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This thesis comprises the usage of two commonly known hinge-binding moieties in drug discovery. First, the quinazoline scaffold of gefitinib (5) was utilized in a macrocyclization strategy to introduce selectivity. In general, the quinazoline hinge-binding moiety is a commonly used scaffold which can be found in 14% of approved kinase inhibitors. The most familiar applications are EGFR inhibitors such as gefitinib (5), erlotinib (6), afatinib, or dacomitinib for the treatment of NSCLC. But other kinases like CDK2, CDK4, or p38 are reported targets as well.
The N-phenylquinazolin-4-amine moiety of gefitinib (5) was conserved however, the residues at the aromatic ring in the linker were modified, the residue targeting the solvent-exposed region was varied, and the linker at the C6 position of the quinazoline was adjusted to enable the macrocyclization. An overview of the structural modifications is shown in Figure 35A.
Kinome-wide screening of gefitinib (5) revealed several off-targets besides EGFR (Figure 35B), making it an excellent starting point for a macrocyclization strategy. Introducing a linker to the N phenylquinazoline-4-amine scaffold and retaining the residues on the aromatic ring as well as the methoxy group targeting the solvent-exposed region improved the selectivity profile and the efficacy towards EGFR WT and its mutants. Truncation of the linker moiety led to the mutant selective macrocycle 26f with an excellent kinome-wide selectivity profile (Figure 35B). An inhibitor that is effective on EGFR mutations while ineffective on the EGFR WT could represent an enhancement of patient treatment, as it potentially causes less side effects. Further studies could determine the effect of the most promising macrocycles in lung cancer cell lines. Additionally, the pharmacokinetic properties could be optimized, e.g. by introducing solubilizing groups, targeting the solvent-exposed region.
The second scaffold comprises the 3-aminopyrazole-based hinge-binding moiety. It is a privileged scaffold in medicinal chemistry for the development of kinase inhibitors. Previous publications report the anti-proliferative and anti-cancer potential of pyrazole-based molecules. They play a crucial role in the treatment of various diseases and cancer types like inflammation disorders, lymphoma, or breast cancer. This scaffold can be found e.g. in the aurora kinase inhibitor tozasertib or in the promiscuous kinase inhibitor 23, published by Statsuk et. al. Rescreening compound 23 in a comprehensive kinase panel against 468 human protein kinases confirmed the unselective behavior with a selectivity score of S35 = 0.56 (Figure 36B), making it a great starting point for further optimizations. The N-(1H-pyrazol-3-yl)pyrimidin-4-amine scaffold was conserved however, the residues targeting the solvent-exposed region were varied and different linkers were attached.
The introduction of different residues at the pyrazole dramatically influenced the selectivity profile of the desired kinases. Ester moieties caused to a favorable combination of selectivity and potency towards the kinase of interest CDK16. The removal of additional residues at the pyrimidine, targeting the solvent-exposed region, increased the efficiency towards CDK16. Further optimization led to the highly potent and selective CDK16 inhibitor 98d (IC50 = 33 nM). NanoBRETTM screening against the complete CDK family revealed a preferred inhibition of the PCTAIRE and PFTAIRE subfamily with cellular IC50 values of 20 nM – 120 nM and 50 nM – 180 nM, respectively. A FUCCI cell cycle assay and viability assessment of 98d confirmed previously published results, reporting a G2/M cell cycle arrest followed by apoptosis and accumulation of p27 through knockout of CDK16 in SCC cells. Consequently, further studies could evaluate the anti-tumor activity of 98d in SCC and NSCLC or elucidate the effect of 98d in AMPK-related macroautophagy. 98d represents a novel tool compound to investigate the understudied kinases of the PCTAIRE family and enable to enlighten the biological role of those kinases.
Macrocyclization of the N-(1H-pyrazol-3-yl)pyrimidin-4-amine core resulted in the selective BMPR2 inhibitor 110a. It showed a good binding affinity towards BMPR2 with a KD value of 205 nM as well as a good potency with an IC50 value of 506 nM. A comprehensive selectivity screen against 468 kinases revealed an excellent selectivity profile with S35 = 0.01. As no BMPR2 inhibitors have been published so far, 110a represents a novel compound that may provide further insights into the canonical BMP pathway, noncanonical signaling, or its impact on BMPR2-associated diseases like PAH.
The introduction of additional residues targeting the solvent-exposed region shifted the selectivity towards the MST kinases. The exchange from the pyrimidine to a quinazoline moiety resulted in the highly potent and selective macrocyclic MST3 inhibitor 113c. NanoBRETTM measurements demonstrated the preferred inhibition of MST3 with IC50 values of 210 nM and 30 nM for intact and lysed cells, respectively. A weaker activity could be seen for MST4 with 1.8 µM and 510 nM, while MST1 and MST2 were not affected. To date, no selective MST3 inhibitors have been published, making 113c a valuable tool compound for further functional studies. As MST3 is influencing the cell cycle progression, 113c could be tested in a further cell cycle assay to elucidate the inhibitory effect of 113c on MST3 and consequently on the cell cycle. Furthermore, the anti-tumor activity of 113c in breast cancer could be determined, as Madsen et. al. reported a high MST3 and MST4 activity triggered by FAM40B mutations.
One of the main things that we as humans do in our lifetime is the recognition and/or classification of all kind of visual objects. It is known that about fifty percentage of the neocortex is responsible for visual processing. This fact tells us that object recognition (OR) is a complex task in our and in the animal brain, but we do it in a fraction of a second.
The main question is: How does the brain exactly do it? Does the brain use some feature extraction algorithm for OR tasks? The hierarchical structure of the visual cortex and studies on a part of the visual cortex called V1 tell us that our brain uses feature extraction for OR tasks by Gabor filters. We also use our previous knowledge in object recognition to detect and recognize the objects which we never saw before. Also, as we grow up we learn new objects faster than before.
These facts imply that the visual cortex of human and other animals uses some common (universal) features at least in the first stages to distinguish between different objects. In this context, we might ask: Do universal features in images exist, such that by using them we are able to efficiently recognize any unknown object? Is it necessary to extract new special features for any new object? How about using existing features from other tasks for this? Is it possible to efficiently use extracted feature of a specific task for other tasks? Are there some general features in natural and non-natural images which can also be used for specific object recognition? For example, can we use extracted features of natural images also for handwritten digit classification?
In this context, our work proposes a new information-based approach and tries to give some answers to the questions above. As a result, in our case we found that we could indeed extract unique features which are valid in all three different kinds of tasks. They give classification results that are about as good as the results reported by the corresponding literature for the specialized systems, or even better ones.
Another problem of the OR task is the recognition of objects, independently of any perception changes. We as humans or also animals can recognize objects in spite of many deformations (e.g. changes in illumination, rotation in any direction or angles, distortion and scaling up or down) in a fraction of a second. When observing an object which we never saw, we can imagine the rotated or scaled up objectin our mind. Here, also the question arises: How does the brain solve this problem? To do this, does the brain learn some mapping algorithm (transformation), independent of the objects or their features?
There are many approaches to model the mapping task. One of the most versatile ones is the idea of dynamically changing mappings, the dynamic link mapping (DLM). Although the dynamic link mapping systems show interesting results, the DLM system has the problem of a high computational complexity. In addition, because it uses the least mean squared error as risk function, the performance for classification is also not optimal. For random values where outliers are present, this system may not work well because outliers influence the mean squared error classification much more than probability-based systems. Therefore, we would like to complete the DLM system by a modified approach.
In our contribution, we will introduce a new system which employs the information criteria (i.e. probabilities) to overcome the outlier problem of the DLM systems and has a smaller computational complexity. The new information based selforganised system can solve the problem of invariant object recognition, especially in the task of rotation in depth, and does not have the disadvantage of current DLM systems and has a smaller computational complexity.
Systematisch verabreichte Chemotherapeutika sind oft uneffektiv bei der Behandlung von Krankheiten des zentralen Nervensystems (ZNS). Eine der Ursachen hierfür ist der unzureichende Arzneistoff-Transport ins Gehirn aufgrund der Blut-Hirn-Schranke. Eine der Strategien für den nicht-invasiven Wirkstoff-Transport ins Gehirn ist die Verwendung von Nanopartikeln. Polybutylcyanoacrylat-Nanopartikel, die mit Polysorbat 80 (Tween® 80) überzogen wurden, können die Blut-Hirn-Schranke passieren und somit Wirkstoffe ins Gehirn transportieren. Wird die Blut-Hirn-Schranke durch einen Hirntumor partiell beschädigt und hierdurch ihre Permeabilität am Ort des Tumors erhöht, können Nanopartikel den Tumor zusätzlich durch den sogenannten EPR-Effekt erreichen. Im ersten Teil der vorliegenden Arbeit wurde die Beladung der Nanopartikel durch Variation der Formulierungparameter mit dem Ziel optimiert, eine Formulierung mit höherer Wirksamkeit für die Therapie von Glioblastom-tragenden Ratten zu entwickeln. Außerdem wurde das Potential von Doxorubicin, das an mit „Stealth Agents“ überzogenen Polybutylcyanoacrylat-Nanopartikel gebunden war, für die Chemotherapie von Hirntumoren untersucht. Im zweiten Teil dieser Studie wurden die Gehirn- und Körperverteilung in gesunden und in Glioblastom-101/8-tragenden Ratten nach i.v.-Gabe von Poly(butyl-2-cyano[3- 14C]acrylat)-Nanopartikeln, die mit Polysorbat 80 beschichtet wurden, und solchen, die noch zusätzlich mit Doxorubicin geladen waren (DOX-14C-PBCA + PS), untersucht. Die Standardformulierung von Doxubicin-Polybutylcyanoacrylat-Nanopartikeln (DOX-NP) wurde durch anionische Polymerisierung von Butylcyanoacrylat in Anwesenheit von DOX hergestellt. Zusätzlich wurden unterschiedliche DOX-NP Formulierungen durch Veränderung der Herstellung produziert. Das therapeutische Potential der Formulierungen wurde in Ratten mit ins Gehirn transplantieren Glioblastom 101/8 untersucht. Neben Polysorbat 80 wurden Poloxamer 188 und Poloxamin 908 als Überzugsmaterial verwendet. Die Resultate ergaben, dass die mit Polysorbat 80 überzogene Standardformulierung am effektivsten war. Die höhere Wirksamkeit von DOX-NP+PS 80 könnte durch die Fähigkeit dieser Träger erklärt werden, den Wirkstoff während eines frühen Stadiums der Tumorentwicklung durch einen Rezeptor-vermittelten Mechanismus, der durch den PS 80-Überzug aktiviert wurde über die intakte Blut-Hirn-Schranke, zu transportieren. Unsere Ergebnisse zeigen auch, dass Poloxamer 188 und Poloxamin 908 den antitumoralen Effekt von DOX-PBCA beträchtlich verbessern. Der anti-tumorale Effekt dieser Formulierungen könnte möglicherweise dem EPR-Effekt zugeschrieben werden. Es ist bekannt, dass die tumorale Arzneistoff-Aufnahme durch den EPR-Effektes für lang-zirkulierende Wirkstoffträger ausgeprägter ist und so mehr Wirkstoff durch die Tumor-geschädigte Blut-Hirn-Schranke gelangt. Unbeschichtete Nanopartikel, Polysorbat 80-beschichtete Nanopartikel oder mit Doxorubicin beladene und mit Polysorbat 80 beschichtete Nanopartikel wurden in gesunden und Tumor-tragenden Ratten injiziert. Diese Nanopartikel-Präparationen zeigten einer unterschiedliche Korpenverteilung in den Ratten. Unbeschichtete Nanopartikel sammelten sich in den RES-Organen an. Mit PS 80 beschichtete NP reduzierten die Aufnahme der NP in Leber und Milz, während sich die Konzentration der NP in der Lunge erhöhte. Diese Beobachtungen deuten darauf hin, dass die Änderung der Oberflächeneigenschaften der NP durch das Tensid, zu einer Interaktion mit unterschiedlichen Opsoninen führt, welches die Aufnahme der NP von verschiedenen phagozitierenden Zellen erleichtert. Hingegen war die Aufnahme der mit DOX beladenen, PS 80-beschichteten Nanopartikel den unbeschichteten Partikel ähnlich. Im Vergleich mit gesunden Ratten und mit Tumor-tragenden Ratten hingegen war die Konzentration der NP im Gehirn von Tumor tragenden Ratten 10 Tage nach der Tumor-implantation signifikant höher. In Anwesenheit des Glioblastoms ist der Transport von NP in das Gehirn das Resultat verschiedener Faktoren: zusätzlich zur Fähigkeit von PS 80-Nanopartikeln, die Blut-Hirn-Schranke zu passieren, extravasieren diese Träger wegen des EPR Effekts über das durch den Tumor undichte Endothelium. Die Konzentration von PS 80 [14C]-PBCA NP war im Glioblastom signifikant höher als mit DOX [14C]-PBCA NP. Dieses Phänomen kann durch die unterschiedliche Mikroumgebung von zerebralem intra-tumoralen und intaktem Gehirngewebe erklärt werde. Insbesondere können sich die positive Ladung der tumoralen Regionen und die positive Ladung der DOX [14C]-PBCA NP negativ beeinflussen. Dennoch waren die Doxorubicin-Konzentration in Glioblastom ausreichend, einen therapeutischen Effekt zu ermöglichen.
Chapter I of this work addressed the piggyBac (PB) transposon system, a non-viral genome engineering tool that is capable of efficiently performing stable integration of DNA sequences into a target cells genome and has already been used in clinical trials. However, the PB transposase has the problematic property of preferentially integrating transposons near transcriptional start sites (TSSs). This increases the likelihood of causing genotoxic effects, limiting its potential use as a tool in clinical applications. It has been shown in the past that the PB transposase shows physical interactions with BET proteins (e.g. BRD4) through Co-IP experiments. Representatives of these proteins are part of the transcriptional activation complex and are abundant at TSSs. Accordingly, it was previously proposed that this interaction is the underlying cause for the biased integration preference. For the first chapter of this thesis, the goal was to disrupt this interaction potentially modifying said integration preference. A secondary structure hypothesized to be mainly responsible for said interaction was extensively mutated resulting in several PB variants that were analyzed for their interaction capacity through a series of Co-IP experiments with BRD4. In total, seven substitutions were identified (E380F, V390K, T392Y, M394R, K407C, K407Q, and K407V) which exhibited reduced interaction capacity with BRD4. Each of the aforementioned mutants were used to generate integration libraries and, through NGS, it was determined if the integration preferences of the respective mutants had changed. In the immediate range 200 base pairs up- and downstream from known TSSs all mutants used exhibited a reduced integration bias. At a wider observation window 3 kbp up- and downstream from TSSs, further mutants with the substitutions M394R, T392Y and V390K showed a reduction in integration frequency of 17.3%, 1.5% and 5.4%, respectively, compared to the wildtype. Of particular note was the M394R mutant, which showed a reduction in all window sizes analyzed with a maximum of 65% less integration preference in the immediate vicinity of TSSs, theoretically generating a safety advantage over the wildtype transposase.
Chapter II was dedicated to the overall safety improvement for transposon-based gene modification and addresses the time point after the transgene has already been integrated and serious side effects may not be preventable. With this in mind, the aim was to develop a novel suicide-switch that can be stably introduced into cells via transposition, and reliably leads to cell death of the modified cells once activated. A system based on CRISPR/Cas9 was developed, where single guide RNAs were used to guide the Cas9 nuclease to Alu elements. These are short, repetitive sequences, which are distributed over the human genome in more than one million copies. Inducing double strand breaks within these elements would lead to genomic fragmentation and cell death. To be inducible, a transcriptional as well as post- translational control mechanism was added. Transcription of the Cas9 nuclease was regulated using a tet-on system, making expression dependent on doxycycline (DOX) supplementation. Furthermore, a version of the Cas9 nuclease called arC9 was used that allows double strand break generation only in the presence of 4-Hydroxytamoxifen (4-HT). Together with an expression cassette for the Alu-specific guide RNA and an expression cassette for the reverse tetracycline controlled transactivator all components were arranged between transposase-specific recognition sequences on a plasmid to allow transposon-system based gene transfer. The system was tested in HeLa cells. First, conditional expression of the arC9 nuclease was confirmed by addition of 1 μg/ml DOX. Second, the suicide-switch was further induced by adding 200 nM 4-HT and protein extracts were assayed for the KAP1 phosphorylation. Only upon induction with DOX and 4-HT phosphorylated KAP1 was detected, indicating DNA damage. Further, extensive growth and survival experiments were conducted to determine the effect of suicide-switch induction on cell proliferation and survival. Between 24 and 48 hours after induction, a halt in cell division was detected, after which extensive cell death was observed. Within 5 days post induction, >99% of all cells were eliminated. In the absence of both inducers, no significant differences in survival were observed compared to control cells line lacking Alu-specific guide RNAs. Microscopic examinations of the <1% surviving cell fraction revealed a senescence-associated phenotype and showed no signs of resumption of the cell division process. Accordingly, the second chapter of this thesis also achieved its goal in developing a functional suicide-switch that can be inserted into human cells via transposition, is highly dependent on the necessary induction signals, and exhibits excellent elimination capabilities in the context tested.
