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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.
Many ethicists consider the rule of nonmaleficence – Do no harm! – to be the most fundamental ethical rule and key to ethics. This rule is taken as the foundation of the present work. I argue that any entity, that can be harmed, ought to be morally considered. Only those entities can be harmed that are inherently goal-directed or striving – in other words, that possess a telos. The reason is that by constantly acting in ways to preserve their being and to prevent their own not-being, goal-directed entities express that they value their own good. To harm such a goal-directed entity therefore means to act against the values and the good of it. The argument so far supports ethical biocentrism, that is, the view that all living, goal-directed beings are harmable, possess interests, and are, thus, morally considerable, while non-living beings are not. Yet, I digress from classical biocentrism since I conclude, based on analysis of evolutionary and biological findings, that the locus of goal-directedness and potential harm is also, if not foremost, situated in genes. Within many species, individual organisms sacrifice themselves for the betterment of their descendants like in praying mantises where males sacrifice themselves and are eaten by the female during copulation. This shows that it is not necessarily the organism as an individual which follows its own interests and goals. Individual organisms are – to a high degree – “directed” by their genes. Even in highly developed animals, genes play a significant role in the goal-directedness of the individuals. An adult human organism, for example, consists of trillions of individual cells. However, all these cells are derived from a single cell – the fertilized egg. Each of our lives begins with a single cell that contains almost all information to finally form our functioning body. Where do all the instructions, the goal-directedness come from to finally form an adult organism if not from the genes contained in this first cell, the zygote? It is the genes of each zygote that contain a set of information for making the appropriate adult. Organisms are largely programmed to do everything necessary to stay in existence, to survive, and finally to pass on their genes successfully – either by reproducing or by helping close relatives that carry a similar set of genes. The main interests of genes lie in their continued existence. This necessitates reproduction since the gene-carrying organisms will inevitably die. Single genes, though, are difficult to morally consider directly since they perform entirely in and through individual organisms. Without the individual organisms, genes cannot survive. The good news for ethics is that the interests of genes and organism usually converge: individual organisms try to survive – as do their genes. In practice, it thus makes much more sense to give moral attention to entire organisms instead of single genes. An advantage of the gene-centric ethical theory proposed here is that the moral relevance of future generations and species can be “directly” justified: Since genes have an interest in their continued existence (in the form of identical copies), they would be harmed if future generations were doomed to inexistence. Within a species with many individuals, each gene is likely to be represented in many organisms. The smaller the gene pool of a species gets, the less likely is the existence of the same gene and, therefore, the less likely is the fulfillment of its fundamental interests. Hence, saving one of the last individuals of an endangered species would be ethically preferable to saving an individual of a populous species. Unfortunately, moral conflicts are abundant – not only concerning biodiversity conservation. We often have to choose between harming either entity A or entity B – for example in the daily questions of food and eating. In such cases, a strictly egalitarian theory (especially an egalitarian biocentric one) would be no real help and without any guiding power. Therefore, on a second level of morality, we have to include additional criteria that help to minimize the overall harm. For these criteria to be objective, universalizable, and thus moral ones, I apply a number of widely accepted ethical principles like the principle of proportionality, impartiality, self-defense, and universalizability. By recurring to these principles, I identify a set of morally relevant criteria for a fair resolution of moral conflict situations which help to minimize the overall harm done. The identified criteria are: (phylogenetic) nearness, endangerment, r- or K-selected species, evolutionary distinctiveness, ability to regrow and to regenerate, pain-susceptibility, and ecosystematic role. In sum, my gene-centric environmental ethical theory provides numerous reasons and arguments for biodiversity conservation – for protecting genes, organisms, species, and ecosystems alike – without neglecting the needs of humans.
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).
