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The central goal of this investigation is to describe the dynamic reaction of a multicellular tumour spheroid to treatment with radiotherapy. A focus will be on the triggered dynamic cell cycle reaction in the spheroid and how it can be employed within fractionated radiation schedules.
An agent-based model for cancer cells is employed which features inherent cell cycle progression and reactions to environmental conditions. Cells are represented spatially by a weighted, dynamic and kinetic Voronoi/Delaunay model which also provides for the identification of cells in contact within the multicellular aggregate. Force-based interaction between cells will lead to rearrangement in response to proliferation and can induce cell quiescence via a mechanism of pressure-induced contact inhibition. The evolution of glucose and oxygen concentration inside the tumour spheroid is tracked in a diffusion solver in correspondence to in vitro or in vivo boundary conditions and a corresponding local nutrient uptake by single cells.
Radiation effects are implemented based on the measured single cell survival in the linear-quadratic model. The survival probability will be affected by the radiosensitivity of the current cycle phase and the local oxygen concentration. Quiescent cells will reduce the effective dose they receive as a consequence of their increased radioresistance. The radiation model includes a fast response to fatal DNA damage through cell apoptosis and a slow response via cell loss due to misrepair during the radiation-induced G2-block.
A simplified model for drug delivery in chemotherapy is implemented.
The model can describe the growth dynamics of spheroids in accordance to experimental data, including total number of cells, histological structure and cell cycle distribution. Investigations of possible mechanisms for growth saturation reveal a critical dependence of tumour growth on the shedding rate of cells from the surface.
In response to a dose of irradiation, a synchronisation of the cell cycle progression within the tumour is observed. This will lead to cyclic changes in the overall radiation sensitivity of the tumour which are quantified using an enhancement measure in comparison to the expected radiosensitivity of he tumour. A transient strong peak in radiosensitivity enhancement is observed after administration of irradiation. Mechanisms which influence the peak timing and development are systematically investigated, revealing quiescence and reactivation of cells to be a central mechanism for the enhancement.
Direct redistribution of cells due to different survival in cell cycle phases, re-activation of quiescent cells in response to radiation-induced cell death and blocking of DNA damaged cells at the G2/M checkpoint are identified as the main mechanisms which contribute to a synchronisation and determine the radiosensitivity increase. A typical time scale for the development of radiosensitivity and the relaxation of tumours to a steady-state after irradiation is identified, which is related to the typical total cell cycle time.
A range of clinical radiotherapy schedules is tested for their performance within the simulation and a systematic comparison with alternative delivery schedules is performed, in order to identify schedules which can most effectively employ the described transient enhancement effects. In response to high-dose schedules, a dissolution of the tumour spheroid into smaller aggregates can be observed which is a result of the loss of integrity in the spheroid that is associated with high cell death via apoptosis. Fractionated irradiation of spheroids with constant dose per time unit but different inter-fraction times clearly reveals optimal time-intervals for radiation, which are directly related to the enhancement response of the tumour.
In order to test the use of triggered enhancement effects in tumours, combinations of trigger- and effector doses are examined for their performance in specific treatment regimens. Furthermore, the automatic identification and triggering in response to high enhancement periods in the tumour is analysed.
While triggered schedules and automatic schedules both yield a higher treatment efficiency in comparison to conventional schedules, treatment optimisation is a revealed to be a global problem, which cannot be sufficiently solved using local optimisation only.
The spatio-temporal dynamics of hypoxia in the tumour are studied in response to irradiation. Microscopic, diffusion-induced reoxygenation dynamics are demonstrated to be on a typical time-scale which is in the order of fractionation intervals. Neoadjuvant chemotherapy with hydroxyurea can yield a drastic improvement of radiosensitivity via cell cycle synchronisation and specific toxicity against radioresistant S-phase cells.
The model makes clear predictions of radiation schedules which are especially effective as a result of triggered cell cycle-based radiosensitivity enhancement. Division of radiation into trigger and effector doses is highly effective and especially suited to be combined with adjuvant chemotherapy in order to limit regrowth of cells.
Respiration is one of the key processes of energy transduction used by the cell. It consists of two components: electron transfer and ATP production. The electron transfer chain converts the energy released from several biochemical redox reactions into an electrochemical proton gradient across membranes. This stored energy is used as the driving force for the production of ATP by the ATP synthase. The mitochondrial electron transfer chain contains four major protein complexes called complexes I-IV, with counting starting at the lower side of the redox potentials. It has been discussed for a long time how these protein complexes are organized in the membranes. Do they diffuse freely in the membrane? Alternatively, do they form a supercomplex built up of several neighboring complexes? The evidence supporting the free diffusion mode is that both electron transfer intermediates (cytochrome c and quinone) behave as “pool”. However, respiratory supercomplexes have been detected in membranes from bacteria, fungi, yeast, plant and animal during the last decade, and sometimes the respiratory complexes are only stable inside a supercomplex. Therefore, the idea of supercomplex formation has become more popular. The argument that the supercomplex arises from solubilization and is a detergent artifact could be rejected because: 1) supercomplexes can be isolated from many organisms in an active form; 2) supercomplexes have been proven to stabilize the individual complexes in some cases; 3) supercomplexes can be very stable after chromatographic isolation in some cases....
The four subunit (SU) aa3 cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. It uses electrons from cytochrome c to reduce molecular oxygen to water. Its binuclear active center, residing in SU I, contains hemeÊa3 and CuB, the latter being liganded by three histidine residues. Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity.
To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT CcO was constructed, carrying the gene for SUÊI on a low copy number plasmid. This rec. WT showed, as expected, no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential overproduction of SUÊI due to plasmid coding and the resulting deficiency in metal inserting chaperones might impair the correct insertion of hemeÊa3 and CuB because of a deficiency in metal inserting chaperones. This in turn might lead to differences in side chain orientation and to changes in the water network. However, slight changes might cause an increased accessibility of the active center for hydrogen peroxide, resulting in an increased catalase activity. The availability of chaperones and therefore the proposed structural reasons for the difference was improved by cloning the genes for the two metal inserting chaperones CtaG and Surf1c on the same plasmid together with SUÊI. This new rec. WT CcO showed in fact a reduced catalase activity. Another WT with a deletion in the chromosomal second, non expressing gene of SU I was analysed to prove plasmid coding as the reason for the difference of the ATCC WT and the rec. WT. This strain showed an increased kcat of the catalase activity as well, additionally pointing to a regulatory effect of the non expressed gene for SU I in the chromosome. To fathom the structural difference of the increased catalase activity, differential scanning calorimetry was used, but no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging, the thermal stability of the rec. WT CcO declined faster than that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.
To characterize the catalase reaction, several known inhibitors were used to probe the contribution of the different metal cofactors in the catalase reaction. In addition variants in aromatic amino acids near the active center were constructed to conclude on a possible reaction mechanism of the catalase activity of CcO. These variants in combination with the wild type forms were analysed for radical signals by EPR-spectroscopy. A radical relevant for the catalase reaction of CcO was found in the F-intermediate of all variants and all wild type forms. This narrow 12 G radical signal was assigned to a porphyrine radical probably involved in the catalase reaction of CcO. Moreover, gas chromatography-mass spectrometry measurements were used to analyse isotopically labelled oxygen produced in the catalase reaction.
As a result of these experiments, a reaction cycle of the catalase activity of CcO is postulated and the structural difference between the ATCC and rec. WT CcO is outlined. The catalase activity appears to be a true catalase activity and not a "pseudocatalase" activity.
The canonical Wnt pathway, also known as Wnt/β-‐catenin pathway, comprises a network of proteins which control diverse developmental and adult processes in all metazoan organisms. The binding of canonical Wnt ligands to a cell surface receptor complex, consisting of frizzled family members and low density lipoprotein receptor-‐ related protein 5 or 6 co‐receptors, triggers a signaling cascade which results in a β-catenin-‐mediated transcriptional activation of different target genes, implicated in cellular proliferation, apoptosis, migration and differentiation. A couple of years ago, several groups including us, iden2fied transient activation of the canonical Wnt-pathway in endothelial cells (ECs) of the developing central nervous system (CNS). In this context, Wnt/β-‐catenin signaling could be demonstrated to be crucial for brain angio genesis as well as for the establishment of the blood-brain barrier (BBB) phenotype in the newly formed vessels.
Gliomas, in particular the glioblastoma (GBM), belong to the group of highly vascularized solid tumors which gain their vascularization due to an angiogenic switch occurring during tumor progression. Interestingly, nuclear localized β-‐catenin could be exclusively detected in the activated endothelium of induced rat gliomas and of human GBM, suggesting a so far unknown and not further characterized involvement of the canonical Wnt pathway in pathological angiogenesis. In order to systematically decipher the precise role of endothelial Wnt/β-‐catenin signaling in tumor angiogenesis, I established
murine GL261 glioma cell lines overexpressing either Wnt1 or Dickkopf (Dkk) 1 in a doxycycline-‐dependent manner, an activator and potent inhibitor of Wnt/β-‐catenin signaling, respectively. In subcutaneous and intracranial transplantations, tumor-derived Wnt1 reduced, while Dkk1 increased GL261 tumor growth without affecting in vitro proliferation, cell cycle or cell death of the established cell lines. Nowadays, it is well accepted that solid tumors are dependent on vascular support allowing them to grow beyond a certain size. In my work I could show that tumor-‐derived Wnt1 targets the tumor vasculature by increasing endothelial Wnt/β-‐catenin signaling, which reduced tumor vessel density and resulted in a more quiescent tumor vasculature. Furthermore, Wnt1-‐expression mediated tight association of smooth muscle cells (SMCs) and pericytes to the tumor endothelium, a phenotype which is unusual for tumor vessels and a described hallmark of tumor vessel normalization. In contrast, inhibition of endothelial Wnt/β-‐catenin signaling by Dkk1 mediated an opposing effect, characterized by endothelial hyper-proliferation and a tumor vasculature with a rough basal lamina distribution and loosely anached mural cells, indicative of a strong angiogenic activity. The described vascular effects in Wnt1-expressing GL261 tumors could be verified by subcutaneous transplantations of a rat glioma cell line constitutively expressing Wnt1. Furthermore, an applied in vivo MatrigelTM plug assay uncovered the reduction in vessel density upon Wnt1 simulation to be tumor cell independent, suggesting an EC-‐autonomous effect. This hypothesis was confirmed by subcutaneous transplantations of parental GL261 cells into mice with genetically generated endothelial β-‐catenin gain-of-function (GOF). The derived GOF tumor from this experiment comprised a quiescent and normalized tumor vasculature and phenocopied the vascular effects observed in Wnt1-expressing tumors.
Our previous work provided evidence that Wnt/β-‐catenin signaling contributes to the BBB phenotype of the developing CNS through the transcriptional regulation of the tight junction protein claudin-‐3. Furthermore, the coverage of pericytes to brain vessels has been described to correlate with BBB integrity. In agreement with these publications, vessels of intracranial Wnt1-‐expressing GL261 tumors retained or regained barrier properties, indicated by a reduced leakage of the tracer Evans blue and endogenous mouse immunoglobulin G and increased junctional localiza2on of the tight junction proteins claudin-‐3, -‐5 and zonula occludens-‐1.
Overall, we detected sustained endothelial Wnt/β-‐catenin signaling to induce a quiescent and normalized tumor vascularization. Interestingly, the Notch signaling pathway has been shown to inhibit the angiogenic tip cell and to promote the quiescent stalk cell phenotype via its ligand Delta-like ligand 4 (Dll4) and the receptors Notch1 and 4. Mechanistically, my work demonstrated for the first time that overactivation of endothelial Wnt/β-‐catenin signaling reactivated expression of Dll4 in the tumor endothelium, which could be shown in vitro to increase Notch signaling and to favor a stalk cell-like gene signature. Furthermore, we uncovered the platelet-derived growth factor subunit B (pdgm) as a novel transcriptional target of Wnt/β-catenin signaling in ECs. Hence endothelial-‐derived PDGF-‐B is known to promote the recruitment of mural cells, the upregulation of this factor might explain the increased SMC/pericyte coverage observed in the tumor vasculature upon sustained endothelial Wnt/β-‐catenin signaling which additionally might promote a cycle of vascular normalization.
Taken together, my work reveals several vascular effects, being mediated by reinforced endothelial Wnt/β-‐catenin signaling during tumor angiogenesis. While a moderate level of canonical Wnt signaling, observed in vessels of human astrocytomas and murine control tumors, is considered to be associated with tumor angiogenesis, dominant activation of this pathway in ECs is shown to limit angiogenesis and to promote a quiescent and normalized tumor vasculature with increased barrier properties. Furthermore, my work discovers pdgm as a novel target of canonical Wnt signaling in ECs.
The work presented in this dissertation therefore not only uncovers the role of endothelial Wnt/β-‐catenin signaling in tumor angiogenesis but additionally reveals this pathway to be a novel modulator in pathological vessel development which might proof to be a valuable therapeutic target for anti-angiogenic and edema glioma therapy.
Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in many cancers and contribute to apoptosis resistance. Therefore, they represent promising anticancer drug targets. Here, we report that small molecule IAP inhibitors at subtoxic concentrations cooperate with monoclonal antibodies against TRAIL receptor 1 (Mapatumumab) or TRAIL receptor 2 (Lexatumumab) to induce apoptosis in neuroblastoma cells in a highly synergistic manner (combination index <0.1). Importantly, we identify RIP1 as a critical regulator of this synergism. RIP1 is required for the formation of a RIP1/FADD/caspase-8 complex that drives caspase-8 activation, cleavage of Bid into tBid, mitochondrial outer membrane permeabilization, full activation of caspase-3 and caspase-dependent apoptosis. Indeed, knockdown of RIP1 abolishes formation of the RIP1/FADD/caspase-8 complex, subsequent caspase activation and apoptosis upon treatment with IAP inhibitor and TRAIL receptor antibodies. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 inhibits IAP inhibitor- and TRAIL receptor-triggered apoptosis. By comparison, over-expression of the dominant-negative superrepressor IκBα-SR or addition of the TNFα-blocking antibody Enbrel does not inhibit IAP inhibitor- and Lexatumumab-induced apoptosis, pointing to a NF-κB- and TNFα-independent mechanism. Of note, IAP inhibitor also significantly reduces TRAIL receptor-mediated loss of cell viability of primary cultured neuroblastoma cells, underscoring the clinical relevance. By demonstrating that RIP1 plays a key role in the IAP inhibitor-mediated sensitization for Mapatumumab- or Lexatumumab-induced apoptosis, our findings provide strong rationale to develop the combination of IAP inhibitors and TRAIL receptor agonists as a new therapeutic strategy for the treatment of human cancer.
According to the standard model of particle physics, the most fundamental building blocks of the known matter are quarks and leptons, while the interactions between these fundamental objects is mediated through bosons. On one hand the leptons can exist in nature as individual particles, while on the other hand quarks appear always as bound states called hadrons. The knowledge that hadrons are built from more fundamental particles dates back to the second half of the 20th century when the work by Gell-Mann and Zweig led to the development of the quark model. The experimental proof that the hadrons are bound objects composed of more elementary particles was done through the study of deep inelastic scattering of electrons off protons. These experiments were done in a similar fashion to the studies of the atomic model led by Rutherford at the beginning of the 20th century. Further experimental analysis led to the conclusion that a large fraction of the proton momentum is not carried alone by the quarks, but by the bosons that mediate the strong interaction called gluons. The cleanest experimental signature for the existence of the gluons came from electron-positron annihilation experiments, where a quark-antiquark pair is created and one of the quarks radiates a hard gluon. Due to confinement neither the quarks nor the gluon can be observed directly, but are measured experimentally as three collimated showers of particles named jets. Since the ground breaking experiments performed at DESY, jets have provided a tool to study the properties of quarks and gluons...
The dissertation focuses on the semiconductor industry to analyze the current state of the international division of labor and its impact on the engineering labor process. Three extensive case studies on design centers of semiconductor companies located in Central and Eastern Europe (CEE) are used to bridge two major gaps in the current academic debate. While the discussion on the development of the international division of labor in manufacturing has already moved towards a more sophisticated perspective that acknowledges a multi-centric structure of international division of labor, on the level of engineering work the hierarchic dichotomy of center and periphery still prevails. Analyzing both location and upgrading processes as well as the labor process the study is able to challenge this perspective. With the focus on CEE the dissertation re-focuses the analysis on a region hitherto not very prominent in research on the international division of labor and the electronics industry. The semiconductor industry with its decade long history of internationalization of both production and product development allows the analysis to focus on local upgrading and control in the labor process that are already stabilized and not anymore distorted by adjustment dynamics of initial phases of internationalization. The study is organized in two major parts representing its two levels of perspective - industry and work. First, the industry perspective with the development of global networks of production and development is used to analyze the industry organization and geographic scope of the developing international division of labor. The Global Production Network approach with its upgrading perspective is combined with research on locational decisions of R&D operations, innovation dynamics and work categories to sketch the shifts in the electronics and semiconductor industry. The study is able to show how a network based industry organization is developing, that is however increasingly driving processes of vertical integration through triangular restructuring. Based on data from field research in CEE in three extensive case studies the focus is put on the upgrading process of chip design centers in global networks of production and development. Using work categories to assess both local upgrading as well as location within global design networks the study is able to show how peripheral operation are able to develop into relatively central design centers. The most important result of the study is its account on processes of integration, through which locally integrated product development teams emerge that comprise of almost all necessary functions for product development. With this the often perpetuated idea of an increasingly modularized and internationalized engineering work is challenged. Simultaneously, a new phase in the process of internationalization is described that is characterized by increased localization, while the integration into and reliance on global networks is growing. Second, the study analyzes the engineering labor process within global networks of production and design of the electronics industry. The Labor Process Theory (especially Friedman's approach) is used to analyze the control in the engineering labor process in chip design centers in CEE. Its main argument is that the labor process in peripheral product design locations in CEE has developed considerably with regards to levels of autonomy in work tasks organization and control structure. The labor process in these formerly peripheral design centers has developed towards a project organization where managerial strategies tend towards responsible autonomy. However, a layered structure of control strategies is used by management, where forms of direct control often undergird strategies of responsible autonomy. The ability to develop an efficient labor process organization is dependent on the ability to reduce the international interface contacts towards the beginning and the end of development projects. This is directly linked to the process of local integration, or functional upgrading, through which the technical and managerial capabilities that are necessary for such a work organization are developed locally. This is the point where the international division of labor and the labor process organization need to be developed in unison through company strategy. However, local worker struggle, mostly through resistance by individual engineers, has also decisive effects on the development of the labor process. Additionally, local factors such as the labor market are central to the analysis advancing a more dialectical perspective on the relations between global and local levels of internationalization. The analysis shows how integrated forms of international division of labor are increasingly developing.
The midbrain DA system comprising dopamine (DA) neurons of the substantia nigra (SN) and the ventral tegmental area (VTA) is involved in various brain functions, including voluntary movement and the encoding and prediction of behaviorally relevant stimuli. In Parkinsonʼs disease (PD), a progressive degeneration of particularly vulnerable SN DA neurons causes a progressive DA depletion of striatal projection sites. As a consequence, motor symptoms such as tremor, hypokinesia and rigidity appear once about 50 % to 70 % of SN DA neurons have been lost. Under physiological conditions, SN DA neurons can encode behaviorally salient events and coordinated movements through tonic and phasic activity and correlated striatal DA release. Burst-activity mediates a phasic, supralinear rise of striatal DA levels and allows to activate coordinated movements via modulation of corticostriatal signals.
In the present dissertation project, pathophysiological adaptations of surviving SN DA neurons after a partial degeneration of the nigrostiatal system have been studied using a 6-hydroxydopamine mouse model of PD. Combining in vivo retrograde tracing techniques with in vitro whole-cell patch-clamp recordings, multifluorescent immunolabeling and confocal microscopy allowed an unambiguous correlation of electrophysiological phenotypes, anatomical positions and neurochemical phenotypes of recorded neurons on a single-cell level. In vitro, neuronal activity of SN DA neurons is characterized by spontaneous, slow pacemaker activity of 1 to 10 Hz and a high degree of spike-timing precision. In vitro current-clamp recordings of surviving SN DA neurons using acute brain slice preparations after a partial, PD-like degeneration of the nigrostriatal DA system showed a significant perturbation of spontaneous pacemaker activity, mirrored by a decreased spike-timing precision compared to controls. Selective pharmacology and whole-cell voltage-clamp recordings served to identify calciumactivated SK channels as molecular effectors of a perturbated pacemaker activity of surviving SN DA neurons. SK channels and have been shown to critically contribute to the spike-timing precision of SN DA neurons. Consistently, in vitro current-clamp recordings after pharmacological blockade of SK channels in vitro caused a significant decrease of spike-timing precision, occluding previously observed differences between surviving SN DA neurons and controls.In addition to in vitro patch-clamp recordings, extracellular single-unit recordings in anaesthetized animals in vivo served to study surviving SN DA neurons embedded in an intact neuronal network after a partial, PD-like degeneration of the nigrostriatal DA system. Combining in vivo single-unit recordings, juxtacellular neurobiotin labeling and multifluorescent immunohistochemistry allowed to directly correlate electrophysiological and neurochemical phenotypes as well as anatomical positions on a single-cell level. In vivo, surviving SN DA neurons showed a significant decrease of spike-timing precision as reflected by an increased irregularity and an augmented burst activity compared to controls.
The present dissertation project provided a unique combination of a neurotoxicological PD mouse model, retrograde tracing techniques and in vitro as well as in vivo electrophysiologiy, allowing to unambiguously correlate electrophysiological adaptations, projection-specific anatomical positions and neurochemical phenotypes of SN DA neurons after a partial degeneration of the nigrostriatal system. Surviving SN DA neurons exhibited a significant deficit of SK channel activity after a partial degeneration of the nigrostriatal DA system. In consequence of a diminished SK channel activity observed in vitro, surviving SN DA neurons exhibited and enhanced burst activity in vivo, providing a plausible mechanism to compensate a striatal DA depletion.
This thesis examines the literary output of German servicemen writers writing from the occupied territories of Europe in the period 1940-1944. Whereas literary-biographical studies and appraisals of the more significant individual writers have been written, and also a collective assessment of the Eastern front writers, this thesis addresses in addition the German literary responses in France and Greece, as being then theatres of particular cultural/ideological attention. Original papers of the writer Felix Hartlaub were consulted by the author at the Deutsches Literatur Archiv (DLA) at Marbach. Original imprints of the wartime works of the subject writers are referred to throughout, and citations are from these. As all the published works were written under conditions of wartime censorship and, even where unpublished, for fear of discovery written in oblique terms, the texts were here examined for subliminal authorial intention. The critical focus of the thesis is on literary quality: on aesthetic niveau, on applied literary form, and on integrity of authorial intention. The thesis sought to discover: (1) the extent of the literary output in book-length forms. (2) the auspices and conditions under which this literary output was produced. (3) the publication history and critical reception of the output. The thesis took into account, inter alia: (1) occupation policy as it pertained locally to the writers’ remit; (2) the ethical implications of this for the writers; (3) the writers’ literary stratagems for negotiating the constraints of censorship.
Time-critical applications process a continuous stream of input data and have to meet specific timing constraints. A common approach to ensure that such an application satisfies its constraints is over-provisioning: The application is deployed in a dedicated cluster environment with enough processing power to achieve the target performance for every specified data input rate. This approach comes with a drawback: At times of decreased data input rates, the cluster resources are not fully utilized. A typical use case is the HLT-Chain application that processes physics data at runtime of the ALICE experiment at CERN. From a perspective of cost and efficiency it is desirable to exploit temporarily unused cluster resources. Existing approaches aim for that goal by running additional applications. These approaches, however, a) lack in flexibility to dynamically grant the time-critical application the resources it needs, b) are insufficient for isolating the time-critical application from harmful side-effects introduced by additional applications or c) are not general because application-specific interfaces are used. In this thesis, a software framework is presented that allows to exploit unused resources in a dedicated cluster without harming a time-critical application. Additional applications are hosted in Virtual Machines (VMs) and unused cluster resources are allocated to these VMs at runtime. In order to avoid resource bottlenecks, the resource usage of VMs is dynamically modified according to the needs of the time-critical application. For this purpose, a number of previously not combined methods is used. On a global level, appropriate VM manipulations like hot migration, suspend/resume and start/stop are determined by an informed search heuristic and applied at runtime. Locally on cluster nodes, a feedback-controlled adaption of VM resource usage is carried out in a decentralized manner. The employment of this framework allows to increase a cluster’s usage by running additional applications, while at the same time preventing negative impact towards a time-critical application. This capability of the framework is shown for the HLT-Chain application: In an empirical evaluation the cluster CPU usage is increased from 49% to 79%, additional results are computed and no negative effect towards the HLT-Chain application are observed.
