised classification leading to refrigerant families and the three unsupervised classifications (environmental, thermodynamic and molecular ones). Therefore, refrigerant classification into families does not imply same classification based upon environmental properties, thermodynamic features and molecular descriptors. A different refrigerant classification was performed, i.e. the one based upon order relationships of refrigerant environmental properties. In this case the Hasse diagram technique, a method based on partial order theory, was applied to the 40 refrigerants characterised by environmental properties. A parameter free procedure for ordering classes based upon order relationships of their elements was developed. For that purpose, the dominance and separability degrees were introduced, first one indicates the extent to which members of one class hold higher descriptor values than the members of another class; while separability degree quantifies the lack of order relationships between two classes. Dominance and separability degrees were related by a theorem. By the application of dominance and separability degrees to refrigerant families three main classes were detected: problematic substances, gathering CFCs, octafluorocyclobutane and bromochlorodifluoromethane; least problematic ones, collecting HCs, CMs, carbon dioxide, trifluoroiodomethane, dimethyl ether and ammonia; and moderately problematic refrigerants, made from HCFCs, HFCs and HFEs. It was found that some HFEs are not dominated by CFCs, which raises the question on the applicability of these substances as environmentally acceptable replacements. METEOR (Method of evaluation by order theory), a procedure for prioritising descriptors and studying its effect on the order relationships of the objects considered was discussed. When applied to the refrigerants, the effect of prioritising ODP, GWP and ALT in the order relationships of these substances was studied. It was found that pentafluorodimethyl ether, a HFE, is one of the most problematic refrigerants under a large range of priorities of the environmental properties considered. Due to the mathematical generality of the methods here introduced, they are not restricted to the analysis of refrigerants but can be used to the study of different sets whose elements are characterised by various attributes.

Experimental Constraints on Silicate Perovskite Forming Reactions and Elastic Properties: Geophysical Implications for Chemical Heterogeneity in the Deep Mantle (2008)

Saikia Ashima

Three experimental investigations have been performed in order to understand how the composition of the mantle may influence mineral stability and elastic properties and how these may influence seismic properties of the deep mantle. The phase relations of calcium perovskite have been studied in high pressure and temperature experiments to examine the effect of its formation on seismic discontinuity features at 520 km depth in the mantle. The effect of varying composition on the compressibility of magnesium silicate perovskite has been examined in order to understand the geophysical consequences of chemical heterogeneity in the lower mantle. Calorimetric measurements of the pyrope-majorite garnet solid solution have been made to provide essential thermodynamic data for modeling the formation reactions of both magnesium and calcium silicate perovskite.

Vulnerability to Drought Risk and Famine: Local Responses and External Interventions among the Afar of Ethiopia, a study on the Aghini Pastoral Community (2008)

Ali Hassen Muhaba

In East African countries drought-related famine has been a number one risk. Ethiopia is among those countries that are repeatedly stricken by recurrent famine. Agricultural and pastoral households have increasingly become vulnerable to famine. The successive Ethiopian governments attributed the recurring famine and hunger to natural events, particularly to droughts. However, though drought triggers famines, it does not necessarily lead to famine disaster in every context. This is the current tone of literature in disaster causation. Each famine has its own specific causes in each context and this requires exploring the causal factors thereof. This study in the Afar region, in north-east Ethiopia, attempts to explain the root causes of vulnerability to famine, and assess the local and external responses. The central argument of the research is that pastoralists’ vulnerability to famine and food crisis has increased overtime because of the complex interplay of multiple factors such as environmental or ecological degradation, socio-economic destabilization, and political processes. It is also stated that despite efforts of internal and external actors, vulnerability of the pastoral groups to famine has increased over time. In that respect the study attempts to explain how these factors have led to an increase of vulnerability and livelihood insecurity among the Afar pastoralists. Three specific arguments are addressed through analyzing both secondary and primary data. These are: The Afar pastoralists’ vulnerability to famine has increased over the past decades because of the combined effects of drought, ecological crisis and external pressures (encroachments, loss of key pastoral resources, violent conflict and political instability). Pastoral households/communities are currently less able to cope with stresses through their traditional coping and adaptive strategies. Consequently, pastoral households/communities have become more dependent on public transfer (food aid) to cope with recurring food crisis. The empirical research used both qualitative and quantitative data in addressing the basic research questions. The outline of the research is structured to suit an approach of presenting discussions at macro and micro levels. An assessment of factors both, at macro (regional/national) and micro (community) levels, is made on the basis of secondary and primary data respectively. Accordingly, factors related to ecological degradation, socio-political processes and recurrent droughts, etc. are examined. The extent of these problems at the macro level is assessed mainly based on secondary data, while the magnitude of these problems at micro (community) level is assessed on the basis of primary data gathered through a household survey, and individual and focus group interviews. Based on the analyses of both primary and secondary data the research attempts to answer the question why the Afar pastoralists’ vulnerability to famine has increased over time and how the local and external actors have responded to recurring famine. The search for explanations of vulnerability to famine focuses on highlighting the interplay of multiple causal factors at different levels within historical socio-political and economic processes overlapping with ecological crisis and recurrent drought. Accordingly the research highlights the major factors that have created vulnerability to famine. These include: External pressures which include state intervention, land alienation, encroachment by cultivators, loss of dry season/drought retreats, curtailment of mobility and unfavourable terms of trade. Stresses which include frequent drought, conflicts, political instability, weakening mutual support systems, lack of trust in formal government institutions due to non-participatory, lack of commitment, patron-client relationship, and corruption. Ecological/environmental crises which are reflected in terms of loss of key pastoral resources (grass, natural fodder vegetation, and water). The conclusion of the research is that famine and the increase of vulnerability are not primarily the consequences of drought, but of external domination and uneven development. This suggests that the genesis of food crisis (famine) must be understood as an interaction of institutional, economic and political variables. Natural events like droughts don’t necessarily lead to famine in all contexts. It is only when livelihood assets are eroded, opportunities are constrained and people are not well-prepared that the consequences of natural events develop into famine or food crisis. This suggests that production or yield failures caused by drought do not become famines unless other conditions are propitious. Therefore, the current approach in social science research with regard to disaster causation is to look at the interrelationship between natural risks and social vulnerability.