This work describes the recent scientific and technical achievements obtained at the high-precision Penning trap mass spectrometer SHIPTRAP. The scientific focus of the SHIPTRAP experiment are mass measurements of short-lived nuclides with proton number larger than 100. The masses of these isotopes are usually determined via extrapolations, systematic trends, predictions based on theoretical models or alpha-decay spectroscopy. In several experiments the masses of the isotopes 252-255No and 255,256Lr have been measured directly. With the obtained results the region of enhanced nuclear stability at the deformed shell closure at the neutron number 152 was investigated. Furthermore, the masses have been used to benchmark theoretical mass models. The measured masses were compared selected mass models which revealed differences between few keV/c² up to several MeV/c² depending on the investigated nuclide and model. In order to perform mass measurements on superheavy nuclei with lower production rates, the efficiency of the SHIPTRAP setup needs to be increased. Currently, the efficiency is 2% and mainly limited by the stopping- and extraction efficiency of the buffer gas cell. The stopping and extraction efficiency of the current buffer gas cell is 12%. To this end, a modified version of the buffer gas cell was developed and characterized with 223Ra ion source. Besides a larger stopping volume and a coaxial injection the new buffer gas cell is operated at a temperature of 40K. The operation at cryogenic temperatures increases the cleanliness of the buffer gas. From extraction measurements and simulations an overall efficiency of 62(3)% was determined which results in an increase by a factor of 5 in comparison to the current buffer gas cell. Aside from high-precision mass measurements of heavy radionuclides the mass differences of metastable isobars was measured to identify candidates for the neutrinoless double-electron capture. Neutrinoless double-electron capture can only occur if the neutrino is its own antiparticle and a physics beyond the standard model exists since the neutrinoless double-electron capture violates the conservation of the lepton number. Due to its expected long half-life this decay has not yet been observed. However, the decay rate is resonantly enhanced if mother and daughter nuclide are degenerate in energy. Suitable candidates for the search of the neutrinoless double-electron capture have been identified with mass difference measurements uncertainties of about 100eV/c². In this work the results of the mass difference measurements of 12 possible candidates are presented.
The metabolomic approach is one part of the "-omics" cascade further comprising genomic, transcriptomic, and proteomic investigations. Since information about the metabolome of the important human pathogenic bacterium Staphylococcus aureus is scarce, the aim of this thesis is the characterization of the exo- and endometabolome of this bacterium on a most global scale. For this, the metabolomic platform consisting of the analytical instruments used for 1H-NMR spectroscopy, HPLC-MS, and GC-MS analysis was applied. First, the requirements for an accurate sampling procedure for the analysis of intracellular metabolites are presented, explaining important pitfalls during the sampling and the subsequent metabolome analysis via HPLC-MS and GC-MS (book chapter I). The challenging task of the metabolite identification is demonstrated, as well as the requirements for absolute quantification of intracellular metabolites. In order to enhance the knowledge about the staphylococcal physiology and the biochemical network, the impact of different stresses and varying cultivation media on the bacterial metabolite pool was investigated in several studies. In article I, a first description of the primary metabolism of growing S. aureus COL cells cultivated aerobically in CDM is provided. This study also monitored the adaptation to glucose starvation on the level of metabolites and proteins. The uptake of all amino acids and the secretion and reuse of overflow metabolites were analyzed in a time-dependent manner. During the switch to a non-growing state, a drastic rearrangement of the amino acid pool in the bacterial cells was detected, and intracellular amounts of glycolytic intermediates were found to decrease in parallel to extracellular glucose exhaustion. During infection processes, S. aureus has to cope with varying levels of oxygen supply, including anaerobic conditions. A global metabolomic approach investigated the adaptation of S. aureus COL to strict anaerobic conditions using CDM as the culture medium. Thereby only linear growth was possible despite the higher uptake rate of glucose compared to aerobically, logarithmically growing cells. In an anoxic environment, S. aureus mainly switched on the less reliable lactic acid fermentation. Only serine and threonine but no alanine were significantly taken up. Subsequent glucose limitation led to energy starvation indicated by a drop in the adenylate energy charge. This was accompanied with an arrest of the fermentative metabolism and declining numbers of colony-forming units without taking advantage of the energy supplying arginine deiminase pathway. Compared to the established CDM, the eukaryotic cell culture medium RPMI 1640 provides more in vivo-like growth conditions. In article II, the growth behavior and the metabolic footprint of the S. aureus strains COL and HG001 were investigated during the aerobic cultivation in RPMI 1640 medium. Both strains are commonly used in laboratory research. The observed uptake and secretion pattern of extracellular metabolites provides important information for infection studies in which this medium is used for the precultivation of S. aureus. The extracellular accumulation of the noncanonical D-amino acid D-isoleucine was an interesting