Unterschiede im Denken und Verhalten zwischen Menschen empirisch zu ermitteln, hat eine lange Tradition in der Differentiellen Psychologie. Forscher dieses Fachgebiets entwickeln spezielle Tests, um Personen hinsichtlich bestimmter psychologischer Merkmale zu klassifizieren. Bekannte Bespiele hierfür sind Intelligenztests, die oft zum Einsatz kommen, um z.B. passende Mitarbeiter für bestimmte Positionen zu selektieren. Dieser differenzielle Ansatz wurde bisher im Bereich der Erforschung neuronaler Grundlagen der Wahrnehmung weitgehend ignoriert. Interindividuelle Unterschiede zwischen Personen wurden meist als Messfehler eingestuft und durch Mittelungsverfahren über die Gruppe herausgerechnet (Kanai and Rees, 2011). Neuere Ergebnisse zeigen jedoch, dass hirnstrukturelle Unterschiede zwischen Personen Unterschiede im Verhalten erklären können (siehe Kanai and Rees, 2011; Kleinschmidt et al., 2012 für einen Überblick). Dieser Ansatz wird mit den hier vorgestellten Studien weiter ausgebaut. Dabei wird der Frage nachgegangen, ob Unterschiede in der Hirnanatomie im Menschen dessen Individualität in der bewussten visuellen Wahrnehmung vorhersagen kann. Insbesondere wird untersucht, inwieweit die Integrationsleistung zwischen den Hirnhälften von spezifischen transkallosalen Faserverbindungen abhängt. Des Weiteren wird überprüft, ob die Größe der frühen visuellen Areale einen Einfluss auf die Reizverarbeitung innerhalb der Hirnhälfte hat. Als Paradigmen verwendeten wir in allen Studien mehrdeutige visuelle Reize. Das besondere an diesen Reizen ist, dass deren Interpretation trotz gleichbleibender physikalischer Darbietung ständig wechselt. Dadurch können Hirnprozesse sichtbar gemacht werden, die unabhängig vom visuellen Reiz mit der bewussten Wahrnehmung einhergehen. Zudem werden die Wechsel zwar von allen Versuchspersonen empfunden, es gibt aber diesbezüglich große Unterschiede zwischen den Beobachtern.
In Kapitel 2 wurden Reize verwendet, die eine Scheinbewegung verursachen (Wertheimer, 1912). Ein passendes Beispiel für dieses Phänomen ist das Daumenkino, bei dem durch die schnelle Abfolge von Standbildern der Eindruck einer Bewegung entsteht. Wir verwendeten in unserer Studie eine spezielle Form der Scheinbewegung, das „Motion Quartet“ (Neuhaus, 1930; Chaudhuri and Glaser, 1991). Bei dieser Form löst die rechteckige Anordnung vierer weißer Quadrate den Eindruck von Bewegung aus. Die Anordnung besteht aus zwei alternierenden Bildern mit jeweils zwei Paaren von diagonal gegenüberliegenden Quadraten (oben links und unten rechts vs. oben rechts und unten links). Die Beobachter sehen entweder eine waagrechte oder eine senkrechte Bewegung. Interessanterweise weiß man aus früheren Studien, dass meistens vertikale Bewegungen wahrgenommen werden, wenn der Abstand zwischen den vier Quadraten gleich ist und die Beobachter den Mittelpunkt des Quartetts fixieren (Chaudhuri and Glaser, 1991). Aufgrund der Organisation des visuellen Systems muss die Sehinformation für waagrecht erscheinende Bewegung über beide Hirnhälften integriert werden, während die senkrecht erscheinende Bewegung nur von einer Hemisphäre verarbeitet wird. Das Quartett erzeugt deshalb in erster Linie senkrechte Bewegung, denn die Kommunikation zwischen den beiden Gehirnhälften braucht länger oder ist aufwändiger als die innerhalb einer Hemisphäre. Allerdings gibt es große Unterschiede zwischen Versuchspersonen, welche Bewegungsrichtung wahrgenommen wird. Chaudhuri und Kollegen hatten bereits zuvor gezeigt, dass jeder Teilnehmer einen individuellen Gleichgewichtspunkt (parity ratio) hat, an dem er beide Bewegungsrichtungen gleich oft wahrnimmt. Dieser Gleichgewichtspunkt spiegelt wieder, wie gut jemand die Informationen aus beiden Hirnhälften integrieren kann. Bei den meisten Teilnehmern muss der waagrechte Abstand kleiner sein als der senkrechte, nur dann ist die Wahrnehmung sowohl waagrechter als auch senkrechter Bewegung ausgeglichen. Unsere Ergebnisse in Kapitel 2 bestätigen die Befunde von Chaudhuri und Glaser (1991) indem sie zeigen, dass der Gleichgewichtspunkt stark zwischen Versuchspersonen variiert. Darüberhinaus zeigen unsere Ergebnisse, dass der individuelle Gleichgewichtspunkt über Monate stabil und damit eine konstante Eigenschaft von Personen ist. Zudem sprechen unsere Befunde dafür, dass der Gleichgewichtspunkt eng mit der Struktur bestimmter Faserverbindungen zusammenhängt. Wie bisherige Studien gezeigt haben, sind jene visuelle Areale, die Bewegung verarbeiten (hMT/V5), hauptsächlich für die Verarbeitung von Scheinbewegung zuständig (Sterzer et al., 2002; Sterzer et al., 2003: Sterzer and Kleinschmidt, 2005; Rose and Büchel, 2005). In unserer Untersuchung fanden wir, dass der geschätzte Durchmesser der Faserverbindungen im Corpus Callosum von eben diesen Regionen den individuellen Gleichgewichtspunkt vorhersagen konnte. Dieser Zusammenhang scheint auf die Bewegungszentren des Sehsystems begrenzt zu sein. Benachbarte kallosale Faserbündel des Sehsystems, die andere visuelle Gebiete miteinander verbinden, sind nicht mit dem Gleichgewichtspunkt assoziiert.
In Kapitel 3 und 4 verwendeten wir einen weiteren mehrdeutigen Stimulus. Hier wurden die Messungen mit dem Phänomen der „Binokularen Rivalität“ (engl. „Binocular Rivalry“) durchgeführt. Dabei werden den beiden Augen sehr unterschiedliche Bilder dargeboten, von denen zu jedem Zeitpunkt nur eine Interpretation bewusst wahrgenommen werden kann. Bei einer bestimmten Variation der Binokularen Rivalität wird die Präsentation der Reize so kontrolliert, dass sich die Änderung des subjektiven Erlebens von einem Bild zum anderen wellenartig ausbreitet (Wilson et al., 2001). Wilson (2001) und Kollegen zeigten bereits in ihrer Studie, dass es bei der Übertragung der Wanderwelle zwischen den Hirnhälften zu einer Verzögerung kommt. Unsere Ergebnisse in Kapitel 3 bestätigen diese Befunde und zeigen zusätzlich, dass diese Verzögerung stark zwischen Beobachtern variiert. Ähnlich wie für den Gleichgewichtspunkt von Kapitel 2 fanden wir auch für diese Verzögerung eine hohe zeitliche Stabilität. Es wurde bereits in vorherigen Studien gezeigt, dass die Ausbreitung der Wanderwelle eng mit der Aktivität im primären visuellen Kortex zusammenhängt (Lee et al., 2005, 2007). Unsere Ergebnisse in Kapitel 3 zeigen, dass die Varianz zwischen Personen für die Verzögerung zum großem Teil durch den Durchmesser der transkallosalen Faserverbindungen des V1 vorhergesagt werden kann. Auch hier bestand kein Zusammenhang zwischen Faserverbindungen benachbarter visueller Areale.Neben der Verzögerung zwischen den Hirnhälften zeigte auch die Ausbreitungsgeschwindigkeit der Wanderwelle innerhalb der Hemisphären eine hohe zeitliche Stabilität. Es stellt sich somit die Frage, ob strukturelle Eigenschaften von bestimmten visuellen Arealen die Ausbreitungsgeschwindigkeit vorhersagen kann. Wie in Kapitel 4 dargestellt, konnten wir einen starken Zusammenhang zwischen der Größe von V1 und der Ausbreitung der Wanderwelle feststellen. Dieser Zusammenhang ist positiv und, wie sich bei Hinzunahme anderer Areale in die Analyse zeigte, spezifisch für den primären visuellen Kortex. Demnach breitet sich die durch den binokularen Wettbewerb erzeugte Wanderwelle umso langsamer über das Sehfeld aus, je größer das Areal bei der entsprechenden Person ist. Die Darstellung in der Abbildung auf der Seite 123 bietet noch einmal einen grafischen Überblick über die oben beschriebenen Ergebnisse dieser Doktorarbeit. Zusammengefasst zeigt diese Arbeit exemplarisch am Beispiel der inter- und intrahemisphärischen Integration auf, wie eng Struktur und Funktion des Gehirns miteinander verknüpft sind. Bei Parametern, die sich experimentell nicht von uns als Forscher variieren lassen, griffen wir auf den Ansatz der differentiellen Psychologie zurück. Dabei nutzten wir die bei Individuen bereits gegebenen Unterschiede aus, um Rückschlüsse auf ganz allgemeine Gesetzmäßigkeiten, wie z. B. der Einfluss der kallosalen Faserdurchmesser und die Oberflächengröße spezifischer Areale auf die Wahrnehmung zu ziehen. Wie wir aufzeigen, formen also schon ganz grundlegende Eigenschaften früher sensorischer Areale unsere Wahrnehmung. Der von uns gewählte Ansatz könnte in zukünftiger Forschung auch auf höhere Funktionen, die uns als Menschen ausmachen, angewandt werden.
Climate and subsequent environmental changes are regarded as one driver of species evolution. Against this background the present study investigates the evolutionary history of the mammalian family Bovidae (Cetartiodactyla, Mammalia), today the most species-rich family of large herbivores on the African continent. Temporal and spatial patterns in that group’s evolution are the focus of the present study and were investigated using methods and data deriving from multiple disciplines (palaeontology, genetics, climatology, conservation biology). The results serve as a validation of macroevolutionary hypotheses of species evolution.
A major proportion of African mammalian fossils can be assigned to that family. Due to their morphological adaptations, bovid species are highly indicative of their habitats. Hence, bovids are of great importance for paleontology. However, a strong taphonomic bias is present in the fossil record of bovids, favoring large and arid- adapted species. Molecular phylogenies of extant species and species distribution modelling combined with climate reconstructions can help to overcome these limitations.
A molecular phylogeny, based on the cytochrome b gene of 136 bovid species served as basis for analysis of temporal patterns. Divergence events were dated using the relaxed molecular clock approach. The tree was time calibrated at 30 nodes using information inferred from the fossil record. Lineage-Through-Time plots and the respective statistical analyses reveal detailed temporal patterns in the evolutionary history of tribes and groups combining arid- and humid-adapted tribes. The resulting pattern shows three distinct phases. Phase 1 (P1) is dominated by speciation events within the humid group, while the second phase (P2) is marked by a dominance of speciation within the arid group. The switch in diversification rates (BDS) from P1 to P2 is dated to 2.8 million years ago. The third phase (P3) shows low diversification rates for all groups, starting around 1.4 million year ago and culminates in a significantly reduced diversification rate for the complete family at 0.8 million years ago. Both transitions are contemporaneous with global climate changes and turnover events in fossil faunal communities.
To investigate the impact of climate changes onto the habitat availability within the last 3 million years and its putative influence on diversification rates, the species distribution modeling method was applied. For 85 African species and subspecies the climate niches were established and grouped into 5 climate-groups based on their climate preferences. For each group the available habitat for the period before and after the BDS was calculated on continental scale using reconstructed climate scenarios. To evaluate the modeled habitat distributions, regional analyses were performed in test areas surrounding well studied fossil sites (Laetoli, Olduvai, Chiwondo Beds, Lothagam, Koobi Fora, West Turkana, Swartkrans, Sterkfontain und Toros-Menalla). Habitat profiles (HP) permitted the comparison of the model based habitat reconstruction with the interpretations of classic paleontological reconstruction. The validity of the habitat modeling has been shown in particular for East African test areas. The reconstructions for the northern and southern fossil sites does not support the modeled habitats in these areas. Yet, the method of habitat- profiling may serve as suitable tool for environmental reconstruction of areas lacking sufficient paleontological material. A comparison of habitat availability before and after the BDS on continental scale identified a significant loss of habitat for humid adapted groups (7-22%) and habitat gain for arid adapted groups (19-173%). The climatically intermediate group experiences a tremendous gain of habitat (3366%). The greatest environmental change was modeled for East Africa, initiated by a progressive regional aridification.
In addition to the distribution modeling for past climate conditions, the geographical distribution was modeled for the future, i.e. for climate scenarios representing the years 2050 and 2080 under a putative climate change scenario (global surface warming). It was shown that in particular the arid groups have to expect a remarkable loss of habitat (41-76%), while a gain of available habitat can be expected for the humid adapted groups (114-577%). The climatically intermediate group suffers the strongest habitat loss (85%). Regions with locally stable climate conditions were detected and may serve as potential refugia and are already today known as Africa’s hot spots of biodiversity.
The results show a positive correlation of high diversification rates and increasing habitat availability. None of the tested speciation hypotheses taken alone explains the observations (e.g., Turnover-pulse Hypothesis, Relay Model). A major element in these hypotheses is the passive fragmentation of populations induced by unfavorable climate changes. In contrast, the Periodic Model (Grubb 1999) considers natural, periodically recurring climate changes and moreover, the active dispersal of individuals and resulting founder events. I added the effect of a superimposed directed climate trend – like the progressive aridification since the late Pliocene in Africa – which leads to a bias in the proportion and probability towards leading edge effects. This Directed Periodic Model explains the patterns found in the evolution of Bovidae.
The combination of a molecular phylogeny and species distribution modeling, together with information inferred from the fossil record, reveals remarkable temporal and spatial patterns in the evolution of bovids, and helps overcome the limitations of the fossil record. The present study highlights the importance of active dispersal and founder populations in speciation processes. A point widely unattended in speciation hypotheses. The fully dated molecular phylogeny is the most densely sampled tree for the family Bovidae to date and may serve as a framework for a connection of present and future population studies, permitting the connection of medium-scale with long- term effects induced by climate and environmental changes.
Introduction: The involvement of platelets in various diseases has been increasingly recognized in the recent decades. This contribution is believed to involve platelet secretion and formation of reactive microparticles. Platelets contain two functionally important forms of vesicles, alpha and dense granules, which are secreted upon activation of platelets. Alpha granules incorporate larger molecules such as adhesive proteins, e.g. P-selectin, vWF and fibrinogen; chemokines like PF4 and RANTES and growth hormones like VEGF and PDGF are among the most important proteins attributed to the involvement of platelets in pathological conditions. In contrast, dense granules contain small molecules like ADP, ATP, serotonin and histamine, and they are more rapidly and completely secreted than alpha granules. Like in all secreting cells, regulated exocytosis in platelets is mediated by “zippering” of three different classes of SNARE proteins. The subtypes of these proteins found to be involved in platelet secretion are SNAP-23, syntaxin-2 and -4 and VAMP-3 and -8. Apart from SNARE proteins, other conserved proteins influencing exocytosis by e.g. acting on SNARE proteins have been described, one of the most important ones being Munc13. Platelets contribute to the progression of atherosclerosis by local deposition of inflammatory mediators like PF4, RANTES and CD40L, which leads to enhanced leukocyte recruitment and plaque formation. In 1865, Armand Trousseau first described the correlation between cancer and thrombotic events. Since the 1960s, an increasing number of studies have found an involvement of platelets also in the progression of cancer, especially in the formation of metastases. Platelets bind to circulating tumor cells and may shield them from NK cell attacks and shear stress. Platelets may also facilitate the interaction of tumor cells with other cell types and the vessel wall. Lastly, they may secrete molecules that influence the tumor cell phenotype and invasiveness.
Aims of this study: We sought to generate and describe genetically modified mouse lines with defective platelet secretion and to employ these mouse lines in murine models of atherosclerosis and tumor progression to study the role of platelet secretion under pathological in vivo conditions.
Results: Clostridial toxins cleave members of the SNARE protein family and can thus completely block exocytosis of neuronal and other cells. We generated three transgenic mouse lines expressing tetanus, botulinum-E or -C light chains and two transgenic mouse lines with dominant-negative mutations of SNAP-23 under the control of the platelet-specific PF4 promotor. None of these constructs was able to interfere with platelet secretion despite expression of the transgene. A functional null mutant of the only Munc13 isoform expressed in platelets, Munc13-4, showed complete lack of dense granule secretion, measured by ATP release, while alpha granule release as determined by PF4 and vWF secretion, was unaltered. Morphology, composition and adhesion of these platelets were also normal. Aggregation in response to U46619 and collagen and formation of large aggregates in flow chamber assays was attenuated. Munc13-4-deficient mice showed a severe defect in bleeding time and no formation of stable aggregates in FeCl3 thrombosis model. In response to B16 melanoma and LLC1 carcinoma cells, Munc13-4 KO platelets also showed complete abrogation of dense granule secretion, whereas alpha granule secretion and binding of platelets to tumor cells was unchanged. Interestingly, wild-type platelets, but not Munc13-4 KO platelets, enhanced transmigration of B16 and LLC1 cells through an endothelial cell layer. Exogenous ATP was able to mimic the effect of wild-type platelets and the ATP-degrading enzyme apyrase blocked platelet-mediated tumor cell transmigration. Platelets incubated with tumor cells secreted large amounts of ATP. Murine endothelial cells showed perturbed adherens junctions identified by irregular VE-cadherin staining and gap formation when incubated with supernatants from tumor cell-activated platelets as well as increased permeability under the same conditions. Addition of apyrase preserved normal endothelial morphology and function. In vivo, primary tumor growth and weight was comparable in wild-type and Munc13-4 KO mice upon B16 or LLC1 flank injection but formation of lung metastases was strongly reduced. Number, but not size of metastases was also reduced upon i.v. injection of B16 and LLC1 cells. We found P2Y2 and P2X4 receptors to be the most abundantly expressed endothelial metabotropic and ionotropic ATP receptors, respectively. Neither knock-down nor inhibition of P2X4 in endothelial cells influenced platelet-mediated transendothelial migration of B16 cells, but knock-down of P2Y2, for which no specific antagonist is available, strongly reduced plateletdependent tumor cell transmigration. When B16 melanoma cells were injected i.v. shortly after FITC-dextran (70 kDa) into wild-type mice, prominent leakage of FITC-dextran was observed three hours post-injection at extraluminal sites in the lung. In contrast, leakage into the lung parenchyma was at basal levels in Munc13-4 KO and P2Y2 KO mice after B16 cell injection. Marginal vascular leakage in Munc13-4 KO mice lacking platelet ATP secretion and in P2Y2 KO mice lacking the main endothelial ATP receptor correlated with strongly reduced extravasation of CFSE-labeled B16 melanoma cells 6 hours post-injection in these mice. Consistently, P2Y2 KO mice showed strongly reduced formation of metastases in the lung after i.v. injection of B16 or LLC1 tumor cells. Bone marrow-transplanted LDLR KO mice reconstituted with Munc13-4-deficient or wildtype bone marrow and subjected to 16 weeks of high fat diet showed no significant difference in atherosclerotic plaque formation in the aorta.
Discussion: We hereby provide a thorough analysis of a mouse line with an exclusive defect in platelet dense granule secretion, thus representing a unique genetic tool to study the role of dense granule secretion in various contexts without interfering with other platelet functions. We also provide evidence how extravasation of circulating tumor cells is facilitated by tumor cell-induced ATP release from platelets. This ATP release destabilizes endothelial barriers and facilitates tumor cell extravasation and formation of metastases in the target organ. Since metastasis is the leading cause of cancer death, pharmacological interference with endothelial P2Y2 receptor function may represent a promising therapeutic strategy.
In this thesis the behavior of banks in financial markets which banks frequently use to obtain short-term as well as long-term financing is studied. In the first chapter we incorporate an interbank market for collateralized lending among banks into a dynamic, stochastic, general equilibrium (DSGE) framework to analyze the impact of variations in the expected value of the collateral on the interbank lending volume. We find that a central bank which decides to lower the haircut on eligible collateral in repurchase agreements is able to stimulate interbank markets. In the second chapter a microeconomic model of bank behavior on the interbank market is set up to analyze the impact of risk-taking behavior of interbank borrowing banks and uncertainty about their balance sheet quality on the lending behavior of interbank lending banks. It is found that the disruptions on the interbank market are the result of optimal behavior on the part of interbank lending banks in response to the uncertainty about the balance sheet quality of an interbank borrowing bank. In the third chapter we use monthly data on German bank bond spreads and regress it on bank-specific risk factors to assess the degree of market discipline in the German bank bond market. The regression results for the whole German bank bond market indicate that the bond spread does not show signs of market discipline. However, a structural break analysis uncovers that since the beginning of the financial crisis the German bank bond market exhibits at least a weak form of market discipline for bonds issued by medium-size and large banks.
In the first part of this work, the development of a novel two-dimensional native gel electrophoretic system (2-D BN/hrCNE) is described. This new system simplifies proteomics and biochemical analysis of mega protein complexes that are dissociated into the constituent complexes during 2-D electrophoresis, thereby reducing the complexity of the system considerably. This technique is exceptionally well suited for the in-gel detection of fluorescence-labeled proteins and the identification of individual enzymes and protein complexes by specific in-gel assays on native gels.
In the second part, a new technique for the native immunoblotting of blue native gels (NIBN) was developed. This new technique allows for the identification of conformation-specific antibodies and the discrimination of antibodies recognizing linear epitopes of denatured proteins. Identification of conformation-specific antibodies is becoming increasingly important not only for the electron microscopic identification of native proteins but also for structural investigations in general. For this purpose, a commonly used protocol for Western blotting of blue native gels was modified in such a way that the native state of proteins and protein complexes was retained throughout the complete protocol. Instead of using the denaturing methanol in Western blotting protocols, mild detergents such as Tween 20, digitonin and Brij 35 were used for the obligatory removal of protein bound Coomassie-dye.
The detection of respiratory complex I by activity staining on the blot membrane demonstrated that all three non-ionic detergents preserved the native state of complex I. The native state of the enzyme on the blot membrane was also monitored and confirmed with the help of a set of conformation-specific antibodies. NIBN can be used as a simple alternative method to the demanding native ELISA to screen for conformation-specific antibodies for structural studies. Unlike the time consuming native ELISA, NIBN does not require introduction of appropriate affinity tags and purification of the target protein by chromatography. Thus, the NIBN technique is especially useful for microscale projects and for proteins not easily accessible to genetic manipulation.
The third part aimed at identification of the immediate protein interaction partners of Cox26, a hydrophobic protein that has been identified by our group as a novel component of yeast respiratory supercomplex. Multi-dimensional electrophoretic techniques were applied to identify non-covalent and covalent protein-protein interactions of Cox26. Three-dimensional electrophoresis (BNE/BNE/SDS-PAGE) gave both qualitative and quantitative information on covalent and non-covalent interactions of Cox26 and subunits of cytochrome c oxidase (complex IV), and showed that most of the Cox26 protein was non-covalently bound to the complex IV moiety of the respirasomes. Four-dimensional electrophoresis (BNE/BNE/SDS/SDS-PAGE) applying reducing and non-reducing conditions revealed that a minor fraction of Cox26 used a single cysteine residue in the center of a predicted transmembrane helix to form a disulfide bond with the Cox2 subunit of complex IV. A structural role of Cox26 protein in the assembly/stability of respiratory strings or patches has been suggested.
The last part of this work focused on the isolation and characterization of native and morphologically intact nucleoids from bovine heart mitochondria, since only a few studies on nucleoid organization and composition have been carried out on mammalian tissues. The nucleoids appeared as distinct bands (apparent mass around 30-36 MDa) in blue native-PAGE on large pore gels. The moderate variation in particle size seems to reflect variations in the binding of loosely nucleoid-associated components like respiratory chain complexes. The estimated 30-36 MDa mass of nucleoids on native gels suggested that each nucleoid contains one mtDNA molecule provided that nucleoids contains equal amounts of DNA, protein and RNA (Miyakawa et al., 1987).
Electron microscopic analysis of native nucleoids, which was performed by Dr. Karen Davies from the Max-Planck-Institute of Biophysics, Department of Structural Biology, Frankfurt, showed homogenous pool of particles with dimensions in 85x100 nm (in negative stain) and 100x150 nm (in cryo-tomography). Some of the nucleoids showed dumbbell-shape indicating dimerization of nucleoids. Recent EM and high-resolution light microscopy analysis of mammalian nucleoids have reported that nucleoids have a size of 70 nm in average. We also observed the same size of 70 nm in cryo-tomogramms when we applied harsher treatment of the native nucleoid particles with dimensions 100x150 nm. This observation is in agreement with published nucleoid sizes from both EM and high-resolution light microscopy, if we assume that native nucleoids have been dissociated under harsher treatment.
The protein composition of bovine heart mt-nucleoids was analyzed by a number of complementary approaches to identify low and highly abundant, easily dissociating and tightly bound proteins, and to rank the 90 most abundant mt-nucleoid proteins. Native and denaturing gel electrophoresis techniques were coupled to LC-MS/MS to achieve a comprehensive protein component analysis. Qualitative MS analysis of highly purified nucleoids identified more than 400 proteins, including well known nucleoid proteins such as mitochondrial transcription factor and mtDNA-binding protein (TFAM), mitochondrial single-stranded DNA-binding protein (mtSSB), mitochondrial DNA polymerase subunit gamma-2 (POLG2) and mitochondrial helicase C26H10ORF2 protein (Twinkle). These proteins were ranked according to Mascot scores, and sorted according to presumed functional properties. A large group of proteins involved in protein synthesis comprised an almost complete set of subunits of mitochondrial ribosomes suggesting that the nucleoids contained significant amounts of mitochondrial ribosomes. Identification of sixty six proteins from the oxidative phosphorylation (OXPHOS) system comprising around 100 proteins in total suggested that OXPHOS proteins are also associated with mt-nucleoids.
