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Development of a reliable platform for Laser-Plasma Accelerator driven Free-Electron Laser Studies
No full textFree-Electron Lasers (FELs) based on start-of-the-art radio-frequency (RF) accelerator facilities have been established as a reliable source of coherent, high-brightness x-ray beams, advancing and pushing the boundaries of a broad range of scientific applications. Their capabilities would unlock significant improvements to applications in medical imaging and semiconductor manufacturing, among others, but to make this technology available to a larger community, significant steps to reduce the cost and footprint of the accelerator facilities are required.
With the emergence of laser-plasma accelerators (LPAs) as powerful, compact alternatives to RF machines, leveraging these sources to drive FELs immediately arose as a promising application. First results of LPA-driven FEL radiation have been achieved by the community over the last few years, but reliable high-gain FEL operation, required to prove the potential of serving as a capable platform for future light source facilities, has yet to be demonstrated.
This dissertation was carried out at the Hundred TeraWatt Undulator (HTU) system, part of the Berkeley Lab Laser Accelerator (BELLA) group at Lawrence Berkeley National Laboratory (LBNL), and driven by the goal to demonstrate reliable operation of an LPA-driven FEL. The work presented here aims to achieve a more repeatable and stable LPA interaction, to enable a first demonstration of LPA-driven FEL lasing on the HTU system, and to develop an experimental platform for subsequent investigations of the LPA-driven FEL performance.
To achieve this goal, a large part of the work conducted as a part of this thesis project went towards improvements of the diagnostic capabilities of the experiment, to obtain new insights into the key parameters of the LPA interaction. These results were used to inform the need for additional stabilization systems to improve system performance, and their subsequent development, installation, and characterization are discussed as well.
The setup and commissioning of new, as well as the upgrade of existing diagnostics enabled the discovery of systematic instabilities of the laser system. Specifically variations of the laser pulse duration and energy, uncovered as part of this work, were shown to significantly impact the LPA performance. Implementing new stabilization capabilities targeted towards these variations, in combination with extensive efforts to further increase long-term reliability of the LPA interaction, yielded successful demonstration and characterization of LPA-driven FEL lasing at 420 nm. Through subsequent improvements to the laser system and operational procedures, first results exceeding 1000-fold gain from an LPA-driven FEL, with higher than 90% reliability, were achieved. During a dedicated follow-up campaign, continuous operation of the LPA-driven FEL over more than eight hours was demonstrated, while maintaining the performance of more than 90% of all shots exhibiting FEL gain. \newline
These results represent an unprecedented level of both shot-to-shot and long-term stability, as well as overall performance for LPA-driven FELs, and serve as a crucial step towards the demonstration of the technology as a viable platform for future compact FEL facilities
Nonlinear Elasticity in Concrete Structures: Toward In-Situ Methods for Integrity Assessment
No full textDecades of research on small-scale concrete specimens—such as cores and beams—have demonstrated that concrete, a highly heterogeneous material, exhibits elastic nonlinear effects, namely the stress- and time-dependence of its elastic properties. These nonlinear effects are highly sensitive to the presence of heterogeneities, even at the milli- or nanoscale. Prior studies have proposed leveraging these nonlinear elastic effects as a nondestructive testing method for detecting small-scale damage in concrete structures. However, formalizing such techniques for real-world applications requires transferring knowledge from controlled laboratory conditions to field settings. This research extends the study of nonlinear elastic effects to a full-scale concrete structure under field conditions. Specifically, it investigates the presence of classical and nonclassical nonlinear elastic behaviours—the acoustoelastic effect and Slow Dynamics, respectively—and their relationship with the relative integrity level of a concrete test bridge. Through an experiment conducted to measure vibrations across multiple frequency bands, this work demonstrates that soft microstructures—associated with varying levels of structural integrity—can be identified using nondestructive, wavefield-based measurement techniques. These findings contribute to the investigations for the development of in-situ methods for early-stage damage detection in existing and new concrete infrastructure
Fish fauna structure, feeding ecology and growth of keystone fish species in the Elbe and Odra estuaries
No full textIn the Elbe and Odra estuaries essential feeding grounds, nursery areas, spawning grounds and transitional habitats for several fish species are found. With climate change and human activities (e. g. channel management, waste water sewage and nutrient inputs) both estuaries have faced several anthropogenic stressors in the past and in the present. These stressors are likely to have an influence on estuarine ecosystems and their food webs. Keystone fish species play an important role in estuarine food webs. With this regard, in this thesis the feeding ecology and growth of keystone fish species in the Elbe and Odra estuaries were analysed. Additionally, in the Elbe estuary, the structure and stock development of the fish fauna, as well as fish health, were evaluated based on stow net catch data and tissue-specific stress responses of the holobiont. Analyses on feeding and growth of the four keystone fish species zander Sander lucioperca (Linnaeus, 1758), smelt Osmerus eperlanus (Linnaeus, 1758), ruffe Gymnocephalus cernua (Linnaeus, 1758) and flounder Platichthys flesus (Linnaeus, 1758) were carried out, as these species occur in high numbers throughout both estuaries and play key roles in the respective aquatic food webs.