outcome. The strain specific metabolic footprint points to noteworthy differences in the biochemical system of both strains. Moreover, this study demonstrates the impact of the cultivation medium on the metabolic status of bacterial cells. Due to increasing resistance against a large number of antibiotics, community- and hospital- acquired infections with S. aureus are of major concern in medical therapy. Thus, greater knowledge about adaptive mechanisms after antibiotic treatment is required. In article III, the response of S. aureus HG001 to antibiotics with varying target sides, such as ciprofloxacin, erythromycin, fosfomycin, vancomycin, and ampicillin, was investigated on the metabolite level. Thereby, the abundances of 176 intracellular metabolites were observed in a time-dependent manner, thus providing the most comprehensive experimental metabolite dataset so far available for S. aureus. None of the antibiotic compounds led to alterations of single metabolite amounts, but mostly entire metabolic pathways were affected. The intermediates of the cell wall biosynthesis were affected by each antibiotic, confirming this pathway as the most potential target for new antibacterial compounds. The metabolite composition of human nasal secretions and human sweat was analyzed, since such secretions present natural habitats of S. aureus during the colonization of typical host sides. The results confirm that the bacteria has to cope with low concentrations of most of the amino acids but large amounts of urea and lactate during host colonization. Considering the supply of amino acids, the results support the usage of the RPMI 1640 medium as a step to more in vivo-like cultivation experiments. Moreover, essential information for future studies about the adaptation of S. aureus to more in vivo growth conditions is provided. Altogether, the metabolomic approach was proven to be an important tool for helping unravel the complex bacterial metabolism and the environmental factors that also play a role in the virulence of Staphylococcus aureus.
Energetic ions are made to collide with atmospheric molecules. Positively charged ions of argon (Ar^+), helium (He^+), hydrogen (H_2^+ ), and protons (H^+) with energies of 50 keV to 350 keV are used as the bombarding ion. The ion beam of desired energy is produced using a linear ion accelerator at the University of Greifswald. The mass and energy distribution of sputtered particles were analysed using an Electrostatic Quadrupole SIMS (EQS) analyser. The target gases used are oxygen (O_2), sulfur hexafluoride (SF_6), and nitrogen (N_2). The ionized and fragmented particles due to collisions have been investigated. We have discovered a new process for negative ion formation in energetic ion collision with O_2 and SF_6 molecules. The process is a two body reaction between the projectile and the molecule without the need for a third particle (such as an external electron). It requires a direct charge transfer from the projectile to the molecule leaving it intact as O_2^- or SF_6^- . The process is experimentally confirmed by using a proton as projectile which does not have an electron to transfer. In comparison with positive ion fractions (O_2^+ , SF_5^+ ), the negative ions fraction is smaller by 2 orders of magnitude. This shows that the two body charge exchange process is weak due to the larger energy transfer required compared to the positive ion forming mechanisms. The two body charge exchange mechanism is not observed for ion collisions with N_2 molecule. No stable negative ion exist for N_2 molecule. The collision cross section for the ion formation during energetic ion – O_2 collision has been determined within the investigated impact energy. For SF_6 molecule the partial ion fraction of the secondary ions are determined for different projectiles involved. This kind of investigation is of great importance mainly in atmospheric physics. Energetic ions are constantly emitted from mass of the energy sources in the universe (e.g. sun). They interact with planetary objects or atmosphere on their way. A deep knowledge about the interaction processes is necessary to understand the ionospheric physics and space exploration. As second part of my thesis, a GaAs(100) surface is bombarded with 150 keV Ar^+ ion beam. From etching the surface to thin film coating, ion bombardment on solid surface found great role in the fabrication process of modern electronic and optical devices. In order to increase the knowledge on sputtering materials and because of profound importance in modern electronics, we choose GaAs(100) as our target. Among the sputtered atoms and ions, small sized cluster ions having more than 6 atoms have been identified. GaAs is a heteroatomic semiconductor containing gallium and arsenic in equal ratio. A preferential phenomenon of ’abundant sputtering’ of gallium compared to little arsenic (GaAs) has been investigated from their mass intensity. The experimental ion counts are compared with theoretically predicted relative abundance. This phenomenon of preferential sputtering is known for atomic species of sputtered GaAs but not for the sputtered cluster ions. The main reasons for this abundant sputtering of one element is attributed to the difference in ion formation energies and surface compositional change taking place during the sputtering process. Another notable characteristics is the preference in charge state among the sputtered ions. For instance, among sputtered atomic ions the ion counts of Ga^+ is 3 orders larger than As^+ ion and As^- is 2 orders larger than Ga^- ion. To get a clue for this behavior, we have investigated the energy distribution of both negatively and positively charged clusters. Different ion formation mechanisms were discussed. The energy distribution of atomic ion is partially explained by using a modified theory given by M. W. Thompson.