Interestingly, TFAM, described as a main mtDNA packaging factor in human and other mammalian cells, was not confirmed here as a major nucleoid component from bovine heart mitochondria. Fluorescence staining of protein spots on 2-D IEF/SDS gels clearly identified TFAM, but according to the stain intensity, this protein did not rank in the list of the 90 most abundant nucleoid proteins. Western blot analysis of sucrose gradient fractions revealed an enrichment of putative TFAM isoform in nucleoid fractions. Unexpectedly, the uncharacterized mitochondrial protein Es1 was identified as the most abundant nucleoid protein in bovine heart nucleoids instead. This implicates that nucleoid organization may differ between species and tissues. A functional characterization of Es1 is required to clarify its role in mammalian nucleoids.
The environmental impact of climate change is meanwhile not only discussed in the scientific community but also in the general public. However, little is known about the interaction between climate change and pollutants like pesticides. A combination of multiple stressors (e.g. temperature, pollutants, predators) may lead to severe alterations for organisms such as changes in time of reproduction, reproductive success and growth performance, mortality and geographic distribution. The questions if aquatic organisms tend to react more sensitive towards incidents under climate change conditions remains. Therefore, within the present thesis the aquatic ecotoxicological profile of the fungicide pyrimethanil, as an exemplarily anthropogenic used contaminant, was examined.
A large test battery of ecotoxicological standard tests and supplement bioassays with non-model species was conducted to investigate if species-specific or life stage-specific differences occur or if temperature alteration may change the impact of the fungicide. Two of the most sensitive species (Chironomus riparius and Daphnia magna) were used to investigate the acute and chronic thermal dependence of pyrimethanil effects. The results clearly depict that the ecotoxicity of pyrimethanil at optimal thermal conditions did not depend on the trophic level, but was species-specific. With regard to EC10 values the acute pyrimethanil toxicity on C. riparius increased with higher temperature (6.78 mg L-1 at 14°C and 3.06 mg L-1 at 26°C). The chronic response of D. magna to the NOEC (no observed effect concentration) of the fungicide (0.5 mg L-1) was examined in an experiment which lasted for several generations under three simulated near-natural temperature regimes (‘cold year, today’ (11 to 22.7°C), ‘warm year, today’ (14 to 25.2°C) and ‘warm year, 2080’ (16.5 to 28.1°C)). A pyrimethanil-induced mortality increase was buffered by the strongly related increase of the general reproductive capacity, while population growth was stronger influenced by temperature than by the fungicide. At a further pyrimethanil concentration (LOEC – lowest observed effect concentration: 1 mg L-1), a second generation could not be established by D. magna under all thermal regimes.
Besides daphnids, the midge C. riparius was used for a second multigeneration study. In a bifactorial test design it was tested if climate change conditions alter or affect the impact of a low fungicide concentration on life history and genetic diversity. The NOAEC/2 (half of the no observed adverse effect concentration derived from a standard toxicity test) was used as a low pyrimethanil concentration to which laboratory populations of the midges were chronically exposed under the mentioned temperature scenarios. During the 140-day-multigeneration study, survival, emergence, reproduction, population growth, and genetic diversity of C. riparius were analyzed. The results reveal that high temperatures and pyrimethanil act synergistically on life history parameters of C. riparius. In simulated present-day scenarios, a NOAEC/2 of pyrimethanil provoked only slight to moderate beneficial or adverse effects. In contrast, an exposure to a NOAEC/2 concentration of pyrimethanil at a thermal situation likely for a summer under the future expactations uncovered adverse effects on mortality and population growth rate. In addition, genetic diversity was considerably reduced by pyrimethanil in the ‘warm year, 2080’ scenario, but only slightly under current climatic conditions. The multigeneration studies under near-natural thermal conditions indicate that not only the impact of climate change, but also low concentrations of pesticides may pose a reasonable risk for aquatic invertebrates in the future. This clearly shows that thermal and multigenerational effects should be considered when appraising the ecotoxicity of pesticides and assessing their future risk for the environment.
In addition to temperature further multiple abiotic and biotic stressors alterate pollutant effects. Moreover, to better discriminate and understand the intrinsic and environmental correlates of changing aquatic ecosystems, it was experimentally unraveled how the effects of a low-dose of pyrimethanil on daphnids becomes modified by different temperatures (15°C, 20°C, 25°C) and in the presence/ absence of predator kairomones of Chaoborus flavicans larvae. The usage of a fractional multifactorial test design provided the possibility to investigate the individual growth, reproduction and population growth rate of Daphnia pulex via different exposure routes to the fungicide pyrimethanil at an environmentally relevant concentration (0.05 mg L-1) - either directly (via the water phase), indirectly (via algae food), dually (via water and food) or for multiple generations (fungicide treated source population).
The number of neonates increased with increasing temperatures. At a temperature of 25°C no significant differences between the individual treatment groups were observed although the growth was overall inhibited due to pyrimethanil. Besides, at 15 and 20°C it is obvious that daphnids which were fed with contaminated algae had the lowest reproduction and growth rate. The obtained results clearly demonstrate that multiple stress factors can modify the response of daphnids to pollutants. The exposure routes of the contaminant are of minor importance, while temperature and the presence of a predator are the dominant factors impacting the reproduction of D. pulex. It can be concluded that low concentrations of pyrimethanil may disturb the zooplankton community at suboptimal temperature conditions, but the effects will become masked if chaoborid larvae are present. Therefore it seems necessary to observe prospectively if the combination of several stress factors like pesticide exposure and suboptimal temperature may influence the life history and sensitivity of several aquatic invertebrates differently.
Besides standard test organisms it is inevitable to conduct test with aquatic invertebrate which are not yet considered regularly in ecotoxicological experiments. For example molluscs represent one of the largest phyla of macroinvertebrates with more than 100.000 species, being ecologically and economically important. Therefore, within the present study embryo, juvenile, half- and full-life cycle toxicity tests with the snail Physella acuta were performed to investigate the impact of pollutants on various life stages. Different concentrations of pyrimethanil (0.06-0.5 or 1.0 mg L-1) assessed at three temperatures (15°C, 20°C, 25°C) revealed that pyrimethanil caused concentration-dependent effects independent of temperature. Interestingly, the ecotoxicity of pyrimethanil was higher at lower temperature for the embryo hatching and F1 reproduction, but its ecotoxicity for the growth of juveniles and the F0 reproduction increased with increasing temperature. More specifically, it could have been observed that especially during the reproduction test high mortality rates occurred at the highest concentration of 1 mg L-1 at all temperatures. Due to high mortality rates no snails were available for the F1 at the highest concentrations (0.5 and 1.0 mg L-1). Compared to the F0, overall more egg masses were produced in the F1, being all fertile and no mortality occurred. For the F1-generation the strongest pyrimethanil effects were detected at 15°C. A comparison of effect concentrations between both generations showed that the F1 is more sensitive than the F0.
These results indicate that an exposure over more than one generation may give a better overview of the impact of xenobiotics. With the establishment of an embryo and reproduction test under different temperatures and various concentrations of pyrimethanil with P. acuta we could successfully show that molluscs can respond more sensitive than model organisms and that both, chemical and thermal stressor strongly influence the behaviour of the pulmonates. It can be concluded that the high susceptibility for the fungicide observed in gastropods clearly demonstrates the complexity of pesticide-temperature interactions and the challenge to draw conclusions for the ecotoxicological risk assessment of pesticides under the impact of global climate change.
The mantle xenoliths collected by kimberlites indicate that the subcratonic mantle underneath the Archean crust is mostly a residue of high degrees of partial melting which was subsequently reenriched. The majority of the xenoliths show cryptic metasomatism and only few modal metasomatism.
Much effort has been put into deciphering different kinds of enrichment processes within the mantle. Here, we take the approach to look into the inventory of subcalcic garnets which stem from cpx-free harzburgites and dunites. These subcalcic garnets, commonly with sinusoidal REE patterns, carry the major budget of the trace elements of their host rock. Thus, they are promising objects to study both depletion and enrichment. Most importantly, the analysis of a single grain subcalcic garnetwill provide almost all important information of the bulk rock. Our aim is to gain detailed information mainly on metasomatism on a craton wide scale by combining major, trace elements and Lu-Hf and Sm-Nd isotopic signatures from subcalcic garnets. Eventually, we will summarize the metasomatic agent(s) and processes and possibly the timing of the enrichment within the lithospheric mantle underneath the Kaapvaal craton.
Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful tool for measuring nanometer distances in spin-labeled systems and recently is increasingly applied to membrane proteins. However, after reconstitution of labeled proteins into liposomes, spin labels often exhibit a much faster transversal relaxation (Tm) than in detergent micelles, thus limiting application of the method in lipid bilayers. In the first part of the thesis, optimization of transversal relaxation in phospholipid membranes was systematically investigated by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin aggregation and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples with low local concentrations does deuteration of acyl chains and buffer significantly prolong Tm. In these cases, values of up to 7 μs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect Tm, either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.
In the second part of the thesis, inhomogeneous distribution of spin-labels in detergent micelles has been studied. A common approach in PELDOR is measuring the distance between two covalently attached spin labels in a macromolecule or singly-labeled components of an oligomer. This situation has been described as a spin-cluster. The PELDOR signal, however, does not only contain the desired dipolar coupling between the spin-labels of the molecule or cluster under study. In samples of finite concentration the dipolar coupling between the spin-labels of the randomly distributed molecules or spin-clusters also contributes significantly. In homogeneous frozen solutions or lipid vesicle membranes this second contribution can be considered to be an exponential or stretched exponential decay, respectively. In this study, it is shown that this assumption is not valid in detergent micelles. Spin-labeled fatty acids that are randomly partitioned into different detergent micelles give rise to PELDOR time traces which clearly deviate from stretched exponential decays. As a main conclusion a PELDOR signal deviating from a stretched exponential decay does not necessarily prove the observation of specific distance information on the molecule or cluster. These results are important for the interpretation of PELDOR experiments on membrane proteins or lipophilic peptides solubilized in detergent micelles or small vesicles, which often do not show pronounced dipolar oscillations in their time traces.
In the third part, PELDOR has been utilized to study the structural flexibility of the Toc34 GTPase homodimer, a preprotein receptor of the translocon of the outer envelope of chloroplasts (TOC). Toc34 belongs to GAD subfamily of G-proteins that are regulated and activated by nucleotide-dependent dimerization. However, the function of Toc34 dimerization is not yet fully understood. Previous structural investigations of the Toc34 dimer yielded only marginal structural changes in response to different nucleotide loads. PELDOR revealed a nucleotide-dependent transition of the dimer flexibility from a tight GDP to a flexible GTP-loaded state. Substrate-binding stabilizes the dimer in the transition state mimicked by GDP-AlFx, but induces an opening in the GDP or GTP-loaded state. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which represents the regulatory mode for dimerizing GTPases.
In the fourth part of the thesis, conformational flexibility and relative orientation of the N-terminal POTRA domains of a cyanobacterial Omp85 from Anabaena sp. PCC 7120, a key component of the outer membrane protein assembly machinery, were investigated by PELDOR spectroscopy. Membrane proteins of the Omp85-TpsB superfamily are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. It has been suggested that the N-terminal POTRA domains (P1 and P2) might have functions in substrate recognition. Molecular dynamics (MD) simulations predicted a fixed orientation for P2 and P3 and a flexible hinge between P1 and P2. The PELDOR distances measured between the P2 and P3 POTRA domains are in good agreement with the structure determined by X-ray, and compatible with the MD simulations suggesting a fixed orientation between these domains. PELDOR constraints between the P1 and P2 POTRA domains imply a rather rigid structure with a slightly different relative orientation of these domains compared with the X-ray structure. Moreover, the large mobility predicted from MD is not observed in the frozen solution. The PELDOR results further highlight the restricted relative orientation of the POTRA domains of the Omp85-TpsB proteins as a conserved characteristic feature that might be important for the processive sliding of the unfolded substrate towards the membrane.
Hepatitis C virus (HCV) assembly and production is closely linked to lipid metabolism. Indeed, lipid droplets (LD) have been shown to serve as a platform for HCV assembly. To investigate the effect of HCV on the host cell proteome, 2D-gelelectrophoresis with subsequent MALDI-TOF mass spectrometry of HCV replicating and the corresponding control cells were done. Based on this analysis, it was found out that HCV-replicating Huh7.5 cells revealed lower amounts of TIP47 (tail interacting protein of 47kD) compared to HCV-negative cells. TIP47, a cytoplasmic sorting factor, has been shown to be associated with lipid droplets. As it is known that HCV-replication and assembly takes place at the so called ”membranous web” that is composed of LDs and rearranged ER-derived membranes, it was tempting to investigate the role of TIP47 in HCV life-cycle. Western blot analysis did reveal that overexpression of TIP47 in HCV replicating Huh7.5 cells leads to decreased amounts of the HCV core protein while the levels of non-structural protein (NS)5A and intracellular HCVgenomes are increased. Moreover, in TIP47 overproducing cells higher amounts of infectious HCV particles are secreted. Vice versa, inhibition of TIP47 expression by siRNA results in a decreased level of intracellular NS5A, increased amounts of intracellular core and less infectious viral particles in the supernatant. In addition, complete silencing of TIP47 by lentiviral transduction abolishes HCV replication that can be restored by transfection of these cells with a TIP47 expression construct. It has been shown recently that apoE binds to NS5A and that this interaction plays an important role for the HCV life cycle (Benga et al., 2010). The C-terminal part of TIP47 harbours a 4 helix bundle motif and displays high homology to the N-terminus of apoE. Therefore, we investigated the interaction of NS5A and TIP47. Confocal double immunofluorescence microscopy revealed that a fraction of NS5A colocalizes with TIP47. Coimmunoprecipitation experiments and a yeast-two-hybrid screening confirmed the interaction between NS5A and TIP47 and deletion of the N-terminal-TIP47-PAT domain abolishes this interaction. From this we conclude that the TIP47-NS5A interaction is required for virus morphogenesis. Moreover, TIP47 can bind to Rab9 and this is relevant for targeting the viral particle out of the cell. In accordance to this, TIP47 was identified to be associated to the viral particle. Mutants of TIP47 that fail to bind Rab9 reveal lower amounts and a changed distribution of the HCV core protein. Furthermore, we could see that the core staining colocalizes with subcellular structures that were identified as autophagosomes using a p62-specific antibody which is a specific autophagosome-marker. Based on this, we hypothized that destruction of the Rab9 binding domain misdirects the viral particle towards the lysosomal compartment.
For the first time it could be shown that TIP47 interacts with NS5A and is associated to the viral particle, therefore plays a crucial role for the virus morphogenesis and secretion of the viral article.
Taken together, these results indicate that TIP47 is an essential cellular factor for the life cycle of HCV Abstract and might be used as target for antiviral treatment, e.g. by targeting the NS5A-TIP47 interaction, based on small molecules that mimic the NS5A-specific sequence that binds to TIP47 which might result in a competition of the TIP47/NS5A interaction.
The universal biological energy currency adenosine triphosphate (ATP) is synthesized by the F1Fo-ATP synthase in most living organisms. The overall structure and function of F-type ATPases is conserved in the different organisms. The F1Fo-ATP synthase consist of two domains; the soluble F1 complex has the subunit stoichiometry α3β3γδε and the membrane embedded Fo complex consists of subunits ab2c10-15 in its simplest form found in bacteria. F1 and Fo both function as reversible rotary motors that are connected by a central stalk (γε) and a peripheral stalk (b2δ).
For ATP synthesis, the electrochemical energy formed by a proton or sodium ion gradient is required. The ion translocation across the Fo subcomplex induces torque in the motor part of the enzyme (cnγε), which causes conformational changes in the α3β3 domain leading to ATP synthesis from ADP and inorganic phosphate (Pi) catalyzed in the β-subunits. ATP hydrolysis causes a reverse torque in the Fo subcomplex triggering uphill ion translocation from cytoplasm to periplasm, and the enzyme functions as an ion pump.
The ATP synthesis mechanism is well understood, since several high-resolution structures of F1 are available. In contrast, the ion translocation mechanism across the membrane, mediated by the Fo subcomplex, is not understood in its structural detail.
Subunit a and the c-ring form an ion pathway, but subunit b is needed to form an active ion translocation pathway in both H+- and Na+-dependent systems. Several high-resolution structures of c-rings have provided insights in the ion translocation mechanism. The different ion translocation models based on biochemical, biophysical and structural analysis are in agreement in the fact that ions are translocated through a periplasmic ion access pathway in subunit a to the middle of the membrane and there to the binding site of a c-subunit. After almost a whole rotation of the c-ring the ion returns into the a-c interface, where it can be released to the cytoplasm. In the different models the cytoplasmic access pathway has been proposed to be located in subunit a, at the a-c interface or within the c-ring. The driving force of torque generation has been proposed to be the pH gradient or membrane potential. Several biochemical studies show that a conserved arginine in helix four of subunit a (R226 in Ilyobacter tartaricus or R210 in Escherichia coli)plays a critical role in the ion translocation. The arginine has been proposed to function as an electrostatic separator between the cytoplasmic and periplasmic pathways and as a mediator of the ion exchange into the c-ring ion-binding site.
Structural data of a related enzyme (V1Vo-ATPase from Thermus thermophilus) has provided insight into the helical arrangement of the ion translocating subunits I and Lring (related to subunit a and the c-ring). These structures indicated a small interface between subunit I and the L-ring, and two four-helix bundles in the N-terminal domain of subunit I were proposed to build the periplasmic and cytoplasmic ion pathways. To comprehend the ion-translocation and torque generation mechanism in F1Fo-ATP synthase, structural data of an intact a-c complex is needed.
The goal of this work was to obtain structural data of subunit a, most preferably in a complex with the c-ring or additionally with subunit b. Therefore, a new purification procedure for the I. tartaricus Fo-subcomplex, heterologously expressed in E. coli cells, was established. The purified Fo was characterized biochemically and by Laserinduced liquid bead ion desorption mass spectrometry (LILBID-MS). These analyses showed that pure and completely assembled Fo containing all its subunits in the correct stoichiometry (ab2c11) was obtained. The purified Fo complex was stable at 4°C for several months and at room temperature in the presence of lipids for several weeks. A lipid analysis was performed by thin-layer chromatography (TLC) to investigate the qualitative lipid composition of I. tartaricus whole lipid extract and various I. tartaricus F1Fo isolates. The whole lipid extract contained PC, PG and PE lipids and probably cardiolipin. PC, PG and PE lipids were bound to wild type I. tartaricus F1Fo, whereas recombinant I. tartaricus F1Fo did not have any bound lipids, but was able to bind the synthetic lipids POPC and POPG if they were provided during the purification.
For subsequent structural studies the purified Fo was subjected to two-dimensional (2D) crystallization trials. Vesicles and sheets tightly packed with protein and crystals with a rare plane group for I. tartaricus c11 (p121) were obtained. The c-ring was visible in the CCD images, and immunogold-labeling revealed the presence of the His-tagged a-subunit in the reconstituted vesicles. Furthermore, atomic force microscopy (AFM) imaging showed protein densities next to the c-rings, which protruded less from the membrane (0.4±0.1 nm) than the c-ring (0.7±0.1 nm). These protein densities presumably belonged to subunit a.
Cryo-electronmicroscopy (cryo-EM) was used to collect data of the p121 crystals and a merged projection density map was calculated to 7.0 Å resolution. The unit cell of the crystals (81 × 252 Å) contained two asymmetric units with three c-rings in each and next to the c11-rings new prominent densities were visible. In each extra density up to 7 transmembrane helices were visible, belonging to the stator subunit a and/or subunit b. To elucidate whether there are conserved elements in the three extra densities non-crystallographic averaging was applied using a single-particle approach.
Six possible arrangements for the c-rings and the extra densities were identified and used for the averaging. The extra densities were enhanced only in one of the possible arrangements. The average showed a four-helix bundle and a fifth helix in close proximity to the c-ring. Two more helices were present in each position but their position was ambivalent. The data obtained in this work provides the first insight in the helical arrangement in the a-c interface of F1Fo-ATP synthase.
Detailed knowledge of reaction mechanisms is key to understanding chemical, biological, and biophysical processes. For many reasons, it is desirable to comprehend how a reaction proceeds and what influences the reaction rate and its products.
In biophysics, reaction mechanisms provide insight into enzyme and protein function, the reason why they are so efficient, and what determines their reaction rates. They also reveal the relationship between the function of a protein and its structure and dynamics.
In chemistry, reaction mechanisms are able to explain side products, solvent effects, and the stereochemistry of a product. They are also the basis for potentially optimizing reactions with respect to yield, enhancing the stereoselectivity, or for modifying reactions in order to obtain other related products.
A key step to investigate reaction mechanisms is the identification and characterization of intermediates, which may be reactive, short-lived, and therefore only weakly populated. Nowadays, the structures of those can in most cases only be hypothesized based on products, side products, and isolable intermediates, because intermediates with a life time of less than a few microseconds are not accessible with the commonly used techniques for structure determination such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.
In this thesis, two-dimensional infrared (2D-IR) spectroscopy is shown to be a powerful complement to the existing techniques for structure determination in solution. 2D-IR spectroscopy uses a femtosecond laser setup to investigate interactions between vibrations - analogous to 2D-NMR, which investigates the interactions between spins. Its ultrafast time resolution makes 2D-IR spectroscopy particularly well suited for the two topics investigated in this thesis: Structure Determination of Reactive Intermediates and Conformational Dynamics of Proteins.
Structure Determination of Reactive Intermediates: The focus of this thesis is using polarization-dependent 2D-IR (P2D-IR) spectroscopy for structure determination of N-crotonyloxazolidinone (referred to as 1), a small organic compound with a chiral oxazolidinone, known as Evans auxiliary, and its reactive complexes with the Lewis acids SnCl4 and Mg(ClO4)2. Chiral oxazolidinones in combination with Lewis acids have frequently been used in stereoselective synthesis for over 30 years. Nevertheless, the detailed mechanisms are in many cases xvi ABSTRACT still mere hypotheses and have not yet been experimentally proven. By accurately measuring the angles between the transition dipole moments in the molecules using an optimized P2D-IR setup and comparing the results to DFT calculations, the conformation of 1 and the conformation and coordination of the main complexes with SnCl4 and Mg(ClO4)2 are unequivocally identified and analyzed in depth. Structural details, such as a slight twist in the solution structure of 1, are detected using P2D-IR spectroscopy; these cannot be inferred from NMR spectroscopy or DFT calculations. In addition to the main Lewis acid complexes, complexes in low concentration are detected and tentatively assigned to different conformations and complexation geometries. The knowledge of those structures is essential for rationalizing the observed stereoselectivities. Additionally, a method is introduced that enables structure determination of molecules in complex mixtures and even in the presence of molecules with similar spectral properties and in high concentration. This work sets the stage for future studies of other substrate-catalyst complexes and reaction intermediates for which the structure determination has not been possible to date.
Conformational Dynamics of Proteins: Exchange 2D-IR spectroscopy allows the investigation of fast dynamics without disturbing the equilibrium of the exchanging species. It is therefore well suited to investigate fast dynamics of proteins and to reveal the speed limit of those. The temperature dependence of the conformational dynamics between the myoglobin substates A1 and A3 in equilibrium is analyzed. The various substates of myoglobin can be detected with FTIR spectroscopy, if carbon monoxide is bound to the heme. From previous studies it is known that the exchange rates at room temperature are in the picosecond time range, well suited to be investigated by 2D-IR spectroscopy. In the temperature range between 0 °C and 40 °C only a weak temperature dependence of the exchange rate in the myoglobin mutant L29I is observed in the present study. The exchange rate approximately doubles from 15 ns-1 at 0 °C to 31 ns-1 at 40 °C. It turned out that the conformational dynamics correlates linearly with the solvent viscosity, which itself is temperature dependent. Comparing our results to measurements at cryogenic temperatures, the linear relation between exchange time constant for this process and the viscosity is shown for the temperature range between -100 °C and 40 °C (corresponding to a viscosity change of 14 orders of magnitude). Thus, it is proven that the dynamics of the conformational switching are mainly determined by solvent dynamics, i.e., the protein dynamics are slaved to the solvent dynamics. This is the first time slaving is observed for such fast processes (in the picosecond time range). The observation implies a long-range structural rearrangement between the myoglobin substates A1 and A3. In addition, the exchange for other mutants and wild type myoglobin is analyzed qualitatively and found to agree with the conclusions drawn from L29I myoglobin.
Savanna regions in West Africa are valuable cultural landscapes and provide a wide range of ecosystem services for human well-being and are frequently affected by human-induced disturbances. Aside from agricultural activities (crop production and animal husbandry), the harvesting of timber and non-timber forest products is crucial for household income, alimentation and medicinal purposes. Most indigenous woody species have undergone increasing anthropogenic pressure as social and economic conditions have changed dramatically during recent decades, resulting in further habitat fragmentation and increased disturbance severity. Human land use activities influence growth conditions for plants by altering various abiotic factors, such as light, nutrient availability and water supply. They are found to alter demographic parameters (e.g., germination, seedling and sapling growth, survival and mortality rates) of woody plant individuals and alter the structure and stability of populations. The degree of anthropogenic disturbance varies between land-cover types, distance to settlements, and protection status. In the context of land-use change, there is an urgent need to better understand and evaluate the impact of land-use on savanna vegetation, particularly on the population biology of common savanna woody species. A major conclusion to be drawn from this thesis is that land use influences savanna vegetation in a complex way and does not necessarily lead to a decline or loss of tree populations and species. It is rather that in a constantly changing landscape, as a result of human-induced disturbances, populations of ubiquitous and some common species can be stable over time. The abundance of some species tends to decline consistently, whereas others benefit from human disturbance. Moreover, the study provides an insight into the structure and dynamics of common, dominant and less dominant savanna woody plants in a communal and a protected area. There is a need for further basic studies to assess the impact of land use and ecological preferences of all species, including repeated density studies that look at survivorship and transition probabilities over a number of seasons as well as longterm in-situ experiments in settlement areas in order to better understand woody plant populations in settlement areas as the few remaining semi-natural sites are likely to decrease in the future. A challenge will be the development of strategies to protect species within a landscape under cultivation.