The food webs in estuarine ecosystems are important biological indicators, therefore the feeding ecology of the four keystone fish species was studied by stomach content analyses (chapter 2). The most important prey of zander in both estuaries were fish and mysids. For smelt amphipods were the most important prey in the Elbe estuary, whereas in the Odra estuary smelt mainly consumed mysids. The most important prey of ruffe in the Elbe estuary were also amphipods, while annelids (lower estuary) and insect larvae (upper estuary) were the dominant prey in the Odra estuary. In the Elbe estuary, flounder fed mainly on copepods, whereas in the Odra estuary they preferred bivalves. In the Elbe estuary higher dietary overlaps were found, particularly between smelt and ruffe, zander and ruffe, as well as zander and smelt. By comparing the present diets of zander, smelt and ruffe from the Elbe estuary with previous studies (1990s), it was noticeable that the consumption of amphipods increased, while mysids and copepods decreased in their diets. These shifts indicated a restructuring of the food web, likely driven by environmental changes, which highlights the vulnerability of estuarine ecosystems.
In chapter 3 we aimed to analyse the growth patterns of the four keystone fish species to identify regional differences from both estuaries and to compare present findings with those from past studies. Age determination on hard structures (otoliths, scales, opercula) and length measurements were carried out. This allowed the determination of size ranges of the individual age groups, which were used to calculate the parameters of the von Bertalanffy growth function. The asymptotic lengths of smelt, ruffe, and flounder were larger in the Elbe estuary compared to the Odra estuary, whereas their growth coefficients were smaller in the Elbe. The growth of zander, smelt and ruffe from the Elbe. estuary were inferior compared to previous studies. The growth of juvenile zander in the Elbe estuary showed a noticeable reduction in the maximum turbidity zone located in the middle estuary. Slower growth at these sites appears to be linked to reduced fish prey intake, as juvenile zander relied more on invertebrates like amphipods, mysids, and decapods. Flounder from the Odra estuary showed smaller asymptotic length compared to other studies from the Baltic, but had a higher growth coefficient in contrast to studies from the Pomeranian Bay and Gulf of Gdańsk. Our findings indicate a connection between environmental changes and fish growth via changes in feeding behaviour, especially in the Elbe estuary.
We created a periodic time series over the last four decades (1984-2022), combining fish species densities data from stow net catches alongside with environmental data in the Elbe estuary. In chapter 4, we showed that the fish fauna of the Elbe estuary has become more similar in guild structure to that of macrotidal estuaries in Europe, with a relative increase in marine-estuarine opportunists and a decrease in diadromous species. Due to an improvement of the water quality in the 1990s, fish densities, especially of smelt, increased until 2010. However, anthropogenic hydromorphological interventions, have led to an increase of suspended particular matter until 2022. Together with a reduced river runoff and poor oxygen condition, these factors acted as stressors for fishes in the estuary. In 2021-2022 mean fish densities dropped by over 91 % compared to 2009-2010. This decline was mainly driven by a reduction of the key species smelt, alongside decreases in twaite shad Alosa fallax (Lacepede, 1803), flounder, ruffe, common bream Abramis brama (Linnaeus, 1758), and other species. In contrast, densities of marine-estuarine opportunist species such as herring Clupea harengus Linnaeus, 1758 and whiting Merlangius merlangus (Linnaeus, 1758) increased. Overall, this time-series provided insights into the strong impact of human intervention in estuarine ecosystems.