In the present work, the photo-protection mechanisms in plants and purple bacteria were investigated experimentally at the molecular level. For this purpose, several spectroscopic methods were combined and applied to elucidate the function of carotenoids, pigments of the photosynthetic apparatus, in photo-protection. The experiments were focused on the mechanisms involved in quenching of singlet and triplet states of the electronically excited (bacterio)chlorophylls. This photosynthetic reaction events occur on an ultrafast time-scale. Measuring such short-lived events, and understanding the underlying principles, demand some of the most precise experiments and exact measurement technologies currently available. This implies certain requirements for the light source used: a suitable wavelength within the absorption band of the sample, sufficient power, and, most importantly, a pulse duration short compared to the studied reaction. Nowadays, we can achieve all this requirements using femtosecond-spectroscopic systems, which produce laser pulses shorter than 100 femtoseconds (fs). Transient absorption spectroscopy provides important information on molecular dynamics interrogating electronic transitions. The technique is based on photochemical generation of transient species with femtoseconds pump pulses and measuring transient absorption changes of the sample using a second, time delayed probe pulse which in this case is a spectrally broad white-light pulse.
The reggie protein family consists of two homologous members, reggie-1 and reggie-2, also termed flotillin-2 and flotillin-1, respectively, that are ubiquitously expressed and evolutionarily well conserved, suggesting an important but so far ill-defined function. In various cell types, both reggies have been found to be constitutively associated with lipid rafts by means of acylation modifications and oligomerization. Lipid rafts are glycosphingolipid- and cholesterol-rich membrane microdomains which have been implicated in several cellular processes including membrane transport and signal transduction through growth factor receptors. However, the molecular details of these processes are still poorly understood. With the observation that reggies colocalize with activated glycosylphosphatidylinositolanchored proteins (GPI-APs) and Fyn kinase in rafts, a role for these proteins in signaling events has been suggested. In agreement with that, we have previously shown that reggie-1 becomes multiply tyrosine phosphorylated by Src kinases in response to epidermal growth factor (EGF) stimulation, pointing to a function for reggie-1 in growth factor signaling. Furthermore, overexpression of reggie-1 enhances spreading on fibronectin substrate in a tyrosine-dependent manner, thus revealing a role for reggie-1 in regulation of actin cytoskeleton through growth factor receptors. Due to the similarity shared by reggie proteins at amino acid level and to their ability to form hetero-oligomeric complexes, the first aim of this study was to analyze the putative tyrosine phosphorylation of reggie-2 in growth factor stimulated cells. Similarly to reggie-1, reggie-2 was found to be multiply tyrosine phosphorylated by Src kinase and to exist in a molecular complex with Src, with the degree of co-immunoprecipitation dependent on the activity of Src. Recent studies from us have also shown that administration of EGF results in the endocytosis of reggie-1 from the plasma membrane into endosomes, which is in line with a proposed role for reggies in membrane trafficking processes. In order to characterize in detail the endocytic mechanism that mediates the uptake of reggie-1, the dependency of reggie-1 endocytosis on clathrin and dynamin was investigated by means of overexpressing a variant form of Eps15 or a dominant negative form of dynamin-2. In either case the translocation of reggie-1 into endosomes in response to EGF was not affected, and this, together with the results that reggie-1 colocalized with cholera toxin (CTX) but not with transferrin receptor (TfnR) during EGF signaling, indicates that reggie-1 is taken up by means of a dynaminindependent, raft-mediated pathway. These findings are very well in line with recent data showing the pathway of entry into cells of reggie-2 as a raft-mediated endocytic pathway. The endocytosis of reggie-2 in response to EGF was also analyzed in this study. Similarly to reggie-1, in growth factor stimulated cells reggie-2 underwent a translocation from the plasma membrane to endosomes where the two reggies were found to colocalize with each other, suggesting that epidermal growth factor signaling might trigger the endocytosis of reggie oligomers. In addition, colocalization with both the late endosomal marker LAMP3/CD63 and epidermal growth factor receptor (EGFR) was detected, again indicating a function for reggies in signal transduction through growth factor receptors. EGFR has been reported to localize in rafts but, although this association is thought to be functional during EGF stimulation, how segregation of EGFR into rafts modulates its endocytosis and signaling is still under debate. Since reggie oligomers have recently been suggested to define a raft subtype, a further aim of this study was to investigate whether the depletion of reggies by means of small interfering RNA could interfere with the signaling and the trafficking through EGFR. Knockdown of reggie-2 resulted in an altered tyrosine phosphorylation of EGFR in response to EGF, while the degree of ubiquitination was not affected. Less efficient phosphorylation of tyrosine residues, especially of those which are docking sites for Grb2 and Shc, led in turn to an impaired activation of p38 and ERK1/2 MAPKs. Depletion of reggie-2 did not affect the early trafficking of activated EGFRs, with receptors being endocytosed and delivered to late endosomes as efficiently as in control cells. This would be in line with the normal degree of ubiquitination observed for EGFR, as ubiquitin moieties have been proposed to represent sorting tags that ensure receptor endocytosis into early endosomes and its proper intracellular trafficking. On the contrary, after prolonged EGF stimulation, depletion of reggie-2 resulted in a decreased downregulation of both receptor-bound ligand and EGFR, and in their accumulation in intracellular vesicles, thus pointing to a role for reggie-2 in the degradative pathway. Taken all together, these data ndicate that the association of EGFR with reggie-microdomains is likely to be important for proper receptor trafficking and signaling.
In der Experimentierhalle der Physik am Campus Riedberg der Goethe – Universität wird gegenwärtig die Beschleunigeranlage FRANZ aufgebaut. FRANZ steht für Frankfurter Neutronenquelle am Stern-Gerlach-Zentrum. Die Anlage bietet vielfältige Experimentiermöglichkeiten in der Untersuchung intensiver, gepulster Protonenstrahlen. Ein Forschungsschwerpunkt an den sekundären Neutronenstrahlen sind Messungen zur nuklearen
Astrophysik. Die Neutronen werden durch einen 2 MeV Protonenstrahl mittels der Reaktion 7Li (p, n) 7Be erzeugt. Die geplanten Experimente erfordern sowohl eine hier weltweit erstmals realisierte Pulsrepetitionsrate von bis zu 250 kHz bei Pulsströmen im 100 mA – Bereich als auch eine extreme Pulskompression auf eine Nanosekunde bei dann auftretenden Pulsströmen im Ampere – Bereich. Daneben ist auch ein Dauerstrich – Strahlbetrieb im mA – Strombereich möglich. Auch viele einzelne Beschleunigerkomponenten wie die Ionenquelle, der Chopper zur Pulsformung, die hochfrequent gekoppelte RFQ-IH-Kombination, der Rebuncher in Form einer CH – Struktur und der Bunchkompressor sind Neuentwicklungen. Mittlere Strahlleistungen von bis zu 24 kW treten im Niederenergiestrahltransportbereich auf, da die Ionenquelle grundsätzlich im Dauerstrich zu betreiben ist, auch bei Hochstrom mit hohen Pulsrepetitionsraten. Der Personen- und Geräteschutz spielt damit auch eine wesentliche Rolle bei der Auslegung des Kontrollsystems für FRANZ. Der Aufbau von FRANZ und seine wesentlichen Komponenten werden in Kapitel 2 erläutert. Die vielen unterschiedlichen Komponenten wie Hochspannungsbereich, Magneten, Hochfrequenzbauteile und Kavitäten, Vakuumbauteile, Strahldiagnose und Detektoren machen plausibel, dass auch das Kontrollsystem für eine solche Anlage speziell ausgelegt werden muss. In Kapitel 4 werden zum Vergleich die Konzepte zur Steuerung und Regelung aktueller, großer Beschleunigerprojekte aufgezeigt, nämlich für die „European Spallation Source ESS“ und für die „Facility for Antiproton and Ion Research FAIR“. In der vorliegenden Arbeit wurde die Ionenquelle als komplexe Beschleunigerkomponente ausgewählt, um Entwicklungen zur Steuerung und Regelung durchzuführen und zu testen. Zum Anfahren und Betreiben der Ionenquelle wurde ein Flussdiagramm (Abb. 5.15) entwickelt und realisiert. Im Detail wurden Untersuchungen zur Abhängigkeit der Heizkathodenparameter von der Betriebsdauer gemacht. Daraus konnte ein Algorithmus zur Vorhersage eines rechtzeitigen Filamentaustausches abgeleitet werden. Weiterhin konnte die Nachregelung des Kathodenheizstromes automatisiert werden, um damit die Bogenentladungsspannung innerhalb eines Intervalls von ± 0.5 V zu stabilisieren. Das Anfahren des Filamentstroms wurde ebenfalls automatisiert. Dazu wird die Vakuumdruckänderung in Abhängigkeit der Filamentstromerhöhung gemessen, ausgewertet und daraus der nächste erlaubte Stromerhöhungsschritt abgeleitet. Auf diese Weise wird der Betriebszustand schneller und kontrollierter erreicht als bei manuellem Hochfahren. Das Ziel eines unbemannten Ionenquellenbetriebs ist damit näher gerückt. In einem ersten Test zur Komponentensteuerung und zur Datenaufnahme wurde ein Ionenstrahl extrahiert und durch den ersten Fokussierungsmagneten – einen Solenoiden – transportiert. Es wurde der Erregungsstrom des Solenoiden sowie die Strahlenergie automatisch durchgefahren, die Daten abgespeichert und daraus ein Kontourplot zum gemessenen Strahlstrom hinter der Fokussierlinse erstellt (Abb. 5). Die vorliegende Arbeit beschäftigt sich nur mit den „langsamen“ Steuerungs- und Regelungsprozessen, während die schnellen Prozesse im Hochfrequenzregelungssystem unabhängig geregelt werden. Neben der Überwachung des Betriebszustandes aller Komponenten werden auch alle für den Service und die Personensicherheit benötigten Daten weggeschrieben. Das System basiert auf MNDACS (Mesh Networked Data Acquisition and Control System) und ist in JAVA geschrieben. MNDACS besteht aus einem Kernel, welcher die Komponententreiber-Software sowie den Netzwerkserver und das graphische Netzwerkinterface (GUI) betreibt. Weterhin gehört dazu das Driver Abstraction Layer (DAL), welches den Zugang zu weiteren Computern oder zu lokalen Treibern ermöglicht. CORBA stellt die Middleware für Netzwerkkommunikation dar. Dadurch wird Kommunikation mit externer Software geregelt, weiterhin wird die Umlegung von Kommunikation im Fall von Leitungsunterbrechungen oder einem lokalen Computerabsturz festgelegt. Es gibt bei FRANZ zwei Kontrollebenen: Über Ethernet läuft die „High Level Control“ und die Datenverarbeitung. Über die „Low Level Control“ läuft das Interlock – und Sicherheitssystem. Die Netzwerkverbindungen laufen über 1 Gb Ethernet Links, womit ein schneller Austausch auch bei lokalen Netzwerkstörungen noch möglich ist. Um bei Stromausfällen das Computersystem am Laufen zu halten, wurde im Rahmen dieser Arbeit ein „Uninterruptable Power Supply“ UPS beschafft und erfolgreich am Hochspannungsterminal getestet.
Capoeta damascina (Teleostei: Cyprinidae) is one of the most common freshwater fish species, found throughout the Levant, Mesopotamia, Turkey and Iran. According to the state of knowledge prior to this study, C. damascina, which is distributed over a wide range of isolated water bodies, was not a well-defined species. It was questionable whether it represents a single species or a complex of closely related species with high intraspecific and comparatively low interspecific variability. The goal of this study was to investigate the taxonomy, systematic position of the C. damascina species complex and the phylogenetic relationships among its members, based on morphological features as well as molecular phylogeny. Samples obtained from throughout the geographic range of this species complex were subjected to comparative morphological analyses in order to define, properly diagnose and separate species within the C. damascina complex. To elucidate phylogenetic relationships among members of the C. damascina species complex, samples were subjected to genetic analyses, using two molecular markers targeting the mitochondrial cytochrome oxidase I (COI, n = 103) and the two adjacent divergence regions (D1-D2) of the nuclear 28S rRNA genes (LSU, n = 65). Based on morphological and molecular genetic data, six closely related species were recognized within the C. damascina complex: C. buhsei, C. caelestis, C. damascina, C. saadii, C. umbla and an undescribed species, Capoeta sp.1. Analyses of the morphometric and meristic data obtained in this study revealed phenotypic variability among the various populations within a species and among the different species. Such differences in morphological characters reflect genetic differences, environmentally induced phenotypic variation or both, as the meristic phenotype of fish is sometimes a consequence of environmental parameters acting on the genotype. Based on phylogenetic analyses, two main lineages were identified within the C. damascina species complex: a western lineage represented by C. caelestis, C. damascina and C. umbla and an eastern lineage represented by C. buhsei, C. saadii and Capoeta sp.1. The close phylogenetic relationships between C. damascina and C. umbla and the sharing of same haplotypes between one specimen of C. damascina from Euphrates and another of C. umbla from Tigris reflect one of three possibilites: recent speciation, mitochondrial introgression or a combination of both. The results obtained in this study indicate that speciation of the above-mentioned six taxa is quite recent and that their dispersal and present-day distribution can be related to Pleistocene events. The drying out of the Persian Gulf, probably during one of the first glacials of the Pleistocene, led the ancestor of the C. damascina species complex in Mesopotamia to reach the rivers of the Gulf and of Hormuz basins and differentiate there, giving rise to the eastern lineage (ancestor of C. buhsei, C. saadii and Capoeta sp.1). As connections presumably existed among the different river drainages and basins in Iran during the wet periods of the Pleistocene, the ancestor of C. buhsei, C. saadii and Capoeta sp.1 was subsequently able to colonize the various Iranian drainages and differentiate there, giving rise to C. buhsei, C. saadii and Capoeta sp.1. After the separation from the eastern lineage, the western lineage, represented by the ancestor of C. damascina, C. umbla and C. caelestis, most likely reached the Levant from the Tigris-Euphrates system during the Pleistocene glacials, when river connections existed in the regions of the upper courses of Ceyhan Nehri (southern Turkey) and some western affluents to the Euphrates. From Ceyhan Nehri, it dispersed into other rivers in southern Turkey during Pleistocene periods of low sea levels until it reached Göksu Nehri and evolved into C. caelestis. The sister population differentiated into C. damascina and C. umbla. Based on the results obtained in this study, it is likely that C. damascina colonized the Levant and southern Turkey during the Pleistocene glacials. This is well supported by the low genetic variability among the C. damascina populations. Direct connections existed among the river drainages in the Levant during the Pleistocene periods of low sea level, thus serving as a pathway for the dispersal of C. damascina. The results of this study provide a coherent picture of the taxonomic position, phylogenetic relationships and evolutionary history of the C. damascina species complex and explain present patterns of distribution considering paleogeographic events.
The subject of this thesis is the experimental investigation of the neutron-capture cross sections of the neutron-rich, short-lived boron isotopes 13B and 14B, as they are thought to influence the rapid neutron-capture process (r process) nucleosynthesis in a neutrino-driven wind scenario.
The 13;14B(n,g)14;15B reactions were studied in inverse kinematics via Coulomb dissociation at the LAND/R3B setup (Reactions with Relativistic Radioactive Beams). A radioactive beam of 14;15B was produced via in-flight fragmentation and directed onto a lead-target at about 500 AMeV. The neutron breakup of the projectile within the electromagnetic field of the target nucleus was investigated in a kinematically complete measurement. All outgoing reaction products were detected and analyzed in order to reconstruct the excitation energy.
The differential Coulomb dissociation cross sections as a function of the excitation energy were obtained and first experimental constraints on the photoabsorption and the neutron-capture cross sections were deduced. The results were compared to theoretical approximations of the cross sections in question. The Coulomb dissociation cross section of 15B into 14B(g.s.) + n was determined to be s(15B;14B(g:s:)+n) CD = 81(8stat)(10syst) mb ; while the Coulomb dissociation cross section of 14B into a neutron and 13B in its ground state was found to be s(14B;13B(g:s:)+n) CD = 281(25stat)(43syst) mb: Furthermore, new information on the nuclear structure of 14B were achieved, as the spectral shape of the differential Coulomb dissociation cross section indicates a halolike structure of the nucleus.
Additionally, the Coulomb dissociation of 11Be was investigated and compared to previous measurements in order to verify the present analysis. The corresponding Coulomb dissociation cross section of 11Be into 10Be(g.s.) + n was found to be 450(40stat)(54syst ) mb, which is in good agreement with the results of Palit et al.