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 study the potential of molecular RT-PCR based methods for diagnostic or epidemiological investigations concerning negative-sense RNA viruses should be demonstrated exemplary for orthobunyaviruses (segmented genome) and lyssaviruses (non segmented genome). The recent discovery of a novel orthobunyavirus from the Simbu serogroup, Schmallenberg virus (SBV), via next generation sequencing and metagenome analysis led to the development of novel molecular detection methods. Due to the potential emergence of further orthobunyaviruses from the Simbu serogroup, a generic pan-Simbu real-time RT-PCR system was developed. This system was able to detect all tested Simbu serogroup viruses. As additional feature a species classification via sequencing is possible. Moreover, the novel pan-Simbu real-time RT-PCR system seems to offer a broad detection spectrum for orthobunyaviruses in general. Hence, this protocol allows a broad screening of samples predominantly for Simbu serogroup virus genomes but also might allow the identification of some related orthobunyaviruses in mammalian or insect samples. A comparison of the pan-Simbu real-time RT-PCR system with diagnostic real-time RT-PCRs revealed an overall higher sensitivity of the diagnostic assays for SBV detection. The diagnostic SBV-S3 assay convinced with the highest sensitivity and reliability for SBV detection. Additionally, the SBV-M1 assay turned out as highly specific for SBV and therefore is a valuable tool for a precise diagnosis in geographical regions where multiple orthobunyaviruses are endemic. Furthermore, the SBV genome diversity in Germany was investigated using a molecular epidemiological approach. Genome variability was extremely high in the N-terminal region of the putative envelope glycoprotein Gc which might have an impact on immunogenicity or host-cell infection. Phylogenetic analyses indicated that sequence variation is independent of host species and geographical distribution. In contrast to SBV as a novel pathogen, rabies encephalitis (caused by the prototype lyssavirus Rabies virus) is known for more than 4000 years. Thus numerous molecular techniques have been developed for lyssavirus detection, considering the diversity of this genus they all have certain limitations as regards their diagnostic range. Results of a lyssavirus ring trial among European laboratories indicate that RT-PCR could be a highly reliable diagnostic tool if at least two independent tests with broad diagnostic range are applied. Another approach suggested that a change from two-step to one-step PCR strategy or a variation of the RT-chemistry may have a remarkable influence on assay performance. However, no ultimate approach or strategy has been found yet, that would facilitate rabies routine diagnosis or epidemiological surveys on molecular grounds. Thus, there is a need for a potent, reliable and practical system for lyssavirus diagnosis and characterization, suitable as a second diagnostic line next to classical techniques like the fluorescent antibody test. For this purpose a diagnostic two level cascade protocol was developed with emphasis on the most relevant European lyssaviruses. On a first level two independent generic pan-lyssavirus screening assays, targeting different genomic regions, were applied. On a second level two probe-based species-specific multiplex PCR systems for the rapid classification of European lyssaviruses were used. All applied assays displayed an overall highly sensitive and specific detection with an excellent reproducibility and repeatability. Moreover, the diagnostic cascade protocol combines all known advantages of the real-time PCR technology including speed and reduced risk of cross-contamination with improved safety of molecular testing based on a double-check strategy for the screening as well as the confirmatory assays. In the frame of the second Bokeloh bat lyssavirus case in a German bat, the capability of real-time PCR for the quantification of viral loads was demonstrated. Another convenient example for the potential of molecular RT-PCR based methods is the epidemiological investigation of the rabies epizootic in Namibian kudu antelopes. Phylogenetic analyses of a 602 bp fragment of the nucleoprotein gene indicated a separate grouping of the Rabies virus (RABV) isolates from kudu apart from RABV isolates from jackals. Full genome sequencing revealed unique mutations in the glycoprotein gene of RABV isolates from kudu, suggesting an independent rabies cycle in Namibian kudu antelopes. All given examples were used to illustrate the application spectrum of molecular RT-PCR based methods for diagnostic or epidemiological purposes. The advantages of molecular techniques were emphasized and in particular real-time RT-PCR systems proved their fitness for purpose and appear to represent standard techniques for the next decade.