In chapter 5, we integrated tissue specific gene expression data from fish host (gill and liver RNAseq) and 16S rRNA metabarcoding of gill mucus microbiota with physiological and abiotic parameters to assess the health of juvenile zander along the Elbe estuary. Liver specific gene expression patterns in fish from highly turbid areas in the middle estuary indicated starvation, which aligned with the compromised body condition. The gill microbiome derived from the freshwater area with local occuring oxygen minimum zones was dominated by potentially pathogenic taxa, including Shewanella, Acinetobacter, Aeromonas and Chryseobacterium. Their presence was found alongside with strong immune responses in the host gill tissue and increased energy demand in the liver tissue, supporting a potentially pathogenic role. Together, these results demonstrate how physiological and microbiome responses in estuarine fish can reflect the cumulative impact of abiotic stressors, which are likely to intensify under ongoing climate change
Experimental investigation on Non-local entanglement in electron-exchange collisions
No full textThe main goal of this experiment is to investigate quantum entanglement between massive particles with the potential for transferring spin polarization via non-local quantum effects. This approach employs electron-exchange scattering with spin-1/2 targets to generate entanglement. By eliminating photon-mediated interactions, this experimental configuration enables direct examination of non-local quantum state transfer between massive particles, whether temporally immediate or subluminal, while providing fundamental insights into entangled quantum systems comprising two massive particles.
The experiment consists of two primary phases. Initially, elastic electron-exchange scattering between free, unpolarized electrons and unpolarized fermionic atoms (sodium) is experimentally achieved to create a tunable entanglement resource. After the collision, the electrons will scatter and calculations show that the maximum degree of entanglement between sodium atoms and electrons occurs when the energy of incoming electrons is 10 eV and they are scattered at 60 degrees angle.
Subsequently, after a well-defined delay period that allows electrons to travel away from the collision center, a nanosecond-pulse laser with circularly polarized light (σ+) is used to excite the sodium atoms. This, in part, transfers the light’s polarization to the entangled atoms. Due to quantum entanglement, these entangled atoms are then predicted to transmit their polarization (or a part of it) to the previously unpolarized, though entangled, electronic ensemble. The key element here is the transfer of polarization from the sodium atoms to the entangled free electrons, even when they are separated by meters. Finally, the spin polarization of the electron ensemble is detected using a Mott polarimeter, where a non-zero measurement would indicate non-local quantum mechanical effects.
Preliminary experimental results align partially with theoretical predictions, suggesting the potential validity of the proposed approach. However, further investigations are needed to fully confirm these findings and establish conclusive evidence of the observed quantum mechanical effects
Messung des inklusiven tt-Wirkungsquerschnitts und Suche nach zusätzlichen Skalaren in tt-Endzuständen mit dem CMS-Experiment
No full textTwo key measurements and two phenomenological studies of top quark pair (tt) production with the CMS experiment at the CERN Large Hadron Collider are presented. They include the first measurement of the tt cross section at the new energy frontier, a detailed analysis of the tt threshold culminating in the first observation of tt bound state effects, and searches for new physics in tt final states, such as heavy scalars, pseudoscalars or Axion-Like Particles.
The inclusive tt production cross section σ(tt) is measured for the first time at the world-record energy of √s = 13.6 TeV, using 1.21 fb^(-1) of early LHC Run 3 data. By combining the dilepton and lepton+jets (l+jets) decay channels and constraining the lepton and b tagging efficiencies in situ, a precision of 3.4%, comparable to previous σ(tt) measurements, is achieved. This constitutes the first measurement of proton-proton scattering at √s = 13.6 TeV worldwide.
Furthermore, a search for heavy spin-0 states decaying to tt using 138 fb^(-1) of LHC Run 2 data at √s = 13 TeV in the dilepton channels is presented. The invariant tt mass m(tt) is combined with spin correlation observables to gain sensitivity to the spin and CP structure of possible new intermediate states.