Function of the ATP-dependent Metalloprotease FtsH during sporulation in Bacillus subtilis (2008)

Thi Thuy Ai Le

The analysis of the function of the ftsH gene of Bacillus subtilis started about ten years ago. It was shown at that time that an ftsH knockout was viable, but exhibited a pleiotropic phenotype. Cells are sensitive to salt and heat shock, exhibit filamentous growth, are difficult to be transformed and are almost unable to sporulate. Despite the severe phenotype caused by the absence of the ftsH gene, the precise functions of this protein remained unclear. This PhD thesis presents data to elucidate the function of ftsH during sporulation. Furthermore, it describes the construction of a cold-inducible expression system. The major finding of this thesis is that the FtsH protease interferes with the synthesis and/or phosphorylation of Spo0A, the master regulator during initiation of sporulation called phase 0. In the ftsH knockout, the amount of Spo0A is greatly reduced, and the small amounts present are inactive. When the wild-type spo0A allele was replaced by an IPTG-inducible allele coding for mutant Spo0A protein being fully active in the absence of phosphorylation (Spo0A-Sad67), spores were formed at a normal rate in an ftsH knockout. Again, this result indicates that FtsH is clearly involved in the formation of active Spo0A and that this protease is only essential during stage 0 of sporulation. To become active, Spo0A needs to be phosphorylated by the multi-component system called phosphorelay. Since no active Spo0A is present in an ftsH knockout, it was hypothesized that FtsH has to degrade one or more negative regulator(s) either preventing the phosphorylation of Spo0A or/and being involving in its rapid dephosphorylation. The further analysis focused on four antagonists of the phosphorelay, three Rap phosphatases being involved in the dephosphorylation of Spo0F~P, and Spo0E which targets Spo0A~P. When a null allele in any one of them was combined with the ftsH knockout, the wild-type amount of Spo0A was restored only in the case of the ftsH spo0E knockout and the sporulation frequency was increased by two to three orders of magnitude in all double knockouts, but remained below 1%. Since overexpression of Spo0E reduces the sporulation frequency and removal of the gene from the genome has an opposite effect, a direct interaction between FtsH and Spo0E was envisaged. In vitro proteolysis assays with purified GST-FtsH and GST-Spo0E showed that Spo0E is indeed a target of FtsH. In contrast, the two homologs of Spo0E, YisI and YnzD, remained stable upon incubation with FtsH. Since all three proteins are distinguished by a C-terminal extension of about 25 amino acids present in Spo0E, but not in the two other phosphatases, these additional amino acids could serve as a target for FtsH. When two mutant versions of Spo0E, Spo0E94 and Spo0E11, with truncated C-terminal ends were analyzed, they turned out to be stable in the presence of FtsH. When the C-terminal 25 amino acids was transferred to YnzD, this fusion protein became unstable when incubated with FtsH. In conclusion, the C-terminal end of Spo0E confers instability to this enzyme. Since a spo0E knockout in a wild-type background does not result in a sporulation frequency close to 100% and a combination of a spo0E and an ftsH knockout raises the sporulation frequency only close to 1%, it can be concluded that there are additional targets for FtsH interfering with the synthesis of active Spo0A. Moreover, it is likely that FtsH also exerts a function late during sporulation. It could be shown that SpoVM, a small peptide essential for spore morphogenesis, inhibits the proteolytic activity of the B. subtilis FtsH protease in vitro. It can be inferred that SpoVM also inhibits activity of FtsH during sporulation, and in the absence of SpoVM, FtsH will degrade at least one protein essential for successful completion of sporulation. When the intracellular proteomes of spoVM+ and spoVM- cells were compared, a total of 83 proteins were identified being either completely absent or present in reduced amounts in the absence of the peptide. Analysis of the expression of the spoVM gene revealed that cells started to synthesize the spoVM transcript at stage 2 while the SpoVM peptide accumulated at stage 4. The 5´ untranslated region of the spoVM transcript has been identified to act as a negative regulator of its own transcription or translation. Furthermore, a cold-inducible expression system has been constructed allowing intra- and extracellular production of recombinant proteins. This expression system makes use of a two-component signal transduction system, which senses changes in the fluidity of the cytoplasmic membrane.