An exciting in vivo function of ATP-sensitive potassium channels in substantia nigra dopamine neurons Ð Implications for burst firing and novelty coding ÐPhasic burst activity is a key feature of dopamine (DA) midbrain neurons. This particular pattern of excitation of DA neurons occurs via a synaptically triggered transition from low-frequency background spiking to transient high-frequency discharges. Burst-firing mediated phasic DA release is critical for flexible switching of behavioural strategies in response to unexpected rewards, novelty and other salient stimuli. However, the cellular and molecular bases of burst signalling in distinct DA subpopulations of the substantia nigra (SN) or the ventral tegmental area (VTA) are unknown.
DA neuron excitability is controlled by synaptic network inputs, neurotransmitter receptors and ion channels, which generate action potentials and determine frequency and pattern of electrical activity in a complex interplay. ATP-sensitive potassium (K-ATP) channels are widely expressed throughout the brain, where in most cases they are believed to act as metabolically-controlled 'excitation brakes' by matching excitability to cellular energy states. However, their precise physiological in vivo function in DA neurons remains elusive.
To study burst firing and the underlying ionic mechanisms with single cell resolution, in vivo single-unit recordings were combined with juxtacellular neurobiotin labelling as well as immunohistochemical and anatomical identification of individual DA neurons. In vivo recordings were performed in adult isoflurane-anaesthetised wildtype (WT) and global K-ATP channel knockout mice, lacking the pore forming Kir6.2 subunit (Kir6.2-/-). In addition, DA cell-selective functional silencing of K-ATP channel activity in vivo was established using virus-mediated expression of dominant-negative Kir6.2 subunits. Careful control experiments ruled out any significant contributions from nonDA neurons as transduction was effectively limited to SN DA neurons rather than affecting those cells that innervate them. Virus-based K-ATP channel silencing in combination with juxtacellular recording and labelling was achieved to define the electrophysiological phenotype of individually identified, virally-transduced DA neurons in vivo.
Single-unit recordings revealed that K-ATP channels Ð in contrast to their conventional hyperpolarising role Ð in a subpopulation of DA neurons located in the medial SN (m-SN) act as cell-type selective gates for excitatory burst firing in vivo. The percentage of spikes in bursts was threefold reduced in Kir6.2-/- compared to WT mice. Classification of firing patterns based on visual inspection of autocorrelation histograms and on a newly developed spike-train-model confirmed the dramatic shift from phasic burst to tonic single-spike oscillatory firing in Kir6.2-/-. This significant decrease of burstiness was selective for m-SN DA neurons and was not exhibited by DA cells in the lateral SN or VTA. Virus-based K-ATP channel silencing in vivo unequivocally demonstrated that the activity of postsynaptic K-ATP channels was sufficient to disrupt bursting in m-SN DA neuron subtypes. Patch-clamp recordings in brain slices indicated an essential role of K-ATP channels for NMDA-mediated in vitro bursting. In accordance with previous studies in DA midbrain neurons, NMDA receptor stimulation triggered burst-like firing in m-SN DA cells in vitro, but only when K-ATP channels were co-activated in these neurons.
K-ATP channel-gated burst firing in m-SN DA neurons might be functionally relevant in awake, freely moving mice. To explore the behavioural consequences of SN DA neuron subtype-selective K-ATP channel suppression, spontaneous open field (OF) behaviour of mice with bilateral K-ATP silencing across the whole SN (medial + lateral) or in only the lateral SN was tested. Analysis of WT and global Kir6.2-/- mice showed reduced exploratory locomotor activity of Kir6.2-/- in a novel OF environment. Remarkably, K-ATP channel silencing in m-SN DA neurons phenocopied this novelty-exploration deficit, indicating that K-ATP channel-gated burst firing in medial but not lateral SN DA neurons is crucial for WT-like novelty-dependent exploratory behaviour.
In summary, a novel role of K-ATP channels in promoting the excitatory switch from tonic to phasic firing in vivo in a cell-type specific manner was discovered. The present PhD thesis provides several important insights into the pivotal function of K-ATP channels in medial SN DA cells, which project to the dorsomedial striatum, for burst firing and its important consequences for context-dependent exploratory behaviour.
In collaboration with two other research groups transcriptional up-regulation of K-ATP channel and NMDA receptor subunits and high levels of in vivo burst firing were detected in surviving SN DA neurons from Parkinson's disease (PD) patients Ð providing a potential link of K-ATP channel activity to neurodegenerative pathomechanisms of PD. Using high-resolution fMRI imaging another study in humans has recently identified distinct DA midbrain regions that are preferentially activated by either reward or novelty. Taken together, these human data and the results of the present PhD thesis suggest that burst-gating K-ATP channel function in SN DA neurons impacts on phenotypes in disease as well as in health.
The role of the Ca2+-dependent protease calpain in the diabetes-associated platelet hyperreactivity
(2012)
Platelets from diabetic patients are characterised by hyperreactivity resulting in exaggerated adhesion, aggregation and thrombus formation which contribute to the development of cardiovascular complications known to be one of the main causes of diabetes-related mortality. One of the mechanisms suggested to be involved in the diabetes-related platelet hyperactivation is the increased [Ca2+]i which leads to the overactivation of Ca2+-dependent proteases, the calpains. Among the calpain isoforms expressed in platelets the two ubquitiously expressed μ- and m-calpain are thought to play an important role in physiological and pathophysiological processes. Particularly μ-calpain is known to be involved in many steps of physiological platelet activation such as aggregation, adhesion, secretion, and signalling. However, we could show that diabetes was associated with an enhanced activation of both μ- and m-calpain in platelets
In the first part of the study we focussed on the characterization of the molecular mechanism regulating calpain activity. Indeed, although Ca2+ is considered to be the main regulator of the proteolytic activity of the conventional calpains, other mechanisms such as the presence of phospholipids and phosphorylation have been reported to affect their activity. Since most studies reported the phosphorylation of m-calpain we were interested to see whether μ-calpain activity might be also affected by phosphorylation. We could show that the activity of μ-calpain was enhanced by the PKC activator PMA suggesting its possible regulation by phosphorylation. However, whether PKC directly targeted μ-calpain remains unclear. Given that substrate recognition is important for a protease to process its substrate and since no common consensus could be attributed to calpain substrates, our next interest was to understand the mechanism regulating the recognition of its substrates by calpain. Since phosphorylation has been reported to protect different proteins from calpain degradation we investigated whether the calpain substrate CD31 could be phosphorylated in platelets and whether this could affect its recognition by calpain. Although we could show that the tyrosine phosphorylation of CD31 was increased after activation of platelets by thrombin and that this effect was attenuated in platelets from diabetic patients, tyrosine phosphorylation of CD31 seemed to have no effect on its sensitivity to calpain-mediated proteolysis.
After the analysis of the mechanism regulating calpain activity as well as its interaction with its substrates, our next interest was the identification of new calpain substrates in platelets. Since a previous study from our group showed that PPARγ agonists could indirectly reverse the diabetes-associated calpain activation we performed DIGE analysis of platelet samples from diabetic patients before and after PPARγ agonist treatment. Using this approach we could identify four novel calpain substrates in platelets: Integrin-linked kinase (ILK), α parvin, CLP36 and septin-5. Next, we assessed the effect of calpain-mediated cleavage on the function of these newly identified proteins. We could show that μ-calpain was essential for the dissociation of ILK from the IPP complex and its activation while m-calpain-mediated cleavage led to its cleavage and inactivation. Functionally, we also showed that μ-calpain was involved in platelet adhesion while m-calpain was important for spreading.
The next protein we analysed was septin-5, a small GTPase known to regulate platelet degranulation by association with other septins and syntaxin-4. We found that the interaction between septin-5 and syntaxin-4 was inhibitory for platelet degranulation. We could demonstrate that the μ-calpain-mediated cleavage dissociated septin-5 from syntaxin 4 and led to increased secretion of platelet α-granules. Next, we investigated the in vivo role of calpain in the diabetes-associated platelet hyperreactivity. We induced diabetes in mice and could reproduce calpain activation in platelets such as that found in human. Indeed, calpain activation in murine platelets also led to the cleavage of several calpain substrates including ILK and septin-5. Moreover, platelets from diabetic mice demonstrated an increased aggregation and thrombus formation in vivo. Treatment of the animals with the calpain inhibitor A-705253 (30 mg/kg/day for 10 days) significantly restored platelet function and substrate cleavage. In conclusion, in this part of the study, we could show that the increased calpain-dependent α-granule secretion and platelet adhesion may account for the enhanced vascular proliferation and thrombus formation in diabetes and calpain inhibition represents a promising way to prevent atherothrombosis development.
In the last part of the study we analysed another enzyme known to play a crucial role in diabetes, the AMPK which is an energy-sensing kinase known to be impaired in diabetes. We could show that the two catalytic subunits AMPK α1 and α2 are expressed in platelets. The AMPKα2 seemed to be the subunit involved in platelet activation since AMPKα2-deficient mice demonstrated a defect in clot retraction and the stabilization of the thrombus while the animals showed a normal bleeding time. Mechanistically, we showed in platelets that the upstream kinase of AMPKα2 is LKB1 which was activated by thrombin stimulation via a PI-3K-dependent pathway. AMPKα2 then phosphorylated the Src-family kinase Fyn, which is responsible for the phosphorylation of its substrate β3 integrin on Tyr747. These data indicate that AMPKα2, by affecting Fyn phosphorylation and activity, plays a key role in platelet αIIbβ3 integrin signalling, leading to clot retraction and thrombus stability. Although the effect of diabetes in the AMPK-dependent pathway could not be investigated we assume that the dysregulation of this pathway may account for the thrombus destabilization and enhanced embolization encountered in diabetes.
In this thesis, various aspects on the theoretical description of ultracold bosonic atoms in optical lattices are investigated. After giving a brief introduction to the fundamental concepts of BECs, atomic physics, interatomic interactions and experimental procedures in chapter (1), we derive the Bose-Hubbard model from first principles in chapter (2). In this chapter, we also introduce and discuss a technique to efficiently determine Wannier states, which, in contrast to current techniques, can also be extended to inhomogeneous systems. This technique is later extended to higher dimensional, non-separable lattices in chapter (5). The many-body physics and phases of the Bose-Hubbard is shortly presented in chapter (3) in conjunction with Gutzwiller mean-field theory, and the recently devised projection operator approach. We then return to the derivation of an improved microscopic many-body Hamiltonian, which contains higher band contributions in the presence of interactions in chapter (4). We then move on to many-particle theory. To demonstrate the conceptual relations required in the following chapter, we derive Bogoliubov theory in chapter (5.3.4) in three different ways and discuss the connections. Furthermore, this derivation goes beyond the usual version discussed in most textbooks and papers, as it accounts for the fact, that the quasi-particle Hamiltonian is not diagonalizable in the condensate and the eigenvectors have to be completed by additional vectors to form a basis. This leads to a qualitatively different quasi-particle Hamiltonian and more intricate transformation relations as a result. In the following two chapters (7, 8), we derive an extended quasi-particle theory, which goes beyond Bogoliubov theory and is not restricted to weak interactions or a large condensate fraction. This quasi-particle theory naturally contains additional modes, such as the amplitude mode in the strongly interacting condensate. Bragg spectroscopy, a momentum-resolved spectroscopic technique, is introduced and used for the first experimental detection of the amplitude mode at finite quasi-momentum in chapter (9). The closely related lattice modulation spectroscopy is discussed in chapter (10). The results of a time-dependent simulation agree with experimental data, suggesting that also the amplitude mode, and not the sound mode, was probed in these experiments. In chapter (11) the dynamics of strongly interacting bosons far from equilibrium in inhomogeneous potentials is explored. We introduce a procedure that, in conjunction with the collapse and revival of the condensate, can be used to create exotic condensates, while particularly focusing on the case of a quadratic trapping potential. Finally, in chapter (12), we turn towards the physics of disordered systems derive and discuss in detail the stochastic mean-field theory for the disordered Bose-Hubbard model.
The ongoing debate on deforestation in the tropics usually points out agriculture and logging as the main causes. The two activities are often linked and the trails created by logging com-panies with their heavy machines are afterwards used by farmers to penetrate deep into the forest and cultivate. Shifting cultivation is a widespread agricultural practice in the tropics and its sustainability is often a matter of controversy. It is necessary to investigate forest recovery after shifting cultivation, analyze its succession stages for comparison with regeneration after natural disturbance, and evaluate its role for discussing the hazards of deforestation.
The study of systems whose properties are governed by electronic correlations is a corner stone of modern solid-state physics. Often, such systems feature unique and distinct properties like Mott metal-insulator transitions, rich phase diagrams, and high sensitivity to subtle changes in the applied conditions. Whereas the standard approach to electronic structure calculations, density functional theory (DFT), is able to address the complexity of real-world materials but is known to have serious limitations in the description of correlations, the dynamical mean-field theory (DMFT) has become an established method for the treatment of correlated fermions, first on the level of minimal models and later in combination with DFT, termed LDA+DMFT.
This thesis presents theoretical calculations on different materials exhibiting correlated physics, where we aim at covering a range in terms of systems --from rather weakly correlated to strongy correlated-- as well as in terms of methods, from DFT calculations to combined LDA+DMFT calculations. We begin with a study on a selection of iron pnictides, a recently discovered family of high-temperature superconductors with varying degree of correlation strength, and show that their magnetic and optical properties can be assessed to some degree within DFT, despite the correlated nature of these systems. Next, extending our analysis to the inclusion of correlations in the framework of LDA+DMFT, we discuss the electronic structure of the iron pnictide LiFeAs which we find to be well described by Fermi liquid theory with regard to many of its properties, yet we see distinct changes in its Fermi surface upon inclusion of correlations. We continue the study of low-energy properties and specifically Fermi surfaces on two more iron pnictides, LaFePO and LiFeP, and predict a topology change of their Fermi surfaces due to the effect of correlations, with possible implications for their superconducting properties. In our last study, we close the circle by presenting LDA+DMFT calculations on an organic molecular crystal on the verge of a Mott metal-insulator transition; there, we find the spectral and optical properties to display signatures of strong electronic correlations beyond Fermi liquid theory.
With the increasing energies and intensities of heavy-ion accelerator facilities, the problem of an excessive activation of the accelerator components caused by beam losses becomes more and more important. Numerical experiments using Monte Carlo transport codes are performed in order to assess the levels of activation. The heavy-ion versions of the codes were released approximately a decade ago, therefore the verification is needed to be sure that they give reasonable results. Present work is focused on obtaining the experimental data on activation of the targets by heavy-ion beams. Several experiments were performed at GSI Helmholtzzentrum für Schwerionenforschung. The interaction of nitrogen, argon and uranium beams with aluminum targets, as well as interaction of nitrogen and argon beams with copper targets was studied. After the irradiation of the targets by different ion beams from the SIS18 synchrotron at GSI, the γ-spectroscopy analysis was done: the γ-spectra of the residual activity were measured, the radioactive nuclides were identified, their amount and depth distribution were detected. The obtained experimental results were compared with the results of the Monte Carlo simulations using FLUKA, MARS and SHIELD. The discrepancies and agreements between experiment and simulations are pointed out. The origin of discrepancies is discussed. Obtained results allow for a better verification of the Monte Carlo transport codes, and also provide information for their further development. The necessity of the activation studies for accelerator applications is discussed. The limits of applicability of the heavy-ion beam-loss criteria were studied using the FLUKA code. FLUKA-simulations were done to determine the most preferable from the radiation protection point of view materials for use in accelerator components.
In dieser Arbeit werden Schmelz- und Anreicherungsprozesse des Erdmantels, sowie Kristallisationsereignisse der Erdkruste zweier ausgewählter Gebiete in Namibia und Spanien mithilfe geochemischer Methoden rekonstruiert und in einen zeitlichen Zusammenhang gebracht. Ein Vergleich der gewonnenen Ergebnisse beider Kompartimente soll dabei weitere Informationen liefern inwieweit Prozesse des Erdmantels und der Erdkruste miteinander verknüpft waren. Insbesondere soll ein weitere Beitrag zur aktuellen Diskussion geliefert werden, bei der sich das sogenannte „pulsed growth“ und „steady accumulation“ Modell gegenüberstehen (siehe Zusammenstellung Pearson et al., 2007). Zudem tragen die neu gewonnenen Daten dazu bei, die regionalen geologischen Gegebenheiten im besonderen Hinblick auf die geotektonische Geschichte besser zu verstehen.
Das Gibeon Kimberlit Feld befindet sich in der tektonischen Einheit des Rehoboth Terranes in Namibia und ist gekennzeichnet von Vulkanismus vor etwa 72.5 Ma (Davies et al., 2001), der Granat Peridotite und krustale Xenolithe mit an die Oberfläche beförderte. Eine klare Einordnung des Rehoboth Terranes in die Gesamtheit des Süd Afrikanischen Plattenverbunds ist noch nicht vollständig geklärt.
Die Südöstliche vulkanische Provinz in Spanien (SEVP) mit besonderem Hinblick auf die Region um Casas de Tallante stellt das zweite Probengebiet für diese Arbeit dar. Vor etwa 2.6 Ma (Bellon et al., 1983) kam es zur Extrusion von alkali-basaltischen Schmelzen, die zahlreiche Spinell / Plagioklas Peridotite mit sich brachten. Tufflagen, sowie die Matrix der Basalte ermöglichen einen Einblick in die untere Kruste der Region.
Untersuchungen der Erdmantelproben aus Namibia auf ihre Haupt- und Spurenelementchemie, sowie Lu-Hf und Sm-Nd Isotopie zeigten, dass zwei verschiedene Manteltypen vorliegen („N“ und „σ“ Typ), die zu einem Zeitpunkt um etwa 850 Ma („N“) und 1.9 Ga („σ“) angereichert wurden. Eine letzte Anreicherung beider Typen fand vermutlich während der Pan–Afrikanischen Orogenese um etwa 450 Ma statt. Die Reinterpretation eines zuvor publizierten Datensatzes (Pearson et al., 2004), suggeriert, dass es zu einer ersten Verarmung der σ Peridotite um etwa 2.9 Ga kam.
Untersuchungen der U-Pb und Hf Isotopie an Zirkonen aus der unteren Kruste des Probengebiets in Namibia ergaben, dass es zur Bildung von juvenilem Krustenmaterial vermutlich bereits im Archaikum kam (wie bereits vorgeschlagen durch z.B. Hoal et al., 1995; Franz et al., 1996), sowie in den Zeiträumen von 2.3 bis 2.7 und 1.5 bis 1.6 Ga, mit jeweils anschließendem krustalem Recycling und Krustenmischung. Eine Übereinstimmung von Mantel- und Krustenevents konnte für die Zeiträume von etwa 1.8, 0.8 - 0.9 Ga und 0.4 – 0.5 Ga gefunden werden. Eine mögliche erste Verarmung des σ Mantels wird bestätigt durch Zirkonalter im Bereich von 2.7 bis 2.9 Ga.
Die Analyse ausgewählter Spinell / Plagioklas Peridotite aus der SEVP, ergaben, dass ein heterogener Mantel mit mindestens 3 verschiedenen Typen vorliegt. Eine Korrelation der Lu-Hf Isotopie von 3 Proben dieses Probensatzes, sowie den Hf Isotopien einer weiteren Probe von Bianchini et al. (2011) suggerieren, dass es eventuell zu einem Verarmungsereignis zu einem Zeitpunkt von etwa 550 Ma kam. Sr Isotopien von Klinopyroxenen und Plagioklasen im Vergleich ergaben, dass die Sr Isotopie der Plagioklase, im Gegensatz zu den Klinopyroxenen, von denen der Alkali Basalte überprägt wurden.
Zirkonanalysen aus Lokalitäten innerhalb der SEVP (U-Pb, Hf) ergaben ein weitreichendes Altersspektrum, beginnend bei etwa 2-3 Ma bis hin ins Archaikum (2.7 bis 2.9 Ga) mit Provenance Ursprung aus Gondwana und dem Arabisch-Nubischen Schild. Die Kombination der U-Pb Altersinformationen mit den entsprechenden Hf Isotopien, zeigten, dass es vermutlich bereits im Archaikum zu juveniler Krustenbildung kam. Zirkone > 100 µm datieren den Zeitpunkt der Eruption der Alkali Basalte mit Altern um etwa 2.6 Ma und Hf Isotopien, die einem leicht verarmten Mantel entsprechen. Ein mögliches Verarmungsereignis im Erdmantel zu einem Zeitpunkt von etwa 550 Ma, ist im Einklang mit Krustenrecycling zu selbigem Zeitpunkt.
Die neugewonnenen Daten dieser Arbeit unterstützten das „pulsed growth“ Modell.
Literatur
Bellon, H., Bordet, P. and Montenat, C., 1983. Chronology of the Neogene Magmatism from Betic Ranges (Southern Spain). Bulletin De La Societe Geologique De France, 25(2): 205-217.
Bianchini, G., Beccaluva, L., Nowell, G.M., Pearson, D.G. and Siena, F., 2011. Mantle xenoliths from Tallante (Betic Cordillera): Insights into the multi-stage evolution of the south Iberian lithosphere. Lithos, 124(3-4): 308-318.
Davies, G.R., Spriggs, A.J. and Nixon, P.H., 2001. A non-cognate origin for the Gibeon kimberlite megacryst suite, Namibia: Implications for the origin of Namibian kimberlites. Journal of Petrology, 42(1): 159-172.
Franz, L., Brey, G.P. and Okrusch, M., 1996b. Steady state geotherm, thermal disturbances, and tectonic development of the lower lithosphere underneath the Gibeon Kimberlite Province, Namibia. Contributions to Mineralogy and Petrology, 126(1-2): 181-198.
Hoal, B.G., Hoal, K.E.O., Boyd, F.R. and Pearson, D.G., 1995. Age constraints on crustal and mantle lithosphere beneath the Gibeon kimberlite field, Namibia. South African Journal of Geology, 98(2): 112-118.
Pearson, D.G., Irvine, G.J., Ionov, D.A., Boyd, F.R. and Dreibus, G.E., 2004. Re-Os isotope systematics and platinum group element fractionation during mantle melt extraction: a study of massif and xenolith peridotite suites. Chemical Geology, 208(1-4): 29-59.
Pearson, D.G., Parman, S.W. and Nowell, G.M., 2007. A link between large mantle melting events and continent growth seen in osmium isotopes. Nature, 449(7159): 202-205.
Die Wechselwirkung zwischen zwei verschiedenartigen Wellenphänomenen in einer Höhe von ca. 10 bis 100 km, der mittleren Atmosphäre, ist das zentrale Thema der vorliegenden Arbeit. Schwerewellen entstehen durch Oszillationen der Luft in einer stabil geschichteten Atmosphäre. Durch die Vielzahl von Schwerewellen-Paketen, die in der Troposphäre durch Gebirge, Gewitter, Fronten und andere dynamische Prozesse angeregt werden, wird Energie und Impuls in die mittleren Atmosphäre transportiert. Durch den turbulenten Zerfall von brechenden Schwerewellen wird auf die mittlere Strömung eine Kraft ausgeübt, welche im Bereich der Mesopause bei ca. 90 km maximal wird. Daraus resultiert die sogenannte interhemispherische residuelle Zirkulation, die in der Mesosphäre den Sommer- mit dem Winterpol verbindet und die beeindruckend kalte Sommer-Mesopause mit Temperaturen von unter −140°C verursacht. Thermische Gezeiten sind ein weiterer wichtiger Teil in der Dynamik der mittleren Atmosphäre. Sie werden durch die Erwärmung der Tagseite der Erde angeregt und sind globale Schwingungen mit Perioden von 24 Stunden und harmonischen Vielfachen. Mit Wind- und Temperatur-Amplituden von bis zu 50 m/s und 30 K dominieren sie die Tagesvariabilität im Mesopausen-Bereich.
In der Mesosphäre wird die Wechselwirkung zwischen Schwerewellen und thermischen Gezeiten wichtig. Dort wird durch die Gezeitenwinde das Brechen von Schwerewellen zeitlich moduliert und eine periodische Kraft erzeugt, welche auf die Gezeiten rückwirkt. Doch selbst unter Zuhilfenahme modernster Hochleistungsrechner kann in komplexen Zirkulationsmodellen nur ein Bruchteil des turbulenten sowie des Wellen-Spektrums aufgelöst werden. Der Effekt der nichtaufgelösten Skalen, wie Turbulenz und Schwerewellen, muss somit in effizienter Weise parametrisiert werden. Üblicherweise wird in Schwerewellen-Parametrisierungen die horizontale und zeitliche Variation des Hintergrundmediums vernachlässigt. Es entsteht eine vertikale Säule, in der sich stationäre Schwerewellen-Züge instantan nach oben ausbreiten. Es ist jedoch äußerst fraglich, inwieweit eine solche Beschreibung, auf der ein Großteil früherer Untersuchungen basiert, für das Ergründen der Schwerewellen-Gezeiten-Wechselwirkung hinreicht. Für diese Arbeit wurde deswegen das Ziel gesetzt, die Defizite der konventionellen Beschreibung der Schwerewellen-Ausbreitung in realistischen Gezeiten zu quantifizieren.
