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In summary, the transcriptome data demonstrated that acute RAP for 7h induces significant changes in the expression of several left atrial genes, including those reflecting ANG II-mediated oxidative stress, tissue remodeling, and energy depletion. Furthermore, the results from the dronedarone study demonstrated that this drug is capable of attenuating most of RAP-induced changes in oxidative stress-related gene expression. Accordingly, the haemodynamic parameters also showed that dronedarone reduced RAP-induced microvascular flow abnormalities. This view is supported by the observation that in the used porcine model of acute AF, dronedarone decreased RAP-dependent PKC phosphorylation, NADPH isoform expression, F2-isoprostane release and IκBα phosphorylation. Additionally, the results of the irbesartan study indicate that ET-1 contributes to AF-dependent atrial fibrosis by synergistic activity with ANG-II to stimulate SGK1 expression and enhance phosphorylation of the SGK1 protein which, in turn, induces CTGF. The latter has been consistently associated with tissue fibrosis. In support of this view, in vitro analyses using HL-1 cells verified CTGF induction after short episodes of RAP and additionally in response to exogenous addition of ET-1. Accordingly, irbesartan was shown to attenuate most of the RAP-dependent changes in atrial or ventricular gene expression.
Because of its antimicrobial properties, nonthermal plasma could serve as an alternative to chemical antisepsis in wound treatment. Therefore, this study investigated the inactivation of biofilm-embedded Pseudomonas aeruginosa SG81 by a surface barrier-discharged (SBD) plasma for 30, 60, 150 and 300 s. In order to optimize the efficacy of the plasma, different carrier gases (argon, argon admixed with 1% oxygen, and argon with increased humidity up to approx. 80%) were tested and compared against 0.1% chlorhexidine digluconate (CHG) exposure for 600 s. The antimicrobial efficacy was determined by calculating the difference between the numbers of colony-forming units (CFU) of treated and untreated biofilms. Living bacteria were distinguished from dead by fluorescent staining and confocal laser scanning microscopy. Both SBD plasmas and CHG showed significant antimicrobial effects compared to the untreated control. However, plasma treatment led to a higher antimicrobial reduction (argon plasma 4.9 log<sub>10</sub> CFU/cm<sup>2</sup>, argon with admixed oxygen 3 log<sub>10</sub> CFU/cm<sup>2</sup>, and with increased gas humidity 2.7 log<sub>10</sub> CFU/cm<sup>2</sup> after 300 s) compared to CHG. In conclusion, SBD plasma is suitable as an alternative to CHG for inactivation of Pseudomonas aeruginosa embedded in biofilm. Further development of SBD plasma sources and research on the role of carrier gases and humidity may allow their clinical application for wound management in the future.
In this thesis we have revisited the formation of the excitonic insulator (EI), which realizes an exciton condensate. In contrast to optically created exciton condensates, the EI forms in thermal equilibrium and is solely driven by the Coulomb attraction between electrons and holes. The EI phase is anticipated to occur near the semimetal-semiconductor (SM-SC) transition at low temperatures. Depending from which side the EI is approached, it forms due to a BCS-type condensation of electron-hole pairs or a Bose-Einstein condensation (BEC) of excitons. The extended Falicov-Kimball model (EFKM) is the minimal model the EI can be described with. This model describes spinless fermions in two dispersive bands (f band and c band), that interact via a local Coulomb repulsion. The EFKM is also used to describe electronic ferroelectricity (EFE). Both phases, the EI and EFE-type ordering, are characterized by a spontaneous f-c hybridization in the EFKM. We have presented the EI phase, the EFE phase, and the orderings they compete with. Moreover, we have determined the ground-state phase diagram of the EFKM. We have focused particularly on the anticipated BCS-BEC crossover within the EI and have analyzed the formation scenarios. The exciton spectrum and the exciton density in the normal phase close to the critical temperature give information about relevant particles and therefore the nature of the transition. We have demonstrated that the whole EI is surrounded by a halo", that is, a phase composed of electrons, holes and excitons. However, on the SM side, only excitons with a finite momentum exist. These excitons appear only in a small number and barely influence the SM-EI transition. This phase transition is driven by critical electron-hole fluctuations, generated by electrons and holes at the Fermi surface. On the SC side, excitons with arbitrary momenta exist. Most notably, we have found the number of zero-momentum excitons to diverge at the SC-EI transition, signaling the BEC of these particles. Within the EI phase, there is a smooth crossover from the BCS regime to the BEC regime. One of the promising candidates to observe the EI experimentally, is the transition-metal dichalcogenide 1T-TiSe2. Strong evidences were found favoring an EI scenario of the charge-density-wave (CDW) formation in this material. However, some aspects point to a lattice instability to drive the CDW transition. We have addressed this issue by analyzing the recently discovered chiral property of the CDW in 1T-TiSe2. We have found that the EI scenario is insufficient to explain a stable, long range chiral charge ordering. Lattice degrees of freedom must be taken into account. In particular, nonlinear electron-phonon coupling and phonon-phonon interaction are crucial. By estimating appropriate model parameters for 1T-TiSe2, we have suggested a combination of excitonic and lattice instability to drive the CDW transition in this material. Experiments in 1T-TiSe2 and other materials suggest that the coupling to the lattice is non-negligible. We have extended therefore the model by an explicit exciton-phonon interaction, and have analyzed crucial effects of this interaction. While the single-particle spectrum is not modified qualitatively, the electron-hole pair spectrum changes significantly. The inclusion of the phonons lead to a massive collective mode in the ordered ground state in contrast to the case for vanishing exciton-phonon coupling, where the mode is acoustic. We have suggested that a gapless collective mode leads to off-diagonal long range order. This questions that the ground state for finite exciton-phonon coupling represents a condensate.
The region surrounding the excitonic insulator phase is a three-component plasma composed of electrons, holes, and excitons. Due to the extended nature of the excitons, their presence influences the surrounding electrons and holes. We analyze this correlation. To this end, we calculate the density of bound electrons, the density of electrons in the correlated state, the momentum-resolved exciton density, and the momentum-resolved density of electron-hole pairs that are correlated but unbound. We find qualitative differences in the electron-hole correlations between the weak-coupling and the strong-coupling regime.
During the past decade, various physical properties of the Yukawa ball, like structure and energy states, were unraveled using experiments. However, the dynamical features served further attention. Therefore, the main aim of my thesis was to investigate and understand how a finite system-represented by Yukawa clusters-evolves from a solid, crystalline structure to a liquid-like system, how it behaves in this phase and in what manner the reordering back into the solid state can be described. As a method of choice to reach this goal, laser heating has been proven successful. Moreover, the special importance of wakefields for dust clusters confined at low neutral gas pressure was addressed. Melting of finite dust clouds can be induced in two ways, either by altering the properties of the ambient plasma or by laser heating. The latter was shown to be a generic melting scenario, allowing to estimate a critical coupling parameter at the melting point. Moreover, the melting transition of finite 3D dust systems was found to be a two-step process where angular order is lost before the radial order starts to diminish at higher energies. Next, the mode dynamics of finite 3D dust ensembles in the solid and the liquid phase was studied. Crystal and fluid modes revealed the main spectral properties of the system. The normal modes are mainly suited to describe crystalline states. Fluid modes were excited naturally and via laser heating, with excitation frequencies almost independent of the coupling parameter in the solid and the liquid-like regime. Tuning the plasma parameters can be used to vary the particle-particle interaction via the ion focus. Both methods, even though assuming equilibrium situations, allowed to hint at these wakefields. The corresponding peaks in the fluid and normal mode spectra were no eigenmodes, confirming the nonequilibrium character of the ion focusing effect. First steps to extend the normal mode theory to achieve the dynamics of wake-affected nonequilibrium dust clusters were presented. Statistical quantities were obtained evaluating long-run experiments and transport coeffcients for finite dust systems were calculated via the instantaneous normal mode technique. Diffusion was found considerably higher for 3D than for 2D dust clusters. Using the configurational entropy, we have shown that in 2D and 3D disorder increases with increasing size of the system, in agreement with simulations. The temperature dependence of the configurational entropy differs for 2D and 3D dust clouds, with a threshold behavior found for finite 2D ensembles only. Finally, using instantaneous normal modes to reveal the total fraction of unstable modes, the predictive connection of Keyes (Phys Rev E 62, p7905, 2000), between transport and disorder was tested and verified for 2D, but not for 3D clusters. The reason for this has to be left open. Finally, laser-mediated recrystallization processes of finite 3D dust clouds were investigated. First, the temporal evolution of the Coulomb coupling parameter was traced during heating and recrystallization. A cooling rate has been determined from the initial phase of recrystallization. This cooling rate is lower than damping by the neutral gas, in agreement with simulations. We have observed a large fraction of metastable states for the final cluster configurations. Further, we have revealed that the time scale for the correlation buildup in the finite 3D ensemble was on even slower scales than cooling. Thus, different time scales can be attributed to the fast emergence of the shells and to the slower individual ordering within the shells.