One of the most important events in human history occurred during the Early Pleistocene: the dispersal of early hominins out of Africa and into Europe and Asia. In Western Europe, the earliest evidences of the genus Homo have been found in the Baza Basin, at the sites of Orce in the SE of the Iberian Peninsula. These sites contain fossils and lithic industry dated approximately as 1.4–1.3 Ma.While hominin remains and artifacts at Orce, as well as the accompanying fauna, have been extensively studied, the properties and evolution of the Early Pleistocene vegetation in the basin remain unknown. The general effect of climate change on the expansion of early hominins from Africa into Eurasia still remains unclear. It is not known if the Early Pleistocene climate changes and the development of glacials periods led to the extirpation of European communities, or if those communities were able to endure and persist through such adverse climatic periods. This open question highlights the need for climate and environmental analyses for the time before, during and after the first presence of Homo in Europe. This PhD thesis contributes to that need by the presentation of the first long pollen record of the Baza Basin, where the oldest hominin sites in Western Europe are found.
This work presents a contribution to the literature on methods in search of lowdimensional models that yield insight into the equilibrium and kinetic behavior of peptides and small proteins. A deep understanding of various methods for projecting the sampled configurations of molecular dynamics simulations to obtain a low-dimensional free energy landscape is acquired. Furthermore low-dimensional dynamic models for the conformational dynamics of biomolecules in reduced dimensionality are presented. As exemplary systems, mainly short alanine chains are studied. Due to their size they allow for performing long simulations. They are simple, yet nontrivial systems, as due to their flexibility they are rapidly interconverting conformers. Understanding these polypeptide chains in great detail is of considerable interest for getting insight in the process of protein folding. For example, K. Dill et al. conclude in their review [28] about the protein folding problem that "the once intractable Levinthal puzzle now seems to have a very simple answer: a protein can fold quickly and solve its large global optimization puzzle simply through piecewise solutions of smaller component puzzles".
The ALICE High-Level-Trigger (HLT) is a large scale computing farm designed and constructed for the purpose of the realtime reconstruction of particle interactions (events) inside the ALICE detector. The reconstruction of such events is based on the raw data produced in collisions inside the ALICE at the Large Hadron Collider. The online reconstruction in the HLT allows the triggering on certain event topologies and a significant data reduction by applying compression algorithms. Moreover, it enables a real-time verification of the quality of the data.
To receive the raw data from the various sub-detectors of ALICE, the HLT is equipped with 226 custom built FPGA-based PCI-X cards, the H-RORCs. The H-RORC interfaces the detector readout electronics to the nodes of the HLT farm. In addition to the transfer of raw data, 108 H-RORCs host 216 Fast-Cluster-Finder (FCF) processors for the Time-Projection-Chamber (TPC). The TPC is the main tracking detector of ALICE and contributes with up to 16 GB/s to over 90% of the overall data volume. The FCF processor implements the first of two steps in the data reconstruction of the TPC. It calculates the space points and their properties from charge clouds (clusters) created by charged particles traversing the TPCs gas volume. Those space points are not only the base for the tracking algorithm, but also allow for a Huffman-based data compression, which reduces the data volume by a factor of 4 to 6.
The FCF processor is designed to cope with any incoming data rate up to the maximum bandwidth of the incoming optical link (160 MB/s) without creating back-pressure to the detectors readout electronics. A performance comparison with the software implementation of the algorithm shows a speedup factor of about 20 compared with one AMD Opteron 6172 Core @ 2.1 GHz, the CPU type used in the HLT during the LHC Run1 campaign. Comparison with an Intel E5-2690 Core @ 3.0 GHz, the CPU type used by the HLT for the LHC Run2 campaign, results in a speedup factor of 8.5. In total numbers, the 216 FCF processors provide the computing performance of 4255 AMD Opteron cores or 2203 Intel cores of the previously mentioned type. The performance of the reconstruction with respect to the physics analysis is equivalent or better than the official ALICE Offline clusterizer. Therefore, ALICE data taking was switched in 2011 to FCF cluster recording and compression only, discarding the raw data from the TPC. Due to the capability to compress the clusters, the recorded data volume could be increased by a factor of 4 to 6.
For the LHC Run3 campaign, starting in 2020, the FCF builds the foundation of the ALICE data taking and processing strategy. The raw data volume (before processing) of the upgraded TPC will exceed 3 TB/s. As a consequence, online processing of the raw data and compression of the results before it enters the online computing farms is an essential and crucial part of the computing model.
Within the scope of this thesis, the H-RORC card and the FCF processor were developed and built from scratch. It covers the conceptual design, the optimisation and implementation, as well as the verification. It is completed by performance benchmarks and experiences from real data taking.
The Late Cretaceous is known to be mostly affected by warm periods interrupted temporarily by a number of cooling events. The reconstruction of the paleoclimatic conditions during a period of high concentration of CO2 in the atmosphere is of great importance for the creation of future climate models. We applied the recently developed method reconstructing the SST from the TEX86 (TetraEther indeX of tetraethers consisting of 86 carbon atoms).
The sample material used for the present study was obtained from the tropical Late Cretaceous southern Tethys upwelling system (Negev/Israel), lasting from the Late Santonian to the Early Maastrichtian (~ 85 to 68 Ma). On the core samples from the Shefela basin, representing the outer belt of the upwelling system and the outcrop profile from the open mine Mishor Rotem (Efe Syncline), representing the inner belt, various bulk geochemical and biomarker studies were performed in this thesis.
Derived from TEX86 data, a significant long-term SST cooling trend from 36.0 to 29.3 °C is recognized during the Late Santonian and the Early Campanian in the southern Tethys margin. This is consistent with the opening and deepening of the Equatorial Atlantic Gateway (EAG) and the intrusion of cooler deep water from the southern Atlantic Ocean influencing the global SSTs and also the Tethys Ocean. Furthermore, the cooler near shore SST usually found in modern upwelling systems could be verified in case of the ancient upwelling system investigated in the present study. The calculated mean SST in the inner belt (27.7 °C) represented in the Efe Syncline was 1.5 °C cooler in comparison to the more seaward located outer belt (Shefela basin).
Moreover, geochemical and biomarker analyses were used to identify both the accumulation of high amounts of phosphate in the PM and good preservation of organic matter (OM) in the lower part of the OSM section. Total organic carbon (TOC) contents are highly variable over the whole profile reaching from 0.6 % in the MM, to 24.5 % in the OSM. Total iron (TFe) varies from 0.1 % in the PM to 3.3 % in the OSM and total sulfur (TS) varies between 0.1 % in the MM and 3.4 % in the OSM. Different correlations of TS, TOC and TFe were used to identify the conditions during the deposition of the different facies types. Natural sulfurization was found to play a key role in the preservation of the OM particularly in the lower part of the OSM. Samples from the OSM and the PM were deposited under dysoxic to anoxic conditions and iron limitation lasted during the deposition of the OSM and the PM, which effected the incorporation of sulfur into OM.
Phosphorus is highly accumulated in the sediments of the PM with a mean proportion of 11.5 % total phosphorus (TP), which is drastically reduced to a mean value of 0.9 % in the OSM and the MM. From the correlation of the bulk geochemical parameters TOC/TOCOR ratio and TP a major contribution of sulfate reducing bacteria to the phosphate deposition is concluded. This interrelation has previously been investigated in recent coastal upwelling systems off Peru, Chile, California and Namibia. This was further supported by the analysis of branched and monounsaturated fatty acids indicating the occurrence of sulfate reducing and sulfide oxidizing bacteria during the deposition.
According to the results from the analysis of n-alkanes and C27- to C29-steranes up to 95 % of the OM was of marine origin.
Organic sulfur compounds (OSC) were a major compound class in the aromatic hydrocarbon fraction and n-Alkyl and isoprenoid thiophenes were the most abundant, with highest amounts found for 2-methyl-5-tridecyl-thiophene (28 µg/g TOC). The relatively high abundance of ββ-C35 hopanoid thiophenes and epithiosteranes is equivalent to an incorporation of sulfur during the early stages of diagenesis.
Moreover, the geochemical parameters δ13Corg, δ15Norg, C/N and the pristane/phytane (Pr/Ph) ratio, were studied for reconstruction of seafloor and water column depositional environments. The high C/N ratio along with relatively low values of δ15Norg (4 ‰ to 6 ‰) and δ13Corg (-29 ‰ to -28 ‰) are consistent with a significant preferential loss of nitrogen-rich organic compounds during diagenesis. Oxygen-depleted conditions lasted during the deposition of the PM and the bottom of the OSM, reflected by the low Pr/Ph ratio of 0.11–0.7. In the upper part of the OSM and the MM the conditions changed from anoxic to dysoxic or oxic conditions. This environmental trend is consistent with co-occurring foraminiferal assemblages in the studied succession and implies that the benthic species in the Negev sequence were adapted to persistent minimum oxygen conditions by performing complete denitrification as recently found in many modern benthic foraminifera.
Furthermore, the anammox process could have influenced the nitrogen composition of the sediments. In this anaerobically process nitrite and ammonia are converted to molecular nitrogen.
Proteostasis stressors that destabilize the cellular proteome, like heat shock, trigger transcription and translational reactions leading to the accumulation of heat shock proteins, also called molecular chaperones. During stress, induction of stress response genes is prioritized so that molecular chaperones and other stress response proteins are synthesized to cope with proteome misfolding and aggregation. In order to promote the selective translation of stress-specific genes, translation of others genes that are nonessential for cell survival has to stop. Nonessential protein-coding mRNAs accumulate in the cytosol with the associated proteins to form granular structures called stress granules (SG). These membrane-less organelles are thought to be involved in cell survival, mRNA stabilization and mRNA triage. They were proposed to form via the liquid-liquid phase separation which can be triggered by the high local concentration of RNA-binding proteins. mRNAs were long thought to simply play a scaffolding role by bringing RNA-binding proteins together and allowing their concentration and local aggregation. Recently, the active role of mRNAs in the SG assembly became apparent, too. For example, the spontaneous assembly of total yeast RNA into granules was observed, and these RNA granules showed a large overlap with SG transcriptome. Furthermore, cytosolic mRNAs can be released from polyribosomes under stress and be exposed to the cytosolic contents as free mRNAs. It has been suggested that this massive increase of free mRNA in the cytosol might overload the capacities of RNA-stabilizing proteins. The remaining free mRNA molecules would then become exposed to misfolded and aggregation-prone proteins and trigger granulation.
We investigated the role of free mRNAs in different stress conditions during the early and chronic phases of stress response and explored their involvement in SGs assembly and amlyoidogenesis. We identified and studied the interactome of a free mRNA probe incubated with heat shocked cell lysate by means of quantitative mass spectrometry. Proteomics analysis allowed us to identify 79 interactors of free mRNA. Among these interactors, we focused on the translation initiation factor eIF2α and on the RNA methyltransferase TRMT6/61A. Both interactions were verified biochemically, which confirmed that the association is enhanced in heat shocked lysate. In vitro reconstitution showed that free mRNA and TRMT6 interact directly. Ex vivo pulldowns revealed that eIF2α and TRMT6/61A interact under stress conditions and that this interaction is RNA-dependent.
TRMT6/61A is a tRNA methytransferase responsible for the methylation of the adenosine 58 at the position 1 producing m1A. However, also mRNAs have been recently found to be methylated by TRMT6/61A. Our bioinformatics analyses revealed that significantly more mRNAs enriched in SG contain the motif for methylation than SG-depleted mRNAs. We hypothesized that m1A methylation of mRNAs could constitute a tag for the mRNAs targeting to SGs. TRMT61A knock-down (KD) cell lines were generated using the CRISPR-Cas9 technique. In TRMT61A KD cells, m1A was significantly reduced on mRNAs, which correlated with an increased sensitivity of the cells to proteostasis stress. KD cells also showed defects in SG assembly. In heat shocked cells, an m1A motif-containing mRNA recovered better after returning to normal temperature than a control mRNA with mutated motif. In addition, we could isolate SGs and analyze their m1A and m6A content by mass spectrometry. While m6A content in SG mRNAs was very similar to cytosolic mRNAs, m1A was almost 8 times enriched in SGs. Thus, we could confirm experimentally the results of the bioinformatics analysis and directly support the hypothesis that m1A is a tag to direct mRNAs for sequestration. Finally, we compared amyloidogenesis in wild-type and TRMT61A KD cell lines. Cells with reduced levels of TRMT61A demonstrated an increased accumulation of transfected Aβ and an impaired aggregate clearance. Various assays led us to conclude that the lack of m1A deposition on mRNAs enhanced RNA co-aggregation with amyloids.
Based on our results, we propose a model explaining the fate of free mRNA during proteostasis stress. Upon polysome disassembly, free mRNA is released and becomes free to interact with other proteins, including the methyltransferase TRMT6/61A. TRMT6/61A methylates the freed mRNAs containing the cognate motif. The m1A tag then targets mRNAs to SGs promoting sequestration. Upon stress release, SGs disassemble, thus releasing rescued mRNAs which could now reenter translation and support cell recovery. On the other hand, non-sequestered mRNAs increasingly co-aggregate with aggregating proteins. Thus, deficiency of the N1-adenine methylation of mRNAs due to the lack of TRMT6/61A increases the amount of unpacked mRNAs. The deposition of m1A on mRNAs could then be a way to protect them during exposure to stress, to limit their co-aggregation with misfolded proteins and to allow a faster recovery upon stress release.
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
This study investigates a historical event that occurred during the Indonesian Revolution as depicted in Indonesian historical films and argues that these films not only attempt to depict the past but also use the past as a means of social commentary, teaching moral insight, and historical reinforcement. The historical films selected are The Long March (Darah dan Do’a) (1950) and Mereka Kembali (1972). Both films deal with the Long March event experienced by the troops of the Siliwangi Division in 1948. These troops were previously assigned to infiltrate Yogyakarta and its surrounding areas. They were instructed to march back to their original base in West Java as a part of the military strategies to confront the Dutch during the Indonesian Revolution, also known as the Indonesian War of Independence. This event became known as the Long March of the Siliwangi Division. This study examines not only the representation of the past or the texts of the films but also the production process, which includes the motivations of the filmmakers and the public reception when the films were screened for the public at the time—in 1950 and 1972, respectively. This approach provides a broader and richer dimension, valuable insights into the behind-the-scenes process of making the selected historical films, and essential information about the public reception of the films. From the production point of view, there are two main reasons for making these historical films: personal reason and social engagement. Further, the military also plays a vital role in these historical film productions. From the historical representation aspect, these two films depict the events of the Long March of the Siliwangi Division as a journey full of various obstacles and difficulties, such as harsh terrain, lack of food, battles against the Dutch, and internal disputes with fellow Indonesians: Darul Islam. From the reception aspect, the audience’s point of view, these films provide several representations that meet their expectations about the Long March of the Siliwangi Division. However, the audience disagrees with some of the other representations. Finally, the study revealed that historical films are potential vehicles for telling, interpreting, entertaining, legitimating and preserving the past. In addition, this study has a vital implication for reopening the tradition of Indonesian film studies and reigniting attention to old films.
Concentration of multivariate random recursive sequences arising in the analysis of algorithms
(2006)
Stochastic analysis of algorithms can be motivated by the analysis of randomized algorithms or by postulating on the sets of inputs of the same length some probability distributions. In both cases implied random quantities are analyzed. Here, the running time is of great concern. Characteristics like expectation, variance, limit law, rates of convergence and tail bounds are studied. For the running time, beside the expectation, upper bounds on the right tail are particularly important, since one wants to know large values of the running time not taking place with possibly high probability. In the first chapter game trees are analyzed. The worst case runnig time of Snir's randomized algorithm is specified and its expectation, asymptotic behavior of the variance, a limit law with uniquely characterized limit and tail bounds are identified. Furthermore, a limit law for the value of the game tree under Pearl's probabilistic modell is proved. In the second chapter upper and lower bounds for the Wiener Index of random binary search trees are identified. In the third chapter tail bounds for the generation size of multitype Galton-Watson processes (with immigration) are derived, depending on their offspring distribution. Therefore, the method used to prove the tail bounds in the first chapter is generalized.
Next-generation DIRC detectors, like the PANDA Barrel DIRC, with improved optical designs and better spatial and timing resolution, require correspondingly advanced reconstruction and PID methods. The investigation of the PID performance of two DIRC counters and the evaluation of the reconstruction and PID algorithms form the core of this thesis. Several reconstruction and PID approaches were developed, optimized, and tested using hadronic beam particles, experimental physics events, and Geant simulations. The near-final design of the PANDA Barrel DIRC was evaluated with a prototype in the T9 beamline at CERN in 2018. The analysis finds excellent agreement between the experimental data and the Geant simulations for all reconstruction algorithms. The best PID performance of up to $5.2 \pm 0.2$ s.d. $\pi$/K separation at 3.5 GeV/c, was obtained with a time imaging PID method. The PANDA Barrel DIRC simulation, as well as the reconstruction and PID algorithms, were evaluated using experimental data from the GlueX DIRC as part of the FAIR Phase-0 program. The performance validation was carried out using physics events of the GlueX experiment and simulations. The initial analysis results of the commissioning dataset show a $\pi$/K separation power of up to 3 s.d. at a momentum of 3.0-3.5 GeV/c, obtained using a geometric reconstruction algorithm.