The analysis is supported by a detailed modeling study of off-shell tt production and of the interference between tt and tW production.
An excess of events over the tt continuum background is observed at low values of m(tt), with spin correlations consistent with a pseudoscalar state. It is interpreted in terms of a pseudoscalar tt bound state ηt, and its cross section is measured to be σ(ηt) = 8.7 ± 1.1 pb using a simplified model inspired by non-relativistic quantum chromodynamics.
The excess is statistically significant at more than five standard deviations, constituting the first observation of tt bound state effects.
Other interpretations of the observed excess are similarly possible. In particular, scenarios with generic pseudoscalar or scalar bosons are explored, and exclusion regions on their coupling to the top quark are derived both for the dilepton channels alone as well as in a combination with a separate analysis of the l+jets channels. In addition, Axion-Like Particles (ALPs) decaying to tt are considered in the case of vanishing tree-level ALP-gluon couplings, while the more generic case is investigated phenomenologically in simulation.Es werden zwei essentielle Messungen und zwei phänomenologische Studien zur Produktion von Top-Quark-Paaren (tt) mit dem CMS-Experiment am CERN Large Hadron Collider vorgestellt.
Sie umfassen die erste Messung des tt-Wirkungsquerschnitts bei der weltweit höchsten Schwerpunktsenergie, eine detaillierte Analyse der tt-Produktionsschwelle, die in die erste Beobachtung von einem gebundenen Zustand des tt-Systems mündet, sowie Suchen nach neuer Physik in tt-Endzuständen, wie etwa schwere Skalar- oder Pseudoskalarbosonen oder Axion-Like Particles.
Der inklusive tt-Produktionsquerschnitt σ(tt) wird zum ersten Mal bei √s = 13.6 TeV gemessen, unter Verwendung von frühen LHC Run 3-Daten mit integrierter Luminosität von 1.21 fb^(-1). Durch Kombination der Dilepton- und Lepton+Jets (l+jets)-Zerfallskanäle von tt und simultane Bestimmung der Lepton- und b-tagging-Effizienzen in situ wird eine Präzision von 3.4% erreicht, die mit früheren σ(tt)-Messungen vergleichbar ist.
Dies stellt die weltweit erste Messung von Proton-Proton-Streuprozessen bei √s = 13.6 TeV dar.
Darüber hinaus wird eine Suche nach schweren Spin-0-Zuständen, die zu tt zerfallen, in den Dilepton-Kanälen mit 138 fb^(-1) Daten von LHC Run 2 bei √s = 13 TeV vorgestellt. Die invariante Masse von tt (m(tt)) wird mit Spinkorrelations-Observablen kombiniert, um die Sensitivität gegenüber dem Spin und der CP-Struktur möglicher neuer intermediärer Zustände zu erhöhen.
Die Analyse wird durch detaillierte Studien zur Modellierung der Off-Shell-tt-Produktion sowie zur Interferenz zwischen tt- und tW-Produktion untermauert.
Ein Überschuss von Ereignissen gegenüber dem tt-Kontinuums-Hintergrund wird bei niedrigen Werten von m(tt) und mit Spinkorrelationen konsistent mit einem pseudoskalaren Zustand beobachtet. Er wird als pseudoskalarer gebundener tt-Zustand ηt interpretiert, und dessen Produktionsquerschnitt wird mithilfe eines vereinfachten, von nichtrelativistischer Quantenchromodynamik inspirierten Modells zu σ(ηt) = 8.7 ± 1.1 pb gemessen.
Der Überschuss ist mit einer Signifikanz von mehr als fünf Standardabweichungen statistisch belegt und stellt somit die erste Beobachtung von gebundenen Zuständen im tt-System dar.
Weitere Interpretationen des beobachteten Überschusses sind ebenfalls möglich.