Die "Ray Tracing"-Methode wird auf die Problemstellung der Schwerewellen-Gezeiten-Wechselwirkung angewendet. In der "Ray Tracing"-Methode werden Schwerewellen-Pakete entlang ihrer Ausbreitungspfade explizit verfolgt und Veränderungen der Schwerewellen-Eigenschaften durch den Einfluss der Hintergrundströmung berücksichtigt. Vom Autor wurde das globale "Ray Tracing"-Modell RAPAGI (RAy PArameterization of Gravity-wave Impacts) entwickelt und mit realistischen Gezeitenfeldern aus dem Zirkulationsmodell HAMMONIA (HAmburg MOdel of the Neutral and Ionized Atmosphere) betrieben. In verschiedenen "Ray Tracing"-Experimenten wird für ein einfaches Schwerewellen-Ensemble gezeigt, wie horizontale Gradienten des Hintergrundmediums sowie dessen Zeitabhängigkeit wesentlichen Einfluss auf die Ausbreitung und Dissipation von Schwerewellen nehmen. Zum einen führt die durch Gezeitenwellen hervorgerufene Transienz zu einer tageszeitlichen Modulation der absoluten Schwerewellen-Frequenz.
Die dadurch induzierten Variationen der horizontalen Phasengeschwindigkeit der Schwerewellen können die anfängliche Phasengeschwindigkeit um bis zu eine Größenordnung übertreffen und folgen dem Verlauf des Hintergrundwindes. Die kritische Filterung von Schwerewellen wird durch diese Modulation abgeschwächt, was im Vergleich zu konventionellen Schwerewellen-Parametrisierungen zu einer im Mittel um 30 % geringeren Kraftwirkung auf die Gezeiten führt. Zum anderen werden durch horizontale Gradienten in der gesamten Hintergrundströmung Schwerewellen-Pakete horizontal abgelenkt. Wellen, die gegen die Hintergrundströmung laufen, werden in der Stratosphäre in die Maxima der Wind-Jets hineingeführt. Durch dieses Verhalten wird analog zum Fermatschen Prinzip der geometrischen Optik die Laufzeit der Schwerewellen in der mittleren Atmosphäre minimiert. Es entsteht eine Fokussierung von Schwerewellen-Feldern, bei gleichzeitiger Zunahme der horizontalen Wellenzahl in den Experimenten im Mittel um ca. 10 %. Dadurch reduziert sich der Schwerewellen-Impulsfluss und die mittlere und ebenfalls die periodische Kraft auf die Hintergrundströmung im Mittel um weitere 20 % bis 30 %. Konventionelle Schwerewellen-Parametrisierungen scheinen somit die Kraftwirkung von brechenden Schwerewellen zu uberschätzen. Aus den Ergebnissen der Arbeit wird klar, dass Schwerewellen-Parametrisierungen nicht "blind" für jede Untersuchung genutzt werden können. Alle Annahmen und Näherungen in Parametrisierungen müssen je nach Zielstellung neu getestet werden.
There is increasing evidence that climate change will have a severe impact on species’ distributions by altering the climatic conditions within their present ranges. Especially species inhabiting stream ecosystems are expected to be strongly affected due to warming temperatures and changes in precipitation patterns. The aim of this thesis was to
investigate how distributions of aquatic insects, i.e., benthic stream macroinvertebrates would be impacted by warming climates. The methods comprised of an ensemble forecasting technique based on species distribution models (SDMs) and climate change scenarios of the Intergovernmental Panel on Climate Change of the year 2080. Future model projections were generated for a wide variety of species from a number of taxonomic orders for two spatial scales: a stream network within the lower mountain ranges of Germany, and the entire territory across Europe. In addition, the effect of the modelling technique on habitat suitability projections was investigated by modifying the choice of study area (continuous area vs. stream network) and the choice of predictors (standard vs. corrected set).
Projections of future habitat suitability showed that potential climate-change impacts would be dependent on species’ thermal preferences, and with a similar pattern for both spatial scales. Future habitat suitability was projected to remain for most or all of the modelled species, and species were projected to track their climatically suitable conditions by shifting uphill along the river continuum within the lower mountain ranges, and into a north-easterly direction across Europe. Cold-adapted headwater and high-latitude species were projected to lose suitable habitats, whereas gains would be expected for warm-adapted river and low-latitude species along the river continuum and across Europe, respectively. Additionally, habitat specialist species in terms of endemics of the Iberian Peninsula were identified as potential climate-change losers, highlighting their restricted habitat availability and therefore vulnerability to warming climates.
The main findings of this thesis underline the high susceptibility of stream macroinvertebrates to ongoing climate change, and give insights into patterns of possible consequences due to changes in species’ habitat suitability. Concerning the methodology, a clear recommendation can be given for future modelling approaches of stream macroinvertebrates by building models within a stream network and with a careful choice of environmental predictors, to reduce uncertainties and thus to improve model projections.
The tumor suppressor programmed cell death 4 (Pdcd4) exerts its function by inhibiting protein translation initiation. Specifically, it displaces the scaffold protein eukaryotic initiation factor 4G (eIF4G) from its binding to the eukaryotic initiation factor 4A (eIF4A). Thereby, Pdcd4 inhibits the helicase activity of eIF4A, which is necessary for the unwinding of highly structured 5’ untranslated regions (UTRs) of messenger RNAs (mRNAs) often found in oncogenes like c-myc to make them accessible for the translation machinery and subsequent protein production. Overexpression of Pdcd4 inhibits tumorigenesis in vitro and in vivo and inversely, Pdcd4 knockout mice show enhanced tumor formation. In line, Pdcd4 is lost in various tumor types and proposed as prognostic factor in colon carcinomas. Unlike most other tumor suppressors that are rendered nonfunctional by mutations (e.g., p53), Pdcd4 loss is not attributable to mutational inactivation. It is regulated via translational repression by microRNAs and increased degradation of the protein under tumor promoting, inflammatory conditions and mitogens. Specifically, proteasomal degradation of Pdcd4 is controlled by p70 S6 Kinase (p70S6K)-mediated phosphorylation in its degron sequence (serines 67, 71 and 76). Stimulation of the PI3K-AKT-mTOR pathway by growth factors, hormones and cytokines initiates p70S6K activity. Phosphorylated Pdcd4 is subsequently recognized by the E3 ubiquitin ligase beta-transducin repeats-containing protein (β-TrCP) and marked with a polyubiquitin tail to be detected by the 26S proteasome for degradation. β-TrCP represents the substrate specific recognition subunit of the ubiquitin ligase complex responsible for protein-protein interaction with Pdcd4 as substrate for ubiquitin transfer and subsequent proteasomal disassembly.
The first part of the present work aimed at identifying novel stabilizers of the tumor suppressor Pdcd4 in a high throughput screen (HTS). As assay design, a fragment of Pdcd4 from amino acid 39 to 91, containing the phosphorylation sensitive degron sequence, was fused to a luciferase reporter gene construct. Stable expression of this Pdcd4(39-91)luciferase (Pdcd4(39-91)luc) fusion protein in HEK 293 cells served as read-out for the Pdcd4 protein amount to be detected in a high throughput compatible cell-based assay. Loss of Pdcd4(39-91)luc was induced by treatment with 12-O-
tetradecanoylphorbol-13-acetate (TPA), a phorbolester, which activates the PI3K signaling cascade leading to degradation of Pdcd4. The cut-off for hit definition was set at >50% activity in rescuing the Pdcd4(39-91)luc signal from TPA-induced degradation. Activity was calculated relative to the difference of DMSO- and TPA-treated cells (ΔDMSO-TPA = RLUDMSO-RLUTPA). Initial screening of a protein kinase inhibitor library (PKI) revealed hit substances expected to show Pdcd4 stabilizing activity by inhibition of kinases involved in Pdcd4 downregulation, e.g., the mTOR inhibitor rapamycin, the PI3K inhibitors wortmannin and LY294002 and the PKC inhibitors GF 109203X and Ro 31-8220.
The Molecular Targets Laboratory (MTL) of the National Cancer Institute (NCI) in Frederick, USA, hosts one of the largest collections of crude natural product extracts as well as a big substance libraries from pure synthetic sources. Screening of over 15 000 pure compounds and over 135 000 natural product extracts identified 46 pure and 42 extract hits as Pdcd4 stabilizers. For nine synthetic and six natural product derived compounds (after bioassay-guided fractionation), dose-dependent activities for recovering the TPA-induced Pdcd4(39-91)luc loss defined IC50s in the low micromolar range. Most importantly, these compounds were confirmed to stabilize endogenous Pdcd4 protein levels from forced degradation as well. This result proved the assay design to be highly representative for endogenous cellular mechanisms regulating Pdcd4 protein stability. The next step was to stratify the hit substances according to their likely mechanism of action to be located either up- or downstream of the p70S6K-mediated phosphorylation of Pdcd4. Therefore, phosphorylation of S6, as proto-typical p70S6K target, was analyzed and uncovered two natural derived compounds to influence p70S6K activity. Four substances did not affect p70S6K phosphorylation activity and were therefore considered to stabilize Pdcd4 by acting downstream, i.e. on the β-TrCP-mediated proteasomal degradation.
In the second part of this work, one of these compounds, namely the sesquiterpene lactone erioflorin, isolated by bioassay-guided fraction from the active extract of Eriophyllum lanatum, Asteraceae, was further characterized in detail with respect to its molecular mechanism of action. Erioflorin dose-dependently protected both Pdcd4(39-91)luc and endogenous Pdcd4 protein from TPA-induced degradation with IC50s of 1.28 and 2.64 μM, respectively. Pdcd4 stabilizing activity was maximal at 5 μM erioflorin. Up to this concentration, erioflorin was verified not to inhibit p70S6K activity. In addition, it was observed that erioflorin rescued Pdcd4(39-91)luc from both, wild type and constitutively active p70S6K-mediated downregulation. Only wild type p70S6K was inhibitable by the mTOR inhibitor rapamycin which served as an upstream acting control. To study the next section of Pdcd4 regulation, i.e. recognition by the E3 ubiquitin ligase β-TrCP, Pdcd4(39-91)luc and endogenous Pdcd4 were immunoprecipitated from whole cell extracts with the corresponding antibodies. In this key experiment, treatment with TPA increased overexpressed β-TrCP binding to both and this coimmunoprecipitation could be strongly reduced by erioflorin treatment. This result strongly pointed to an inhibitory mechanism of the β-TrCP specific binding to Pdcd4 by erioflorin. In addition, erioflorin disrupted the binding of in vitro transcribed/translated β-TrCP to Pdcd4 in an in vitro interaction assay to exclude nonspecific intracellular signals. Furthermore, polyubiquitination of Pdcd4 was decreased by erioflorin treatment as well. To clarify questions regarding specificity of erioflorin for the E3 ubiquitin ligase β-TrCP, stability of another important β-TrCP target was explored, i.e. the tumor suppressor inhibitor of kappa B alpha (IκBα). Indeed, the tumor necrosis factor alpha (TNFα)-mediated loss of IκBα could be prevented by erioflorin cotreatment. On the other hand, the E3 ubiquitin ligase von Hippel Lindau protein (pVHL) was left unaffected as its target hypoxia inducible factor 1 alpha (HIF-1α) could not be stabilized from oxygen-dependent degradation by erioflorin treatment. These results argued strongly for erioflorin being a specific inhibitor of β-TrCP-mediated protein degradation. Functional consequences of erioflorin treatment were investigated by observing its influence on the transcriptional activities of the transformation marker activator protein 1 (AP-1, an indirect downstream target of Pdcd4) and nuclear factor κB (NF-κB which is directly inhibited by IκBα). Indeed, erioflorin showed significant inhibition of AP-1 and NF-κB reporter constructs at 5 μM, a concentration for which an impact on cell viability was excluded. Finally to characterize the significance of erioflorin in a cell-based tumorigenesis assay, the highly invasive colon carcinoma cell line RKO was tested in a two dimensional migration assay. Erioflorin was discovered to significantly lower cell migration in a wound closure assay.
In conclusion, development of a high throughput compatible cell-based reporter assay successfully identified novel substances from pure synthetic and natural product derived background as potent stabilizers of the tumor suppressor Pdcd4. In addition, this work aimed at elucidating the detailed mechanism of action of the sesquiterpene lactone erioflorin from Eriophyllum lanatum, Asteraceae. Erioflorin was discovered to inhibit the E3 ubiquitin ligase β-TrCP, thereby preventing protein degradation of tumor suppressors like Pdcd4 and IκBα. This may offer the possibility to more specifically target protein degradation and generate less adverse side effects by blocking a particular E3 ubiquitin ligase compared to general proteasome inhibition.
Identification of translationally deregulated proteins during inflammation-associated tumorigenesis
(2012)
The translation of mRNAs into proteins is an elaborate and highly regulated process. Translational regulation primarily takes place at the level of initiation. During initation the eukaryotic initiation factors (eIFs) form a complex that binds to the 5’end of the mRNA to scan for a start codon. Once recognized, the ribosome is recruited to the mRNA and protein synthesis starts. Initiation of translation can basically occur via two distinct mechanisms, i.e. cap-dependent and cap-independent that is mediated via internal ribosome entry sites (IRESs). The former is mediated by a 5’cap structure composed of a 7-methylguanylate which is added to every mRNA during transcription and recruits the initiation complex. IRES-dependent translation involves elements within the 5’untranslated region (UTR) of the mRNA that mostly bind IRES trans-acting factors (ITAFs) which associate either with the initiation complex or with the ribosome itself and consequently allow for internal initiation of translation.
During tumorigenesis the demand for proteins is increased due to rapid cell growth, which consequently requires enhanced translation. Many factors that regulate translation are overexpressed in tumors. Moreover, signaling pathways that trigger translation or further hyperactivated by the surrounding tumor microenvironment. This environment is largely generated by infiltration of immune cells such as macrophages that secrete cytokines and other mediators to promote tumorigenesis. As the effects of inflammatory conditions on the translation of specific targets are only poorly characterized, my study aimed at identifying translationally deregulated targets during inflammation-associated tumorigenesis.
For this purpose, I cocultured MCF7 breast tumor cells with conditioned medium of activated monocyte-derived U937 macrophages (CM). Polysome profiling and microarray analysis identified 42 targets to be regulated at the level of translation. The results were validated by quantitative PCR and one target - early growth response 2 (EGR2) - was chosen for in depth analysis of the mechanism leading to its enhanced translation.
In order to identify upstream signaling molecules causing enhanced EGR2 protein synthesis the cytokine profile of CM was analyzed and the impact of several cytokines on EGR2 translation was examined. Preincubation of CM with neutralizing antibodies revealed that lowering interleukin 6 (IL-6) had only little effect, whereas depletion of IL 1β significantly reduced EGR2 translation. This finding was corroborated by the fact that treatment with recombinant IL-1β enhanced EGR2 translation to virtually the same extend as CM. Further experiments revealed that this effect was mediated via the p38-MAPK signaling cascade.
Interestingly, I observed that the mTOR inhibitor rapamycin, which reduces cap-dependent translation, specifically stimulated EGR2 translation. This result argued for an IRES-dependent mechanism that might account for EGR2 translation. The use of bicistronic reporter assays verified this hypothesis. In line with the above mentioned results, CM, IL-1β and p38-MAPK induced EGR2-IRES activity.
Since IRESs commonly require ITAFs to mediate translation initiation, the binding of proteins to the 5’UTR was analyzed using mass spectrometry. Among others, several previously described ITAFs, such as polypyrimidine tract-binding protein (PTB) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) were identified to directly bind to the EGR2-5’UTR. Furthermore, overexpression of hnRNP-A1 enhanced EGR2-IRES activity whereas a dominant negative form of hnRNP-A1 significantly decreased it, thus, showing its importance for EGR2 translation.
In summary, my data provide evidence that EGR2 expression can be controlled by IRES-dependent translational regulation, which is responsive to an inflammatory environment. The identified mechanism may not be exclusive for one target but might be representative for gene expression regulation mechanisms during tumorigenesis. This is of special interest for the treatment of cancer patients and development of more specific therapies to reduce tumor outcome.
Chemical contamination of the environment and thus of aquatic ecosystems is steadily increasing. Whenever environmental pollutants enter a water body, they affect not only the water, but also the sediment. Substances that bind to sediment particles can be stored for a long time, whereby sediments act as sinks for some contaminants. Therefore, sediment
assessments often more accurately describe the contamination of a water body than investigations of the water itself. Among environmental chemicals, endocrine disrupting compounds (EDCs) have gained more and more attention in recent years. Since they interfere with endocrine systems and may disturb reproduction, they endanger the survival of populations or even species. Hazardous substances enter the aquatic environment by different pathways, with sewage treatment plants (STPs) belonging to the most important contamination sources.The main objective of this work is a comprehensive sediment assessment of predominantly small surface waters in the German federal state of Hesse. The 50 study sites, located in 44 different creeks and small rivers, are situated in the densely populated and economically important Frankfurt/Rhine-Main area, as well as in rural and less urbanized regions.
Chemical analytical data, provided by the Hessian Agency for the Environment and Geology (HLUG), indicated different contamination levels of the study sites. In order to investigate the general toxicity of the sediment samples, the oligochaete Lumbriculus variegatus and the midge Chironomus riparius were exposed to whole sediments and apical endpoints regarding biomass, survival, and reproduction were determined. In further experiments, special attention was paid to the contamination with endocrine active compounds. For this purpose, the reproductive success of the New Zealand mudsnail Potamopyrgus antipodarum was analyzed after exposure to whole sediments. Additionally, a yeast-based reporter gene assay was applied with sediment eluates to assess the estrogenic and androgenic activity of the samples. Biotest results were compared with chemical analysis data to investigate whether the test organisms reflect the measured pollution of the study sites and if the observed effects can be explained by chemical contamination.
Five study sites, all located less than 1 km downstream of a STP discharger, were selected for further investigations based on the results of the sediment monitoring. The sediments from these sites were conspicuous due to their general toxic and/or estrogenic activity. In order to investigate whether the observed effects can be ascribed to the effluents, an active biomonitoring study was conducted with the mudsnail P. antipodarum and the zebra mussel Dreissena polymorpha, exposed at study sites located up- and downstream of the discharger.
In addition to endocrine activity, genotoxic effects were investigated using the comet assay and the micronucleus assay. Endocrine activity was examined based on the reproductive output of P. antipodarum and the content of vitellogenin-like proteins in D. polymorpha. Yeast-based reporter gene assays were used to estimate the endocrine potential (estrogen, anti-estrogen, anti-androgen, dioxin-like) of sediment and water samples.
22% of the 50 sediments showed ecologically relevant effects in the biotests with L. variegatus and C. riparius. Only one sediment caused a relevant effect on both test organisms, while the other ten positively tested sediments affected either L. variegatus or C. riparius, probably due to differences in inter-species sensitivities. This suggests that a combination of different biotests is necessary for a comprehensive evaluation of sediment toxicity. 78% of the sediments caused a significantly increased number of embryos in P. antipodarum, which could be ascribed to estrogenic contamination of the sediment samples. An increase in the number of embryos by 60%, as observed in this study, and an associated increase in population size may result in the displacement of other, less competitive species.
In the in vitro tests, 66% of the sediments showed estrogenic activity and 68% showed androgenic activity. Maximum observed values were 40.9 ng EEQ/kg sediment (EEQ = estradiol equivalent) for estrogenic and 93.4 ng TEQ/kg sediment (TEQ = testosterone equivalent) for androgenic activity. Natural and synthetic hormones as well as alkylphenols were the major contributors to the total estrogenicity of environmental samples in several other studies, and are likely responsible for a large part of the estrogenic activity in this case as well. Similarly, androgenic activity is mainly due to natural steroids and their metabolites.
Bioassay results reflect the analytically measured contamination levels at the study sites only very infrequently. This can be ascribed to the occurrence of integrated effects of chemical mixtures present in the sediments. Additionally, effects of substances not included in the analytical program or of substances present in concentrations below the detection limit of the chemical analytical investigations as well as varying bioavailabilities might be relevant. The fact that a large part of the observed effects cannot be explained by the chemical contamination demonstrates the need for effect studies in ecotoxicological sediment assessments.
In order to identify possible causes for the effects observed in the sediment monitoring, e.g. contamination sources, the area types (urban fabrics, arable lands, pasturages, etc.) of the catchment areas belonging to the study sites were analyzed. No significant differences were found between the area profiles of the sampling sites with and without effects in the biotests.
The results indicate that the contamination responsible for the observed effects can be ascribed to different sources. Furthermore, study sites whose sediments exerted significant effects in biotests were located in anthropogenic as well as in predominantly natural areas. The active biomonitoring study at STPs revealed genotoxic and endocrine effects only sporadically.
However, in the in vitro tests considerable endocrine activities of sediment and water samples were determined. No conclusive picture emerges as to whether the observed effects occur more frequently downstream of the dischargers, and thus could be attributed to a contamination by sewage. This indicates that contamination sources other than STP dischargers, for example agricultural runoff, may contribute to the observed effects. Weaker effects and biological activities downstream of a discharger compared to an upstream site might be ascribed to a dilution effect by the effluents. A comparison of the measured in vitro estrogenicity with exposure studies described in the literature shows that adverse effects in aquatic organisms can be expected at the EEQ concentrations determined in the present study.
The results of the sediment monitoring and the STP study revealed a widespread endocrine pollution of small surface waters in Hesse. The fact that the bioassay results only rarely reflect study site contamination as determined by chemical analysis demonstrates the need for effect studies in comprehensive sediment assessments. In some cases STP dischargers increased, in other cases they decreased the observed in vivo effects and in vitro activity of environmental samples. Transferring the results obtained in laboratory studies to the field, adverse effects on aquatic ecosystems can be expected. The study illustrates the need for restrictive measures that contribute to the removal or reduction of environmental pollutants.
For the identification of substances that have so far not been linked to adverse effects on the environment, methods such as effect-directed analyses (EDA) or toxicity identification evaluation (TIE) should be increasingly applied in future studies. Furthermore, bioassays for the assessment of endocrine activity should be implemented in standardized monitoring programs.
From the dawn of civilization, a multitude of religious has developed each very complex. These great differences among religions make it difficult to find a least common denominator or to talk at length of religion in general. On the other hand, focusing on just one specific religion often causes us to ignore or underestimate some of the very broad traits that religions seem to share. The fact remains that we will make no headway into the question of what makes a specific religion a religion if we do not seek some characteristics common to all religions.
Religion is usually associated with the supernatural or the divine.505 However, the notion of a supernatural realm does not occur in the non-theistic schools of Buddhism and functions in very different ways, say, in Taoism, Hinduism, and Islam. These shortcomings suggest that religion is notoriously difficult to define. To me, religion is any action taken through every aspect of our being to release us from our weakness or imperfection, and bring us closer to the divine reality. In other words, it is a human inner desire or activity to unite with an absolute being.
The examination of the intellectual dimension of religion that is, its key beliefs is most beneficial when it is guided and informed. Since epistemology is the theory of knowledge, one would therefore expect epistemological discussions of religion to concentrate on the question whether one could have knowledge of religious beliefs. Religious epistemology is simple to say that it is the epistemology of distinctively religious beliefs, but that will not be helpful in the absence of a definition of religious beliefs.
Philosophers of religion might consider the epistemology of religious belief, pondering questions about the sources and justifications of religious knowledge. Fundamental questions regarding the nature of knowledge are likely to arise in any culture. After all, everyone has some stake in distinguishing truth from error, wisdom from ignorance, and the path to knowledge from the path to ignorance. Epistemologists have discussed, in addition to the defining conditions and the sources of knowledge, the extent of human knowledge. They have asked how far human knowledge can extend. Many philosophers find it obvious that we know at least some things, if only things about personal experiences or household physical objects. Others have claimed, however, that we really have no knowledge. Such philosophers admit that people typically feel confident that they have some knowledge, but these philosophers insist that our apparent cases of knowledge are mere illusions. Many epistemologists aim not to set knowledge beyond our reach or to escape the quest for knowledge but rather to make many of our ordinary claims to knowledge more secure by explaining knowledge. They seek to explain what knowledge consists in and how we get it.506
Therefore, a good deal of the task of general epistemology is to understand the nature of the various truth-relevant merits which beliefs can possess, the necessary and sufficient conditions for beliefs to possess those merits, and the virtues of mind and practice requisite and apt for their presence merits such as being reliably formed, being warranted, being entitled, being scientific, being rational, being justified and indeed being true.