The central aim of this thesis was the investigation of protein/polyanion interaction using circular dichroism (CD) spectroscopy, enzyme immune assay (EIA), isothermal titration calorimetry (ITC) and flow cytometry (FC). A further aim was to understand why an endogenous protein becomes immuno-genic when forming a complex. The focus was on the protein platelet factor (PF4), which gained wide interest in the clinical field, due to its role in the life-threatening, immune-driven, adverse drug effect heparin-induced thrombocytopenia (HIT). PF4 is a small homotetrameric chemokine with several basic amino acids on its surface, forming a positively charged ring. The antibodies that are formed during HIT recognize an epitope exposed on PF4, when it is in a complex with heparin at a certain molar ratio at which, PF4 tetramers are aligned on the heparin and forced into close approximation. The main results and conclusions of the thesis are summarized below: 5.1 Evolutionary Conservation of PF4 (Paper I – PF4/Evolution) By carrying out an amino acid sequence survey we found that the positively charged amino acids contributing to the heparin binding site on the surface of PF4 and related proteins are highly conserved in all vertebrates, including fish species. PF4 interacts with the phospholipid lipid A, the innermost part of the lipopolysaccharide (LPS) of Gram negative bacteria. We showed that the shorter the sugar chain of the O antigen, outer and inner core of the LPS were the more PF4 was binding. The interaction of PF4 with lipid A is inhibited by heparin, suggesting that the amino acids known to contribute to heparin binding are also involved in binding to lipid A. 5.2 PF4 Interaction with Polyanions (PA) of varying Length and Degree of Sulfation (Paper II – PF4/PA) CD spectroscopy was found to be a powerful technique to monitor structural changes of PF4 caused by binding to various clinically relevant polyanions. Therefore PF4 was titrated with different PA to investigate the dependencies: i. impact of the PF4:PA molar ratio, ii. degree of polymerization of the PA and iii. degree of sulfation of the PA. In all cases, exposure of HIT-relevant epitope(s) was only observed for PA that also induced changes in secondary structure of PF4. A comparison of results of an immune ¬assay with CD spectroscopic data showed that the extent of complex anti¬genicity correlates well with the magnitude of changes in PF4 secondary structure, and that the structural changes of PF4 have to exceed a certain threshold to achieve PF4/PA complex antigenicity. These findings allowed us to calculate expectation intervals for complex antigenicity solely using CD spectroscopic data. To our knowledge, this was the first demonstration that the capability of drugs to induce antigenicity of PF4 can be assessed without the necessity of in vivo studies or the use of antibodies obtained from immunized patients specific for the antigens. The antigenicity of PF4 in complex is not restricted to negative charges originating from sulfate groups, PA with phosphate groups are also capable (binding to phospholipids). We investigated inorganic polyphosphates (polyP) with a chain length of 75 Pi and showed that the induced secondary structural changes are even higher compared to the changes induced by the different heparins and that the PF4/P75 complexes are antigenic as well. 5.3 PF4 Interaction with defined oligomeric Heparins (Paper III – PF4/defined Heparins) We tested highly purified, monodisperse heparins. In contrast to the clinically relevant but relatively undefined (high polydispersity index) glycosamino glycans reported in paper II (PF4/PA). The defined heparins induced higher secondary structural changes. Here we showed for the first time that strong conformational changes during PF4/PA complex formation are necessary but not sufficient for to the expression of the anti-PF4/heparin antibody binding site. Also, the size of the complexes is not the only prerequisite for anti-PF4/heparin antibody binding (tested by atomic force microscopy). By ITC we found that antigenicity is only induced if the PF4/PA complex has a high binding enthalpy and the complex formation leads to a negative change in entropy. 5.4 PF4/Polyphosphates (polyP) Complex Antigenicity and Interaction with Escherichia coli (E. coli, Paper IV – PF4/polyP) PolyP with chain lengths of 45 Pi and 75 Pi induced remarkable secondary structural changes in the PF4 molecule, thereby exposing the epitope recognized by anti-PF4/heparin antibodies. The induced conformational changes were similar to the changes induced by the defined heparins. Again a high binding enthalpy was observed but here in connection with a positive change in entropy. Further we showed that polyP (≥45 Pi) enhance PF4 binding to the surface of Gram negative E. coli at intermediate concentration and disrupt the binding at elevated polyP concentrations. The increased amounts of PF4 on the bacterial surface also improved the binding of anti-PF4/heparin antibodies and thereby the phagocytosis of the bacteria by poly¬morpho¬nuclear leucocytes. 5.5 Nucleic acid based Aptamers induce structural Changes in the PF4 Molecule (Paper V – PF4/Aptamer) Nucleic acids are another class of molecules containing phosphate groups. Especially after cell damage their extra¬cellular concentration can be locally quite high (>2 mg/ml). We found that certain aptamers form complexes with PF4 and thereby inducing anti-PF4/aptamer antibodies which cross-react with PF4/heparin complexes. Moreover by CD spectroscopy we showed that the protein C-aptamer caused similar secondary structural changes of PF4 like heparin, but already at much lower concentration. The maximally induced changes by the protein-C aptamer were even higher and persisted over a broader concentration range. 5.6 Protamine Interaction with Heparin (Paper VI – PS/Heparin) After the intensive investigation of the complex formation between PF4 and many different classes of PA we assessed another protein for structural changes upon complex formation with heparin. Protamine (PS) a protein in routinely used in post-cardiac surgery to reverse the anticoagulant effects of heparin was found to unfold but not to refold with increasing concentration of PA in solution. 5.7 Conclusion and Outlook When starting this thesis, it was believed that repetitive structures formed by PF4 on a heparin chain mold the epitope recognized by antibodies inducing HIT. These repetitive structures might exhibit similarities with viral capsids and are therefore recognized by the immune system of some patients. We found that induced by the close approximation PF4 changes its conformation, thereby exposing a neoepitope. The conserved positively charged amino acids of the heparin binding site and the involvement of these amino acids in the binding to lipid A confirm our hypothesis of PF4 as part of an ancient immune-mediated host defense mechanism. As possible consequence of the “primitive mechanism of defense” the highly variable O-antigens of LPS might have significantly contributed to an efficient escape mechanism by hiding the structures that made the bacteria vulnerable. In turn polyP might be an adaption of the host improve pathogen recognition by PF4 and further by antibodies inducing phagocytosis of the PF4-marked objects. Although shown only for PF4 and PS, our findings might be applicable to other proteins that also express epitopes upon changes in their secondary structure. Our physicochemical methods may further be applied: i. to drug development for the prediction of antigenicity induced by polyanionic drugs, ii. to guide the development of synthetic heparins and other polyanion based drugs, e.g. aptamers, that do not lead to HIT and iii. to provide relevant aspects for other biological functions of heparins.