Insbesondere werden Szenarios mit generischen pseudoskalaren oder skalaren Bosonen untersucht, und Ausschlussregionen
hinsichtlich ihrer Kopplungen an das Top-Quark werden sowohl für die Dilepton-Kanäle allein als in Kombination mit einer separaten Analyse der l+jets-Kanäle berechnet. Zusätzlich werden zu tt zerfallende Axion-Like Particles (ALPs) betrachtet: einerseits im Fall verschwindender ALP-Gluon-Kopplungen, andererseits im allgemeinen Fall, der phänomenologisch in Simulationen untersucht wird
Strukturuntersuchungen zur ribosomalen Qualitätskontrolle und zur antibiotischen Hemmung während der Initiierung der Translation
No full textDer Translationsstillstand stört den zellulären Proteinsyntheseprozess und führt zu unvollständigen und potenziell schädlichen Proteinprodukten. Um diesem Problem zu begegnen, haben Zellen ribosomenassoziierte Qualitätskontrollmechanismen (RQC) entwickelt, die blockierte ribosomale Komplexe abbauen. Einer der ersten Schritte in diesem Prozess besteht darin, die ribosomalen Untereinheiten zu trennen und die Peptidyl-tRNA von der großen Untereinheit freizusetzen. Während die RQC- Wege in Eukaryonten gut erforscht sind, ist über das bakterielle System noch viel unbekannt.
In Bacillus subtilis haben jüngste Studien gezeigt, dass RqcH und RqcP zusammenarbeiten, um Polyalanin-Schwänze an Peptidyl-tRNA-50S-Komplexe zu binden und sie so für den Abbau zu markieren. Die genauen Faktoren und Bedingungen, die bei der Spaltung dieser blockierten bakteriellen Ribosomen eine Rolle spielen, sind jedoch noch nicht genau bekannt. Im Gegensatz dazu enthält Escherichia coli, dem RqcH fehlt, Hsp15, ein Homolog von RqcP, obwohl beide an unterschiedliche Regionen des Ribosoms binden sollen. In unserer Studie haben wir herausgefunden, dass Hitzeschocks zur Dissoziation von translatierenden Ribosomen führen, wodurch Peptidyl-tRNA-50S-Komplexe entstehen, die Substrate für Hsp15 sind. Wir haben die Struktur des nativen Hsp15-Peptidyl-tRNA-50S- Komplexes mit einer Auflösung von 3,0 Å gelöst und dabei festgestellt, dass Hsp15 ähnlich wie RqcP bindet. Diese Entdeckung steht im Gegensatz zu früheren Berichten, die eine andere Bindungsstelle für Hsp15 in E. coli nahelegen. Darüber hinaus haben wir erste Hinweise darauf gefunden, dass PrfH ein möglicher Kandidat für das Recycling von Hsp15-Peptidyl-tRNA-50S-Komplexen sein könnte.
Wir untersuchten auch die Peptidyl-tRNA-Hydrolase (Pth), die nachweislich das naszierende Peptid aus dem 50S-Peptidyl-tRNA-Komplex während des ribosomalen Abbaus freisetzt. Trotz mehrerer Versuche ist es uns nicht gelungen, die Struktur der an den 50S-Peptidyl-tRNA-Adr1-Komplex gebundenen Pth aufzulösen. Diese Bemühungen bilden jedoch eine Grundlage für künftige Studien und tragen zu einem besseren Verständnis der möglichen Rolle von Pth im Ribosom bei. Ein wesentlicher
Teil dieser Arbeit konzentriert sich auf die Initiierung der Translation, den entscheidenden ratenbegrenzenden Schritt der Proteinsynthese. Dieser Prozess wird von mehreren Antibiotika wie Kasugamycin, Edein und dem kürzlich entdeckten Tetrapeptid GE81112 angegriffen. Angesichts der zunehmenden Bedrohung durch Antibiotikaresistenzen ist es von entscheidender Bedeutung zu verstehen, wie diese Inhibitoren funktionieren, was dazu beitragen könnte, ihre Wirksamkeit als antimikrobielle Mittel zu verbessern. In früheren Strukturstudien wurden Kasugamycin, Edein und GE81112 an 30S-Untereinheiten gebunden erfasst, aber diese Strukturen wurden nicht im Zusammenhang mit aktiven Translationsinitiationskomplexen bestimmt, und ihre Auflösung war begrenzt (3,3-13 Å). Darüber hinaus haben Unstimmigkeiten zwischen diesen Strukturen und den zugehörigen biochemischen Daten dazu geführt, dass ihre Mechanismen unklar sind. In dieser Studie haben wir mit Hilfe der Kryo-Elektronenmikroskopie (Kryo-EM) die Strukturen von Kasugamycin, Edein und GE81112, die an 30S- Initiationskomplexe gebunden sind, bei Auflösungen zwischen 2,0 und 2,9 Å aufgelöst. Diese Ergebnisse zeigen unterschiedliche Bindungsstellen für Edein und GE81112 im Vergleich zu früheren kristallographischen Studien. Die hochaufgelösten Strukturen stimmen mit früheren biochemischen Daten überein und bieten neue Einblicke in die molekularen Wechselwirkungen dieser Inhibitoren. Wir fanden heraus, dass Kasugamycin und Edein den 30S-Initiationskomplex einfangen, bevor die Initiator-tRNA die mRNA entschlüsselt, während GE81112 den 30S- Initiationskomplex nach der tRNA-Akkommodation einfängt. Obwohl diese Inhibitoren an eine konservierte Stelle der 30S-Untereinheit binden, blockieren sie aufgrund ihrer unterschiedlichen chemischen Strukturen verschiedene Schritte der Translationsinitiation.
