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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.