The extraction of raw materials in mining, as for example copper, generally requires a separation of the natural resources quarried. In most cases complex ores, mixtures of different minerals and gangue have to be separated in order to enable an economic processing. In particular for the extraction of sulfides, oxides, carbonates, phosphates, but also of coal, froth flotation is mainly used for this purpose, therefore it is considered as the most important separation process in raw material industries. Several billion tons of ores are processed annually. The principle of flotation is based on the surface properties of the mixtures components and the separation efficiency, which decisively determines the required amount of water and various chemicals, if nothing else, is an important criterion in mineral exploration and it also significantly influences the environmental impact of mineral processing. The aim of, this work was to investigate the influence, of, low-temperature plasmas on the mineral surface and, based on the acquired knowledge, to develop and verify strategies that would increase the efficiency of flotation processes through plasma pre-treatment of mineral mixtures. Since these studies are unprecedented, the results presented can be classified as a contribution to application-oriented basic research. Powder of the sulfide minerals, pyrite (FeS2), chalcopyrite (CuFeS2), chalcocite (Cu2S) and molybdenum sulfide (MoS2), were treated with plasmas of a radiofrequency and a microwave discharge and the resulting surface modifications were investigated by structure analysis such as X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). During the plasma process, the argon/oxygen and argon/hydrogen process gas mixtures used were analyzed by mass spectrometry (MS), taking into account the quantity of gaseous reaction products released, in order to estimate the rate at which chemical reactions occur. Furthermore, Langmuir and thermal probes, as well as different methods of optical emission spectrometry (OES) were utilized, which enabled a characterization of the discharges used with regard to different plasma parameters. It has been shown that sulfur dioxide (SO2) in Ar/O2 plasmas and hydrogen sulfide (H2S) in Ar/H2 plasmas are the only reaction products which can be detected by MS during the mineral treatments. Thus, the resulting sulfur rate loss could be time-resolved determined by means of additional calibrations with calibrating gases. Especially at Ar/O2-MW plasma treatments two fundamental mechanisms of mineral modification could be separated by time. Pure plasma-surface interactions at the beginning and, additionally, thermally induced reactions in during the evolution of the treatments. Comparisons regarding the relative sulfur loss during plasma-surface interactions between the investigated minerals have shown a strong influence of the process parameters whereas, under identical conditions, CuFeS2 reacted up to eight and nineteen times faster reacted than FeS2 or Cu2S. This result represents the basis of the strategy to optimize the flotation of the minerals investigated: The selective generation of oxides on the surface of one component in a mixture of sulphide minerals. In particular, at the separation of CuFeS2/FeS2 mixtures by using the oxide collectors Flotinor Fs-2 in a micro flotation cell, a high selectivity could be achieved. The recovery of CuFeS2 amounted to 100 % while less than 10 % of FeS2 was recovered and no other modifying reagents were used. XPS and XRD analyses indicate the possibility that metal oxide are created upon the CuFeS2 surface, while the formation of iron sulfates upon the FeS2 surface prevented the oxide collector adsorption. An increased intensity of the plasma treatment leads to an increased sulphate formation also on CuFeS2, whereas the recovery, and thus the selectivity of the flotation, was reduced again. It could be shown that this effect can be utilized for the separation of, CuFeS2/MoS2 mixtures by using both, oxide and sulfide collectors, because sulfates are not formed on molybdenum sulfide during Ar/O2 plasmas treatments. By means of the plasma diagnostics used the energy input onto the substrate, the gas temperature and the degree of dissociation of molecular gases were estimated and correlations regarding the surface modification have been worked out. Thereby, the region investigated within the parameter space could be enlarged due to the use of different excitation frequencies, 13.56 MHz and 2.45 GHz, and additional insights have been provided. Further studies, beyond the scope of this work, are, nevertheless, required in order to generate a more comprehensive picture of plasma-mineral interactions and to enable an optimal application of the obtained results.
The systemic renin-angiotensin system (RAS) is an endocrine system that is mainly known to regulate blood pressure, fluid and electrolyte balance as well as volume homeostasis in the body through different active metabolites, the angiotensin (Ang) peptides. In addition, local renin-angiotensin systems have been discovered in various tissues, including the islet of Langerhans. Starting with angiotensinogen, the precursor of all angiotensin peptides which is cleaved into the decapeptide Ang I by renin, the RAS is divided into three axes. The main classical RAS axis is composed of angiotensin converting enzyme (ACE), angiotensin (Ang) II, and the Ang II type 1 receptor (AT1R), whereas the two alternative RAS axes comprise either ACE2, Ang-(1-7) and the receptor Mas or the aminopeptidase N (APN), Ang IV and the insulin-regulated aminopeptidase (IRAP). The activation of the main ACE/Ang II/AT1R RAS axis has been associated with metabolic syndrome, type 2 diabetes mellitus, and islet dysfunction. The detrimental effects resulting from the pathological activation of this axis have been shown to be attenuated or even abolished by the pharmacological inhibition of components of the main RAS axis. However, the impact of the two alternative ACE2/Ang-(1-7)/Mas and APN/Ang IV/IRAP RAS axes on islet function is less well understood. Previous studies mainly focused on the possible protective actions of Ang-(1-7) via the receptor Mas in insulin-sensitive tissues and on well known risk factors of metabolic syndrome (insulin resistance, hyperglycemia, obesity, hypertension and dyslipidemia). Thus, the impact of this axis on β-cell function and, in particular, insulin production and release was examined in the present study. Glucose and fatty acids have been subjects of diabetic research because they are established pathophysiologically relevant features of the metabolic syndrome and are known to harm β cells, phenomena which are referred to as gluco- or lipotoxicity, respectively. The pathophysiologically relevant factors glucose, saturated fatty acid (FA) palmitic acid (PA), and the methyl ester of the omega-3 fatty acid docosahexaenoic acid (DHA-ME) were used in the present study to characterize the local β-cell RAS as well as β-cell function under pathophysiological conditions. Results of the present work demonstrate the expression of selected components of the RAS in isolated murine islets of Langerhans and the rat insulinoma cell line BRIN-BD11 under basal conditions. The alternative ACE2/Ang-(1-7)/Mas and APN/Ang IV/IRAP RAS axes were activated by high glucose in BRIN-BD11 cells after 24 h. Coincidently with these findings insulin production was found to be increased. In contrast, the expression of components of the main ACE/Ang II/AT1R RAS axis and the Ang II type 2 receptor (AT2R) were not affected under the same conditions (Härdtner et al., 2013). Both FAs, PA and DHA-ME were shown to alter the expression of components of the renin-angiotensin system in BRIN-BD11 cells. PA increased the expression of AT1R, the receptor of the main RAS axis, and of AT2R, whereas that of the receptor of the alternative ACE2/Ang-(1-7)/Mas RAS axis, Mas, appeared to be down-regulated at basal low glucose concentrations (5.5 mM). These effects were accompanied by a dose-dependent reduction of the insulin production and secretion. In contrast, DHA-ME augmented the expression of components of the ACE2/Ang-(1-7)/Mas axis and IRAP at low glucose concentrations, an effect which could be partially enhanced under high glucose conditions (25 mM). At basal glucose concentrations DHA-ME reduced the insulin secretion, whereas it was increased under high glucose conditions. However, the insulin mRNA amount remained unaffected by DHA-ME. Additionally, in contrast to glucose and palmitic acid, DHA-ME significantly increased the production of reactive oxygen species, at least hydrogen peroxide after 30 min. Expression alterations of components of the alternative ACE2/Ang-(1-7)/Mas RAS axis by glucose and PA correlated strongly with the corresponding insulin secretion and production. Therefore, an involvement of the ACE2/Ang-(1-7)/Mas RAS axis in the regulation of insulin secretion and production was hypothesized and validated in primary islets of Langerhans of both Mas-deficient and wild-type mice. Islets were exposed to the preferred natural ligand for Mas, Ang-(1-7), or to its pharmacological agonists or antagonists, respectively....