Stability of flat galaxies (2008)

Roman Firt

In this thesis we investigate the existence and properties of stationary solutions of the flat Vlasov-Poisson system. This system of partial differential equations can be used as a model of extremely flat astronomical objects and is a combination between the two-dimensional motion of particles and the three-dimensional interaction through their gravitational potential.

Existence and stability of stellardynamic models (2008)

Achim Schulze

We examine existence and stability of stationary solutions to the Vlasov-Poisson system. This system is used in stellardynmaics to describe the evolution of galaxies where collissions are neglected and the evolution is determined by the self-consistent gravitational field which is created by the particles, e.g. the stars . In the first part we examine steady states which decsribe static shells under the influence of a fixed point mass. These solutions can be used as a model for a galaxy with a massive black hole in its center. For the Vlasov--Poisson system under the influence of such a point mass, we prove a global existence result. In the second part, we construct axially symmetric solutions depending on Jacobis integral. The presented results are in accordance with the numerical examinations of the P.O. Vandervoort.

The Effect of Zr-Doping and Crystallite Size on the Mechanical Properties of TiO2 Rutile and Anatase (2008)

Eva Susanne Holbig

TiO2 is an important technological material, used as white pigment, as wide band gap semiconductor in electrochemical dye solar cells, for photocatalysis and in photochemical energy-conversion processes. The most abundant phases are rutile, anatase and brookite. In addition, there are a number of metastable low density modifications. The compression behavior of anatase and rutile was studied for TiO2 and Ti0.9Zr0.1O2 starting materials with crystallite size in the micro- and also nanometer range. Quenched samples of rutile, anatase and high pressure polymorphs synthesized at up to 10 GPa adopt 10 mol% ZrO2, Zr-doped TiO2 starting materials therefore have the composition Ti0.9Zr0.1O2. Compression experiments were carried out in the diamond anvil cell and samples were characterized by in-situ X-ray diffraction, X-ray absorption and Raman spectroscopic measurements. A sol-gel route was developed for the synthesis of nanoscale anatase Tix:Zr1-xO2 with x=0.90 and 1.0, which was annealed at 1000°C to microscale rutile. In hydrothermal experiments, nanoscale anatase Ti0.9Zr0.1O2 was used as starting material for the synthesis of microscale Zr-doped anatase. Neither the incorporation of Zr nor the decrease of crystallite size to the nanometer range modifies the bulk modulus of rutile. These results are different from those of anatase, where a decreasse of crystallite size and doping with Zr leads to an increase of the bulk modulus. Second order EoS fits (K0’=4) resulted in a bulk modulus of microscale anatase of K0=178 GPa and K0=179 GPa. The nanoscale counterpart shows much higher values of K0=237 GPa and K0=243 GPa. In this study, it was found that microscale anatase Ti0.90Zr0.10O2 has K0=195 GPa, which is comparable to undoped material. Largest values were found for nanoscale anatase Ti0.90Zr0.10O2 with K0=258 GPa. Zr-doping thus reduces the compressibility of nanoanatase, even though ZrO2 polymorphs are more compressible than the corresponding TiO2 forms. For the Zr-doped nanoanatase, XRD analysis showed a significant change in compression behavior at pressures 4 GPa, suggested as a consequence of deviatoric stresses during experimental compression of the nanoscale material. Computations on supercells with different distances of neighboring Zr-atoms suggested cluster formation of Zr in the (Ti,Zr)O2 anatase. The resulting structural distortions can further augment the change in compression behavior. Zr-doped nanoanatase becomes stiffer upon multiple compression cycles. While the bulk modulus of the first compression was 211 GPa, after the sample was decompressed, the second compression showed a bulk modulus of 249 GPa. We suggest that partial pressure induced amorphization plays an important role for the observed stiffening. Anatase and rutile TiO2 transform to the MI phase upon compression. The transition pressure increases with a decreasing crystallite size from 12 GPa for microscale material to 18 GPa for crystallite size of 12 nm. For anatase, smaller particles transform to an amorphous phase at pressures of 20–24 GPa. Zr-doping does not seem to vary the transformation pressure. Ab-initio all-electron density functional electronic structure simulations on the ground state energetics of the TiO2 phases rutile, anatase, brookite, TiO2II and MI-phase were performed using the projector augmented wave and the linear augmented plane wave methods along with local density approximation (LDA) and two types of generalized gradient approximations (GGA), using the formulations by Perdew, Bunge and Enzerhoff, referred to as PBE, and by Wu and Cohen, reffered to as WC. The zero pressure volumes are predicted smaller by <3% in LDA computations and larger by 8 and 0.4% in PBE and WC computations. The stable structure at 0 GPa is baddeleyite for LDA computations and anatase for GGA computations, contradicting experimental results that determine rutile as the most stable phase. Rutile appears to have the highest energy in LDA computations and intermediate energy in GGA computations.