The mainstream law and economics approach has dominated positive analysis and normative design of economic regulations. This approach represents a form of applied neoclassical and new institutional economics. Neoclassical and/or new institutional economic theories, models, and analytical concepts are applied automatically to economic regulatory problems.
This automatic application of neoclassical economics to economic regulatory problems loses sight of the valid insights of non-neoclassical schools of economic thought and theories, which may illuminate important aspects of the regulatory problems. This thesis, therefore, advocates an integrated law and economics approach to economic regulations. This approach identifies the relevant insights of neoclassical and non-neoclassical schools of thought and theories and refines them through a process of cross-criticism. In this process, the insights of each school of thought are subjected to the critiques of other schools of thought. The resulting refined insights, which are more likely to be valid, are then integrated consistently through various techniques of integration.
Not only does neoclassical (micro and macro) law and economics overlook the valid insights of non-neoclassical schools of thought, it is also highly reductionist. It ignores the interdependencies of legal institutions, highlighted mainly by the comparative capitalism literature, and the structural interlinkages among socio-economic actors, highlighted by economic sociology and complexity economics. Rather, it takes rational individuals and their interactions subject to the constraint of isolated institution(s) as its unit of analysis. In place of this reductionist perspective, the thesis argues for a systemic approach to economic regulations. This systemic perspective replaces the reductionist unit of neoclassical regulatory analysis with a systemic unit of analysis that consists of the least non-decomposable actors’ network and its associated least non-decomposable institutional network. Then, the thesis develops an operationalized and replicable systemic framework for systemic analysis and design of institutional networks.
Both the systemic and integrated approaches are theoretically consistent and complementary. The systemic approach is in essence a way of thinking that requires a broad and rich informational basis that can be secured by using the integrated approach. Due to their complementarity, they give rise to what I call “the integrated and systemic law and economics approach.” The thesis operationalizes this approach by setting out well-defined replicable steps and applying them to concrete regulatory problems, namely, the choice of a corporate governance model for developing countries and the development of a normative theory of economic regulations. These concrete applications demonstrate the critical bite of the integrated and systemic approach, which reveals significant shortcomings of mainstream law and economics’ answers to these regulatory questions. They also show the constructive potential of the integrated and systemic approach in overcoming the critiques advanced to the neoclassical regulatory conclusions.
The operationalized integrated and systemic approach is both a law and economics as well as a law and development approach. It does not only provide an alternative to mainstream law and economics analysis and design of economic regulations. It also fills a significant analytical lacuna in the law and development literature that lacks an analytical framework for analysis and design of context-specific legal institutions that can promote economic development in developing economies.
The thesis deals with the analysis and modeling of point processes emerging from different experiments in neuroscience. In particular, the description and detection of different types of variability changes in point processes is of interest.
A non-stationary rate or variance of life times is a well-known problem in the description of point processes like neuronal spike trains and can affect the results of further analyses requiring stationarity. Moreover, non-stationary parameters might also contain important information themselves. The goal of the first part of the thesis is the (further) development of a technique to detect both rate and variance changes that may occur in multiple time scales separately or simultaneously. A two-step procedure building on the multiple filter test (Messer et al., 2014) is used that first tests the null hypothesis of rate homogeneity allowing for an inhomogeneous variance and that estimates change points in the rate if the null hypothesis is rejected. In the second step, the null hypothesis of variance homogeneity is tested and variance change points are estimated. Rate change points are used as input. The main idea is the comparison of estimated variances in adjacent windows of different sizes sliding over the process. To determine the rejection threshold functionals of the Brownian motion are identified as limit processes under the null of variance homogeneity. The non-parametric procedure is not restricted to the case of at most one change point. It is shown in simulation studies that the corresponding test keeps the asymptotic significance level for a wide range of parameters and that the test power is remarkable. The practical applicability of the procedure is underlined by the analysis of neuronal spike trains.
Point processes resulting from experiments on bistable perception are analyzed in the second part of the thesis. Visual illusions allowing for than more possible perception lead to unpredictable changes of perception. In the thesis data from (Schmack et al., 2015) are used. A rotating sphere with switching perceived rotation direction was presented to the participants of the study. The stimulus was presented continuously and intermittently, i.e., with short periods of „blank display“ between the presentation periods. There are remarkable differences in the response patterns between the two types of presentation. During continuous presentation the distribution of dominance times, i.e., the intervals of constant perception, is a right-skewed and unimodal distribution with a mean of about five seconds. In contrast, during intermittent presentation one observes very long, stable dominance times of more than one minute interchanging with very short, unstable dominance times of less than five seconds, i.e., an increase of variability.
The main goal of the second part is to develop a model for the response patterns to bistable perception that builds a bridge between empirical data analysis and mechanistic modeling. Thus, the model should be able to describe both the response patterns to continuous presentation and to intermittent presentation. Moreover, the model should be fittable to typically short experimental data, and the model should allow for neuronal correlates. Current approaches often use detailed assumptions and large parameter sets, which complicate parameter estimation.
First, a Hidden Markov Model is applied. Second, to allow for neuronal correlates, a Hierarchical Brownian Model (HBM) is introduced, where perception is modeled by the competition of two neuronal populations. The activity difference between these two populations is described by a Brownian motion with drift fluctuating between two borders, where each first hitting time causes a perceptual change. To model the response patterns to intermittent presentation a second layer with competing neuronal populations (coding a stable and an unstable state) is assumed. Again, the data are described very well, and the hypothesis that the relative time in the stable state is identical in a group of patients with schizophrenia and a control group is rejected. To sum up, the HBM intends to link empirical data analysis and mechanistic modeling and provides interesting new hypotheses on potential neuronal mechanisms of cognitive phenomena.
This thesis is structured into 7 chapters:
• Chapter 2 gives an overview of the ultrashort high intensity laser interaction with matter. The laser interaction with an induced plasma is described, starting from the kinematics of single electron motion, followed by collective electron effects and the ponderamotive motion in the laser focus and the plasma transparency for the laser beam. The three different mechanisms prepared to accelerate and propagate electrons through matter are discussed. The following indirect acceleration of protons is explained by the Target Normal Sheath Acceleration (TNSA) mechanism. Finally some possible applications of laser accelerated protons are explained briefly.
• Chapter 3 deals with the modeling of geometry and field mapping of magnetic lens. Initial proton and electron distributions, fitted to PHELIX measured data are generated, a brief description of employed codes and used techniques in simulation is given, and the aberrations at the solenoid focal spot is studied.
• Chapter 4 presents a simulation study for suggested corrections to optimize the proton beam as a later beam source. Two tools have been employed in these suggested corrections, an aperture placed at the solenoid focal spot as energy selection tool, and a scattering foil placed in the proton beam to smooth the radial energy beam profile correlation at the focal spot due to chromatic aberrations. Another suggested correction has been investigated, to optimize the beam radius at the focal spot by lens geometry controlling.
• Chapter 5 presents a simulation study for the de-neutralization problem in TNSA caused by the fringing fields of pulsed magnetic solenoid and quadrupole. In this simulation, we followed an electrostatic model, wherethe evolution of both, self and mutual fields through the pulsed magnetic solenoid could be found, which is not the case in the quadrupole and only the growth of self fields could be found. The field mapping of magnetic elements is generated by the Matlab program, while the TraceWin code is employed to study the tracking through magnetic elements.
• Chapter 6 describes the PHELIX laser parameters at GSI with chirp pulse amplification technique (CPA), and Gafchromic Radiochromic film RCF) as a spatial energy resolver film detector. The results of experiments with laser proton acceleration, which were performed in two experimental areas at GSI (Z6 area and PHELIX Laser Hall (PLH)), are presented in section 6.3.
• Chapter 7 includes the main results of this work, conclusions and gives a perspective for future experimental activities.
Characterization of mouse NOA1 : subcellular localizaion, G-Quadruplex binding and proteolysis
(2013)
Mitochondria contain their own protein synthesis machinery with mitoribosomes that are similar to prokaryotic ribosomes. The thirteen proteins encoded in the mitochondrial genome are members of the respiratory chain complexes that generate a proton gradient, which is the electromotoric force for ATP synthesis.
NOA1 (Nitric Oxide Associated Protein-1) is a nuclear encoded GTPase that positively influences mitochondrial respiration and ATP production. Although a role in mitoribosome assembly was assigned to NOA1 the underlying molecular mechanism is poorly understood. This work shows that the multi-domain protein NOA1 serves multiple purposes for the function of mitochondria. NOA1 is a dual localized protein that makes a detour through the nucleus before mitochondrial import. The nuclear shuttling is mediated by a nuclear localization signal and the now identified nuclear export signal. SELEX (Systemic Evolution of Ligands by Exponential Enrichment) analysis revealed a G-quadruplex binding motif that characterizes NOA1 as ribonucleoprotein (RNP). G-quadruplex binding was coupled to the GTPase activity and increased the GTP hydrolysis rate. The sequence of localization events and the identification of NOA1 being a RNP lead to the discussion of an alternative import pathway for RNPs into mitochondria. The short-lived NOA1 contains ClpX recognition motifs and is specifically degraded by the mitochondrial matrix protease ClpXP. NOA1 is the first reported substrate of ClpXP in higher eukaryotes and augments the contribution of the ClpXP protease for mitochondrial metabolism. To assess the direct action of NOA1 on the mitoribosome co-sedimentation assays were performed. They showed that the interaction of NOA1 and the mitoribosome is dependent on the GTPase function and the nascent peptide chain. In vitro, NOA1 facilitated the membrane insertion of newly translated and isotope labeled mitochondrial translation products into inverted mitochondrial inner membrane vesicles. In conclusion, NOA1 is a G-quadruplex-RNP that acts as mitochondrial membrane insertion factor for mtDNA-encoded proteins.
This thesis provides a comprehensive model of the molecular function of NOA1 and is the basis for future research. The identification of NOA1 as ClpXP substrate is a major contribution to the field of mitochondrial research.
Starting from the first observation of the halo phenomenon 20 years ago, more and more neutron-rich light nuclei were observed. The study of unstable nuclear systems beyond the dripline is a relatively new branch of nuclear physics. In the present work, the results of an experiment at GSI (Darmstadt) with relativistic beams of the halo nuclei 8He, 11Li and 14Be with energies of 240, 280 and 305 MeV/nucleon, respectively, impinging on a liquid hydrogen target are discussed. Neutron/proton knockout reactions lead to the formation of unbound systems, followed by their immediate decay. The experimental setup, consisting of the neutron detector LAND, the dipole spectrometer ALADIN and different types of tracking detectors, allows the reconstruction of the momentum vectors of all reaction products measured in coincidence. The properties of unbound nuclei are investigated by reconstructing the relative-energy spectra as well as by studying the angular correlations between the reaction products. The observed systems are 9He, 10He, 10Li, 12Li and 13Li. The isotopes 12Li and 13Li are observed for the first time. They are produced in the 1H(14Be, 2pn)12Li and 1H(14Be, 2p)13Li knockout reactions. The obtained relative-energy spectrum of 12Li is described as a single virtual s-state with a scattering length of as = -22;13.7(1.6) fm. The spectrum of 13Li is interpreted as a resonance at an energy of Er = 1.47(13) MeV and a width of Gamma ~ 2 MeV superimposed on a broad correlated background distribution. The isotope 10Li is observed after one-neutron knockout from the halo nucleus 11Li. The obtained relative-energy spectrum is described by a low-lying virtual s-state with a scattering length as = -22.4(4.8) fm and a p-wave resonance with Er = 0.566(14) MeV and Gamma = 0.548(30) MeV, in agreement with previous experiments. The observation of the nucleus 8He in coincidence with one or two neutrons, as a result of proton knockout from 11Li, allows to reconstruct the relative-energy spectra for the heavy helium isotopes, 9He and 10He. The low-energy part of the 9He spectrum is described by a virtual s-state with a scattering length as = -3.16(78) fm. In addition, two resonance states with l 6= 0 at energies of 1.33(8) and 2.4 MeV are observed. For the 10He spectrum, two interpretations are possible. It can be interpreted as a superposition of a narrow resonance at 1.42(10) MeV and a broad correlated background distribution. Alternatively, the spectrum is being well described by two resonances at energies of 1.54(11) and 3.99(26) MeV. Additionally, three-body energy and angular correlations in 10He and 13Li nuclei at the region of the ground state (0 < ECnn < 3 MeV) are studied, providing information about structure of these unbound nuclear systems.
Remodeling of extracellular matrix (ECM) is an important physiologic feature of normal growth and development. In addition to this critical function in physiology many diseases have been associated with an imbalance of ECM synthesis and degradation. In the kidney, dysregulation of ECM turnover can lead to interstitial fibrosis, and glomerulosclerosis. The major physiologic regulators of ECM degradation in the glomerulus are the large family of zinc-dependent proteases, collectively refered to matrix metalloproteinases (MMPs). The tight regulation of most of these proteases is accomplished by different mechanisms, including the regulation of MMP gene expression, the processing and conversion of the inactive zymogen by other proteases such as serine proteases and finally the inhibition of active MMPs by endogenous inhibitors of MMPs, denoted as tissue inhibitors of metalloproteinases (TIMPs). Namely, the MMP-9 has been shown to be critically involved in the dysregulation of ECM turnover associated with severe pathologic conditions such as rheumatoid arthritis or fibrosis of lung, skin and kidney. In the present work I searched for a possible modulation of MMP-9 expression and/or activity in glomerular mesangial cells which are thought as key players of many inflammatory and non-inflammatory glomerular diseases. I found that various structurally different PPARalpha agonists such as WY-14,643, LY-171883 and fibrates potently suppress the cytokine-induced MMP-9 expression in renal MC. Furthermore, I demonstrate that the inhibition of MMP-9 expression by PPARalpha agonists was paralleled by a strong increase of cytokine-induced iNOS expression and subsequent NO formation, suggesting that PPARalpha-dependent effects on MMP-9 expression level primarily result from alterations in NO production which in turn reduces the MMP-9 mRNA half-life. Searching for the detailed mechanism of NO-dependent effects on MMP-9 mRNA stability, I found that NO either given from exogenous sources or endogenously produced increases the MMP-9 mRNA degradation by decreasing the expression of the mRNA stabilizing factor HuR. Furthermore, I demonstrate a reduction in the RNA-binding capacity of HuR containing complexes to MMP-9 ARE motifs in cells treated with NO. Since the reduction of HuR expression can be mimicked by the cGMP analog 8-Bromo-cGMP, I suggest that NO reduces in a cGMP-dependent manner the expression of HuR. Finally, I elucidated the modulatory effect of extracellular nucleotides, mainly ATP, on cytokine-triggered MMP-9 expression. Interestingly, I found that in contrast to NO, gamma-S-ATP the stable analog of ATP potently amplifies the IL-beta mediated MMP-9 expression. The increase in mRNA stability was paralleled by an increase in the nuclear-cytosolic shuttling of the mRNA stabilizing factor HuR. Furthermore, I demonstrate an increase in the RNA-binding capacity of HuR containing complexes to the 3'-UTR of MMP-9 by ATP. In summary, the data presented here may help to find new targets (posttranscriptional regulation) that could be used to manipulate or modulate the expression of not only MMP-9 but also other genes regulated on the level of mRNA stability.
This dissertation examines the language of politics of leading figures of the ex-Free Aceh Movement or Gerakan Aceh Merdeka‟ (GAM) leading figures in selected Aceh media during the 2012 gubernatorial election campaign. By analyzing their symbolic acts, topic selection, campaign methods, and campaign locations as reported in five selected Aceh media, I demonstrate the process of ex-GAM political rhetoric in the post-conflict election setting. The mixed method approach used in the dissertation includes the following: grounded, content analysis, case study, and rhetorical analysis. Data were collected from three local daily broadsheet newspapers (Serambi Indonesia, Rakyat Aceh and Harian Aceh) and two online news portals (The Atjeh Post and The Globe Journal). The research found that ex-GAM political rhetoric was deeply divided into two opposing political camps: the Irwandi camp as the incumbent independent and the PA camp as the challenger from a newly-established local political party. Both camps highlighted the importance of expressive symbolic acts, such as explaining and making promises in their campaigns. Irwandi introduced more varied, specific, practical, and concrete topics in its rhetoric with diverse and less formal campaign methods that appealed to those who envision a prosperous future in Aceh. By contrast, PA employed general, unrealistic, and abstract topics in its rhetoric with less attractive and more formal campaign methods that appealed to emotional and historical romanticism. In relation to ex-GAM political rhetoric and campaign locations, both focused on the traditional regions in northeast Aceh or in the conflict-sensitive regions where most of their loyalists reside, and on big regencies/cities where the potential voters live. However, during the campaign Irwandi appeared less frequently in ethnically diverse locations, whereas PA appeared more in non-traditional regions such as the previously isolated and less-developed regions in the central highlands and southeast regions of Aceh. In this way they highlighted the rhetoric of economic and infrastructure development and that of unity in diversity taking into account the multi-ethnicity of the voters. In terms of overall media appearances, except in The Atjeh Post, where Muzakir Manaf from PA was dominant, Irwandi Yusuf was the most-reported ex-GAM during the election campaign in the selected media. Finally, the available evidence seems to suggest that Irwandi‘s language of politics was rhetorically more attractive than that of other ex-GAM leaders.