Objective: Epithelioid sarcoma (ES) presents unique clinical features in comparison to other sarcoma subtypes. Data regarding the benefits of chemotherapy are very limited. Combination regimens using gemcitabine and docetaxel (Gem/Doce) have proven to be effective, especially in uterine and nonuterine leiomyosarcoma. Yet, there is no available data on the efficacy of Gem/Doce in ES. Methods: A retrospective analysis of the three participating institutions was performed. Twenty-eight patients with an ES diagnosis presented at one of the participating institutions between 1989 and 2012. Of this group, 17 patients received chemotherapy. Results: Patients' median overall survival (OS) after the beginning of palliative chemotherapy was 21 months, and the 1-year OS was 87%. Twelve patients received Gem/Doce with a clinical benefit rate of 83%. The median progression-free survival (PFS) was 8 months for all patients receiving Gem/Doce. The best response was complete remission in 1 patient and partial remission in 6 patients. All 6 patients receiving Gem/Doce as a first-line treatment showed measurable responses with a median PFS of 9 months. Conclusions: In this retrospective study, Gem/Doce was an effective chemotherapeutic regimen for ES. Prospective studies are needed to better assess the effects of this combination drug therapy.
Magnetic reconnection is a fundamental plasma process where a change in field line connectivity occurs in a current sheet at the boundary between regions of opposing magnetic fields. In this process, energy stored in the magnetic field is converted into kinetic and thermal energy, which provides a source of plasma heating and energetic particles. Magnetic reconnection plays a key role in many space and laboratory plasma phenomena, e.g. solar flares, Earth’s magnetopause dynamics and instabilities in tokamaks. A new linear device (VINETAII) has been designed for the study of the fundamental physical processes involved in magnetic reconnection. The plasma parameters are such that magnetic reconnection occurs in a collision-dominated regime. A plasma gun creates a localized current sheet, and magnetic reconnection is driven by modulating the plasma current and the magnetic field structure. The plasma current is shown to flow in response to a combination of an externally induced electric field and electrostatic fields in the plasma, and is highly affected by axial sheath boundary conditions. Further, the current is changed by an additional axial magnetic field (guide field), and the current sheet geometry was demonstrated to be set by a combination of magnetic mapping and cross-field plasma diffusion. With increasing distance from the plasma gun, magnetic mapping results in an increase of the current sheet length and a decrease of the width. The control parameter is the ratio of the guide field to the reconnection magnetic field strength. Cross-field plasma diffusion leads to a radial expansion of the current sheet at low guide fields. Plasma currents are also observed in the azimuthal plane and were found to originate from a combination of the field-aligned current component and the diamagnetic current generated by steep in-plane pressure gradients in combination with the guide field. The reconnection rate, defined via the inductive electric field, is shown to be directly linked to the time-derivative of the plasma current. The reconnection rate decreases with increasing ratio of the guide field to the reconnection magnetic field strength, which is attributed to the plasma current dependency on axial boundary conditions and the plasma gun discharge. The above outlined results offer insights into the complex interaction between magnetic fields, electric fields, and the localized current flows during reconnection.
Governance and Management of Tourism in two Biosphere Reserves in Ecuador: Galapagos and Sumaco
(2014)
Tourism is multi-faceted phenomenon and various stakeholders, levels of government, and sectors are closely interlinked. Governmental and non-governmental institutions, local communities, diverse professionals, and different sectors and tourists do show various perspectives about the management of tourism. In this dissertation, a comprehensive analysis on the current situation of tourism in two biosphere reserves (Galapagos and Sumaco) in Ecuador is presented. Tourism is considered as one of the key strategies to promote environmental conservation and socio-economic development of local communities living in these places. UNESCO biosphere reserves are protected areas of extraordinary natural and cultural value, conceived as places for reconciliation between conservation and development. The concept of sustainable tourism can be seen as an umbrella that unites various forms of tourism in these protected areas (e.g. ecotourism, community-based tourism). The primary objective was to evaluate the main factors of success and failure in the management of tourism and its contribution to biodiversity conservation. Thus, a detailed review of the legal, political, and institutional framework of the country and a description of the two biosphere reserves (physical, biogeographic, demographic, socio-economic, institutional and legal elements) was given. Data collection for the case studies (Galapagos and Sumaco) was performed by the combination of qualitative and quantitative methods. As for qualitative tools, in-depth semi-structured interviews with the main stakeholders in both biosphere reserves were applied. Experts and specialists in the management of protected areas, as well as representatives of different sectors directly and indirectly associated with tourism were interviewed. Moreover, surveys were applied to two different target groups. Tourists were surveyed in order to establish their socio-demographic profile, travel preferences, and degree of knowledge about sustainable tourism, as well as their perception of the sustainability of tourism in the destinations visited. Furthermore, residents in both reserves were surveyed in order to find out about their socio-demographic characteristics, their main economic activities for supporting themselves and their attitude towards tourism, their level of knowledge about biosphere reserves, and their perception about advantages or disadvantages of living in a biosphere reserve. In general, although tourism is a concept that could be developed in accordance with the environment in these natural areas, in the case of Galapagos it is one of the main risk factors for the conservation of biodiversity in the archipelago due to the increasing number of tourists. In contrast, in Sumaco tourism could be an important sustainable alternative to mining, oil extraction and hydroelectric projects, which are the current threats to the conservation of the area. From the environmental point of view, there are serious problems in both reserves, mainly linked to the contamination of water resources. Such pollution imposes risks to the health to both, residents and tourists. The lack of freshwater is particularly critical in Galapagos, given its insular habitat. The invasion of exotic species in Galapagos is one of the most serious threats to the conservation, while deforestation in combination with illegal logging and mining activities is the greatest danger in Sumaco. In terms of sociocultural factors, migration processes undoubtedly shape the attitudes and values of the current population in both reserves. In Galapagos most residents are immigrants, whereas in Sumaco a significant portion are Kichwa people who belong to the area’s native population. In general, the inhabitants in both areas have not yet developed a true environmental awareness. The distribution of human population groups is also associated with the different types of tourism. In both reserves, tourist services offered by local communities have low quality standards and are targeted on a market segment consisting of tourists with a low budget. Thus, the community revenues obtained from tourism are generally only a small percentage of the total tourism market. This situation is much more noticeable in Galapagos where large companies that operate luxury cruises and hotels gain most of their revenues from tourism. Many of them have their headquarters in the main cities of Ecuador and abroad, which means that they pay their taxes in those cities and not in the places where they operate. Inequality in the distribution of the economic benefits of tourism leads to a situation of frustration and discontentment among the residents of the regions involved. Despite this situation, tourism is still a profitable business and residents try to make the most of it, no matter the cost impacts. Temporary and illegal tourism activities is often the normal state of the things: unregistered houses that offer rooms for tourists, taxi drivers who offer tours without being in possession of permits, tourist boat owners, tour guides and even large tourist companies that operate without legal licenses. This situation inevitably leads to a decrease of the quality of services, an uncontrolled increase of business, a consequent dumping of prices and the overall decline of the destination. Ultimately, this means fewer tourists, more environmental degradation and less economic benefits for residents. In this context, there is a serious conflict between the local community and large foreign companies. Governance and management of tourism are essential elements to ensure its development in a sustainable way. The country has a large number of laws, rules, regulations, and plans that regulate the development of sustainable tourism legally and institutionally, especially in protected areas. However, monitoring and law enforcement are major constraints for achieving proper management of tourism. This problem is particularly noticeable in small communities such as Galapagos and Sumaco where interpersonal relationships of kinship and friendship are close, which complicates control and regulation. There are some serious limitations regarding the technical and logistical capacity of the institutions responsible for controlling and managing the tourist activity; they consist mainly in the lack of staff and funding. Galapagos, given its special status, has increased the number of funding managers and staff members for controlling and management, but in many cases, these people are not fully qualified for their positions. Sumaco has only few tourism experts who can help to develop the tourism. The coordination and planning among all stakeholders involved in tourism is still a work in progress to ensure proper management of the tourist destinations. In any case, local communities are developing important initiatives in both biosphere reserves. After all, sometimes planning, coordination and local activities do not necessarily coincide with the national agenda. In general, in both biosphere reserves it is necessary to ensure higher standards of education, both formal and informal. Also it is urgent that the government as a regulating entity ensures the equitable participation of local communities in tourism benefits, control, monitoring, and law enforcement. Moreover, there is the need to encourage and ensure integrated planning of tourism in the different levels of government: local, regional, and national. Sustainable tourism means that the protection of cultural heritage and natural resources is granted at least the same level of importance as the involved economic interests. The unique nature that currently attracts tourists to Galapagos and Sumaco should be preserved not only for ensuring the continuity of the tourism activity itself, but also in order to safeguard biodiversity and natural resources for the coming generations. Responsible tourism practices which care about their social and environmental impacts are not necessarily opposed to economic interests. On the contrary, they need each other for a long-term development.