Epistemology in Western philosophical tradition has until recently offered a prominent definition of knowledge that analyzes knowledge into three essential components: justification, truth, and belief. According to this analysis, propositional knowledge is, by definition, justified true belief. Epistemologists typically focus on propositional knowledge. Knowledge that something is the case, as opposed to knowledge of how to do something. The content of propositional knowledge can be expressed in a proposition, that is, what is meant by a declarative sentence. Knowledge how to do something is by contrast, a skill or competence in performing a certain task. Within the last few decades, philosophers have discovers that these three conditions are not really sufficient for knowledge; something else is required. Genuine knowledge requires not only truth and belief, but also the satisfaction of the belief condition be appropriately related to the satisfaction of the truth condition. That is, on the traditional approach, genuine knowledge requires that a knower have an “adequate indication” that a believed proposition is true. The required “adequate indication”507 of truth, according to Plato, Kant, and many other philosophers, is evidence indicating that a proposition is true. These philosophers thus hold that knowledge must be based on evidence, or justifying reason.
The kind of justification crucial to knowledge is called epistemic justification. Even if knowledge requires justification, a justified belief can be false. In allowing for justified false beliefs, contemporary epistemologists endorse fallibilism about justification. Reformed epistemologists contend that belief in the existence of God can, in some circumstances, have an epistemic status high enough to render it worthy of acceptance even if it has no support from the arguments of natural theology or from any other beliefs. The views of Alston and Mavrodes are sometimes said to display an affinity with Reformed epistemology, and Nicholas Wolterstorff has made significant contributions to its development. But Plantinga has clearly been the leading contemporary advocate of this school of thought in religious epistemology. During the earlier phase of their development, Plantinga concentrated on defending the view that theistic belief can be in certain conditions, is justified or rationally held even in the absence of any propositional evidence or supporting argument.508
For the conviction that theistic belief is properly basic, foundationalists carry out a reconstructive project that would put our revised doxastic structures on foundations so firm that they could withstand rather than ignore skeptical challenges. That is, for the classical foundationlist theistic belief requires support from propositional evidence or argument if it is to be rationally or justifiably held. However, it is fairly clear that belief in God’s existence does not satisfy this criterion; it is neither self-evident nor incorrigible nor evident to the sense for humans at any time in this life.
The question of justification attracts philosophers especially in contemporary epistemology. And controversy of this question focuses on the meaning of ‘justification’ as well as on the substantive conditions of a belief’s being justified in a way appropriate to knowledge. William Alston, for instance, has introduced a non-deontological normative concept of justification that relies mainly on the notion of what is epistemically good from the view-point of maximizing truth and minimizing falsity. Alston link epistemic goodness to a belief’s being based on adequate grounds in the absence of overriding reason to the contrary. But for some epistemologists “Epistemic justification” means simply “evidential support” of certain sort.509 To say that s is epistemically justifiable to some extent for you is, on this view, just to say that s is supportable to some extent by your overall evidential reason. According to epistemologist, knowledge entails beliefs, so that I cannot know that such and such is the case unless I believe that such and such is the case.
Robert Audi mentioned that knowledge arises in experience. It is constituted by conclusively justified true belief, meaning that: the believer may be justified by psychological certain of the true proposition in question and this proposition is so well-grounded as to be itself propositionally certain. And knowledge constitute by such belief may be called epistemic certainty. When we come to religious knowledge, Audi says that religious propositions are simply beyond the scope of human knowledge. But the point is why would it be thought that no religious propositions are known? The most common ground for holding this view is namely, that religious propositions, such as that God exists, cannot be known either a priori or on the basis of experience. The concept of justification or evidence would occur with the concept of belief in a more complex analysis of the concept of knowledge.510
In recent decades, questions of knowledge seem to have been marginalized by question of justification. As a matter of fact, however, epistemological discussion of religious belief, at least since the Enlightenment has tended to focus, not on the question whether religious belief has warrant, but whether it is justified. More precisely, it has tended to focus on the question whether those properties are enjoyed by theistic belief - the belief that there exists a person like the God of traditional Christianity, Judaism, and Islam: an almighty, all knowing, wholly benevolent and loving spiritual person who has created the world. The main epistemological question about religious belief, therefore, has been the question whether or not religious belief in general and theistic belief in particular is rational or rationally acceptable, or whether it is justified?
In its primary sense, rationality is a normative concept that philosophers have generally tried to characterize in such a way that, for any action, belief, or desire, if it is rational we ought to choose it. Rationality is the use of reason to reach a certain level of reasonableness or unreasonableness. Many positive characterizations of rational beliefs have been proposed by beliefs that are either self-evident or derived from self-evident beliefs by a reliable procedure and beliefs that are consistent with the overwhelming majority of one’s beliefs.511 Analytic epistemology in the twentieth century was conducted as if there were a part from truth itself - just one truth-relevant merit in beliefs, that one typically called either “justification or rationality;”512 and since there was a great deal of disagreement on the answer to that question, there was a great flowering of theories of justification, or theories of rationality.
According to Nicholas Wolterstorff, there are a large number of distinct truth-relevant merits in beliefs, and that neither “Justification” nor “rationality”513 picks out single such merit, both are highly ambiguous terms, each picking out a number of distinct merits. Religious beliefs are formed or evoked by experience of some sort, or by believing what is said in some discourse, or by reflection on the implications of some complex of beliefs that one has previously acquired. Here, we notice that there are no rational activities to understand or justify the experience.
Richard Swinburne considered the nature of actual belief. He saw how in a sense all beliefs given rise to action and must be based on evidence. But he knows that not all beliefs are rational beliefs. For him a beliefs will fail to be rational if it is based on evidence in the wrong way or if it is based on the wrong sort of evidence. According to Swinburne beliefs are rational in so far as they are based on investigation which was, in the believer’s view, adequate, and if the believer believes it to be rational. Swinburne understand by religious beliefs as about transcendent reality, including his belief about whether or not there is a God, and his beliefs about what properties God has (what God is like) and what actions he has performed.514
According to John Hick, the issue is not whether it can be established as an item of indubitable public knowledge that God (or the divine or the Transcendent) exists, or most probably exists, but whether it is rational for those who experience some of life’s moments theistically to believe that God exists, and to proceed to conduct their lives on that basis. Hick looks at a rational belief in general way. For him “belief” can mean a proposition believed or it can be defined as an act or state of believing. The idea of evidence normally presupposes a gap between an observed fact, or body of facts, and an inferred conclusion. Therefore, our ordinary moment - to moment perceptual beliefs contradict the principle that all rational believing must be based upon adequate evidence. It is not that they are based upon inadequate evidence, but rather that the model of evidence-inference-belief does not apply here. Ordinary perceptual beliefs arise directly out of our experience, and it is entirely appropriate, proper, and rational to form these beliefs in this way. The relationship between experience and belief has been much debated in recent work in the philosophy of religion. This discussion has focused upon specifically theistic belief and Hick discusses also in these terms and his argument is that it is rational to believe in the reality of God.
Alvin Plantinga argues on these manifestation-experiences that they are properly basic.515 That is to say, it is as rational for religious persons to hold these basic religious beliefs as it is for all of us to hold our basic perceptual beliefs. But, more basically, it is the biblical assumption translated into philosophical terms. According to Plantinga this experience is what justifies me in holding (the belief) that is the ground of my justification, and by extension, the ground of the belief itself. He then applies this principle to religious beliefs.
In philosophy, experience is generally what we perceive by the senses what we learn from others, or whatever comes from external sources or from inner reflection. In the sense, experience is associated with observation and experiment. Empiricism stresses that our knowledge must be based on experience, but rationalism claims that experience is a potential source of error and prefers rational certainty to mere empirical generalization. In ordinary usage, for every experience there must be something experienced that is independent of the subject of experience. But in philosophy, the relation between experience as a state of consciousness and independent objects of experience becomes a focus of debate. There are many different kinds of experiences, but it is religious experiences that interest me here.
From the point of view of epistemology the modifications of consciousness consisting our apparently perceptual experience are of importantly different kinds. In addition to true perceptions there are misperceptions, illusions, and hallucinations. Therefore, if anyone misled by any of these forms of perceptual error, he or she is then deluded. In each case the delusion consists in a mistaken implicit belief about the cause of the experience: Applying this concept of delusion to the realm of religious experience, we have to ask whether those who assume that their experience of living in God’s presence is caused by their being in God’s presence are believing truly or are, on the contrary, under a delusion.
In modern philosophy of mind a major theme which bears on many theoretical issues, concerns the alleged privacy of an experience as an event knowable only to its possessor and the possibility of public access to that experience. There is much philosophical debate concerning precisely how perception is to be analyzed. In particular, questions are raised concerning the status of the phenomenon. But there is general agreement that in perception, objects present themselves to us in ways that enable us to know them. Similarly, in religious experience God presents himself in ways that enable us to know him and his actions. For Alston there are, it seems, important differences between ordinary perceptual or sense experience and religious experience. Sense perception is a common experience, whereas religious experience is less common, perhaps, even rare, sense perception yields a great deal of information about the world, whereas religious experience yields apparently little information about God, all humans have the capacity for sense perception, but many seem not to have the capacity for religious experience. These differences, however, do not show that religious experience has a structure unlike perception. For one thing, neither the frequency of an experience nor the amount of information it yields tells us anything about its structure.
On the other hand, the limitation of the rationalist way is that the only truths capable of being strictly proved are analytic and ultimately tautological. But we cannot by logic alone demonstrate any matter of fact and existence; these must be known through experience. For sure if nothing were given through experience in its various modes, we should never have anything to reason about. This is as true in religion as in other fields. If God exists, God is not an idea but a reality outside us, in order to be known to men and women, God must therefore become manifest in some way within their experience. This conclusion is in line with the contemporary revolt against the rationalist assumptions which have dominated much of western philosophy since the time of Descartes.516 Descartes held that we can properly be said to know only truths that are self-evident or that can be reached by logical inferences from self-evident premises.
Therefore, those who stress faith and attack reason often place a great deal of emphasis on religious experience. However, religious experience is by no means a purely emotional “happening”; rather, it involves concepts and beliefs about the being that is experienced. If we tried to separate religious experiences from such concepts and beliefs - from the religious belief-system, as we shall call it - then there would be no way saying who or what is that is experienced, or of explaining what sort of difference the experience ought to make to the person who has it. However, such a religious belief system needs to be understood; at least to some degree - it is hard to see how understanding it is not going to involve the use of reason.
For Rationalisms, in order for a religious belief-system to be properly and rationally accepted, it must be possible to prove that the belief-system is true. Rationalism in this sense implies a reliance on reason, or intelligence, in deciding our beliefs and actions. The central idea of strong rationalism was stated forcefully by W. K. Clifford. According to his opinion, no religious belief-system is capable of meeting the high standard of proof that should govern all of our believing, and so a reasonable (and moral) person must do without religious beliefs. Among the objections to Christian belief, as well as to Judaic and Muslim, Characteristic of the modern intelligentsia is the objection that it is no longer rational, if ever it was, to believe that God exists. Therefore, the rational person will have to make his way in the world without supposing that there exists any God in whom he could trust.517
However, according to Nicholas Wolterstorff, to believe in God is our fundamental human obligation. Central to Christianity, Judaism, and Islam alike is the conviction that we as human beings are called to believe in God to trust in him, to rely on him, to place our confidence in him. Central also is the conviction that only by believing in God can the deepest stirrings of the human heart be satisfied. Duty and fulfillment here coalesce. The rationality of trusting someone presupposes the rationality of believing that person exists.
John Locke was among the first to formulate articulately the evidentialist challenge to theistic belief. Reason is reasoning for Locke, and clearly he thinks of it as one among others of our belief-forming processes. Faith is another belief-forming process. It, by contrast, consists in accepting something “as coming from God.”518 However, for Locke it still belongs to reason to judge of the truth of its being a revelation, and of the signification of the words wherein it is delivered.
For evidentialist, one’s belief that God exists is rational only if it is formed or sustained by good inference by inferring it from others of one’s beliefs, which in fact provide adequate evidence for it. But, Wolterstorff, see no reason what so ever to suppose that by the criterion offered the evidentialist challenge is tenable. He see no reason to suppose that people who hold as one of their immediate beliefs that God exists always have adequate reason to surrender that belief or ought to believe that they do. However, for him those abstract and highly general theses of evidentialism no longer look very interesting, once we regard them in the light of the criterion offered. Therefore, for him the fact that it is not rational for some person to believe that God exists does not follow that he ought to give up that belief.
But can we accept the Principle of Credulity? One problem is that whereas there is a fundamental uniformity about the way we report both ordinary perceptual experiences and the beliefs about objects of those experiences, there is quite a diversity of reports about religious experiences and the claim based on them. Person give incompatible descriptions of the Reality experienced. Therefore, where perceptions yield conflicting testimony, we must turn to other experiences and rational arguments to determine the truth of the various claims. That is, where there are different accounts, additional considerations must be introduced to help decide which, if any, of the religious experiences are veridical. Although the reports provide a prima facie ground for their acceptance, not all beliefs based on such experiences are true. Just as we at times doubt perceptual claims for good reason, we might do the same for claims based on religious experience.
According to Hick, religion constitutes our varied human response to transcendent reality. Religious experience then is structured by religious beliefs which are implicit within religious experience. But the question is if this complex of experience and beliefs that takes place in different shapes within the different traditions, is to be regarded simply as a human creation or as our response to a transcendent reality, even if a response whose particular forms always involve the creative activity of the human imagination. Of course the problem of terminology is obvious as we see in many parts of philosophy, and without explanatory gloss none of the available descriptive labels for these two possibilities is entirely adequate.
Much of the philosophical discussion of religious diversity continues to centre on the work of John Hick.519 He is not interested in the question of what can justifiably be affirmed in the face of such diversity, rather, he is primarily concerned with the question of which justified response is most reasonable. Moreover, on this question, he leaves no doubt as to his opinion:
519 See John Hick, An Interpretation of Religion: Human response to the Transcendent, (New Haven: Yale University and London: Macmillan press, 1989), 172.
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religious pluralism is by far the most plausible explanation for the pervasive religious diversity we encounter.
Many Westerners will best understand the emergence of inclusivism and pluralism in terms of the history of Christianity. For most of its history, Christianity has been resolutely exclusivist. In late antiquity, it was a new religion, struggling to establish itself in the face of criticism and persecution. It is not surprising to find it making exclusive claims on behalf of its charismatic founder, Jesus of Nazareth. Of course, Christianity is not the only religion to have fostered exclusivist attitudes. In their more militant movements, Muslims have done the same. Some Jews cherish an ethnically exclusive identity as God’s chosen people, and some Hindus revere the Vedas as a source of absolute truth, Buddhists often see in the teachings of Gautama the only dharma that can liberate humans from illusion and suffering.
Hick has set forth a philosophically sophisticated pluralistic hypothesis that may avoid problems of this sort.520 As he sees it, each of the major religious traditions offers a path to salvation or liberation that involves a transformation of human existence from self-centeredness to reality- centeredness.
Religious plurality is simply a fact. There are religious traditions that differ deeply in terms of their doctrines, practices, institutions, scriptures, experiences, and hope. According to pluralism, a single ultimate religious reality is being differently experienced and understood in all the major religious traditions; they all, as far as the philosophers can tell, offer equally effective paths to salvation or liberation.
According to Harold Coward, Judaism is an appropriate tradition in which to begin the stay of religious pluralism and the world religions. The experience of being a minority group in other cultures, which becoming more common place for all the world religions as religious pluralism spreads, has been the norm for Judaism for countless generations. From the biblical period to the present, Judaism has had to formulate its beliefs and practices in the face of challenges from other cultures and religions. The viewpoint of the modern Jew opens the way for relations with Christianity, Islam, and perhaps Hinduism; however, Buddhism - especially Mahayana Buddhism may prove to be in a separate category. The Buddhist consciousness in which no transcendent God is recognized and the Mahayana awareness of the Divine in the
520 Ibid.
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secular may be judged by the Jewish philosopher as a modern idolatry. Therefore, perhaps the most serious challenge for Judaism comes in its response to Buddhism. As long as a religion is founded on the experience of a transcendent God, Judaism seems to be able to enter into spiritual partnership with it. But if that experience does not hold true for the Buddhist - if it is not a transcendent God that is being experienced - can the Jew still embrace the Buddhist as a spiritual partner? This question has yet to be faced by Judaism.521
The relationship between Christianity and the other religions is one of the key issues in Christian self-understanding. Perhaps pluralism is so pressing a challenge because of the exclusivist missionary approaches adopted by Christianity over the past several hundred years.
In the rapidly expanding body of literature resulting from the encounter with other religions, many Christian theologians are concluding that Christian theology cannot continue to be formulated in isolation from the other religions, and that in fact future developments in Christian theology will be the direct result of serious dialogue with the other religions. Although the Churches are altering their ecclesiology so as to open the way for serious dialogue with other religions, the fundamental Christology that underlies traditional ecclesiology has not yet changed.
Through out the centuries the basic Islamic approach to other religions was to search for some fundamental structures that were harmonious with Islam but which lay hidden beneath the other religion’s deviations from true Islam: A major obstacle for understanding other religions was the lack of accurate information. However, Islam has essentially reached the truth toward which all other religions are evolving. Christianity, it seems, has also virtually reached this goal. It is possible that various nations or cultures will reach the truth in their own way. The Qur’an itself teaches that every community in every age has had its prophet. However, modern education will offer Muslims an opportunity to understand each religion in terms of its own culture, history, world view, and claims to truth. This will have an effect on Islamic self-perception.
Therefore, the religious pluralism of the modern world will force Islam, finding itself in much the same position as the other traditions, to come to grips with them rather provincial
521 See Harold Coward, Pluralism: Challenge to World Religions, Harold Coward, 1-12.
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character of some of its past views of other religions.
According to Hinduism, there is one divine reality that manifests itself in many forms. The various religions are simply different revelations of the one divine reality. Hinduism sees itself as being a very open and tolerant religion. But because it asserts that the Vedas are the most perfect revelation of divine truth, Hinduism also sees itself as providing the criteria against which the revelations of all other religions must be tested. Nevertheless, the Hindu view that there is one Divine, which may be reached by many paths, has proven through out the centuries to be a powerful influence upon Hinduism’s interaction with the other religions.522 Therefore, the Hindu contribution to the modern challenge of religious pluralism is to encourage the inquiring spirit and devotion to truth that is larger than any individual tradition.
The Buddhist attitude to other religions has been described as “critical tolerance”523 combined with a missionary goal. Buddhism has demonstrated a remarkable degree of tolerance and flexibility throughout the course of its expansion. Unlike some other religious expansions, the spread of Buddhism has been accomplished more through the dissemination of ideas than by migration of peoples.
Buddhism rejects the worship of God or gods and the performance of religious rituals as a means to release. It also rejects speculations about ultimate beginnings, especially about whether the self and the world were eternal, and a number of speculations about the ultimate state of the self in the future. The tolerant but critical attitude of the Buddha toward the plurality of religious views is shaped into a rigorous philosophic approach by the Madhyamika Buddhists. If, as the Buddha discovered, the goal of religion is compassion, then, say the Madhyamika, the biggest obstacle to realizing that goal is attachment to our own religious beliefs in such a way as to make them absolute. Based on this understanding, the Madhyamika Buddhist’s attitude towards other religions is one of openness and indeed a “missionary desire”524 to enter into dialogue.
The inherent desire to conceptualize and share religious experience is too deeply ingrained in
522 Ibid., 63-80.
523 See K. N. Jayatilleke, The Buddhist Attitude to Other Religions (Kandy, Sri Lanka: Buddhist Publication Society, 1975). And see also Harold Coward Pluralism: Challenge to World Religions, 81.
524 See Harold Coward, Pluralism: Challenge to World Religions, 88.
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human nature to render silence an acceptable answer. In fact the Madhyamika himself has been far from silent. His prescription of silence was only intended to apply to claims of absolute knowledge. As long as the limitation is honored, then discussion, including theological discussion, could take place.
The dialogical approach opens the way for the meeting of Christians with Jews, Muslims, and Hindus. However, the theocentric premise could become an obstacle to meaningful encounters with Buddhists and Advaita Vedantists. Therefore, an unresolved problem for all of these approaches is the Buddhist and even those with considerable exposure to Buddhism and Hinduism seem almost willfully to turn a blind eye to this problem. One possible exception might be found in Tillich’s formulation of the “god above gods” as the “ground of being.”525
Dialogue starts from the assumption that each religion has its absolute claims which cannot be relativized. No amount of reformulation will do away with the difference. But, by letting our theologizing be influenced by others we will be forced to greater honesty and deeper spirituality. The prerequisite for dialogue is not the harmonizing of all beliefs but the recognition that each spiritual person has a committed and absolute conviction, and that these convictions are different.
Therefore, the expected outcome is not the homogenization of particular religions but the mutual deepening of spiritual experience within each particular religion, which may lead to glimpses of a common transcendent reality. This shift in perspective had the effect of drawing attention to the universal nature of religious experience in its many different traditions. In addition to turning attention away from metaphysics, rationalism, or revelation, the focus on the humanity of religion has had the effect of highlighting some of the limitations in human nature that must be taken seriously in all future religion.
The brain is characterized by its immune privileged state. However, recent studies suggest an extended contribution of hematopoietic cells to the brain. After transplantation of genetically labeled bone marrow into bone marrow depleted mice, not only labeled blood cells but also labeled neurons and other non-hematopoietic cells can be observed. Initially interpreted as transdifferentiated hematopoietic stem cells, this contribution later was identified as cell fusion of hematopoietic cells and neurons. Our lab previously addressed the question whether these fusion events also occur under non-invasive conditions. A Cre-LoxP based transgenic mouse line was used to irreversibly label all hematopoietic cells. In these mice, Cre expression is controlled by a hematopoietic promoter, thus causing recombination and subsequent marker gene expression restricted to blood cells. Interestingly, contribution of these hematopoietic cells to non-hematopoietic tissues was observed, but fusion could be excluded as the underlying mechanism. The Cre mRNA or protein seems to reach the non-hematopoietic cells from an external source. Extracellular vesicles, specifically exosomes, are increasingly recognized as a vehicle for the intercellular transfer of cellular components such as proteins or mRNAs. However, if they contribute to signaling between tissues in vivo is completely unknown and would represent a major paradigm shift for intercellular communication. Therefore, the aim of this PhD study is to investigate whether an exosomal transfer between the hematopoietic system and the brain exists. To confirm the previous results, a second Cre-LoxP mouse line that expresses the Cre recombinase under a different hematopoietic promoter is used additionally. Both mouse lines are screened for recombination and show comparable numbers and types of different non-hematopoietic cells. Besides hepatocytes and cells in lung and intestine, recombined Purkinje neurons in the cerebellum are detectable. To assess the influence of inflammation on these recombination events, different lesions such as peripheral tumors or peritonitis are applied to the mice. Inflammatory stimuli strongly increase the numbers of recombined Purkinje neurons. These neurons remain mononuclear, indicating that fusion does not occur. Also in human cerebellar material, no evidence for inflammation induced cell fusion is detectable. To screen for Cre recombinase containing exosomes, exosome purification protocols such as differential ultracentrifugation and sucrose gradient fractioning, are applied. The exosomal content is analyzed with nested PCR and western blot. Hematopoietically expressed Cre mRNA is detectable in blood plasma and hematopoietic cell culture conditioned medium. Further analysis reveals that this Cre mRNA but no Cre protein is contained in exosomes. The exosomal ability to induce recombination is investigated by injections into Cre reporter mice. After direct cerebellar injection, exosomes are sufficient to induce recombination of Purkinje neurons. Brain tissue of mice that received an inflammation is analyzed further to reveal other recombined cell types. The main immune cells of the brain, microglia, are not recombined. Mainly neuronal cell types are recombined in different areas of the brain. The observations made in this study are consistent with the hypothesis that a previously unrecognized way to communicate RNA based signals between the immune system and the brain exists. Specifically neurons are target cells for the uptake of hematopoietic exosomes and seem able to translate exosomal mRNA into functional protein. Microglial cells are neither involved as target cells, nor do they release Cre containing exosomes. By using the Cre-LoxP system, in vivo tracing of exosomes could be achieved for the first time. With this knowledge, other exosomal routes can be uncovered in future. The discovery of the exosomal transfer between the blood and the brain enables further research about the relevance of this signaling pathway. It will be important to investigate its role especially in the context of neural malfunctions and further studies might help to find new therapeutical approaches.
The adaptive immune system protects against daily infections and malignant transformation. In this, the translocation of antigenic peptides by the transporter associated with antigen processing (TAP) into the ER lumen is an essential step in the antigen presentation by MHC I molecules. The heterodimeric ATP-binding cassette transporter (ABC) TAP consist of the two halftransporters TAP1 and TAP2. Each monomer contains an N-terminal transmembrane domain (TMD) and a conserved C-terminal nucleotide-binding domain (NBD). Together, the TMDs build the translocation core and the NBDs bind and hydrolyze ATP, energizing the peptide transport. TAP features an asymmetry in the two ATP-binding sites that are built of several conserved motifs. One motif is the D-loop with the consensus sequence SALD. The highly conserved aspartate of the D-loop of TAP1 reaches into the canonic ATP-binding site and contacts the Walker A motif and the H-loop of the opposite NBD, while the Asp of D-loop of TAP2 is part of the non-canonic ATP-binding site.
To examine this ABC transport complex in mechanistic detail, a purification and reconstitution procedure was established with the function of TAP being preserved. The heterodimeric TAP complex was purified via a His10-tag at TAP1 in a 1:1 ratio of the subunits. Nucleotide binding to the purified transporter was elucidated by tryptophan quenching assays and the affinity constants for MgADP and MgATP were determined to be 1.0 μM and 0.7 μM, respectevely. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. Furthermore, TAP was reconstituted into proteoliposomes and the activity was tested by peptide transport and ATP hydrolysis. Interestingly, the kinetic parameters of the transporter in the reconstituted state are comparable to the data gained for TAP in microsomes.