The future heavy-ion experiment CBM (FAIR/GSI, Darmstadt, Germany) will focus on the measurements of very rare probes, which require the experiment to operate under extreme interaction rates of up to 10 MHz. Due to high multiplicity of charged particles in heavy-ion collisions, this will lead to the data rates of up to 1 TB/s. In order to meet the modern achievable archival rate, this data ow has to be reduced online by more than two orders of magnitude.
The rare observables are featured with complicated trigger signatures and require full event topology reconstruction to be performed online. The huge data rates together with the absence of simple hardware triggers make traditional latency limited trigger architectures typical for conventional experiments inapplicable for the case of CBM. Instead, CBM will employ a novel data acquisition concept with autonomous, self-triggered front-end electronics.
While in conventional experiments with event-by-event processing the association of detector hits with corresponding physical event is known a priori, it is not true for the CBM experiment, where the reconstruction algorithms should be modified in order to process non-event-associated data. At the highest interaction rates the time difference between hits belonging to the same collision will be larger than the average time difference between two consecutive collisions. Thus, events will overlap in time. Due to a possible overlap of events one needs to analyze time-slices rather than isolated events.
The time-stamped data will be shipped and collected into a readout buffer in a form of a time-slice of a certain length. The time-slice data will be delivered to a large computer farm, where the archival decision will be obtained after performing online reconstruction. In this case association of hit information with physical events must be performed in software and requires full online event reconstruction not only in space, but also in time, so-called 4-dimensional (4D) track reconstruction.
Within the scope of this work the 4D track finder algorithm for online reconstruction has been developed. The 4D CA track finder is able to reproduce performance and speed of the traditional event-based algorithm. The 4D CA track finder is both vectorized (using SIMD instructions) and parallelized (between CPU cores). The algorithm shows strong scalability on many-core systems. The speed-up factor of 10.1 has been achieved on a CPU with 10 hyper-threaded physical cores.
The 4D CA track finder algorithm is ready for the time-slice-based reconstruction in the CBM experiment.
Air-sea feedbacks between the Mediterranean Sea and the atmosphere on various temporal and spatial scales play a major role in the Mediterranean regional climate system and beyond. The Mediterranean Sea is a source of moisture due to excess evaporation and, on a long-term average, is associated with a warming of the lower atmosphere in contact with the sea surface due to heat loss at the air-sea interface. The complex air-sea interactions and feedbacks in the Mediterranean basin strongly modulate the sea surface fluxes and favor several cyclogenetic activities under certain meteorological conditions. Examples of such cyclonic activities are medicanes (Mediterranean hurricanes) and Vb-cyclones. Medicanes are mesoscale, marine, and warm-core Mediterranean cyclones that exhibit some similarities to tropical cyclones, while Vb-cyclones are extra-tropical cyclones, that propagate from the Western Mediterranean Sea and travel across the Eastern European Alps into the Central European region. Extremely strong winds and heavy precipitation associated with these cyclones can lead to severe destruction and flooding. Changes in the intensity and frequency of these cyclones are also projected under changing future climate conditions, where the Mediterranean region has been identified as a hotspot in terms of rising temperatures.
The development of high-resolution regional climate models (RCMs) has progressed our understanding of the processes characterizing the Mediterranean climate. However, large uncertainties still exist regarding the estimates of air-sea fluxes, which, in turn, affect the simulation of the Mediterranean climate. Several factors can be attributed to such discrepancies, such as data quality, temporal and spatial resolution, and the misrepresentation of physical processes. To overcome some of these inconsistencies and deficiencies of the existing climate simulations, a new high-resolution atmosphere-ocean regional coupled model (AORCM) has been developed to simulate the air-sea feedback mechanisms. This coupled model incorporates the coupling of RCM COSMO-CLM (CCLM) and the regional ocean model NEMO-MED12 for the Mediterranean Sea (MED) as well as NEMO-NORDIC for the North- and Baltic Sea (NORDIC). Several experiments were performed using both the coupled and uncoupled models to investigate the impact of air-sea interactions and feedbacks on sea surface heat fluxes, wind speed, and on the formation of Mediterranean cyclones (i.e., medicanes and Vb-cyclones). These experiments were performed using different horizontal atmospheric grid resolutions to analyze the effect of resolution on sea surface heat fluxes, wind speed, and the development of medicanes.
The results of the present study indicate that a finer atmospheric grid resolution ([is as appreciated as]9 vs. [is as appreciated as]50 km) improved the wind speed simulations (particularly near coastal areas) and subsequently improved the simulations of the turbulent heat fluxes. Both parameters were better simulated in the coupled simulations than in the uncoupled simulations, but coupling introduced a warm SST bias in winter. Radiation fluxes were slightly better represented in coarse-grid simulations than in fine-grid simulations. However, the higher-resolution coupled model could reproduce the observed net outgoing total surface heat flux over the Mediterranean Sea. In addition to that sub diurnal SST variations have a strong effect on sub-daily heat fluxes and wind speed but minor effects at longer timescales. Regarding the impact of atmospheric grid resolution ([is as appreciated as]50, 25, and [is as appreciated as]9 km) and ocean coupling on medicanes, it was detected that the coupled model with a finer atmospheric grid ([is as appreciated as]9 km) was able to not only reproduce most medicane events, but also improved the track length, warm core, and wind speed compared to the uncoupled model. The coupled model with the coarse-grid ([is as appreciated as]50 and [is as appreciated as]25 km) did not show any improvement in simulating medicanes compared to the uncoupled model. The spectral nudging technique, applied on the wind components above 850 hPa in the interior domain to keep large-scale circulation close to the driving data (i.e., ERAInterim reanalysis), improved the accuracy of the times and locations of generated medicanes, but no improvement was found in the track length and intensity.
Concerning the role of the Mediterranean Sea coupling on Vb cyclones, the investigation showed that atmosphere-ocean coupling had an overall positive impact, although with a strong case-by-case variation, on the trajectories and intensity of Vb-cyclones as a result of the variation in moisture source for each event. In general, all model configurations could replicate Vbcyclones, their trajectories, and associated precipitation fields. The average structure of the precipitation field was best represented in the coupled simulations. Coupling of the North- and Baltic Seas also showed an improvement in some of the simulated Vb-cyclones.
The atmosphere-ocean coupling showed an overall positive impact on the simulation of sea surface heat fluxes and Mediterranean cyclones (medicanes and Vb-cyclones). Moreover, the representation of sea surface heat fluxes, wind speed, and medicane features was more realistic when using a finer atmospheric grid resolution (less than 10 km). The present study suggests that the combination of a finer atmospheric grid resolution together with atmosphere-ocean coupling is advantageous in simulating the Mediterranean climate system.
Nearly 170 million people are chronically infected with HCV and thus at risk of developing liver cirrhosis and hepatocellular carcinoma. Although new and effective oral antiviral drugs are available, there is still the need for a preventive vaccine. In addition, in light of the high number of patients who are chronically infected with HCV the development of a therapeutic vaccine will present a support or even an alternative to the expensive medications.
To induce HCV-specific immune responses in a vaccine model, the HBV capsid is used as a carrier to deliver HCV antigens. Due to its icosahedral structure, the HBV capsid is highly immunogenic and helps to elicit a strong B cell response against the delivered antigens. In addition, the translocation motif (TLM) from the HBV surface protein is fused to the core protein. The TLM conveys membrane-permeability to the carrier capsid, enabling antigen transfer into the cytoplasm, and thus allows immunoproteasomal processing and MHC class I-mediated presentation of the antigen. To load the capsid with foreign antigens, a strep-Tag/streptavidin system is utilized. Recombinant capsids and antigens were purified from the E. coli production system. Detailed characterization of the carrier capsid demonstrated the proper assembly, adequate thermal stability and the successful loading of the foreign antigens onto the capsid surface.
As a further step, seven different HCV-derived proteins were produced and purified for the coupling on the surface of TLM-core particles. The characterization of their immunogenicity using this system is being performed.
Using ovalbumin as a model antigen, which is coupled to the carrier capsids via strep-Tag/streptavidin binding, shows that this system is suitable to efficiently deliver antigens into the cytoplasm of antigen-presenting cells (APCs), leading to the activation of APCs. This activation was assessed by measuring the secretion of IL-6 and TNF-α, in addition to the upregulation of activation markers (CD40, CD80, CD69, and MHC class I). Upon activation, the APCs were able to activate ova-specific CD8+ T cells measured by secreted IFN-γ, which was up to 20-folds more than IFN-γ secreted upon incubation with free ovalbumin. These data indicate that the TLM-capsid is suitable to serve as a carrier to deliver foreign antigens into the cytoplasm of APCs leading to MHC class I-mediated presentation and induction of an antigen-specific CTLs response.
Gepulste dipolare EPR-Spektroskopie ist eine wertvolle Methode, um Abstände von 1.5 bis 10 nm zwischen zwei Spinmarkern zu messen. Diese Information kann für Strukturbestimmungen hilfreich sein, wo traditionelle Methoden wie Kristallstrukturanalyse und NMR nicht angewendet werden können. Zusätzlich ist es möglich, Änderungen in Konformation und Flexibilität zu verfolgen. Für diese Studien haben sich stabile Nitroxidradikale als Spinmarker etabliert. Diese werden spezifisch durch die site-directed spin labelling Methode (SDSL) kovalent an das zu untersuchende Biomolekül gebunden. In den letzten Jahren wurden weitere Spinmarker für Abstandsbestimmungen mittels EPR-Spektroskopie entwickelt. Besonders interessant sind Triarylmethylradikale (im Folgenden abgekürzt als Trityl) und paramagnetische Metallzentren.
Im Vergleich zu Nitroxidradikalen hat das Tritylradikal einige Vorteile: Eine höhere Stabilität in einer reduzierenden Umgebung wie im Inneren von Zellen, längere Elektronenspin-Relaxationszeiten bei Raumtemperatur und ein schmaleres EPR-Spektrum. Deswegen ist dieses organische Radikal ein alternativer Spinmarker, der besonders gut für die Forschung von Biomolekülen in einer nativen Umgebung unter physiologischen Bedingungen geeignet ist. Auch paramagnetische Metallzentren sind weniger reduktionsempfindlich als Nitroxidradikale. Zusätzlich sind diese Spinmarker interessant in biologischen Fragestellungen. Zum Beispiel besitzen zahlreiche Enzyme paramagnetische Manganzentren als Cofaktoren. Zudem kann Magnesium, ein wesentlicher Cofaktor in Enzymen, Nukleinsäuren und Nukleotid-Bindungsdomänen der G- und Membranproteine, oft durch das paramagnetische Mangan ersetzt werden. Um Abstandsmessungen an Biomolekülen, die nur ein Metallzentrum besitzen, durchzuführen, können zusätzliche Spinmarker in Form eines Nitroxid-, Tritylradikals oder eines anderen paramagnetischen Metallkomplexes mithilfe der SDSL-Methode kovalent gebunden werden.
Nitroxidradikale, Tritylradikale und Metallzentren haben deutlich unterschiedliche EPR-spektroskopische Eigenschaften, welche oft als orthogonale Spinmarker bezeichnet werden. Solche Spinmarker sind nützlich für die Untersuchung von verschiedenen Untereinheiten bei makromolekularen Komplexen. Somit können die intramolekularen Abstände innerhalb einer Untereinheit sowie intermolekularen Abstände zwischen den unterschiedlichen Untereinheiten mit nur einer einzigen Probe bestimmt werden. Zusätzlich können die orthogonalen Marker sehr effektiv genutzt werden, um Metallzentren in Biomolekülen mithilfe der Trilateration-Strategie genau zu lokalisieren.
Die hier vorliegende Doktorarbeit beschäftigt sich mit der Nutzung dieser neuen Spinmarker für Abstandsmessungen. Solche Spinmarker sind noch kaum erforscht, obwohl sie für biologische Anwendungen eine große Rolle spielen könnten.
Das erste Ziel dieser Doktorarbeit war eine Studie über Tritylradikale mithilfe der dipolaren EPR-Spektroskopie. Zu diesem Zweck wurden sowohl double quantum coherence (DQC) und single frequency technique for refocussing dipolar couplings (SIFTER) Experimente als auch Hochfrequenz pulsed electron electron double resonance (PELDOR) Experimente mit einem Trityl-Modellsystem durchgeführt. Dabei wurden die Besonderheiten der unterschiedlichen dipolaren Spektroskopiemethoden mit diesem Spinmarker untersucht, um die Empfindlichkeit und Robustheit für die Abstandsmessungen zu optimieren.
Das zweite Ziel war eine Studie über den Einfluss der Hochspin-Multiplizität des Mangans auf die Abstandsbestimmungen. Für diesen Zweck wurde zuerst ein Modellsystem mit einem orthogonalen Mn2+ Ion und Nitroxidradikal mithilfe der PELDOR-Spektroskopie untersucht. Anschließend wurde ein weiteres Modellsystem mit zwei Mn2+-Ionen untersucht, um PELDOR und relaxation-induced dipolar modulation enhancement (RIDME) Experimente bezüglich ihrer Empfindlichkeit und Robustheit sowie Genauigkeit der Datenanalyse zu optimieren.
Das Trityl-Modellsystem wurde in der Arbeitsgruppe von Prof. Sigurdsson synthetisiert. Die EPR Messungen wurden bei zwei verschiedenen Mikrowellenfrequenzen (34 und 180 GHz) durchgeführt. Es wurde gezeigt, dass die Auswahl der optimalen Methode von den EPR-spektroskopischen Eigenschaften des Systems bei den jeweiligen Mikrowellenfrequenzen abhängig ist. Das EPR-Spektrum des Trityls ist bei 34 GHz so schmal, dass das ganze Spektrum von einem üblichen Mikrowellenpuls angeregt werden kann. In diesem Fall sind die DQC und SIFTER Experimente am besten geeignet. Der mit diesen Methoden bestimmte Abstand von 4.9 nm ist in guter Übereinstimmung mit Werten aus der Literatur. Es wurde festgestellt, dass die SIFTER Messung eine höhere Empfindlichkeit als DQC besitzt, da das Signal-zu-Rausch Verhältnis um den Faktor vier größer ist. Außerdem ist die SIFTER-Methode experimentell weniger anspruchsvoll, da ein deutlich kürzerer Phasenzyklus für die Mikrowellenpulse benötigt wird. ...
The development of the atrioventricular (AV) canal and the cardiac valves is tightly linked and a critically regulated process. Anomalies in components of the involved pathways can lead to congenital valve malformations, a leading cause of morbidity and mortality in neonates. Myocardial Bmp as well as endocardial Notch and Wnt signaling have been identified as critical factors for the induction of EMT during the formation of the endocardial cushions and cardiac valves. Of these, canonical Wnt signaling positively regulates endocardial proliferation and EMT but negatively regulates endocardial differentiation. Further, elevated Wnt signaling leads to the ectopic expression of myocardial Bmp ligands suggesting a high level of integration of the involved pathways and crosstalk amongst the different cardiac tissues.
Here we have identified a novel role for Id4 as a mediator between Bmp and Wnt signaling. Id4 belongs to the Id family of proteins and is known to be involved in bone and nervous system development. We found that in zebrafish, id4 is expressed in the endocardium of the AV canal at embryonic stages and throughout the atrial chamber in addition to AV canal, in adults. Using transcription activator-like effector nucleases (TALENs) we established an id4 mutant allele. Our analysis shows that id4 mutant larvae are susceptible to retrograde blood flow, and show aberrant expression of developmental valvular markers. These include expanded expression domains of markers like bmp4, cspg2a and Alcam. In contrast, valve maturation as assessed by the expression of spp1 is considerably reduced in id4 mutants. Using conditional transgenic systems, along with elegant in vivo imaging of transgenic reporter lines, we further found that id4 is a transcriptional target of Bmp signaling, and it is capable of dose dependently restricting Wnt signaling in the endocardium of the Atrioventricular Canal.
Taken together, our data identifies Id4 as a novel player in Atrioventricular Canal and valve development. We show that Id4 function is important in valve development acting downstream of Bmp signaling by restricting endocardial Wnt to allow valve maturation
Batten disease refers to neuronal ceroid lipofuscinoses (NCLs), which are inherited lysosomal storage diseases with diverse ages of onset and cause progressive neurodegeneration. The most common NCL is Juvenile NCL (JNCL), which begins in early childhood and is characterized by lysosomal accumulation of subunit c of the mitochondrial ATP synthase (subunit c). JNCL is caused by mutations in the gene CLN3. This gene encodes the CLN3 protein, a transmembrane protein of unknown structure. Localization of CLN3 is ambiguous, and its exact cellular function is not known. Thereby, it is unclear what mechanisms lead to neurodegeneration in JNCL. Models of JNCL present disturbed membrane bound organelles and cytoskeleton as well as impaired autophagy and lysosomal function. The JNCL gene defect that most patients harbor is deletion of the exons 7 and 8 of CLN3. In the Cln3Δex7/8/Δex7/8 mouse model of JNCL, this deletion has been introduced to the mouse Cln3 gene.