In the PhD-thesis a conditional random field approach and its implementation is presented to predict the interaction sites of protein homo- and heterodimers using the spatial structure of one protein partner from a complex. The method includes a substantially simple edge feature model. A novel node feature class is introduced that is called -change in free energy-. The Online Large-Margin algorithm is adapted in order to train the model parameters given a classified reference set of proteins. A significantly higher prediction accuracy is achieved by combining our new node feature class with the standard node feature class relative accessible surface area. The quality of the predictions is measured by computing the area under the receiver operating characteristic.
Non-thermal atmospheric pressure plasma has drawn more and more attention to the field of wound healing research during the last two decades. It is characterized by a unique composition, which includes amongst others free radicals, ions and electrons. Furthermore, non-thermal plasma exhibits temperatures that are below those inducing thermal cell damage. Next to its well-established anti-bacterial properties, plasma can have lethal as well as stimulating effects on mammalian cells. Therefore, the medical application of non-thermal plasma on chronic wounds seems to be a promising tool to enable healing processes. However, less is known about the plasma-mediated induction of intracellular signaling pathways in human immune cells, which play a leading part in the process of wound recovery and removal of pathogens. Therefore, this thesis examined the cellular effects of a non-thermal atmospheric pressure plasma treatment on human immune cells using the argon plasma jet kinpen 09. Here, the CD4+ T helper cell line Jurkat, the monocyte cell line THP-1 as well as the corresponding primary cells were investigated. First, cell survival and apoptosis induction was assessed in response to non-thermal plasma treatment by growth curves and flow cytometric assays. On the one hand it could be shown that primary cells are more susceptible to plasma treatment than the respective cell lines. On the other hand, monocytes responded less sensitive to plasma exposure than lymphocytes. Furthermore, this thesis outlined the impact of non-thermal plasma treatment on the gene expression level of immune cells. Therefore, DNA microarray analysis was performed with the cell lines Jurkat and THP-1. It became obvious that plasma exposure modulated the expression of several genes in both cell types. Differential expression of distinct target genes was further validated by quantitative PCR in the immune cell lines. Here, elevated gene expression levels of JUN and FOS in Jurkat cells and increased transcription of JUND in THP-1 cells in response to plasma treatment were made visible. JUN, FOS and JUND are components of the transcription factor AP-1, which is involved amongst others in gene expression of IL-8 and HMOX-1. Consequently, transcriptional induction of the inflammatory cytokine IL-8 as well as the enzymes HMOX-1 and GSR was detected in plasma-treated THP-1 cells. In addition, alterations in the protein activation levels were analyzed in plasma-treated Jurkat, THP-1 cells and primary monocytes. Since some of the identified target genes are known to be associated with the MAPK pathways, the regulation of these cascades was further investigated by western blot analysis. In all investigated cell types the pro-proliferative signaling molecules ERK 1/2 and MEK 1/2 as well as the pro-apoptotic signaling proteins p38 MAPK and JNK 1/2 were activated in a plasma treatment time dependent manner. In contrast to Jurkat and primary monocytes, the anti-apoptotic HSP27 was only induced in THP-1 cells in response to plasma exposure. Moreover, modulation of cytokine production and secretion was examined in the different immune cell types and co-cultured THP-1 and HaCaT keratinocytes by ELISA or flow cytometry. While Jurkat cells showed no plasma-mediated regulation of cytokine expression, THP-1 cells revealed an increased IL-8 secretion after long plasma time duration (360 s). Additionally, the intracellular expression levels of IL-6 and IL-8 were modulated in primary monocytes by plasma exposure. While short plasma treatment caused no alteration of the number of cells expressing IL-8 an up-regulation of the intracellular IL-6 level occurred after 30 s of plasma treatment. Long plasma treatment times resulted in a significant decrease of the intracellular IL-8 and IL-6 production levels. Furthermore, co-cultured THP-1 and HaCaT cells as well as mono-cultured THP-1 and HaCaT cells were examined regarding their cytokine secretion profile. Here, cells treated with plasma (180 s) as well as LPS and plasma (180 s and LPS) were compared with untreated cells. IL-6, IL-8 and GM-CSF secretion was induced by both plasma and plasma combined with LPS treatment in mono-cultivated HaCaT cells and co-cultured cells. Though, the highest cytokine secretion levels were reached in the plasma and LPS exposed co-culture. In contrast, mono-cultivated THP-1 cells only showed an increased secretion of IL-6, IL-8 and TNFa after incubation with plasma together with LPS exposed medium. In conclusion, this study revealed for the first time the non-thermal plasma-modulated expression of numerous genes and cytokines and the activation state of various signaling cascades in human immune cells. Thus, it contributes to gain a better understanding of the immune-modulatory impacts of plasma that might promote the wound healing process.
The impact of inbreeding under different environmental conditions and of artificial selection on cold tolerance was investigated in laboratory populations of the tropical butterfly Bicyclus anynana. The investigation focused on (1) the effects of inbreeding on several fitness-related traits and whether inbred individuals are more susceptible to stress, (2) interactions between inbreeding, genetic adaptation to cold stress and environmental conditions, (3) the effects of artificial selection and inbreeding in the adult stage in other developmental stages, and (4) the effect of inbreeding depression on the heat shock response. Environmental conditions are not constant over time; consequently organisms have to deal with environmental changes. Besides naturally fluctuating conditions, human-induced climate change may increase temperature changes as well as the severity of heat or cold waves. Temperature-stress resistance describes an organism’s ability to cope with stressful temperatures. Enhanced resistance to temperature stress can be reached by phenotypic plasticity or genetic adaptation. Plastic organisms are able to react fast to changing environmental conditions, whereas genetic adaptation is more important for long-term adaptation. Natural habitats may also be affected by human impact, causing habitat loss or fragmentation and changes in population structure. A decrease in the population size may result in inbreeding and inbreeding depression (ID). Consequences of inbreeding are well documented, and inbred individuals are predicted to be more sensitive to environmental stress than outbred individuals. The long term persistence of species and populations depends on their ability to adapt to novel conditions which in turn depends on genetic diversity. Therefore, studies of temperature resistance and its evolution in relation to inbreeding are very important. First a higher susceptibility of inbred individuals to environmental stress was determined in different populations of B. anynana. Inbreeding depression was revealed for several fitness-related traits, but not for immunity traits or heat tolerance. Temperature affected most traits, revealing the importance of temperature on ectotherms; just two hours of thermal stress affected important reproductive, life-history and immunity traits already. Importantly though, no evidence were found that inbred individuals are more susceptible to stressful temperatures than outbred individuals. Genetic adaptation and phenotypic plasticity can interact with one another, resulting in genotype-environmental interactions (G x E). The hypotheses tested here were that some genotypes are more plastic than others and that lines with increased cold stress resistance are less plastic with regard to cold resistance than control lines. To induce plastic responses the exposed lines differed in cold tolerance and inbreeding to different temperatures as well as different feeding regimes and measured fitness-related traits. Several interactions were detected in which a selection regime was involved, but these interactions did not show a clear overall pattern. In summary though, findings were that marginal impacts of directional selection and inbreeding on plastic responses and suggest that, at least for my study organism, the genetic architecture of fitness-related traits is not connected with the architecture of plastic responses. The next investigation concerned with the manifestation of genetic adaptation to produce one specific phenotype across development stages and possible trade-offs. The assumption tested was that there is a genetic link between different developmental stages to produce one definite phenotype by imposing selection in the adult stage only. Lines selected for increased cold resistance in the adult stage were used and increased cold resistance throughout all developmental stages was expected. However, higher cold resistance was found only in the adult stage and not in developmental stages. This could be either the result of a resource allocation trade-off between different stages or that there is no cold resistance phenotype. Thus, if selection takes place in the adult stage it does not affect the others. In the last experiment investigation was directed to determine whether there are negative inbreeding effects on the heat shock protein (HSP) response. Under stressful conditions, organisms produce the HSPs and they act as chaperons required for refolding and repairing of stress degraded proteins. Testing was oriented to find if inbreeding as a genetic stressor´ provokes a higher HSP expression and if there is evidence for higher temperature stress susceptibility on inbred individuals. Findings indeed showed a stronger HSP up-regulation in control compared to inbred lines with a negative inbreeding impact occurrence, which may causally underlie inbreeding depression.