To characterize the functional importance of the D-loop, D-loop mutants of either TAP1 or TAP2 were analyzed. Strikingly, TAP containing a mutated D-loop in TAP1 (D674A) shows an ATP-hydrolysis independent peptide translocation. Accordingly, the MHC I surface expression is similar to the wildtype situation. However, the same mutation in TAP2 (D638A) results in an ATPase dependent peptide transport similar to wildtype, whereas TAP containing mutations in both subunits leads to an inactive transporter. Although all D-loop mutants showed no altered peptide binding activity, the TAP1 mutant is inactive in peptide-stimulated ATPase activity. Strikingly, ATP or ADP binding is strictly required for the peptide translocation. Experiments carried out in proteoliposomes demonstrate that wildtype TAP can export peptides against their gradient when low peptide concentrations are offered. In contrast, the D674A mutant can facilitate peptide translocation along their concentration gradient in the two directions. At high peptide concentrations, TAP is trapped in a transport incompetent state induced by trans-inhibition. In conclusion, a TAP mutant that uncouples solute translocation from ATP hydrolysis was created. Since this passive substrate movement is strictly dependent on binding of ATP or ADP, an active transporter was turned into a “nucleotide-gated facilitator”.
In a cysteine cross-linking approach the conformational changes of TAP during peptide transport and the flexibility of the nucleotide binding domains were examined. Single cysteines were introduced in the D-loops of TAP1 and TAP2. Cross-linking by copper-phenantroline (CuPhe) was possible for all combinations. However, by adding ATP, ADP or peptide to the TAP complex no differences in the cross-linking efficiency were detected. By CuPhe cross-linking TAP was trapped in a conformation, in which the peptide binding site was not accessible. To complete a transport cycle, a flexibility of at least 17.8 Å of the NBDs is needed, since TAP cross-linked by CuPhe (2.0 Å) or bismaleimidoethane (BMOE, 8.0 Å) was transport inactive but when TAP was cross-linked by 1,11-bismaleimido-triethyleneglycol (BM[PEG]3, 17.8 Å) transport activity was preserved.
5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Here, the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD) was investigated. In the present study, the identification of two truncated transcripts and four novel 5-LO splice variants containing premature termination codons (PTC) was reported. The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
RT-PCR analysis of different cell types revealed the existence of a large number of 5-LO splice variants. The most interesting splice variants were observed in BL41-E95A cells, which give a raise to novel 5-LO protein isoforms. This leads to the hypothesis of a novel regulatory mechanism in which the dimerization of 5-LO with 5-LO isoforms might regulate the 5-LO activity.
The 5-LO protein expression was reduced on translational level in UPF1 knock down cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry based proteomics study was started to identify compartment specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis demonstrated that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (~ 21%) but not in the soluble fraction (< 1%). Western blot data confirmed the trend of the proteomics analysis. This data suggest that UPF1 is a critical gene expression regulator in a compartment specific way. During differentiation by TGFβ and calcitriol the majority of UPF1 regulated proteins was adjusted to normal level. It appears that that not only the NMD mechanism alters its composition during differentiation. Also the gene expression regulation on translational level by UPF1 seems to be also cell differentiation dependent. An interesting group of UPF1 target genes represent the downregulated proteins. qRT-PCR analysis of randomly chosen genes revealed no effect on mRNA expression upon UPF1 knockdown, suggesting that UPF1 positively influences the translation of these genes. Computational sequence analysis identified a conserved C-rich sequence which might be a hnRNP E2-binding site. hnRNP E2 has been characterized as a translational repressor in myeloid cells. Western blot analysis revealed a differentiation independent up regulation of hnRNP E2 by UPF1 knockdown. Additionally, microRNA-328 (miR-328) has been described as an RNA decoy modulating hnRNP E2 regulation. Due to this, stem loop qRT-PCR showed an up regulation of miR-328 in TGFβ and calcitriol differentiated MM6 cells. Based on this data we suggest a model in which downregulation of UPF1 increases hnRNP E2 expression, leading to translation inhibition. During differentiation, miRNA-328 is upregulated thereby competing with hnRNP E2 leading to an efficient translation
A new era in experimental nuclear physics has begun with the start-up of the Large Hadron Collider at CERN and its dedicated heavy-ion detector system ALICE. Measuring the highest energy density ever produced in nucleus-nucleus collisions, the detector has been designed to study the properties of the created hot and dense medium, assumed to be a Quark-Gluon Plasma.
Comprised of 18 high granularity sub-detectors, ALICE delivers data from a few million electronic channels of proton-proton and heavy-ion collisions.
The produced data volume can reach up to 26 GByte/s for central Pb–Pb
collisions at design luminosity of L = 1027 cm−2 s−1 , challenging not only the data storage, but also the physics analysis. A High-Level Trigger (HLT) has been built and commissioned to reduce that amount of data to a storable value prior to archiving with the means of data filtering and compression without the loss of physics information. Implemented as a large high performance compute cluster, the HLT is able to perform a full reconstruction of all events at the time of data-taking, which allows to trigger, based on the information of a complete event. Rare physics probes, with high transverse momentum, can be identified and selected to enhance the overall physics reach of the experiment.
The commissioning of the HLT is at the center of this thesis. Being deeply embedded in the ALICE data path and, therefore, interfacing all other ALICE subsystems, this commissioning imposed not only a major challenge, but also a massive coordination effort, which was completed with the first proton-proton collisions reconstructed by the HLT. Furthermore, this thesis is completed with the study and implementation of on-line high transverse momentum triggers.
Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and by their plasticity promote all phases of tumor development. Mechanisms of macrophage (M Phi) attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced immunosuppressive interleukin 10 (IL-10) production, are largely unknown. However, a high apoptosis index within tumors and strong M Phi infiltration correlate with poor prognosis. Thus, I aimed at identifying signaling pathways contributing to generation of TAM-like M Phi by using supernatant of apoptotic cancer cells (ACM) as stimulus.
To distinguish novel factors involved in generating TAM-like M Phi, I used an adenoviral RNAi-based approach. The primary read-out was production of IL-10. However, mediators modulating IL-10 were re-validated for their impact on regulation of the cytokines IL-6, IL-8 and IL-12. Following assay development, optimization and down-scaling to a 384-well format, primary human M Phi were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, followed by activation to a TAM-like phenotype using ACM. I identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of 22 targets regulating IL-10 and IL-6. Principal validation of five targets of the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors. Among those, IL-4 receptor-alpha and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion.
One protein identified in the screen, the nerve growth factor (NGF) receptor TRKA was chosen for in-depth validation, based on its involvement in IL-10, IL-6 and IL-12 secretion from ACM-stimulated human M Phi. TRKA possesses a cardinal role in neuronal development, but compelling evidence emerges suggesting participation of TRKA in cancer development. First experiments using pharmacological inhibitors principally confirmed the involvement of TRKA in IL-10 secretion by ACM-stimulated M Phi and revealed PI3K/AKT and to a lesser extend MAPK p38 as important signaling molecules downstream of TRKA activation. Signaling through TRKA required the presence of its ligand NGF, as indicated by NGF neutralization experiments. NGF was not induced by or present in ACM, but was constitutively secreted by M Phi. Interestingly, M Phi responded to authentic NGF with neither AKT and p38 phosphorylation nor IL-10 production. TRKA is well known to be transactivated by other receptors and in neurons its cellular localization is decisive for its function. Inhibitors of common transactivation partners did not influence IL-10 production by human M Phi. Rather, ACM-treatment provoked pronounced translocation of TRKA to the plasma membrane within 10 minutes as observed by immunofluorescence staining. Consequently, I was intrigued to clarify mechanisms of TRKA trafficking in response to ACM.
The bioactive lipid sphingosine-1-phosphate (S1P) has been previously identified as important apoptotic cell-derived mediator involved in TAM-like M Phi polarization. Indeed, I observed S1P and src kinase involvement in ACM-mediated IL-10 induction. Furthermore, inhibition of S1P receptor (S1PR) signaling or src kinase activity prevented TRKA translocation, whereas a TRKA inhibitor or anti-NGF did not block TRKA trafficking to the plasma membrane in response to ACM. Thus, autocrine secreted NGF activated TRKA to promote IL-10 secretion, which required previous S1PR/src-dependent translocation of TRKA to the plasma membrane. Following the detailed analysis of IL-10 regulation, I was interested whether other TAM phenotype markers were influenced by ACM and whether their expression was regulated through TRKA-dependent signaling. Five of six markers were up-regulated on mRNA level by ACM, and secretion of IL-6, IL-8 and TNF-alpha was triggered. S1PR-signaling was essential for induction of all but one marker, whereas TRKA signaling was only required for cytokine secretion. Interestingly, none of the investigated TAM markers was regulated identically to IL-10, emphasizing a tight and exclusive regulation machinery of this potent immunosuppressive cytokine.
Finally, I aimed to validate the in vitro findings in human ACM-stimulated M Phi. Therefore, I isolated murine TAM as well as other major mononuclear phagocyte populations from primary oncogene-induced breast cancer tissue. Indeed, TRKA-dependent signaling was required for spontaneous cytokine production selectively by primary murine TAM. Besides IL-10, the TRKA pathway was decisive for secretion of IL-6, TNF-alpha and monocyte chemotactic protein-1, indicating its relevance in cancer-associated inflammation.
In summary, my findings highlight a fine-tuned regulatory system of S1P-dependent TRKA trafficking and autocrine NGF signaling in TAM biology. Both factors, S1P as well as NGF, might be interesting targets for future cancer therapy.
Um der Erkennung durch das körpereigene Immunsystem entkommen, weisen Tumore Modifikationen in ihrer Mikroumgebung auf. Zu diesen gehören u. a. veränderte Sauerstoffkonzentrationen im Tumorkern und die Freisetzung biochemischer Faktoren aus Tumorzellen, welche die Funktion von Tumor-assoziierten Phagozyten, wie z.B. Dendritischen Zellen (DC) beeinflussen. DC sind professionelle Antigen-präsentierende Zellen, die eine Spezialisierung in verschiedene funktionale Subtypen aufweisen. Myeloische DC (mDC) sind besonders effizient in Hinsicht auf die Präsentation von Antigenen, wohingegen plasmazytoide DC (pDC) regulatorisch auf das Immunsystem einwirken. Beide Subtypen spielen eine wichtige Rolle bei der Karzinogenese.
Während humane mDC, zur therapeutischen Verwendung, ex vivo aus Monozyten hergestellt werden können, war dies für humane pDC bisher nicht möglich. Ein war deshalb ein erstes Ziel dieser Arbeit, ein Protokoll zur Generierung humaner pDC aus humanen Monozyten zu entwickeln. Diese wurden mittels des Wachstumsfaktors Fms-related tyrosine kinase 3 ligand (Flt3-L) zu pDC-Äquivalenten differenziert, welche als monocyte-derived pDC (mo-pDC) bezeichnet wurden. In der Tat zeigten mo-pDC ein für humane pDC charakteristisches Oberflächenmarkerprofil und wiesen, im Vergleich zu mDC, eine geringe Kapazität zur Induktion der Proliferation autologer T Zellen und zur Phagozytose apoptotischer Zellen auf. Mo-pDC erwarben im Verlauf ihrer Differenzierung aus Monozyten eine kontinuierlich erhöhte Expression des pDC-spezifischen Transkriptionfaktors E2-2 und seiner spezifischen Zielgene. Der wichtigste funktionale Parameter von pDC ist die Produktion großer Mengen von Interferon-α (IFN-α). Mo-pDC sezernierten, nach vorheriger Aktivierung mit Tumornekrosefaktor-α (TNF-α) oder wenn zu ihrer Differenzierung neben Flt3-L auch Vitamin D3 oder all-trans-Retinolsäure verwendet wurde, ebenfalls große Mengen IFN-α. Wurden mo-pDC unter Hypoxie, einem prominenten Faktor der Tumormikroumgebung, generiert, so waren die Expression des spezifischen Transkriptionsfaktors E2-2 und die Freisetzung von IFN-α stark vermindert. Diese Daten zeigten zunächst, dass mo-pDC für das Studium von Differenzierung und Funktion humaner pDC eingesetzt werden können.
Weiterhin lieferten sie Hinweise auf eine veränderte Differenzierung humaner pDC unter Hypoxie. In einem nächsten Schritt wurde folglich untersucht, ob Hypoxie auch die Differenzierung von pDC aus deren physiologischen Vorläufern beeinflusst. Wurden Knochenmarkszellen der Maus mit Flt3-L unter Normoxie oder Hypoxie kultiviert, so war die Differenzierung zu pDC unter Hypoxie in der Tat unterdrückt. Dies war abhängig von der Hypoxie-induzierten Aktivität des Hypoxie-induzierten Faktors 1 (HIF-1), da die Flt3-Linduzierte Differenzierung von murinen Knochenmarkszellen, in denen die Expression von HIF-1 in pDC-Vorläuferzellen ausgeschaltet war, unter Hypoxie normal verlief.
Zusammenfassend kann also gesagt werden, dass Hypoxie, durch Aktivierung von HIF-1, Differenzierung und Funktion von pDC unterdrückt. Dieser Mechanismus könnte zu ihrer beschriebenen Dysfunktion in humanen Tumoren beitragen.
Neben Hypoxie sind viele andere Faktoren an der Immunsuppression in Tumoren beteiligt.
Eine Komponente der Mikroumgebung in Tumoren ist das Vorhandensein apoptotischer Tumorzellen. Apoptose von Tumorzellen findet, im Kontrast zur generellen Sicht von Tumoren als Apoptose-resistente Entitäten, auch in unbehandelten Tumoren im Überfluss statt. Apoptotische körpereigene Zellen unterdrücken unter physiologischen Bedingungen das Immunsystem. Deshalb könnte das Freisetzen von apoptotischem Material oder die Sekretion von Faktoren aus sterbenden Tumorzellen einen starken Einfluss auf die Funktion von Tumor-assoziierten DC und die damit verbundene Aktivierung von tumoriziden Lymphozyten haben. Eine diesbezügliche Studie war das zweite Ziel der vorliegenden Arbeit. Humane mDC wurden zu diesem Zweck mit Überständen lebender, apoptotischer oder nekrotischer humaner Brustkrebszellen aktiviert und anschließend mit autologen T Zellen ko-kultiviert. Danach wurde das zytotoxische Potential der ko-kultivierten T Zellen analysiert. Interessanterweise unterdrückte die Aktivierung mit Überständen apoptotischer Tumorzellen die DC-vermittelte Generierung tumorizider T Zellen durch die Ausprägung einer Population von regulatorischen T Zellen (Treg), die durch die gleichzeitige Expression der Oberflächenmoleküle CD39 und CD69 charakterisiert war. Die Ausprägung der CD39-und CD69-exprimierenden Treg Zell-Population war abhängig von der Freisetzung des bioaktiven Lipids Sphingosin-1-Phosphat (S1P) aus apoptotischen Zellen, welches durch den S1P-Rezeptor 4 zur Freisetzung des immunregulatorischen Zytokins IL-27 aus mDC führte.
Neutralisierung von IL-27 in AC-aktivierten Ko-Kulturen von mDC und T Zellen blockierte die Generierung von CD39- und CD69-exprimierenden Treg Zellen und resultierte folglich in der Aktivierung zytotoxischer T Zellen. Weiterhin war die Bildung von Adenosin in den Ko-Kulturen für die Unterdrückung zytotoxischer T Zellen vonnöten. Erste Experimente lieferten Hinweise auf eine direkte Interaktion von CD69- und CD39-exprimierenden Treg Zellen mit CD73-exprimierenden zytotoxischen T Zellen. CD39 und CD73 werden für die Bildung von Adenosin aus ATP benötigt, weswegen die Interaktion von Treg Zellen und zytotoxischen T Zellen die Adenosin-Produktion fördern könnte.
Zusammenfassend zeigen die hier präsentierten Befunde wie Faktoren der
Tumormikroumgebung die Funktion von humanen DC Subtypen beeinflussen können. Ein Verständnis der zugrundeliegenden Mechanismen kann wertvolle Informationen für die Wahl effektiver Immuntherapien oder Chemotherapien liefern und so die Therapie humaner Tumore unterstützen.
With increasing heterogeneity of modern hardware, different requirements for 3d applications arise. Despite the fact that real-time rendering of photo-realistic images is possible using today’s graphics cards, still large computational effort is required. Furthermore, smart-phones or computers with older, less powerful graphics cards may not be able to reproduce these results. To retain interactive rendering, usually the detail of a scene is reduced, and so less data needs to be processed. This removal of data, however, may introduce errors, so called artifacts. These artifacts may be distracting for a human spectator when gazing at the display. Thus, the visual quality of the presented scene is reduced. This is counteracted by identifying features of an object that can be removed without introducing artifacts. Most methods utilize geometrical properties, such as distance or shape, to rate the quality of the performed reduction. This information used to generate so called Levels Of Detail (LODs), which are made available to the rendering system. This reduces the detail of an object using the precalculated LODs, e.g. when it is moved into the back of the scene. The appropriate LOD is selected using a metric, and it is replaced with the current displayed version. This exchange must be made smoothly, requiring both LOD-versions to be drawn simultaneously during a transition. Otherwise, this exchange will introduce discontinuities, which are easily discovered by a human spectator. After completion of the transition, only the newly introduced LOD-version is drawn and the previous overhead removed. These LOD-methods usually operate with discrete levels and exploit limitations of both the display and the spectator: the human.
Humans are limited in their vision. This ranges from being unable to distinct colors at varying illumination scenarios to the limitation to focus only at one location at a time. Researchers have developed many applications to exploit these limitations to increase the quality of an applied compression. Some popular methods of vision-based compression are MPEG or JPEG. For example, a JPEG compression exploits the reduced sensitivity of humans regarding color and so encodes colors with a lower resolution. Also, other fields, such as auditive perception, allow the exploitation of human limitations. The MP3 compression, for example, reduces the quality of stored frequencies if other frequencies are masking it. For representation of perception various computer models exist. In our rendering scenario, a model is advantageous that cannot be influenced by a human spectator, such as the visual salience or saliency.
Saliency is a notion from psycho-physics that determines how an object “pops out” of its surrounding. These outstanding objects (or features) are important for the human vision and are directly evaluated by our Human Visual System (HVS). Saliency combines multiple parts of the HVS and allows an identification of regions where humans are likely to look at. In applications, saliency-based methods have been used to control recursive or progressive rendering methods. Especially expensive display methods, such as pathtracing or global illumination calculations, benefit from a perceptual representation as recursions or calculations can be aborted if only small or unperceivable errors are expected to occur. Yet, saliency is commonly applied to 2d images, and an extension towards 3d objects has only partially been presented. Some issues need to be addressed to accomplish a complete transfer.
In this work, we present a smart rendering system that not only utilizes a 3d visual salience model but also applies the reduction in detail directly during rendering. As opposed to normal LOD-methods, this detail reduction is not limited to a predefined set of levels, but rather a dynamic and continuous LOD is created. Furthermore, to apply this reduction in a human-oriented way, a universal function to compute saliency of a 3d object is presented. The definition of this function allows to precalculate and store object-related visual salience information. This stored data is then applicable in any illumination scenario and allows to identify regions of interest on the surface of a 3d object. Unlike preprocessed methods, which generate a view-independent LOD, this identification includes information of the scene as well. Thus, we are able to define a perception-based, view-specific LOD. Performance measures of a prototypical implementation on computers with modern graphic cards achieved interactive frame rates, and several tests have proven the validity of the reduction.
The adaptation of an object is performed with a dynamic data structure, the TreeCut. It is designed to operate on hierarchical representations, which define a multi-resolution object. In such a hierarchy, the leaf nodes contain the highest detail while inner nodes are approximations of their respective subtree. As opposed to classical hierarchical rendering methods, a cut is stored and re-traversal of a tree during rendering is avoided. Due to the explicit cut representation, the TreeCut can be altered using only two core operations: refine and coarse. The refine-operation increases detail by replacing a node of the tree with its children while the coarse-operation removes the node along with its siblings and replaces them with their parent node. These operations do not rely on external information and can be performed in a local manner. These only require direct successor or predecessor information. Different strategies to evolve the TreeCut are presented, which adapt the representation using only information given by the current cut. These evaluate the cut by assigning either a priority or a target-level (or bucket) to each cut-node. The former is modelled as an optimization problem that increases the average priority of a cut while being restricted in some way, e.g. in size. The latter evolves the cut to match a certain distribution. This is applied in cases where a prioritization of nodes is not applicable. Both evaluation strategies operate with linear time complexity with respect to the size of the current TreeCut.
The data layout is chosen to separate rendering data and hierarchy to enable multi-threaded evaluation and display. The object is adapted over multiple frames while the rendering is not interrupted by the used evaluation strategy. Therefore, we separate the representation of the hierarchy from the rendering data. Due to its design, this overhead imposed to the TreeCut data structure does not influence rendering performance, and a linear time complexity for rendering is retained. The TreeCut is not only limited to alter geometrical detail of an object. The TreeCut has successfully been applied to create a non-photo-realistic stippling display, which draws the object with equal sized points in varying density. In this case the bucket-based evaluation strategy is utilized, which determines the distribution of the cut based on local illumination information. As an alternative, an attention drawing mechanism is proposed, which applies the TreeCut evaluation strategies to define the display style of a notification icon. A combination of external priorities is used to derive the appropriate icon version. An application for this mechanism is a messaging system that accounts for the current user situation.
When optimizing an object or scene, perceptual methods allow to account for or exploit human limitations. Therefore, visual salience approaches derive a saliency map, which encodes regions of interest in a 2d map. Rendering algorithms extract importance from such a map and adapt the rendering accordingly, e.g. abort a recursion when the current location is unsalient. The visual salience depends on multiple factors including the view and the illumination of the scene. We extend the existing definition of the 2d saliency and propose a universal function for 3d visual salience: the Bidirectional Saliency Weight Distribution Function (BSWDF). Instead of extracting the saliency from 2d image and approximate 3d information, we directly compute this information using the 3d data. We derive a list of equivalent features for the 3d scenario and add them to the BSWDF. As the BSWDF is universal, also 2d images are covered with the BSWDF, and the calculation of the important regions within images is possible.
To extract the individual features that contribute to visual salience, capabilities of modern graphics card in combination with an accumulation method for rendering is utilized. Inspired from point-based rendering methods local features are summed up in a single surface element (surfel) and are compared with their surround to determine whether they “pop out”. These operations are performed with a shader-program that is executed on the Graphics Processing Unit (GPU) and has direct access to the 3d data. This increases processing speed because no transfer of the data is required. After computation, each of these object-specific features can be combined to derive a saliency map for this object. Surface specific information, e.g. color or curvature, can be preprocessed and stored onto disk. We define a sampling scheme to determine the views that need to be evaluated for each object. With these schemes, the features can be interpolated for any view that occurs during rendering, and the according surface data is reconstructed. These sampling schemes compose a set of images in form of a lookup table. This is similar to existing rendering techniques, which extract illumination information from a lookup. The size of the lookup table increases only with the number of samples or the image size used for creation as the images are of equal size. Thus, the quality of the saliency data is independent of the object’s geometrical complexity. The computation of a BSWDF can be performed either on a Central Processing Unit (CPU) or a GPU, and an implementation requires only a few instructions when using a shader program. If the surface features have been stored during a preprocess, a reprojection of the data is performed and combined with the current information of the object. Once the data is available, the computation of the saliency values is done using a specialized illumination model, and a priority for each primitive is extracted. If the GPU is used, the calculated data has to be transferred from the graphics card. We therefore use the “transform feedback” capabilities, which allow high transfer rates and preserve the order of processed primitives. So, an identification of regions of interest based on the currently used primitives is achieved. The TreeCut evaluation strategies are then able to optimize the representation in an perception-based manner.
As the adaptation utilizes information of the current scene, each change to an object can result in new visual salience information. So, a self-optimizing system is defined: the Feedback System. The output generated by this system converges towards a perception-optimized solution. To proof the saliency information to be useful, user tests have been performed with the results generated by the proposed Feedback System. We compared a saliency-enhanced object compression to a pure geometrical approach, common for LOD-generation. One result of the tests is that saliency information allows to increase compression even further as possible with the pure geometrical methods. The participants were not able to distinguish between objects even if the saliency-based compression had only 60% of the size of the geometrical reduced object. If the size ratio is greater, saliency-based compression is rated, on average, with higher score and these results have a high significance using statistical tests. The Feedback System extends an 3d object with the capability of self-optimization. Not only geometrical detail but also other properties can be limited and optimized using the TreeCut in combination with a BSWDF. We present a dynamic animation, which utilizes a Software Development Kit (SDK) for physical simulations. This was chosen, on the one hand, to show the universal applicability of the proposed system, and on the other hand, to focus on the connection between the TreeCut and the SDK. We adapt the existing framework, and include the SDK within our design. In this case, the TreeCut-operations not only alter geometrical but also simulation detail. This increases calculation performance because both the rendering and the SDK operate on less data after the reduction has been completed.