The actin cytoskeleton consists of filaments formed through polymerization of actin and provides a framework which defines cellular morphology and also facilitates cell motility, cytokinesis, and cell surface remodeling. Rho GTPases are signaling proteins which regulate the assembly and dynamics of the actin cytoskeleton and play an important role in neuronal morphology. Rho GTPases need to be membrane-anchored in order to become active and initiate a signaling cascade. Their membrane anchorage is achieved through their geranylgeranyl tails, which they acquire through prenylation. Protein prenylation refers to the attachment of a geranylgeranyl or farnesyl group to the C-terminus of a protein. The enzyme geranylgeranyl transferase (GGTase) catalyzes geranylgeranylation, whereas geranylgeranyl pyrophosphate (GGPP) is the donor of the geranylgeranyl group. Cells produce GGPP as well as cholesterol and other lipids through the mevalonate pathway (MVA pathway).
The aim of this study was to analyze how the JNCL gene defect affects cellular morphology, especially the actin cytoskeleton and Rho GTPases, and the MVA pathway which is connected with Rho GTPase activation. These important cellular components play crucial roles in neurons and are implicated in other neurodegenerative diseases, but have received little attention in JNCL. The immortalized CbCln3Δex7/8/Δex7/8 cerebellar precursor cell line from Cln3Δex7/8/Δex7/8 mice was used for the experiments and provides a genetically accurate, neuronal cell model of JNCL. CbCln3Δex7/8/Δex7/8 cells present subunit c accumulation only when aged at confluency, but sub-confluent cells display other phenotypes. The experiments of this study were performed both with confluency-aged and sub-confluent cells. Filamentous actin was visualized, and protein levels as well as membrane localization of several small Rho GTPases was analyzed biochemically. Also the protein levels of GGTase and the key enzymes of the mevalonate pathway were determined.
Staining pattern of filamentous actin was disturbed in confluency-aged CbCln3Δex7/8/Δex7/8 cells. Additionally it was found out that these cells did not grow to wild-type size and exhibited an elongated peroxisomal morphology. Rho GTPases had reduced total levels and showed a tendency of decreased membrane localization. Levels of GGTase and the MVA pathway enzymes were altered. Results of sub-confluent CbCln3Δex7/8/Δex7/8 cells were similar with the exception of HMG-CoA reductase, which is the rate-limiting enzyme of the MVA pathway: while its level in confluency-aged CbCln3Δex7/8/Δex7/8 cells was increased, at sub-confluency it showed a reduced level. Also, in contrast with the confluency-aged cells, Rho GTPases presented a tendency of increased membrane localization.
The results of this study reveal that the accurate JNCL gene defect alters cellular morphology and the activity of the MVA pathway in neuronal cells. Small cell size and disrupted architecture of the actin cytoskeleton are confirmed as neuronal JNCL phenotypes, and the peroxisome is introduced as a novel cellular component affected in JNCL. Through defects in endocytosis, autophagy, lysosomal and mitochondrial function, and cytoskeleton, the JNCL gene defect may prevent cells from growing to wild-type size. The JNCL gene defect may attenuate the MVA pathway via mitochondrial dysfunction and/or upregulation of degradative processes. Attenuation of the MVA pathway may contribute to impaired membrane rafts, which are an established phenotype of JNCL cells. As indicated by reduced GGTase level and supported by downregulation of lipid production through the MVA pathway, the JNCL gene defect might also decrease prenylation of proteins.
In the recent past, we are making huge progress in the field of Artificial Intelligence. Since the rise of neural networks, astonishing new frontiers are continuously being discovered. The development is so fast that overall no major technical limits are in sight. Hence, digitization has expanded from the base of academia and industry to such an extent that it is prevalent in the politics, mass media and even popular arts. The DFG-funded project Specialized Information Service for Biodiversity Research and the BMBF-funded project Linked Open Tafsir can be placed exactly in that overall development. Both projects aim to build an intelligent, up-to-date, modern research infrastructure on biodiversity and theological studies for scholars researching in these respective fields of historical science. Starting from digitized German and Arabic historical literature containing so far unavailable valuable knowledge on biodiversity and theological studies, at its core, our dissertation targets to incorporate state-of-the-art Machine Learning methods for analyzing natural language texts of low-resource languages and enabling foundational Natural Language Processing tasks on them, such as Sentence Boundary Detection, Named Entity Recognition, and Topic Modeling. This ultimately leads to paving the way for new scientific discoveries in the historical disciplines of natural science and humanities. By enriching the landscape of historical low-resource languages with valuable annotation data, our work becomes part of the greater movement of digitizing the society, thus allowing people to focus on things which really matter in science and industry.
1 Purpose of the Study:
The purpose of this retrospective study was to assess the volumetric changes of our institutional pediatric neuroblastoma in response to various therapeutic protocols.
2 Materials and Methods:
A retrospective study was conducted on children with neuroblastoma from different anatomical locations including suprarenal, paraspinal, pelvic, mediastinal and cervical neuroblastoma primaries. These children underwent tumor-stage based therapeutic protocols in Johann Wolfgang Goethe University Hospital, Frankfurt am Main, Germany, between January 1996 and July 2008. The study included 72 patients (44 males and 28 females). Patient demographics (age and gender), disease-related symptoms, laboratory results (tumor biomarkers including ferritin, neuron specific enolase, and urine catecholamine) and histopathological reports were collected from the electronic medical archiving system and subsequently analyzed.
Patients were classified into following groups according the anatomical origin of the primary neuroblastoma into:
1) Suprarenal neuroblastoma Group: This group included patients with neuroblastoma arising from the suprarenal gland. This group composed of 54 patients with male to female ratio (32:22).
2) Paravertebral neuroblastoma Group: This group composed of 6 male patients.
3) Mediastinal neuroblastoma Group: This group included patients with mediastinal neuroblastoma and composed of 3 patients (1 male and 2 females).
4) Pelvic neuroblastoma Group: This group included patients with pelvic neuroblastoma and composed of 6 patients (3 males and 3 females).
5) Cervical neuroblastoma Group: This group included patients with cervical neuroblastoma and composed of 2 male patients.
3 Results:
The mean volume of all suprarenal neuroblastoma group involved in the study before therapy was 176.62 cm3 (SD: 234.15) range: 239.4-968.9cm3. The mean initial volume of all suprarenal neuroblastoma group who underwent observation protocol was 86.0378 cm3 (SD: 114.44) range: 5.2-347.94cm3. Volumetric evaluation of suprarenal neuroblastoma following observation (Wait and See) protocol revealed continuous reduction of the tumor volumes in a statistically significant manner during the follow up periods up to 12 months with p value of less than 0.05. The volumetric changes afterwards were statistically insignificant.
The mean initial volume of all suprarenal neuroblastoma group who underwent primary surgery protocol was 42.4 cm3 (SD: 28.5) range: 7.5-90cm3. Complete surgical resection of the tumor was not feasible in all lesions due to local tumor extension and / or infiltration with the associated risk of injury of nearby organs or structures. However statistical analysis of the volumetric changes in the successive follow up periods did not reveal statistical significance.
Volumetric estimation of the tumor in the subsequent follow up periods revealed significant changes within the period first (3-9 month periods). The changes afterwards were statistically non significant. On the other hand, the mean initial volume of all suprarenal neuroblastoma group who underwent combined chemotherapy and Stem cell transplantation protocol only without surgical interference was 99.98cm3 (SD:46.2) range: 48.48-160.48 cm3. In this group the volumetric changes were variable and difference in volumes in follow up was statistically non significant during the follow up period.
The mean initial volume of all abdominal paravertebral neuroblastoma group was 249.197cm3 (SD: 249.63) range: 9.6-934cm3. The mean initial volume of all pelvic neuroblastoma group was 118.88cm3 (SD: 50.61) range: 73.4-173.4cm3. The mean initial volume of all mediastinal neuroblastoma group was 189.7cm3 (SD: 139.057) range: 10.7-415 cm3. The mean initial volume of all cervical neuroblastoma group was 189.7cm3 (SD: 139.057) range: 10.7-415 cm3. The volumetric measurements in the corresponding follow up periods according to the therapeutic protocol of abdominal paravertebral neuroblastoma, pelvic neuroblastoma, mediastinal and cervical neuroblastoma revealed significant change in the tumor volume within the early 3-6 months from the initial therapy while subsequently the tumor volumetric changes were statistically non significant.
4 Conclusion:
In conclusion, the role of MRI volumetry in the evaluation of tumor response is dependent on the risk adapted concept of neuroblastoma with the combination of different imaging modalities as well the therapeutic protocol. MRI Volumetry in addition to new protocols such as Whole-body imaging and 3D visualization techniques are gaining more importance and acceptance.
Specific functions of biological systems often require conformational transitions of macromolecules. Thus, being able to describe and predict conformational changes of biological macromolecules is not only important for understanding their impact on biological function, but will also have implications for the modelling of (macro)molecular complex formation and in structure-based drug design approaches. The “conformational selection model” provides the foundation for computational investigations of conformational fluctuations of the unbound protein state. These fluctuations may reveal conformational states adopted by the bound proteins. The aim of this work is to incorporate directional information in a geometry-based approach, in order to sample biologically relevant conformational space extensively. Interestingly, coarse-grained normal mode (CGNM) approaches, e.g., the elastic network model (ENM) and rigid cluster normal mode analysis (RCNMA), have emerged recently and provide directions of intrinsic motions in terms of harmonic modes (also called normal modes). In my previous work and in other studies it has been shown that conformational changes upon ligand binding occur along a few low-energy modes of unbound proteins and can be efficiently calculated by CGNM approaches. In order to explore the validity and the applicability of CGNM approaches, a large-scale comparison of essential dynamics (ED) modes from molecular dynamics (MD) simulations and normal modes from CGNM was performed over a dataset of 335 proteins. Despite high coarse-graining, low frequency normal modes from CGNM correlate very well with ED modes in terms of directions of motions (average maximal overlap is 0.65) and relative amplitudes of motions (average maximal overlap is 0.73). In order to exploit the potential of CGNM approaches, I have developed a three-step approach for efficient exploration of intrinsic motions of proteins. The first two steps are based on recent developments in rigidity and elastic network theory. Initially, static properties of the protein are determined by decomposing the protein into rigid clusters using the graph-theoretical approach FIRST at an all-atom representation of the protein. In a second step, dynamic properties of the molecule are revealed by the rotations-translations of blocks approach (RTB) using an elastic network model representation of the coarse-grained protein. In the final step, the recently introduced idea of constrained geometric simulations of diffusive motions in proteins is extended for efficient sampling of conformational space. Here, the low-energy (frequency) normal modes provided by the RCNMA approach are used to guide the backbone motions. The NMSim approach was validated on hen egg white lysozyme by comparing it to previously mentioned simulation methods in terms of residue fluctuations, conformational space explorations, essential dynamics, sampling of side-chain rotamers, and structural quality. Residue fluctuations in NMSim generated ensemble is found to be in good agreement with MD fluctuations with a correlation coefficient of around 0.79. A comparison of different geometry-based simulation approaches shows that FRODA is restricted in sampling the backbone conformational space. CONCOORD is restricted in sampling the side-chain conformational space. NMSim sufficiently samples both the backbone and the side-chain conformations taking experimental structures and conformations from the state of the art MD simulation as reference. The NMSim approach is also applied to a dataset of proteins where conformational changes have been observed experimentally, either in domain or functionally important loop regions. The NMSim simulations starting from the unbound structures are able to reach conformations similar to ligand bound conformations (RMSD < 2.4 Å) in 4 out of 5 cases of domain moving proteins. In these four cases, good correlation coefficients (R > 0.7) between the RMS fluctuations derived from NMSim generated structures and two experimental structures are observed. Furthermore, intrinsic fluctuations in NMSim simulation correlate with the region of loop conformational changes observed upon ligand binding in 2 out of 3 cases. The NMSim generated pathway of conformational change from the unbound structure to the ligand bound structure of adenylate kinase is validated by a comparison to experimental structures reflecting different states of the pathway as proposed by previous studies. Interestingly, the generated pathway confirms that the LID domain closure precedes the closing of the NMPbind domain, even if no target conformation is provided in NMSim. Hence, the results in this study show that, incorporating directional information in the geometry-based approach NMSim improves the sampling of biologically relevant conformational space and provides a computationally efficient alternative to state of the art MD simulations.
Cold target recoil ion momentum spectroscopy (COLTRIM) has been employed to image the momentum distributions of continuum electrons liberated in the impact of slow He2+ on He and H2. The distributions were measured for fully determined motion of the nuclei that is as a function of the impact parameter and in a well de ned scattering plane The single ionization (SI) of H2 leading to H2+ recoil ions in nondissociative states (He2+ + H+ -> He2+ + H+ + e-) and the transfer ionization (TI) of H2 leading to H2 dissociation into two free protons (He2+ H2 -> He+ + H+ + H+ + e-) were investigated. Similar measurements have been carried out for He target, the corresponding atomic two electron system, i.e. the single ionization (SI) (He2+ + He -> He+ + He2+ e- and the transfer ionization (TI) (He2+ + He -> He+ + He2+ + e-). These measurements have been exploited to understand the results obtained for H target. In comparing the continuum electron momentum distributions for H2 with that for He, a high degree of similarity is observed. In the case of transfer ionization of H2, the electron momentum distributions generated for parallel and perpendicular molecular orientations revealed no orientation dependence. The in scattering plane electron momentum distributions for the transfer ionization of H2 by He2+ and for the transfer ionization of He by He2e showed that the salient feature of these distributions for both collisions systems consists in the appearance of two groups of electrons with difeerent structures. In addition to the group of the saddle electrons forming two jets separated by a valley along the projectile axis we nd a new group of electrons moving with a velocity higher than the projectile velocity These new fast forward electrons result from a narrow range of impact parameters and appear as image saddle in the projectile frame. In contrast to the transfer ionization of He, the fast forward electrons group disappears in the in scattering plane electron momentum distribution generated for the single ionization of He. Instead of this group another new group of electrons appear These electrons exhibit an amount of backscattering These backward elec trons appear as image saddle in the target frame The structures that the saddle electrons show are owing to the quasi molecular nature of the collision process For the TI of H2, the TI of He and the SI of He, a pi-orbital shape of the electron momentum distribution is observed This indicates the importance of the rotational coupling 2-p-theta -> 2p-pi in the initial promotion of the ground state followed by further promotions to the continuum The backward electrons as well as the fast forward electrons are not discussed in the theoretical literature at all. However, a number of obvious indications of the existence of the backward and fast forward electrons could be seen in the experimental works of Abdallah et al. as well as in the theoretical calculations of Sidky et al One might speculate that electrons which are promoted on the saddle for some time during the collision could finally swing around the He+ ion in the way out of the collision, i.e. either around the projectile in the forward direction as in the TI case forming the fast forward electrons or around the recoil ion in the backward direction as in the SI case forming the backward electrons. This might be a result of the strong gradient, and hence the large acceleration of the screened He+ potential.
The high energy loss of heavy ions in matter as well as the small angular scattering makes heavy ion beams an excellent tool to produce almost cylindrical and homogeneously excited volumes in matter. This aspect can be used to pump short wavelength lasers. In an experiment performed at the GSI (Gesellschaft für Schwerionenforschung, Darmstadt, Germany) ion accelerator facility in December 2005 the well-known KrF* excimer laser was pumped with an intense high energy uranium beam. Pulses of an uranium beam with initial particle energy of 250 MeV per nucleon, provided by heavy-ion-synchrotron SIS-18, were delivered to the HHT-target station and then stopped inside a gas laser cell. The maximum beam intensity reached in the experiment was 2,5·109 particles per pulse, which resulted in 34 J/g specific energy deposited in the laser gas. By applying electron cooling and a bunch compression technique at SIS-18, the beam pulses were compressed down to 110 ns (FWHM). A mixture of an excimer laser premix gas (95,5% Kr + 0,5% F2) and a buffer gas (Ar 4.8) was used as the laser gas in proportions of 35/65 and 60/40, respectively. The gas pressure inside the laser cell was varied in the range of 1,2÷2 bar in continues flow mode. The experimental setup consisted of a 1 m long stainless steel tube with a number of diagnostic viewports and two mirror adjustment units. The optical cavity was formed by a flat, Alcoated mirror at the beam entrance and a second dielectrically coated, highly reflective mirror with 3 m radius of curvature at a distance of 1,3 m. A beam of heavy ions has been used to pump a short wavelength gas laser for the first time. Laser effect on the KrF* laser transition (λ = 248 nm) has been successfully demonstrated. Laser threshold for this specific setup was reached with a beam intensity of 1,2·109 particles per pulse. Laser action has been clearly proofed by the following methods: appearance of the laser line, spectral narrowing of the laser line, temporal narrowing of the laser signal, non-linear response of the laser output intensity on the pumping power, and cavity disalignment effect. An energy of the laser pulse of about 2 mJ was measured for an ion beam intensity of 2·109 particles per pulse. The time delay of the onset of the laser emission with respect to the pumping pulse was measured as a function of ion beam intensity. The dependence of spontaneous emission spectra on the gas pressure in a range of 1,3÷2 bar was observed and the optimal gas pressure for laser experiments in the sense of laser efficiency was concluded. As a next step in studying short wavelength lasers pumped with heavy ion beams it is planned to reduce the laser wavelength down to the VUV region of the spectrum, and to proceed to the excimer lasers of the pure rare gases: Xe2 * (λ = 172 nm), Kr2 * (λ = 146 nm), Ar2 * (λ = 126 nm), Ne2 * (λ = 83 nm) and He2 * (λ = 80 nm). We believe that the use of heavy ion beams as a pumping source may lead to new pumping schemes on the higher lying level transitions and considerably shorter wavelengths (XUV and X-ray spectral region), which rely on the high cross sections for multiple ionization of the target species.