Bats are ancient mammals that evolved more than 50 million years ago. There are 1,240 different species (> 20% of mammalian species) described so far, which represent one of the most abundant, diverse and widely distributed mammalian groups. Bats are the only mammals which actively fly and therefore can migrate to different areas of the world. It has been increasingly recognized that bats are reservoirs for more than 100 virus species, and several are associated with animal and human epidemics. As natural hosts of rabies virus (RABV) and related lyssaviruses, bats have become a focus of research not only in South America and Africa, but also in Europe and North America. Bats are also considered to be unique in their potential to host emerging and re-emerging zoonotic viruses. To evaluate and reduce the potential risk of rabies transmission to humans or carnivore hosts (like fox, raccoon and dog etc.), active and passive surveillance studies of bat have been performed. Using these approaches diverse lyssaviruses have been detected in bats. However, these studies did not explain the rarely discovered epidemics and the underlying resistance or immune mechanisms in bats as natural hosts for lyssaviruses. Probably, bats are more resistant to lyssavirus infections than other animals. This hypothesis is introducing the research questions of the present thesis: (1) How do the innate immune responses protect bats from fatal outcome of lyssavirus infections? Interferon (IFN) responses which can be induced by the recognition of viruses by pattern recognition receptors act as the first line of defense against lyssavirus infections. Therefore, type I and type III IFNs from European bats were cloned and functionally characterized in this thesis (Chapter 2 and 4). (2) How do the lyssaviruses adapt to escape the host defenses by counteracting the IFN-mediated immune responses? And how do the bats control the viral replication via the IFN responses? To explore the complicated interactions and understand how European bats (Eptesicus serotinus, Myotis myotis and Nyctalus noctula) interact with European bat lyssaviruses (EBLV-1 and 2), a natural host related model for investigations of the bat´s immune system and the virus-host interactions has to be established. Since all of 52 identified European bats species are endangered and strictly protected, stable cell lines from different tissues of M. myotis for in vitro studies were developed and used for molecular and functional studies (Chapter 3 and 4). The data obtained from this thesis revealed that: (a) European bat IFNs do have similar but also distinct molecular characteristics compared with other mammalian IFNs (Chapter 2 and 4); (b) Both investigated bat type I IFNs, IFN-Kappa; and IFN-Omega; present potent anti-lyssaviral activities and display a pathogen associated pattern in the tested cell line (Chapter 2); (c) The established immortalized M. myotis cell lines are differently susceptible to lyssaviruses and contain a functional IFN-mediated signaling cascade (Chapter 3); (d) Bat type III IFN-Lambda;s display cell-type specific functions due to the distinct expression of the IFN-Lambda; receptor (Chapter 4); (e) In bat cell lines a possible evasive strategy of lyssavirus is based on the counteraction of IFN production and/or IFN-mediated defensive pathways (Chapter 3); (f) The higher resistance of brain derived cell line MmBr compared to other cell lines to lyssavirus infection indicates the natural ability of bat´s central nervous system to control the growth of neurotropic virus, which might be an essential reason for the nonclinical outcome (Chapter 3). Overall, the present thesis provides first insights into IFN-mediated innate immune responses against RABV and EBLVs infection in their natural reservoir hosts and an useful toolbox for comparative analysis of virus-host interactions.
High-throughput expression data have become the norm in molecular biology research. However, the analysis of expression data is statistically and computationally challenging and has not kept up with their generation. This has resulted in large amounts of unexplored data in public repositories. After pre-processing and quality control, the typical gene expression analysis workflow follows two main steps. First, the complexity of the data is reduced by removing the genes that are redundant or irrelevant for the biological question that motivated the experiment, using a feature selection method. Second, relevant genes are investigated to extract biological information that could aid in the interpretation of the results. Different methods, such as functional annotation, clustering, network analysis, and/or combinations thereof are useful for the latter purpose. Here, I investigated and presented solutions to three problems encountered in the expression data analysis workflow. First, I worked on reducing complexity of high-throughput expression data by selecting relevant genes in the context of the sample classification problem. The sample classification problem aims to assign unknown samples into one of the known classes, such as healthy and diseased. For this purpose, I developed the relative signal-to-noise ratio (rSNR), a novel feature selection method which was shown to perform significantly better than other methods with similar objectives. Second, to better understand complex phenotypes using high-throughput expression data, I developed a pipeline to identify the underlying biological units, as well as their interactions. These biological units were assumed to be represented by groups of genes working in synchronization to perform a given function or participate in common biological processes or pathways. Thus, to identify biological units, those genes that had been identified as relevant to the phenotype under consideration through feature selection methods were clustered based on both their functional annotations and expression profiles. Relationships between the associated biological functions, processes, and/or pathways were investigated by means of a co-expression network. The developed pipeline provides a new perspective to the analysis of high-throughput expression data by investigating interactions between biological units. Finally, I contributed to a project where a network describing pluripotency in mouse was used to infer the corresponding network in human. Biological networks are context-specific. Combining network information with high-throughput expression data can explain the control mechanisms underlying changes and maintenance of complex phenotypes. The human network was constructed on the basis of orthology between mouse and human genes and proteins. It was validated with available data in the literature. The methods and strategies proposed here were mainly trained and tested on microarray expression data. However, they can be easily adapted to next-generation sequencing and proteomics data.