The selected simulation type is a soft-body simulation. Soft-bodies are deformable in a certain degree but retain their internal connection. An example is a piece of cloth that smoothly fits the underlying surface without tearing apart. Other types are rigid bodies, i.e. idealistic objects that cannot be deformed, and fluids or gaseous materials, which are well suited for point-based simulations. Any of these simulations scales with the number of simulation nodes used, and a reduction of detail increases performance significantly. We define a specialized BSWDF to evaluate simulation specific features, such as motion. The Feedback System then increases detail in highly salient regions, e.g. those with large motion, and saves computation time by reducing detail in static parts of the simulation. So, detail of the simulation is preserved while less nodes are simulated.
The incorporation of perception in real-time rendering is an important part of recent research. Today, the HVS is well understood, and valid computer models have been derived. These models are frequently used in commercial and free software, e.g. JPEG compression. Within this thesis, the Tree-Cut is presented to change the LOD of an object in a dynamic and continuous manner. No definition of the individual levels in advance is required, and the transitions are performed locally. Furthermore, in combination with an identification of important regions by the BSWDF, a perceptual evaluation of a 3d object is achieved. As opposed to existing methods, which approximate data from 2d images, the perceptual information is directly acquired from 3d data. Some of this data can be preprocessed if necessary, to defer additional computations during rendering. The Feedback System, created by the TreeCut and the BSWDF, optimizes the representation and is not limited to visual data alone. We have shown with our prototype that interactive frame rates can be achieved with modern hardware, and we have proven the validity of the reductions by performing several user tests. However, the presented system only focuses on specific aspects, and more research is required to capture even more capabilities that a perception-based rendering system can provide.
Global climate change and land use change will not only alter entire ecosystems and biodiversity patterns, but also the supply of ecosystem services. A better understanding of the consequences is particularly needed in under-investigated regions, such as West Africa. The projected environmental changes suggest negative impacts on nature, thus representing a threat to the human well-being. However, many effects caused by climate and land use change are poorly understood so far. Thus, the main objective of this thesis was to investigate the impact of climate and land use change on vegetation patterns, plant diversity and important provisioning ecosystem services in West Africa. The three different aspects are separately explored and build the chapters of this thesis. The findings help to improve our understanding of the effects of environmental change on ecosystems and human well-being. In the first study, the main objectives were to model trends and the extent of future biome shifts in West Africa that may occur by 2050. Also, I modelled a trend in West African tree cover change, while accounting for human impact. Additionally, uncertainty in future climate projections was evaluated to identify regions with reliable trends and regions where the impacts remain uncertain. The potential future spatial distributions of desert, grassland, savanna, deciduous and evergreen forest were modelled in West Africa, using six bioclimatic models. Future tree cover change was analysed with generalized additive models (GAMs). I used climate data from 17 general circulation models (GCMs) and included human population density and fire intensity to model tree cover. Consensus projections were derived via weighted averages to: 1) reduce inter-model variability, and 2) describe trends extracted from different GCM projections. The strongest predicted effect of climate change was on desert and grasslands, where the bioclimatic envelope of grassland is projected to expand into the Sahara desert by an area of 2 million km2. While savannas are predicted to contract in the south (by 54 ± 22 × 104 km2), deciduous and evergreen forest biomes are expected to expand (64 ± 13 × 104 km2 and 77 ± 26 × 104 km2). However, uncertainty due to different GCMs was particularly high for the grassland and the evergreen forest biome shift. Increasing tree cover (1–10%) was projected for large parts of Benin, Burkina Faso, Côte d’Ivoire, Ghana and Togo, but a decrease was projected for coastal areas (1–20%). Furthermore, human impact negatively affected tree cover and partly changed the direction of the projected climate-driven tendency from increase to decrease. Considering climate change alone, the model results of potential vegetation (biomes) showed a ‘greening’ trend by 2050. However, the modelled effects of human impact suggest future forest degradation. Thus, it is essential to consider both climate change and human impact in order to generate realistic future projections on woody cover. The second study focused on the impact and the interplay of future (2050) climate and land use change on the plant diversity of the West African country Burkina Faso. Synergistic forecasts for this country are lacking to date. Burkina Faso covers a broad bioclimatic gradient which causes a similar gradient in plant diversity. Thus, the impact of climate and land use change can be investigated in regions with different levels of species richness. The LandSHIFT model from the Centre of Environmental System research CESR (Kassel, Germany) was adapted for this study to derive novel regional, spatially explicit future (2050) land use simulations for Burkina Faso. Additionally, the simulations include different assumptions on the technological developments in the agricultural sector. Oneclass support vector machines (SVMs), a machine learning method, were performed with these land use simulations together with current and future (2050) climate projections at a 0.1° resolution (cell: ~ 10 × 10 km). The modelling results showed that the flora of Burkina Faso will be primarily negatively impacted by future climate and land use changes. The species richness will be significantly reduced by 2050 (P < 0.001, paired Wilcoxon signed-rank test). However, contrasting latitudinal patterns were found. Although climate change is predicted to cause species loss in the more humid regions in Southern Burkina Faso (~ 200 species per cell), the model projects an increase of species richness in the Sahel. However, land use change is expected to suppress this increase to the current species diversity level, depending on the technological developments. Climate change is a more important threat to the plant diversity than land use change under the assumption of technological stagnation in the agricultural sector. Overall, the study highlights the impact and interplay of future climate and land use change on plant diversity along a broad bioclimatic gradient in West Africa.Furthermore, the results suggest that plant diversity in dry and humid regions of the tropics might generally respond differently to climate and land use change. This pattern has not been detected by global studies so far. Several of the plant species in West Africa significantly contribute to the livelihoods of the population. The plants provide so-called non-timber forest products (NTFPs), which are important provisioning ecosystem services. However, these services are also threatened by environmental change. Thus, the third study aimed at developing a novel approach to assess the impacts of climate and land use change on the economic benefits derived from NTFPs. This project was carried out in cooperation with Katja Heubach (BiK-F) who provided data on household economics. These data include 60 interviews that were conducted in Northern Benin on annual quantities and revenues of collected NTFPs from the three most important savanna tree species: Adansonia digitata, Parkia biglobosa and Vitellaria paradoxa. The current market prices of the NTFPs were derived from respective local markets. To assess current and future (2050) occurrence probabilities of the three species, I calibrated niche-based models with climate data (from Miroc3.2medres) and land use data (LandSHIFT) at a 0.1° resolution (cell: ~ 10 × 10 km). Land use simulations were taken from the previous study on plant diversity. Three different niche-based models were used: 1) generalized additive models (regression method), 2) generalized boosting models (machine learning method), and 3) flexible discriminant analysis (classification method). The three model simulations were averaged (ensemble forecasting) to increase the robustness of the predictions. To assess future economic gains and losses, respectively, the modelled species’ occurrence probabilities were linked with the spatially assigned monetary values. Highest current annual benefits are obtained from V. paradoxa (54,111 ± 28,126 US$/cell), followed by P. biglobosa (32,246 ± 16,526 US$/cell) and A. digitata (9,514 ± 6,243 US$/cell). However, in the prediction large areas will lose up to 50% of their current economic value by 2050. Vitellaria paradoxa and Parkia biglobosa, which currently reveal the highest economic benefits, are heavily affected. Adansonia digitata is negatively affected less strongly by environmental change and might regionally even supply increasing economic benefits, in particular in the west and east of the investigation area. We conclude that adaptive strategies are needed to create alternative income opportunities, in particular for women that are responsible for collecting the NTFPs. The findings provide a benchmark for local policy-makers to economically compare different land use options and adjust existing management strategies for the near future. Overall, this thesis improves our understanding of the impacts of climate and land use changes on West African vegetation patterns, plant diversity and provisioning ecosystem services. Climate change had spatially varying impacts (positive and negative effects) on the vegetation cover and plant diversity, while predominantly negative effects resulted from human pressure. Regional contrasting impacts of environmental change were also found considering the provisioning ecosystem services.
This dissertation is concerned with the role of prosody and, specifically, linguistic rhythm for the syntactic processing of written text. My aim is to put forward, provide evidence for, and defend the following claims:
1. While processing written sentences, readers make use of their phonological knowledge and generate a mental prosodic-phonological representation of the printed text.
2. The mental prosodic representation is constructed in accordance with a syntactic description of the written string. Constraints at the interface of syntax and phonology provide for the compatibility of the syntactic analysis and the (mental) prosodic rendition of the sentence.
3. The implicit prosodic structure readers impose on the written string entails phonological phrasing and accentuation, but also lower level prosodic features such as linguistic rhythm which emerges from the pattern of stressed and unstressed syllables.
4. Phonological well-formedness conditions accompany and influence the process of syntactic parsing in reading from the very beginning, i.e. already at the level of recognizing lexical categories. At points of underspecified syntactic structure, syntactic parsing decisions may be made on the basis of phonological constraints alone.
5. In reading, the implicit local lexical-prosodic information may be more readily available to the processing mechanism than higher-level discourse structural representations and consequently may have more immediate influence on sentence processing.
6. The process of sentence comprehension in reading is conditioned by factors that are geared towards sentence production.
7. The interplay of syntactic and phonological processes in reading can be explained with recourse to a performance-compatible competence grammar.
The evidence from three reading experiments supports these points and suggests a model of grammatical competence in which constraints from various domains (syntax, semantics, pragmatics, discourse structure, and phonology) interact in providing the possible structural, i.e. grammatical descriptions.
The importance of RNA in molecular and cell biology has long been underestimated. Besides transmitting genetic information, studies of recent years have revealed crucial tasks of RNA especially in gene regulation. Riboswitches are natural RNA-based genetic switches and known only for ten years. They directly sense small-molecule metabolites and regulate in response the expression of the corresponding metabolic genes. Within recent years, artificial riboswitches have been developed that operate according to user-defined demands. Hence, they represent powerful tools for synthetic biology.
This study focused on the development of engineered catalytic riboswitches for conditional gene expression in eukaryotes. A self-cleaving hammerhead ribozyme was linked to a tetracycline binding aptamer in order to regulate ribozyme cleavage allosterically with tetracycline. By integrating such a hybrid molecule into a gene of interest, mRNA cleavage and thereby gene expression is controllable in a ligand dependent manner. The linking domain between ribozyme and aptamer was randomised. Tetracycline inducible ribozymes were isolated after eleven cycles of in vitro selection (SELEX). 80% of the analysed ribozymes show cleavage that strongly depends on tetracycline. In the presence of 1 μM tetracycline, their cleavage rates are comparable to that of the parental hammerhead ribozyme. In the absence of tetracycline, cleavage rates are inhibited up to 333-fold. The allosteric ribozymes bind tetracycline with similar affinity and specificity as the parental aptamer. Ribozyme cleavage is fully induced within minutes after addition of tetracycline. Interestingly, the isolated linker domains exhibit structural consensus motives rather than consensus sequences.
When transferred to yeast, three switches reduced reporter gene expression by 30 - 60% in the presence of tetracycline; none of them controlled gene expression in mammalian cells. In vitro selected molecules do not necessarily retain their characteristics when applied in a cellular context. Therefore, high throughput screening and selection systems have been developed in mammalian cells. The screening system is based on two fluorescent reporter proteins (GFP and mCherry). 1152 individual constructs of the selected ribozyme pool were tested, but none of them reduced reporter gene expression significantly in the presence of tetracycline. The selection system employs a fusion peptide encoding two selection markers (Hygromycin B phosphotransferase and HSV thymidine kinase) facilitating both negative and positive selection. 6.5 x 104 individual constructs of the selected ribozyme pool are currently under investigation.
Nuclear Magnetic Resonance ("NMR") is a powerful and versatile technique relying on nuclei that posses a spin. Since its discovery more than 6 decades ago, NMR and related techniques has become a tool with innumerable applications throughout the fields of Physics, Chemistry, Biology and Medicine. Numerous Nobel Prizes have been awarded for work in the field and a multi billion dollar industry has developed on its basis.
One of NMR's major shortcomings is its inherent lack of sensitivity. Because it relies on the Boltzmann populations of spin states with a minuscule Zeeman splitting, this is particularly true for room temperature experiments.
As a result, in an enormous technological effort to enlarge the Zeeman splitting NMR magnets have been moving to higher and higher magnetic fields. However, even for proton spins possessing the largest magnetic moment of all nuclei, the degree of polarization that can be achieved in the strongest spectroscopic magnets available today (~24 T) at room temperature is merely ~ 8*(10 exp (-5)). In other words, this low polarization theoretically allows a sensitivity enhancement of 104 towards full polarization.
Since Magnetic Resonance Imaging ("MRI") is based on the same principle, it shares this problem with NMR. Furthermore, for technical and physiological reasons full body MRI tomographs do not reach the magnetic field strengths of spectroscopic NMR magnets, making this even more of an issue for MRI.
In consequence, MRI is chiefly restricted to detecting protons, while both MRI and NMR detection of 13C (or other low nuclei) under physiological conditions, i.e. low natural abundance of 13C and a low concentration of the respective substance, suffer from long acquisitions times that are necessary to obtain adequate signal to noise ratios ("SNR").
However, this drawb of NMR can be overcome. The enormous potential sensitivity increase of four orders of magnitude can - at least partially - be exploited by several hyperpolarization techniques, creating entirely new applications and fields of research.
These hyperpolarization techniques comprise chemical approaches like Parahydrogen Induced Polarization ("PHIP") or Photochemically Induced Dynamic Nuclear Polarization ("Photo-CIDNP"), as well as physical techniques like optically pumped (noble) gases13, 14 or Dynamic Nuclear Polarization ("DNP"), which will be the focus of this work. A hyperpolarized substance will render a larger signal without being physically or chemically altered in any other way. It is therefore "marked" without any marker, making it an agent free contrast agent for MRI.
DNP is a technique, in which hyperpolarization of nuclear spins is achieved by microwave (\MW") irradiation of unpaired electron spins in radicals, which are coupled to these nuclei, e.g. 1H, 13C or 15N. The electron spin population is perturbed if the microwave irradiation is resonant with the electron spin transition, which affects the polarization of hyperfine-coupled close nuclei. For large microwave power (i.e. saturating the electron spin transition) the orders of magnitude larger thermal electron spin polarization is effectively transferred to these nuclear spins in the sample. For proton spins the maximum polarization gain amounts to 660, whereas for 13C the sensitivity gain can be as large as 2600. In contrast to e.g. PHIP, which is restricted to specific reaction precursors, DNP is not limited to specific nuclei or hyperpolarization target molecules, making it a very versatile technique. DNP has been first proposed by Overhauser in 1953,15 and experimentally observed shortly thereafter in metals16 and liquids,17 both being systems with mobile electrons. In the 1960s and 70s, DNP was used as a spectroscopic tool in liquids, thoroughly mapping the effect in the low field regime. As well, several other transfer mechanisms were discovered, which are active in the solid state with localized electrons, namely the solid effect the cross effect and thermal mixing. The theory for all three of these mechanisms predicts reduced transfer efficiencies at higher magnetic fields. This fact and the lack of high frequency microwave sources to excite electron spins at magnetic field strengths above 1 T, effectively relegated DNP to a position of an interesting scientifi curiosity.
In the early 1990s, DNP came to a renaissance, when DNP was performed at high field in solid state magic angle spinning ("MAS") experiments using high power gyrotron microwave sources. This pioneering work sparked a surge of new developments and applications.
As well, this success triggered attempts to investigate also the potential of DNP in the liquid state at high magnetic fields, e.g. at 3.4 T35{38 and 9.2 T. To date, DNP can be considered one of the "hot topics" in the field of magnetic resonance, bringing about special issue in magnetic resonance journals and DNP sections on magnetic resonance conferences.
This thesis deals with the development of an in-bore liquid state DNP polarizer for MRI applications operating in ow through mode at a magnetic field strength of 1.5 T. Following this introductory chapter, the theoretical background necessary to understand and interpret the experimental results is explained in chapter 2. Subsequently, chapter 3 deals with the issue of performing liquid state DNP at high magnetic fields and its challenges. The chapter comprises a quick overview of the necessary hardware, the experimental findings for various samples and the interpretation of these findings. along with the ramifications for the aim of this work. Chapter 4 deals with the issue of increasing sensitivity and contrast in MRI, in particular by means of DNP. The chapter illustrates the development of our polarizer by presenting the hardware that was developed and demonstrating its performance under various conditions. As well, several alternative approaches are introduced and compared to our approach. Finally, chapter 5 summarizes the findings and gives an outlook on further developments.
The main purpose of the Transition Radiation Detector (TRD) located in the central barrel of ALICE (A Large Ion Collider Experiment) is electron identification for separation from pions at momenta pt > 1 GeV/c, since in this momentum range the measurements of the specific energy loss (dE/dx) of the Time Projection Chamber (TPC) is no longer sufficient. Furthermore, it provides a fast trigger for high transverse momentum charged particles (pt > 3 GeV/c) and makes a significant contribution to the optimization of the tracking of reaction products in heavy-ion collisions. Its whole setup comprises 18 supermodules out of which 13 are presently operational and mounted cylindrically around the beam axis of the Large Hadron Collider (LHC). A supermodule contains either 30 or 24 chambers, each consisting of a radiator for transition radiation creation, a drift and an amplifying region followed by the read-out electronics. In total, the TRD is an array of 522 chambers operated with about 28 m3 of a Xe-CO2 [85-15%] gas mixture. During the work of this thesis, the testing, commissioning, operation and maintenance of detector parts, the gas system and its online quality monitor, improvements on the detector control user-interface and studies about a new pre-trigger module for data read-out have been accomplished. The TRD gas system mixes, distributes and circulates the operational gas mixture through the detector. Its overall optimization has been achieved by minimizing gas leakage, surveying, controlling, maintaining and continuously improving it as well as designing and carrying out upgrades. Gas quality monitors of the type \GOOFIE" (Gas prOportional cOunter For drIfting Electrons) can be used in gaseous detectors as on-line monitors of the electron drift velocity, gain and gas properties. One of these devices has been implemented within the TRD gas system, while another one surveys the gas of the TPC. Both devices had to be adapted to the specific needs of the detectors, were under constant surveillance and control, and needed to be further developed on both hardware and software side. To improve the operation of the TRD, modifications on its DCS software (Detector Control System) used for monitoring, controlling, operating, regulating and configuring of hardware and computing devices have been carried out. The DCS is designed to enable an operator to interact with equipment through user interfaces that display the information from the system. The main focus of this work was laid on the optimization of the usability and design of the user interface. The front-end electronics of the TRD require an early start signal (\pre-trigger") from the fast forward detectors or the Time-Of-Flight detector during the running periods. The realization of a new hardware concept for the read-out of the TRD pre-trigger system has been studied and first tests were performed. This new module called PIMDDL (Pre-trigger Interface Module Detector Data Link) is meant to acquire all data necessary to simulate and predict the full pre-trigger functionality, and to verify its proper operation. Furthermore, it shall provide all functionalities of the so-called Control Box Bottom as well as keep the functionalities of the already existing PIM (Pre-trigger Interface Module) in order to combine and replace these two modules in the future.
Grave visitation and concepts of life after death : a comparative study in Frankfurt and Hong Kong
(2012)
Grave visitation is a tradition common to many cultures. Yet, this sensitive topic is rarely addressed in cross cultural comparisons. Why do people visit the graves of their parents? What do they do in the cemetery? Could there be a similar set of intentions behind the diverse customs? By examining the visiting patterns in Frankfurt and Hong Kong, this research is aimed at comparing the concepts of life after death that underlie the practice. Phenomenologically oriented, this is an exploratory study based on qualitative interviews. Integrated with in-depth semi-structure interviewing and thematic analysis, the project covered twelve cases in each city. Research participants were purposefully selected. Data analysis was conducted according to the analytical framework approach. After identifying and clustering of themes, three central and interlocking issues were found: 1. the grave as a new home that connects the living and the dead; 2. death and the interpretation of hope; and 3.intergenerational reciprocity and continuing bonds. Though the images of life after death were ambiguously depicted, grave tending reflected shared expectations of the world beyond. Most significantly, visits to the graves strengthened the ties between the living and the dead, revealing a longing for a continued bond regardless of the forms of burial. At the end, this research illustrated not only the meanings of death but also the notion of religiosity through evaluating the secularisation thesis. Emphasising the dynamics of tradition and personal experience, this contextual reading of current death rituals serves as an original source for religious dialogue and education.
Die Bromeliaceae umfassen mehr als 3.100 fast ausschließlich neotropische Arten. Bekannt für ihre außergewöhnliche ökologische Vielseitigkeit haben sich Bromelien erfolgreich in terrestrischen und epiphytischen Lebensräumen ausgebreitet.
Eine umfassende Untersuchung des Gefährdungsgrades aller Bromelienarten Panamas und Costa Ricas stand bisher noch aus und ist insbesondere aufgrund des großen Reichtums an Lebensräumen, der beide Länder auszeichnet, und den vielfältigen Veränderungen geboten.
Im Rahmen der vorliegenden Arbeit wurden während der insgesamt etwa achtmonatigen Feldarbeit 54 Exkursionen in Westpanama durchgeführt und Belege von 61% (126 Arten) der für Panama bekannten Arten gesammelt.
Auf der Basis der Feldarbeit und der in verschiedenen Herbarien durchgeführten Studien (Überprüfung und Digitalisierung von > 8.000 Aufsammlungen) wurden Diversität, Endemismus, Areale und räumliche Muster der Artenvielfalt der Bromeliaceae in Panama und Costa Rica erfasst, dokumentiert und analysiert.
Nur drei der derzeit bekannten acht Unterfamilien der Bromeliaceae finden sich in Panama und vier in Costa Rica. Zwanzig Arten werden hier erstmals für Panama gemeldet. Sechs bisher für Panama gemeldete Bromelienarten wurden als irrtümlich gemeldet identifiziert. Die Flora der Bromeliaceae umfasst nun 16 Gattungen und 206 Arten in Panama sowie 18 Gattungen und 199 Arten in Costa Rica.
33 Arten sind endemisch in Panama, 32 Arten in Costa Rica und 36 Arten sind auf das Gebiet beider Länder beschränkt. Die Gattung Werauhia hat ihr Diversitätszentrum in Panama (47 von insgesamt 87 Arten) und Costa Rica (59/87 Arten) und ist gleichzeitig die artenreichste Gattung in beiden Ländern.
In Panama treten 113 Arten (54,9 %) zwischen 1.000 und 2.000 Höhenmetern auf. Die Art mit der niedrigsten Höhengrenze ist Pitcairnia halophila, die am höchsten angetroffene Art ist Werauhia ororiensis.
Für jede der für Panama und Costa Rica (259 Arten) gemeldeten Bromelienarten wurde eine Verbreitungskarte erstellt; für die in beiden Ländern auftretenden 191 Arten wurde darüber hinaus die potenzielle Verbreitung modelliert.
In Panama ist der prämontane Regenwald mit 138 Arten (einschließlich 25 der insgesamt 33 endemischen Arten) die Holdridge-Vegetationszone mit der höchsten Anzahl an Bromelien. In Costa Rica hat der untere Bergregenwald einen besonders hohen Anteil endemischer Bromelien (13 von insgesamt 32 Arten).
In Panama und Costa Rica beherbergen mittlere Höhenlagen den größten Artenreichtum der Bromeliaceae mit Maximalwerten von etwa 125 Arten im Osten Costa Ricas und in Westpanama. Einige Regionen Panamas verfügen nicht über ausgewiesene Schutzgebiete, weisen jedoch einen hohen Artenreichtum an Bromelien auf (z.B. Teile Westpanamas, El Valle de Anton und benachbarte Gebiete sowie die Serranía de Cañazas).
In der hier vorgestellten Klassifizierung des Gefährdungsgrades gemäß den Richtlinien der IUCN werden für Panama 32 Arten als vom Aussterben bedroht (CR), 36 Arten als Stark Gefährdet (EN) und 36 Arten als Gefährdet (VU) eingestuft. In Costa Rica wird Aechmea aquilega als Ausgestorben (EX) eingeschätzt. Vier Arten werden als vom Aussterben bedroht (CR), 30 Arten als Stark Gefährdet (EN) und 39 Arten als Gefährdet (VU) klassifiziert.
In Panama wurden 184 Arten (89% der insgesamt 206 Arten) in Schutzgebieten nachgewiesen. 122 Arten (59%) wurden sowohl innerhalb als auch außerhalb und 19 Arten (9%) nur außerhalb von Schutzgebieten nachgewiesen. In Costa Rica kommen 182 Bromelienarten (91% der insgesamt 199 Arten) in Schutzgebieten vor, 168 Arten (84%) wurden sowohl innerhalb als auch außerhalb und 14 Arten (7%) nur außerhalb von Schutzgebieten nachgewiesen.
Die Schätzungen zeigen, dass die zu erwartende Gesamtzahl der Bromelienarten in Panama zwischen 224 und 250 Arten liegt, und die zu erwartende Gesamtzahl der Bromelienarten in Costa Rica liegt zwischen 207 und 221 Arten. Den Ergebnissen der Modellierung zufolge wird für eine Anzahl bisher nur für Costa Rica gemeldeter Arten das Auftreten in Panama mit erheblicher Wahrscheinlichkeit prognostiziert (z.B. Guzmania blassi, Werauhia ampla), wie auch umgekehrt das Vorkommen bisher nur für Panama bekannter Arten in Costa Rica (z.B. Aechmea strobilina, Pitcairnia kressii).
Der Erhalt der bestehenden Schutzgebiete sollte ein vorangiges Ziel sein. Darüber hinaus ist es wünschenswert, einige dieser Gebiete auszudehnen und neue Schutzgebiete auszuweisen, um biologisch hochdiverse Gebiete mit einem hohen Anteil endemischer Arten zu schützen.