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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.