In Belize, which is well known for the Belize Barrier Reef and its offshore atolls, coastal lagoons are frequent morphological features along the coast. They represent transitional environments between siliciclastic and carbonate settings. In order to shed light into the Holocene evolution of coastal lagoon environments, five localities along the central coast of Belize were selected as coring sites. These include four coastal lagoons and one marsh area, namely Mantatee Lagoon, Mullins River Beach, Colson Point Lagoon, Commerce Bight Lagoon and Sapodilla Lagoon. A total of 26 sediment cores with core lengths ranging from 109 cm to 500 cm, were drilled using a Lanesky-vibracorer. Overall, 73 m of Holocene sediments and Pleistocene soil were recovered. Together with 58 radiocarbon dates the sediments reveal details on the sediment architecture and depositional features of the localities.
Marine inundation of the mainland and coastal lagoon formation started around 6 kyrs cal BP.
As a response to sea-level rise during the Holocene transgression, facies retrograded towards the coast, as seen in marginal marine overlying brackish mollusk faunas. Evidence for late Holocene progradation of facies due to sea-level stagnation is largely lacking. The occurrence of landward thinning sand beds, hiatuses and marine fauna in lagoonal successions are indications of event (overwash) sedimentation. Sediments recovered are largely of Holocene age (<7980 cal BP), overlying Pleistocene sections. Analyses of sediment composition and texture, radiocarbon dating and mollusk shell identification were used to describe and correlate sedimentary facies.
XRD analyses have identified quartz as the dominant mineral, with the Maya Mountains as main source of coastal lagoon sediments. The most common sedimentary facies include peat and peaty sediment, mud, sand, and poorly sorted sediments. Pleistocene soil forms the basement of Holocene sediments. Holocene mud represents lagoon background permanent sedimentation.
Peats and peat-rich sequences were deposited in mangrove swamp environments, whereas sandy facies mainly occur in the shoreface, beach, barriers, bars, barrier spits and overwash deposits. Facies successions could be identified for each locality, but it has proven difficult to correlate the stratigraphic sequences, especially among localities. These differences among the five locations studied suggest that apart from regional influence such as sea-level rise, local environmental factors such as small-scale variation in geomorphology and resulting facies heterogeneity, connectivity of the lagoon with the sea, antecedent topography and river discharge, were responsible for coastal sedimentation and lagoon development in the Holocene of Belize.
Faunal composition and distribution patterns of mollusk assemblages from 20 shell concentrations in cores collected in coastal lagoons, a mangrove-fringed tidal inlet and the marginal marine area (shallow subtidal) show considerable variation due to environmental heterogeneity and the interplay of several environmental factors in the course of the mid-late Holocene (ca. 6000 cal BP to modern). The investigated fauna ≥2 mm comprises 2246 bivalve, gastropod and 11 scaphopod specimens. Fifty-three mollusk species, belonging to 42 families, were identified. The bivalve Anomalocardia cuneimeris and cerithid gastropods are the dominant species and account for 78% of the total fauna. Diversity indices are low in concentrations from lagoons and relatively high in the marginal marine and tidal inlet areas.
Based on cluster analysis and nonmetric multidimensional scaling (NMDS), seven lagoonal assemblages and three marginal marine/tidal inlet assemblages were defined. A separation between lagoonal and marginal marine/tidal inlet assemblages seen in ordination indicates a lagoon-onshore gradient. The statistical separation among lagoonal assemblages demonstrates environmental changes during the Holocene evolution of the coastal lagoons, which is probably related to the formation of barriers and spits. The controlling factors of species distribution patterns are difficult to figure out, probably due to the heterogeneity of the barrier-lagoon systems and the interaction of paleoecological and paleoenvironmental factors. In addition to the taxonomic analysis, a taphonomic analysis of 1827 valves of A. cuneimeris from coastal lagoons was carried out. There is no relationship between depth and age of shells and their taphonomic condition. Size-frequency distributions and right-left valve ratios of A. cuneimeris suggest that valves were not transported over long distances but were deposited parautochthonously in their original habitat. Shells from tidal inlet and marginal marine environments were also predominantly deposited in their original habitats.
Since the Belize coast was repeatedly affected by hurricanes and the paleohurricane record for this region is poor, the sediment cores have been examined in order to identify storm deposits.
The paleohurricane record presented in this study spans the past 8000 years and exhibits three periods with increased evidences of hurricane strikes occurring at 6000-4900 cal yr BP, 4200-3600 cal yr BP and 2200-1500 cal yr BP. Two earlier events around 7100 and 7900 cal yr BP and more recent events around 180 cal yr BP and during modern times have been detected. Sand layers, redeposited corals and lagoon shell concentrations have been used as proxies for storm deposition. Additionally, hiatuses and reversed ages may indicate storm influence. While sand layers and corals represent overwash deposits, the lagoon shell concentrations, which mainly comprise the bivalve Anomalocardia cuneimeris and cerithid gastropods, have been deposited due to changes in lagoon salinity during and after storm landfalls. Comparison with other studies reveals similarities with one record from Belize, but hardly any matches with other published records. The potential for paleotempestology reconstructions of the barrier-lagoon complexes along the central Belize coast differs depending on geomorphology, and deposition of washovers in the lagoon basins is limited, probably due to the interplay of biological, geological and geomorphological processes.
IL-12-related cytokines produced by dendritic cells are considered to be major inducers of adaptive immune system activation upon innate antigen-sensing. IL-23 specifically is currently being discussed to support the differentiation of potentially auto-aggressive Th17 cells. Prostaglandins as bystander cell products are known to modulate the translation of this process. While previous studies focused therefore on IL-12, ignoring the existence of new IL-12-related cytokines IL-23 and IL-27, this study analysed effects of prostaglandin E2, D2 and 15d-PGJ2 on the secretion pattern of these subunits in the murine immature Langerhans cell line XS52 and the murine immature myeloid dendritic cell line JawsII under TLR4 (LPS) and TLR9 (CpG) stimulation as well as effects of prostaglandins on the murine Th1 cell line IF12 in coculture and upon Con A treatment. In serial semi-quantitative RT-PCR of the IL-12 related cytokines of the XS52 cell line and the JawsII cell line, the p40 subunit was upregulated in both DC cell lines upon TLR-stimulation, the IL-23p19 subunit constantly expressed in XS52 and upregulated in JawsII upon TLR-stimulation, while the IL-27p28 subunit was only weekly expressed under additional stimulating aCD40 Ab treatment. IL-12p35 could only be detected in the immature myeloid cell line. The protein expression of the p40 subunit was measured in Western blot assays following SDS-PAGE under reducing conditions in XS52. The Western blot-based antibody specification allowed the establishment of a p40-specific ELISPOT assays, where overadditive upregulation of the number of LPS-stimulated spot forming XS52 cells was observed under stimulation with PGE2 while PGD2 depressed the number of LPS-stimulated cytokine secreting cells. Contrary IL-12p40 could not be detected in supernatants of the JawsII cell line. Both DC cell lines were further tested for differential response towards different TLR stimulation described as a defining feature of DC subsets. While subunit expression on transcription level did not differ, only LPS-treatment led to constant IL-12p40 expression in supernatants of XS52. CpG-treatment of XS52 cells led to constantly high IL-12p40 levels under additional aCD40 Ab treatment. In IFN-g ELISPOT assays, prostaglandin effects were further analysed in IF12 Th1 cells upon Con A treatment or alternatively upon treatment in a coculture model with the syngeneic cell line XS52 and the T lymphocyte-specific protein ovalbumin. While PGE2 depressed the amount of activated Th1, PGD2 showed no effect. In conclusion, a coculture model has been generated that allows the analysis of DC and TC interactions. The importance of prostaglandins as differential regulators in time- and tissue-dependence in inflammatory processes has been demonstrated. These results accord with recent observations of an upregulation of IL-23 secretion upon PGE2 treatment.
Das Projekt anan ist ein Werkzeug zur Fehlersuche in verteilten Hochleistungsrechnern. Die Neuheit des Beitrags besteht darin, dass die bekannten Methoden, die bereits erfolgreich zum Debuggen von Soft- und Hardware eingesetzt werden, auf Hochleistungs-Rechnen übertragen worden sind. Im Rahmen der vorliegenden Arbeit wurde ein Werkzeug namens anan implementiert, das bei der Fehlersuche hilft. Außerdem kann es als dynamischeres Monitoring eingesetzt werden. Beide Einsatzzwecke sind
getestet worden.
Das Werkzeug besteht aus zwei Teilen:
1. aus einem Teil namens anan, der interaktiv vom Nutzer bedient wird
2. und aus einem Teil namens anand, der automatisiert die verlangten Messwerte erhebt und nötigenfalls Befehle ausführt.
Der Teil anan führt Sensoren aus — kleine mustergesteuerte Algorithmen —, deren Ergebnisse per anan zusammengeführt werden. In erster Näherung lässt anan sich als Monitoring beschreiben, welches (1) schnell umkonfiguriert werden (2) komplexere Werte messen kann, die über Korrelationen einfacher Zeitreihen hinausgehen.
This dissertation investigates several aspects of nominal modification in Ògè, an understudied language of Benue-congo spoken in Àkókó Northwest in Nigeria. The study focuses on two areas of nominal modification namely, Nominal Attributive Modifiers (NAMs) and the strategies of number marking.
The discussion and analysis of NAMs in the language reveal that Ògè belongs to the group of languages which lacks adjectives as a lexical category. NAMs are nominal and they
are derived from an existing lexical category namely, verbs. Predicative modifiers and NAMs have forms that are similar to the long and short forms (LF & SF) of adjectives in languages in which adjectives form an open class, for example, Russian, SerBoCroatian (BCS) and German.
Based on the Minimalist program, the dissertation reveals that unlike Russian, BCS, and German in which the discrepancies between the two forms of adjectives are related to definiteness (as in the case of BCS) and Agree, the discrepancies in the two forms of modifiers in Ògè are related to the fact that Ògè lacks adjectives and resorts into the nominalization of stative verbs in order to derive attributive forms. Using the analyses of adjuncts according to Truswell (2004) and Zeijlstra (2020), the dissertation proposes that NAMs are adjuncts in a modification structure while they are heads in possessive and genitive constructions. In addition, I propose that NAMs are attributive-only modifiers which modify the NP rather than
the DP.
The dissertation also investigates the strategies of number marking in Ògè. Unlike languages in which number marking is obligatory in the nominal domain (Hebrew, German, English),
nouns in Ògè are not always marked for number. This means that nouns in Ògè have general number. The general number nature of nouns in Ògè is like that of the nouns in modifying plural marking languages namely, Halkomelem, Korean, Yucatec Maya and Yorùbá. However, I argue that unlike the modifying plural marking languages in which the Number Phrase (NumP) is not projected, NumP is projected in the nominal spine of Ògè, claiming that NumP bears an
interpretable number feature which values the uninterpretable number feature in D. Argument in support of this comes from the interpretation of the noun in the presence of òtúro (an element which translates to the plural definite interpretation of the noun). I analyze òtúro as a plural determiner which occupies the D-head in the syntax of Ògè. The dissertation argues following Alexiadou (2019) that the locus of the occurrence of the marker of plurality in the nominal spine does not depend on its interpretation as a plural morpheme, rather, the locus of the occurrence of the element that is sensitive to the plural interpretation of the noun depends on other parameters which are definiteness, specificity and animacy.
Floodplains and other wetlands depend on seasonal river flooding and play an important role in the terrestrial water cycle. They influence evapotranspiration, water storage and river discharge dynamics, and they are the habitat of a large number of animals and plants. Thus, to assess the Earth’s system and its changes, a robust understanding of the dynamics of floodplain wetlands including inundated areas, water storages, and water flows is required.
This PhD thesis aims at improving the modeling of large floodplains and wetlands within the global-scale hydrological model WaterGAP, in order to better estimate water flows and water storage variations in different storage compartments. Within the scope of this thesis, I have developed a new approach to simulate dynamic floodplain inundation on a global-scale. This approach introduces an algorithm into WaterGAP, which has a spatial resolution of 0.5 degree (longitude and latitude) globally. The new approach uses subgrid-scale topography, based on high-resolution digital elevation models, to describe the floodplain elevation profile within each grid cell by applying a hypsographic curve. The approach comprises the modeling of a two-way river-floodplain interaction, the separate downstream water transport within the river and the floodplain – both with temporally and spatially different variable flow velocities – and the floodplain-groundwater interactions. The WaterGAP version that includes the floodplain algorithm, WaterGAP 2.2b_fpl, estimates floodplain and river water storage, inundated area and water table elevation, and also simulates backwater effects.
WaterGAP 2.2b_fpl was applied to model river discharge, river flow velocity, water storages, water heights and surface water extent on a global-scale. Model results were comprehensively validated against ground observations and remote sensing data. Overall, the modeled and observed data are in agreement. In comparison to the former version WaterGAP 2.2b, the model performance has improved significantly. The improvements are most remarkable in the Amazon River basin. However, the seasonal variation of surface water extent and total water storage anomalies are still too low in many regions on the globe when compared to observations. A detailed analysis of the simulated results suggests that in the Amazon River basin the introduction of backwater effects is important for realistically simulating water storages and surface water extent. Future efforts should focus on the simulation of water levels in order to better model the flow routing according to water slope. To further improve the model performance in specific regions, I recommend that the globally constant model parameters that affect inundation initiation, river-floodplain interaction, DEM correction for vegetation, and backwater amount at basin or subbasin-scale be adjusted.
Seed dispersal is a key ecosystem function for plant regeneration, as it involves the movement of seeds away from the parental plants to particular habitats where they can germinate and transition to seedlings and ultimately adult plants. Seed dispersal is shaped by a diversity of abiotic and biotic factors, particularly by associations between plants and climate and between plants and other species. Due to the ongoing loss of biodiversity and changing global conditions, such interactions are prone to change and pose a severe threat to plant regeneration. One way to address this challenge is to study associations between plant traits and abiotic and biotic factors to understand the potential impacts of global change on plant regeneration. Plant communities have long been analyzed through the lens of vegetative traits, mainly ignoring how other traits interact and respond to the environment. For instance, while associations between vegetative traits (e.g., specific leaf area, leaf nitrogen content) and climate are well studied, there are few case studies of reproductive traits in relation to trait-environment associations in the context of global change.
Thus, the overarching aim of this dissertation is to explore how trait-environment associations, with a special focus on reproductive traits, can improve our understanding of the effect that global change may have on seed dispersal, and ultimately on plant regeneration. To this end, my research focuses on studying associations between plant traits and abiotic and biotic factors along an elevational gradient in both forests and deforested areas of tropical mountains. This dissertation addresses three principal research objectives.
First, I investigate the extent to which reproductive (seed and fruit traits) and vegetative traits (leaf traits) are related to abiotic and biotic factors for communities of fleshy-fruited plants in the Ecuadorian Andes. I used multivariate analyses to test associations between four (a)biotic factors and seven reproductive traits and five vegetative traits measured on 18 and 33 fleshy fruited plant species respectively. My analyses demonstrate that climate and soil conditions are strongly associated with the distribution of both reproductive and vegetative traits in tropical tree communities. The production of “costly” vs. “cheap” seeds, fruits and leaves, i.e., the production of few rewarding fruits and acquisitive leaves versus the production of many less-rewarding fruits and conservative leaves, is primarily limited by temperature, whereas the size of plant organs is more related to variation in precipitation and soil conditions. My findings suggest that associations between reproductive and vegetative traits and the abiotic environment follow similar principles in tropical tree communities.