Accelerated drug release tests are essential for quality control (QC) of long acting (non-oral) controlled release formulations. Real-time release experiments are usually required for product development, to understand the mechanism of release and to establish a correlation with in vivo release. Ideally, the accelerated test should maintain the biorelevant aspect of the in vitro method and the mechanism of release should not change under accelerated test conditions. At the same time adequate discriminatory ability is a prerequisite as the accelerated test should be able to discriminate between batches with respect to manufacturing variables that can impact on bioavailability. The objective of this thesis was to develop accelerated drug release tests for intravaginal rings (IVRs) and to gain a mechanistic understanding of the principles that facilitate in vitro drug release under accelerated test conditions. A detailed evaluation of the in vitro release characteristics of the formulations under real-time test conditions was considered as a prerequisite for developing predictive accelerated tests. Two formulations were subject of this study, in which the mechanism of release is primarily governed by drug diffusion. One formulation was the commercially available Nuvaring®, a combined hormonal contraceptive IVR that releases etonogestrel and ethinylestradiol with a constant rate over a duration of 3 weeks. The second formulation was a prototype of an investigational IVR that is supposed to be bioequivalent to the marketed formulation. The Nuvaring® provides an example of a reservoir system in which a membrane mediates diffusion, resulting in release rates that are almost constant with time, whereas the investigational IVR is a matrix-type IVR. In these devices drug release is driven by Fickian diffusion through a homogeneous matrix and decays with time. Both IVRs are based on different grades of polyethylene vinyl acetate (PEVA). Accelerated drug release tests were performed at elevated temperature and in hydro-organic solvents since these two parameters were expected to increase drug diffusion through the semicrystalline EVA copolymer. Release experiments with IVRs or endcapped segments were performed in an incubator shaker. The devices were placed in stoppered flasks containing an adequate release medium that was continuously shaken and completely replaced at predetermined time points. Release experiments with endcapped segments were also performed in a small volume version of UPS apparatus 7 (the Reciprocating Holder). Results from release experiments in these two setups were in general comparable when the release from segments was standardized to release per ring with respect to the mass ratio (segment/IVR). Real-time drug release in an aqueous release medium at a temperature of 37 °C from the Nuvaring® was slightly affected by variations in the in vitro test conditions, i.e. media volume and composition (addition of solubility enhancing agents). These variations, however, did not affect the release kinetics that continued to be zero-order with exception of the initial burst. In contrast, real-time drug release from the matrix IVR was affected by the steroid solubility in the release medium, increased with increasing media volume and reached a maximum in release media containing solubility enhancing agents, resulting in distinct release kinetics. Interestingly the steroid solubility had a distinct influence on the release rate under conditions that are commonly assumed to provide sink conditions. Even under experimental conditions that provided minimum drug solubility, the concentration of ethinylestradiol in the receptor medium never exceeded 3 % of the saturation solubility. Accelerated drug release from both IVRs could be observed after exposure to elevated temperature and/or hydro-organic release media. Overall, increased drug release in different hydro-organic media correlated with polymer swelling. The higher swelling capacity of the investigational IVR, when compared with the Nuvaring®, was accounted for a stronger degree of acceleration in different hydro-organic release media. These observations were in agreement with literature sources that report that swelling as well as diffusivity in EVA copolymers increases with increasing VA content, which is lower in the rate-controlling membrane of the Nuvaring®. For the investigational IVR a good correlation between accelerated and real-time release profiles could be obtained if changes in steroid solubility under accelerated conditions were taken into consideration. For example, a good correlation could be observed between accelerated release profiles in hydro-organic media and real-time release profiles in media containing surfactants that provide maximum drug solubility and thus eliminate boundary layer effects. This observation appears reasonable since hydro-alcoholic release media also enhance steroid solubility in the receptor compartment. In case of the Nuvaring® variations in the in vitro test parameters under real-time test conditions did not affect the release kinetics. For this IVR, the mechanism of release was maintained in hydro-organic release media and at elevated temperature. The quantitative relationship between the zero-order release constants and the test temperature could be described by the Arrhenius equation, indicating that accelerated release is governed by an increase in drug diffusion. Validation of the accelerated method with a prototype of the investigational IVR with a different drug load demonstrated that the accelerated methods were able to detect formulation changes with similar discriminatory ability as the real-time test. However, the temperature-controlled accelerated method was less sensitive to detect changes in the release characteristics of a Nuvaring® that have been induced by preliminary heat-treatment, indicating that the accelerated method may be less sensitive to detect changes in IVRs that are a result of physical aging. When the aim is to develop an accelerated method for batch release it is therefore crucial to validate the accelerated method with appropriate samples from non-conforming batches that are out of specification under real-time test conditions and have been obtained by small but deliberate variations in the critical process parameters. In both formulations the degree of acceleration could be further increased by combining the effect of hydro-organic release media with an increase in temperature. Under these test conditions the ability to differentiate between the different prototypes of the investigational IVR was maintained. Moreover, in both IVRs the mechanism of release was not affected by an additional increase in temperature when compared with release profiles in hydro-organic solvents. In conclusion, the results of this study indicate that both temperature and hydro-organic release media are valid parameters to accelerate drug release from delivery systems in which the mechanism of release is primarily governed by diffusion through dense (inert) polymer matrices (i.e. inserts, implants). A correlation between real time and accelerated release will be facilitated if drug release under real-time test conditions is independent of the test parameters. To assure the outcome of the test with respect to quality and safety it is crucial to validate the accelerated method with appropriate batches.
In this thesis several methods of protein engineering were applied to explore and increase enantioselectivity and thermostability of certain carboxylesterases and to better understand the relationship between sequence, structure and function. For example, we were able to confirm the observed conservation of motifs like GX/GGGX and GXSXG, which was reported earlier. Yet, even more details were revealed and some were designated in numbers. However, the numbers may vary when even more sequences will be available, but the trend should remain the same. The power of the ABHDB lies in the information available throughout the very diverse and quite large superfamily. Structural equal positions can be easily compared and analysed regarding mutations, correlated mutations, prevalence etc., and visualization is simplified through direct output with YASARA software. The ABHDB was the first structural alignment of such a large number of known enzymes of the alpha/beta-hydrolase fold superfamily. With methods of rational protein engineering we were able to show that there is little flexibility of the GGG(A)X motif for the eukaryotic enzyme PLE 1 and the natural motif appears to be a good solution for high activity and enantioselectivity of PLE 1 in the conversion of tertiary alcohol esters. In a focused directed evolution approach, we were able to identify variants of BsteE with moderate, but significantly increased enantioselectivity in the kinetic resolution of tetrahydrofuran-3-yl acetate, and hence, were able to proof that the concept of ‘small but smart’ libraries is an efficient way to find improved mutants, while the screening effort was reduced. Moreover, we were able to show that the domain exchange enhanced the thermostability of BsubE, while expression level and activity were maintained or increased, respectively. Despite the great achievements and possibilities at present, we are not yet in the position to directly modify the gene to alter the structure in a complete predictable fashion to improve functional properties as imagined by Ulmer (1983). Nevertheless, substantial changes can be targeted and as demonstrated in this work, several broadly applicable methods are at hand. Furthermore, bioinformatics tools play an essential role in planning of experiments, analysis and interpretation.