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.
Simulations of Short Model Peptides and Practically Relevant Modeled Titanium Implant Surfaces
(2014)
One of the aims of this work was to generate a non restrained force field model including carbon contamination to make the adsorption simulations more realistic and comparable with experimental data. Another purpose was to find out how the special recognition of small linker proteins on titanium dioxide is working. During this work a fixed and a non restrained rutile (100) model was used and critical properties were observed which are not only related to the surface. The rigid water layers on top of the oxide are very important for the protein and peptide adsorption. Therefore the first discussing object were the properties of the water layers and how they can be influenced. The charge distribution on the surface was found to have a big effect on them. Depending on the charges of the surface atoms or the functional groups, resulting out of the hydroxylation equilibrium, precisely the first water layer gets more rigid or smother. This has a big effect on biomolecule adsorption. The peptides need to penetrate these water layers to generate direct interaction points. The correct description of the surface in molecular dynamic simulations therefore has a high influence on the results. The better the model is the better the findings are comparable with experimental ones. Additionally carbon contamination was mimicked by using a monolayer of pentanol molecules. This fits very good with experimental data (e.g. contact angle) and make the oxide model more hydrophobic. Interaction of proteins and peptides in experiments or in medical use are often observed under normal air conditions, which means that the scaffold is i) hydroxylated by water and ii) carbon contaminated in a short period of time. Therefore investigations were done to find out how the contamination influences the adsorption of a formally know good or bad binding peptide (TiOBP1; TiOBP2). It was found that the TiOBP1 is able to bind the different surface modifications very well which coincides with observations made in experiments. The way of adsorption (direct or indirect) depends on the water layers properties. The first layer on high charged surface models is that rigid, that the peptide is not able to adsorb in a direct way. On the carbon contaminated oxide model the adsorption is possible by reducing the flexibility of the secondary structure motive. In the case of TiOBP2 adsorption on the clean surface model results in only weak binding or even in no interaction. Whereas on the carbon contaminated dioxide the once know bad binder is able to interact with the Pentanol monolayer. No direct adsorption is observed but the hydrophobic side chains have the possibility to orient themselves according to the hydrophobic layer without changing significantly in the secondary structure motive. An additional test peptide (minTBP) adsorbs without being affected by the contamination. This raises the question if the distribution of hydrophobic to hydrophilic amino acids has influence on the adsorption ability according to clean and contaminated surface. For experimental application it could be of interest to generated peptides (GEPI´s) which bind both surface types without changing the secondary structure motives then as we know functionality is based on these structures. In the case of the PHMB polymer adsorption was observed depending on the hydroxylation ratio and therefore on the charge density of the rutile (100) surface. After analysis of the simulations takeaways from experiments could be substantiated. The PHMB interacts with the negative charged surface via the first water layer as a film. So the new force field model describing the rutile (100) titanium dioxide surface with additional carbon contamination model of one monolayer pentanol fits the experimental data very well. The adsorption studied on this surfaces indicates that the contamination as expected makes the surface more hydrophobic and influences the adsorption behavior of the tested peptides especially the secondary structure of TiOBP1. This indeed enhances experimental investigations. Peptides which e.g. link organic and inorganic parts should be good adsorbing on clean and contaminated surfaces by keeping their functionality. Furthermore experimental data can be substantiated by using atomistic simulations like in the case of PHMB adsorption.