Second, I assess how climate and microhabitat conditions affect the prevalence of endozoochorous plant species in the seed rain of tropical montane forests in southern Ecuador. I analyzed seed rain data for an entire year from 162 traps located across an elevational gradient spanning of 2000 m. I documented the microhabitat conditions (leaf area index and soil moisture next to each seed trap) at small spatial scale as well as the climatic conditions (mean annual temperature and rainfall in each plot) at large spatial scale. After a one-year of sampling, I counted 331,838 seeds of 323 species/morphospecies. My analyses demonstrate that the prevalence of endozoochorous plant species in the seed rain increases with temperature across elevations and with leaf area index within elevations. These results show that the prevalence of endozoochory is shaped by the interplay of both abiotic and biotic factors at large and small spatial scales.
Third, I examine the potential of seed rain to restore deforested tropical areas along an elevational gradient in southern Ecuador. For this chapter, I collected seed rain using 324 seed traps installed in 18 1-ha plots in forests (nine forest plots) and in pastures (nine deforested plots) along an elevational gradient of 2000 m. After a sampling period of three months, I collected a total of 123,039 seeds of 255 species/morphospecies from both forests and pastures along the elevational gradient. I did not find a consistent decrease in the amount and richness of seed rain between forests and pastures, but I detected a systematic change in the type of dispersed seeds, as heavier seeds and a higher proportion of endozoochorous species were found in forests compared to pastures at all elevations. This finding suggests that deforestation acts as a strong filter selecting seed traits that are vital for plant regeneration.
Understanding the role that trait-environment associations play in how plant communities regenerate today could serve as a basis for predicting changes in regeneration processes of plant communities under changing global conditions in the near future. Here, I show how informative the measurement of reproductive traits and trait environment associations are in facilitating the conservation of forest habitats and the restoration of deforested areas in the context of global change.
Rhythmic changes in environmental lighting conditions have ever been the most reliable environmental cue for life on earth. Nature has therefore selected a genetically encrypted endogenous clock very early in evolution, as it provided cells and subsequently organisms with the ability to anticipate persevering periods of light and darkness. Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. The mammalian endogenous master clock, which synchronizes the body to environmental time, is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. As an integral part of the time-coding system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic nocturnal synthesis and immediate release of the hormone melatonin. In contrast to the transcriptional regulation of melatonin synthesis in rodents, a post-translational shaping is indicated in the human pineal gland. Another important mediator of circadian time and seasonality to the body is the pituitary gland. The aim of this work was to elucidate regulation of melatonin synthesis in the human pineal gland. Furthermore, presence and regulation of clock genes in the human pineal and pituitary gland, and in the SCN were analyzed. Therefore, human tissue, taken from regular autopsies, was analyzed simultaneously for different parameters involved in melatonin biosynthesis and circadian rhythm generation. Presented data demonstrate that post-mortem brain tissue can be used to detect the remnant profile of pre-mortem adaptive changes in neuronal activity. In particular, our results give strong experimental support for the idea that transcriptional mechanisms are not dominant for the generation of rhythmic melatonin synthesis in the human pineal gland. Together with data obtained for clock genes and their protein products in the pituitary, data presented here offer 1) a new working hypothesis for post-translational regulation of melatonin biosynthesis in the human pineal gland, and 2) a novel twist in the molecular competence of clock gene proteins, achieved by nucleo-cytoplasmic shuttling in neuronal and neuroendocrine human tissue. Furthermore, in this study, oscillations in abundance of clock gene proteins were demonstrated for the first time in the human SCN.
Interactional niche in the development of geometrical and spatial thinking in the familial context
(2016)
In the analysis of mathematics education in early childhood it is necessary to consider the familial context, which has a significant influence on development in early childhood. Many reputable international research studies emphasize that the more children experience mathematical situations in their families, the more different emerging forms of participation occur for the children that enable them to learn mathematics in the early years. In this sense mathematical activities in the familial context are cornerstones of children’s mathematical development, which is also affected by the ethnic, cultural, educational and linguistic features of their families. Germany has a population of approximately 82 million, about 7.2 million of whom are immigrants (Statisches Bundesamt 2009, pp.28-32). Children in immigrant families grow up with multiculturalism and multilingualism, therefore these children are categorized as a risk group in Germany. “Early Steps in Mathematics Learning – Family Study” (erStMaL-FaSt) is the one of the first familial studies in Germany to deal with the impact of familial socialization on mathematics learning. The study enables us to observe children from different ethnic groups with their family members in different mathematical play situations. The family study (erStMaL-FaSt) is empirically performed within the framework of the erStMaL (Early Steps in Mathematics Learning) project, which relates to the investigation of longitudinal mathematical cognitive development in preschool and early primary-school ages from a socio-constructivist perspective. This study uses two selected mathematical domains, Geometry and Measurement, and four play situations within these two mathematical domains.
My PhD study is situated in erStMaL-FaSt. Therefore, in the beginning of this first chapter, I briefly touch upon IDeA Centre and the erStMaL project and then elaborate on erStMaL-FaSt. As parts of my research concepts, I specify two themes of erStMaL-FaSt: family and play. Thereafter I elaborate upon my research interest. The aim of my study is the research and development of theoretical insights in the functioning of familial interactions for the formation of geometrical (spatial) thinking and learning of children of Turkish ethnic background. Therefore, still in Chapter 1, I present some background on the Turkish people who live in Germany and the spatial development of the children.
This study is designed as a longitudinal study and constructed from interactionist and socio-constructivist perspectives. From a socio-constructivist perspective the cognitive development of an individual is constitutively bound to the participation of this individual in a variety of social interactions. In this regard the presence of each family member provides the child with some “learning opportunities” that are embedded in the interactive process of negotiation of meaning about mathematical play. During the interaction of such various mathematical learning situations, there occur different emerging forms of participation and support. For the purpose of analysing the spatial development of a child in interaction processes in play situations with family members, various statuses of participation are constructed and theoretically described in terms of the concept of the “interactional niche in the development of mathematical thinking in the familial context” (NMT-Family) (Acar & Krummheuer, 2011), which is adapted to the special needs of familial interaction processes. The concept of the “interactional niche in the development of mathematical thinking” (NMT) consists of the “learning offerings” provided by a group or society, which are specific to their culture and are categorized as aspects of “allocation”, and of the situationally emerging performance occurring in the process of meaning negotiation, both of which are subsumed under the aspect of the “situation”, and of the individual contribution of the particular child, which constitutes the aspect of “child’s contribution” (Krummheuer 2011a, 2011b, 2012, 2014; Krummheuer & Schütte 2014). Thereby NMT-Family is constructed as a subconcept of NMT, which offers the advantage of closer analyses and comparisons between familial mathematical learning occasions in early childhood and primary school ages.
Within the scope of NMT-Family, a “mathematics learning support system” (MLSS) is an interactional system which may emerge between the child and the family members in the course of the interaction process of concrete situations in play (Krummheuer & Acar Bayraktar, 2011). All these topics are addressed in Chapter 2 as theoretical approaches and in Chapter 3 as the research method of this study. In Chapter 4 the data collection and analysis is clarified in respect of these approaches...
Construction and commissioning of a setup to study ageing phenomena in high rate gas detectors
(2014)
In high-rate heavy-ion experiments, gaseous detectors encounter big challenges in terms of degradation of their performance due to a phenomenon dubbed ageing. In this thesis, a setup for high precision ageing studies has been constructed and commissioned at the GSI detector laboratory. The main objective is the study of ageing phenomena evoked by materials used to build gaseous detectors for the Compressed Baryonic Matter (CBM) experiment at the future Facility for Antiproton and Ion Research (FAIR).
The precision of the measurement, e.g., of the gain of a gaseous detector, is a key element in ageing studies: it allows to perform the measurement at realistic rates in an acceptable time span. It is well known the accelerating ageing employing high intensity sources might produce misleading results. The primary objective is to build an apparatus which allows very accurate measurements and is thus sensitive to minute degradations in detector performance. The construction and commissioning of the
setup has been carried out in two steps. During the first step of this work, a simpler setup which already existed in the detector laboratory of GSI had been utilised to define all conditions related to ageing studies. The outcome of these studies defined the properties and characteristics that must be met to build and operate a new, sophisticated and precise setup. The already existing setup consisted of two identical Multi Wire Proportional Chambers (MWPCs), a gas mixing station, an 55Fe source, an x-ray generator, an outgassing box and stainless steel tubing. In a first step, the gain and electric field configuration of the MWPCs were simulated by a combination of a gas simulation (Magboltz) and electric field simulation program (Garfield). The performance and operating conditions of the chambers have been thoroughly characterised before utilising them in first preparatory ageing test. The main diagnostic parameter in ageing studies is the detector gain, thus it is mandatory for precise ageing studies to minimise the systematic and statistical variation of the pressure and temperature corrected gain. To achieve the required accuracy, several improvements of the chamber design and the gas system have been implemented. In addition, the temperature measurement has been optimised. During the preparatory tests, several ageing studies have been carried out. The ageing effect of seven materials and gases have been carried out during these tests: RTV-3145, Ar/CO2 gas, Durostone flushed with Ar/Isobutane gas, Vetronit G11, Vetronit G11 contaminated with Micro 3000 and Gerband 705. The results of these studies went into the design of the new sophisticated ageing setup. For example some tests revealed that there was, even after cleaning, a certain level of contamination with "ageing agents" in the existing setup, which made it imperative to ensure a very high level cleanness of all components during the construction of the setup. The curing period of some testing samples like glues or the gas flow rate were found to be very important factors that must be taken into account to obtain comparable results. Very important changes in the chamber design have been made, i.e., the aluminium-Kapton cathodes used in MWPCs have been replaced with multi-wire planes and the fibreglass housing of the chamber has been changed to metal. The second step started with building the new setup which was designed based on the findings from the first step. The new ageing setup consists of three MWPCs, two moving platforms, an 55Fe source, a copper-anode x-ray generator, two outgassing boxes, both flexible and rigid stainless steel tubes. Before fabrication of the chambers, simulations of their electric field and the gain have been done using Magboltz and Garfield programs. After that, the chambers were installed and tested. A 0.3% peak-to-peak residual variation of the corrected gain has been achieved. Finally, the complete setup has been operated with full functionality in no-ageing conditions during one week. This test revealed very stable gain in all three chambers. After that two materials (Gerban 705 and RTV-3145) have been inserted in the two outgassing boxes and tested. They revealed an ageing rate of about 0.3%/mC/cm and 3%/mC/cm respectively. The final test proves the stability and accuracy of the ageing measurements carried out with the ageing setup at the detector laboratory at GSI which is ready to conduct the envisaged systematic ageing studies.
Inhibitor of Apoptosis (IAP) proteins are expressed at high levels in many cancers and contribute to apoptosis resistance. Therefore, they represent promising anticancer drug targets. Here, we report that small molecule IAP inhibitors at subtoxic concentrations cooperate with monoclonal antibodies against TRAIL receptor 1 (Mapatumumab) or TRAIL receptor 2 (Lexatumumab) to induce apoptosis in neuroblastoma cells in a highly synergistic manner (combination index <0.1). Importantly, we identify RIP1 as a critical regulator of this synergism. RIP1 is required for the formation of a RIP1/FADD/caspase-8 complex that drives caspase-8 activation, cleavage of Bid into tBid, mitochondrial outer membrane permeabilization, full activation of caspase-3 and caspase-dependent apoptosis. Indeed, knockdown of RIP1 abolishes formation of the RIP1/FADD/caspase-8 complex, subsequent caspase activation and apoptosis upon treatment with IAP inhibitor and TRAIL receptor antibodies. Similarly, inhibition of RIP1 kinase activity by Necrostatin-1 inhibits IAP inhibitor- and TRAIL receptor-triggered apoptosis. By comparison, over-expression of the dominant-negative superrepressor IκBα-SR or addition of the TNFα-blocking antibody Enbrel does not inhibit IAP inhibitor- and Lexatumumab-induced apoptosis, pointing to a NF-κB- and TNFα-independent mechanism. Of note, IAP inhibitor also significantly reduces TRAIL receptor-mediated loss of cell viability of primary cultured neuroblastoma cells, underscoring the clinical relevance. By demonstrating that RIP1 plays a key role in the IAP inhibitor-mediated sensitization for Mapatumumab- or Lexatumumab-induced apoptosis, our findings provide strong rationale to develop the combination of IAP inhibitors and TRAIL receptor agonists as a new therapeutic strategy for the treatment of human cancer.
Mitochondial NADH:ubiquinone oxidoreductase (complex I) the largest multiprotein enzyme of the respiratory chain, catalyses the transfer of two electrons from NADH to ubiquinone, coupled to the translocation of four protons across the membrane. In addition to the 14 strictly conserved central subunits it contains a variable number of accessory subunits. At present, the best characterized enzyme is complex I from bovine heart with a molecular mass of about 980 kDa and 32 accessory proteins. In this study, the subunit composition of mitochondrial complex I from the aerobic yeast Y. lipolytica has been analysed by a combination of proteomic and genomic approaches. The sequences of 37 complex I subunits were identified. The sum of their individual molecular masses (about 930 kDa) was consistent with the native molecular weight of approximately 900 kDa for Y. lipolytica complex I obtained by BN-PAGE. A genomic analysis with Y. lipolytica and other eukaryotic databases to search for homologues of complex I subunits revealed 31 conserved proteins among the examined species. A novel protein named “X” was found in purified Y. lipolytica complex I by MALDI-MS. This protein exhibits homology to the thiosulfate sulfurtransferase enzyme referred to as rhodanese. The finding of a rhodanese-like protein in isolated complex I of Y. lipolytica allows to assume a special regulatory mechanism of complex I activity through control of the status of its iron-sulfur clusters. The second part of this study was aimed at investigating the possible role of one of these extra subunits, 39 kDa (NUEM) subunit which is related to the SDRs-enzyme family. The members of this family function in different redox and isomerization reactions and contain a conserved NAD(P)H-binding site. It was proposed that the 39 kDa subunit may be involved in a biosynthetic pathway, but the role of this subunit in complex I is unknown. In contrast to the situation in N. crassa, deletion of the 39 kDa encoding gene in Y. lipolytica led to the absence of fully assembled complex I. This result might indicate a different pathway of complex I assembly in both organisms. Several site-directed mutations were generated in the nucleotide binding motif. These had either no effect on enzyme activity and NADPH binding, or prevented complex I assembly. Mutations of arginine-65 that is located at the end of the second b-strand and responsible for selective interaction with the 2’-phosphate group of NADPH retained complex I activity in mitochondrial membranes but the affinity for the cofactor was markedly decreased. Purification of complex I from mutants resulted in decrease or loss of ubiquinone reductase activity. It is very likely that replacement of R65 not only led to a decrease in affinity for NADPH but also caused instability of the enzyme due to steric changes in the 39 kDa subunit. These data indicate that NADPH bound to the 39 kDa subunit (NUEM) is not essential for complex I activity, but probably involved in complex I assembly in Y. lipolytica.
The utilization of Ginkgo biloba in medicinal practice dates back to 1505 A.D. Ironically, the mechanisms of action of Ginkgo are not fully clarified till now. Nowadays, Ginkgo biloba leaf extracts are mainly indicated for mild to moderate cerebrovascular insufficiency and different forms of dementia. The fact that it is an herbal extract composed of several different components indeed adds to the intricacy of finding its mechanisms of actions. Indisputably, many scientists tried to elucidate the mechanisms of actions of Ginkgo. The first step to achieve this goal was to standardize the leaf extract. The standardized Ginkgo leaf extract contains 22-27 % flavonol glycosides, 2.8-3.4 % of ginkgolide A, B and C, as well as approximately 2.6-3.2 % bilobalide and below 5 ppm ginkgolic acids. A widespread standardized Ginkgo extract is the EGb 761, which was utilized in the current work. One of the earliest proposed mechanisms is the ability of the Ginkgo extract to act as an anti-oxidant, which could be explained by its high flavonoid contents. However, without doubt EGb 761 encompasses other characteristics which distinguish it from other herbal extracts that are also rich in flavonoids. Since free radicals and reactive oxygen species are highly associated with the mitochondrial functions, examination of the effect of EGb 761 on mitochondrial functions was lately addressed. Moreover, this was encouraged as the link between Alzheimer’s disease [AD] and the mitochondria started to emerge. Previously, our group observed mitochondrial protective actions of EGb 761 on cell culture in vitro. Furthermore, anti-apoptotic effects were previously described for EGb 761. However, only very few studies addressed the single constituents and their effect on mitochondrial functions. Flavonoids were studied in several other plant extracts and their radical scavenging activity is unquestionable, but EGb 761 has anti-apoptotic actions which may be attributed to its terpenoid fraction. Exclusively found in the Ginkgo plant, are the ginkgolides and therefore their actions are not yet fully elucidated. Moreover, those who attempted to address these constituents concentrated on one or two candidates, for example bilobalide or ginkgolide B and ignored the rest. Unfortunately, this led to incomplete results, and one couldn’t compare the relative activities of all EGb 761 components in order to state whether all the components are effective or not. ...