Late Quaternary evolution and carbon cycling of tropical peatlands in equatorial Southeast Asia
(2014)
Peatlands are an important component in the global carbon cycle as they act as both long-term sinks for carbon dioxide and significant sources for methane. Over the Holocene period (the past 11,700 years) continuous CO2 uptake by peat accumulation exceeded methane emissions in northern peatlands and resulted in a net-radiative cooling effect on the global climate.Although 11% of the global peatland area is located in the tropics, the role of tropical peatlands in the global carbon cycle and in influencing the Earth’s radiative budget has not been resolved. Climate-carbon cycle models have thus far not included tropical peatlands because reliable data on their past rates of carbon uptake and release are not available. In this thesis this problem has been approached by reconstructing peatland expansion and rates of carbon storage and release over the Late Quaternary (Latest Pleistocene and Holocene) for the largest tropical peatland area, which is located in equatorial SoutheastAsia (i.e. Sumatra, Borneo, Peninsular Malaysia). Peat accumulation in the tropics remains an enigmatic phenomenon, because the constantly high temperatures of 26-27°C should theoretically drive rapid soil carbon turnover and thus not enable the accumulation of peat. Therefore this thesis also explores the mechanisms that cause peat formation in the SoutheastAsian tropics as well as the drivers behind changing rates of carbon accumulation. Carbon dynamics were analyzed at the regional scale (103–105 km2) of SoutheastAsia over millennial timescales (paper, I, II) and at the local scale (101–102 m2) of a peatland site on annual to centennial timescales (paper III, IV). Paper I presents the first systematic classification of the nearly 160,000 km2 SoutheastAsian lowland peatlands (below 70 m a.s.l.) into geographic peatland types. The peatlands were divided into 1) coastal peatlands of PeninsularMalaysia, Sumatra, and Borneo (~130,000 km2) and into inland peatlands (~30,000 km2) of 2) Central Kalimantan (southern Borneo), 3) the Kutai basin (eastern Borneo), and 4) the Upper Kapuas basin (western Borneo). Coastal peatlands formed by primary mire formation directly on freshly exposed marine or mangrove soils with the lowering of the sea level during the Late Holocene. In contrast, inland peatlands formed via paludification on either terrestrial sand soils (Central Kalimantan) or by both paludification and terrestrialization (Kutai basin, Upper Kapuas basin). The sequence of peatland initiation was established by applying the common cumulative basal date frequency approach (paper I). This method revealed clear differences in the timing of peatland initiation: 1) the Upper Kapuas peatlands are the oldest postglacial peat formations and date from 20,000-13,000 cal BP (calendar years before present), 2) inland Central Kalimantan peatlands date from 14,500-9000 cal BP, 3) the Kutai peatlands date from 8300-4900 cal BP, and 4) and the coastal peatlands date from 7700-200 cal BP. Coastal peatlands have a Holocene average carbon accumulation rate of 77 g C m-2 yr-1, being recognized as the globally most effective terrestrial ecosystems in terms of long-term carbon sequestration. Except for the Kutai peatlands, the Holocene average carbon accumulation rates of inland peatlands are significantly lower (20-30 g C m-2 yr-1) and very similar to the average long-term rates of northern peatlands. Fluctuations in past rates of carbon accumulation of SoutheastAsian peatlands could for the first time be linked to paleoclimatic changes, primarily variations in moisture availability (paper I, II). Hydroclimatic influences on carbon accumulation rates were related to shifts in the mean position of the Intertropical Convergence Zone, changes in the intensity of theAustral-Asian monsoon system, and variations in the frequency of the El Niño- Southern Oscillation. In contrast, peatland initiation and expansion was driven by sea-level change (paper I, II). The deglacial rise in sea-level is identified as the primary driver for inland peatland formation in Borneo, because the rising sea-level 1) lowered the hydrological gradients in the SoutheastAsian island archipelago inducing rising ground and surface water levels on these islands, and 2) led to higher atmospheric moisture availability due to the associated expansion of marine water masses on the shelf floor. Paper II shows that inland peatland initiation and expansion was most extensive during deglacial meltwater pulses, when the rate of sea-level rise exceeded 10 mm yr-1. Only when the rate of sea-level rise had slowed down to a threshold of 2.4 mm yr-1 by ~7000 cal BP could peat accumulation along the coasts keep up with the sea-level rise and coastal peatlands could form. Hydro-isostatic adjustment of the Sunda Shelf led to a sea-level lowering by ca. 5 m over the past 4500 years. Falling sea levels exposed extensive marine areas that were rapidly colonized by peat swamp forests.Anewly 140 developed method for the reconstruction of past peatland area based on transfer functions (paper II) reveals that 70%of the peatlands of Sumatra and Kalimantan only formed during the past 4000 years.Moreover, this new transfer function approach shows that the common basal dates approach overestimated the extent of peatlands in the past. This method, in general, leads to higher rates of reconstructed cumulative peat carbon uptake for the past. By combining reconstructed peatland areas and mean rates of carbon accumulation over millennial timescales from each peatland type the carbon uptake of all peatlands from Sumatra and Kalimantan could be quantified for the past 15,000 years (paper II). Carbon uptake remained below 1 Teragram (Tg) C yr-1 from 15,000-5000 cal BP because the total area of peatlands was less than 30,000 km2. Rapid peatland expansion driven by the lowering of sea-level over the past 5000 years increased carbon uptake on Sumatra and Kalimantan to over 7 Tg C yr-1 and resulted in an exponential growth of the regional peat-carbon reservoir to a size of over 20 Pg C. SoutheastAsian peatlands therefore had no significant role in the Late Pleistocene and Early Holocene global carbon cycle. However, because of their rapid expansion after 5000 cal BP by over 100,000 km2 the peatlands of SoutheastAsia became a globally important carbon sink during the Late Holocene and likely caused an atmospheric CO2 drawdown of 1-2 ppm (paper II). This previously unrecognized biospheric carbon sink partly compensated for contemporaneous terrestrial carbon losses associated with the desertification of Northern Africa. The mechanisms that enable high rates of carbon accumulation of coastal peatlands were explored in a peat core study presented in paper III. Here the use of a new coring technique for the tropics and the application of noninvasive geophysical measurements were employed to derive a high-resolution record of carbon accumulation rates. This study provides the first description of peatland pools for SoutheastAsia, which form as tip-up pools from falling trees such as Shorea albida. Based on a pollen and macrofossil record a fossil tip-up pool could be identified in the core and an associated carbon accumulation rate of 100 to over 900 g C m-2 yr-1 determined. Thus tip-up pools function as local hot spots for carbon accumulation, fundamentally different from northern hemisphere peatland pools, which act as net-carbon sources. From a time-series of aerial photographs the rate of tree fall and thus pool formation was determined at 0.4 tree ha-1 yr-1 (paper III).Asimulation model indicates that up to 60%of the peat deposited in peat domes of Borneo is derived from filled up fossil pools – changing the paradigm that Southeast Asian peatlands mainly form from belowground biomass and providing an explanation for the rapid carbon accumulation of these ecosystems. The climate impact of peatlands is, however, not only related to their capacity to rapidly store carbon, because peatlands also release the strong greenhouse gas methane – a by-product of anaerobic decomposition.Ametaanalysis of methane emission data from SoutheastAsian peatlands (paper IV) shows that their average annual methane release of 3 g CH4 m-2 yr-1 is lower than the average annual release of ~9 g CH4 m-2 yr-1 from northern peatlands, although the higher tropical soil temperatures should lead to significantly higher emissions. The limited degree of anaerobic decay is explained by the recalcitrance of the deposited biomass, which contains high amounts of lignin and tannin, providing another explanation for rapid carbon accumulation. Low anaerobic decomposition together with high rates of carbon accumulation imply that limits to vertical peat bog growth in SoutheastAsia are not set by cumulative anaerobic decay as in northern raised bogs. Instead peat bog growth is limited by aerobic decomposition related to water-table lowering as shown by a derived linear relationship between the amount of released CO2 from aerobic peat decomposition and the mean annual depth of the peatland water-table (paper IV). The climatic effect of Southeast Asian peatlands was determined by the global warming potential (GWP) method, which compares carbon uptake with methane emissions in terms of CO2-equivalents. The low methane emissions and high carbon accumulation rates of coastal peatlands result in a net annual uptake of 1340 kg CO2- equiv. ha-1 yr-1 over a 100 year GWP time-horizon. Under natural conditions coastal Southeast peatlands exert a significant net cooling effect on the global climate in contrast to northern peatlands, which have a warming effect or act climatic neutral on this time frame. It can be concluded that the tropical peatlands of SoutheastAsia are the strongest carbon sinks among all peatlands globally with a notable influence on the Earth’s radiative budget. However, today an estimated 90,000 km2 of peatlands in SoutheastAsia is drained for agriculture (e.g. oil palm plantations) and deforestation. These drained peatlands release annually over 140 Tg C yr-1 from aerobic peat 141 decomposition. Drainage also facilitates the regular spread of peat fires in this region, which on average release around 75 Tg C yr-1. Ongoing total carbon losses (~220 Tg C yr-1) exceed the natural carbon uptake by a factor of 25 and demonstrate that the entire SoutheastAsian peatland region has recently switched from a globally important carbon sink to a globally significant source of atmospheric CO2 (paper II, IV).