Characterization of SsoSSB, Sso1450, Sso2001 Proteins and Analysis of CRISPR and cas Genes from Sulfolobus solfataricus P2 (2008)

Dong Han

Following the complete sequencing of the genome of Sulfolobus solfataricus (Sso) P2, this organism has been widely used as a model strain for crenarchaea. The present work concentrated on the characterization of a newly discovered single-stranded binding (SSB) protein from Sso P2 and the computational and experimental analyses of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (cas) genes, respectively. The DNA-binding properties of SsoSSB, the organization of the CRISPR loci and the biochemical properties of some of the cas gene products of Sso P2 were studied. Size-exclusion chromatography indicated that SsoSSB exists as a monomer in solution. Using fluorescence anisotropy as a method to study the interaction of SsoSSB to DNA, it can be shown that SsoSSB binds as a monomer to small oligonucleotides. The approximate binding site size is 4- 6 nt per protein molecule as determined by native electrophoresis. SsoSSB shows a more than 10 fold higher binding affinity to single-stranded (ss) DNA as compared to double-stranded (ds) DNA, which is consistent with former reports. The dissociation constant could be determined to be in a low nanomolar range. Furthermore, SsoSSB preferentially binds to pyrimidine-rich ssDNA as compared to purine-rich DNA. This property is similar to that observed for human replication protein A (RPA). The clustering of repeat sequences in CRISPR loci and the associated cas genes have emerged recently as a new genomic feature of Archaea and of some Bacteria. Formerly, the cas genes have been predicted to encode repair proteins. In the present work, five CRISPR loci and their cas genes in Sso P2 were analyzed with respect to their genomic organization. The repeats of the CRISPR loci show highly conserved sequences at regular intervals, separated by spacer sequences of similar size. Most of the cas gene groups flanking a CRISPR locus contain homologous genes that were also found at other CRISPR loci. The cas genes could be grouped by gene location and gene order. Mostly, in each group they were head-to-tail arranged implying a functional relation. The operons of the cas genes sso1996-2002, sso1438-1443 and sso1398-1403 could be shown to contain Transcription Factor B recognition element (BRE), TATA-box, Shine-Dalgarno and terminator sequences specific for Sulfolobus. Most cas genes could not be expressed in a soluble form in E.coli, even when the expression conditions were widely varied. Refolding of the insoluble proteins was then undertaken. sso1442, sso1996 and sso1997 could be expressed in partially soluble form in E.coli, however catalytical activities could not be identified for these proteins. Refolding of Sso1999, a putative helicase, yielded a soluble protein. However no helicase and ATPase activity could be detected in the renatured Sso1999. Defined biochemical activities could be only assigned to the proteins Sso1450 and Sso2001. In the latter case, the sso2001 gene was fused with an esterase gene from Alicyclobacillus acidocaldarius, and was co-expressed in a soluble form. The enzymatic screening indicated that Sso2001 harbored a nuclease activity. Further experiments showed that Sso2001 was an endonuclease with specificity for cleavage near G residues. The nuclease activity was optimal at the neutral pH range with another activity peak at pH 3. Specific point mutations introduced in Sso2001 indicate that this protein was not a HD-family nuclease as previously predicted. The protein Sso1450 (COG1518), which is considered to be a marker protein of the CRISPR and Cas system, bound nucleic acids, including ssDNA, dsDNA and RNA, with high affinity. The dissociation constant of binding to DNA oligonucleotides was in the nanomolar range. EMSA experiments indicated an aggregation of Sso1450 on the DNA substrates. Interestingly, Sso1450 promoted the annealing of complementary ssDNAs. This finding supported a role of Sso1450 in the recombination of repeat sequences of the CRISPR system as suggested by Koonin’s group (Makarova et al., 2006). The CRISPRs were thought to play a major role in a newly discovered genome immune system in prokaryotes.

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