Darüber hinaus untersuchten wir den Hemmungsmechanismus von Streptomycin, das traditionell als Elongationshemmer bekannt ist, während der Translationsinitiation. Die Kryo-EM ergab, dass Streptomycin die Interaktionen zwischen den Initiationsfaktoren IF1 und IF3 stört, was zu einer verstärkten Dissoziation von IF3 von 30S-Initiationskomplexen führt und möglicherweise die Bildung von unproduktiven 70S-Initiationskomplexen beschleunigt.
Zusammenfassend lässt sich sagen, dass diese Arbeit unser Verständnis der bakteriellen RQC-Mechanismen, insbesondere bei Gram-negativen Bakterien, verbessert hat. Sie wirft auch ein Licht darauf, wie wichtige Inhibitoren auf die Initiierung der Translation abzielen, und liefert Erkenntnisse, die bei der Bekämpfung von Antibiotikaresistenzen von Nutzen sein könntTranslational stalling disrupts the cellular protein synthesis process, leading to incomplete and potentially harmful protein products. To address this, cells have developed ribosome-associated quality control (RQC) mechanisms that dismantle stalled ribosomal complexes. One of the first steps in this process involves separating the ribosomal subunits and releasing the peptidyl-tRNA from the large subunit. While the RQC pathways in eukaryotes are well-studied, much remains unknown about the bacterial system.
In Bacillus subtilis, recent studies have shown that RqcH and RqcP function together to attach polyalanine tails to peptidyl-tRNA-50S complexes, marking them for degradation. However, the precise factors and conditions involved in splitting these stalled bacterial ribosomes are not well understood. In contrast, Escherichia coli, which lacks RqcH, contains Hsp15, a homolog of RqcP, although they reportedly bind to different regions of the ribosome. In our study, we found that heat shock leads to the dissociation of translating ribosomes, producing peptidyl-tRNA-50S complexes, which are substrates for Hsp15. We solved the structure of the native Hsp15-peptidyl-tRNA-50S complex at a resolution of 3.0 Å, revealing that Hsp15 binds similarly to RqcP. This discovery contrasts with earlier reports suggesting a different binding site for Hsp15 in E. coli. Additionally, we introduce preliminary evidence suggesting that PrfH may serve as a putative candidate for recycling Hsp15-peptidyl-tRNA-50S complexes.
We also investigated Peptidyl-tRNA hydrolase (Pth), which has been shown to release the nascent peptide from the 50S-peptidyl-tRNA complex during ribosomal disassembly. Despite several attempts, we were unable to resolve the structure of Pth bound to the 50S-peptidyl-tRNA-adr1 complex. However, these efforts provide a foundation for future studies and contribute to a better understanding of Pth’s potential role in the ribosome.
A significant part of this work focuses on translation initiation which is a key rate- limiting step in protein synthesis. This process is targeted by several antibiotics, such
as kasugamycin, edeine, and the recently discovered tetrapeptide GE81112. Given the growing threat of antibiotic resistance, it is essential to understand how these inhibitors function, which could help improve their efficacy as antimicrobial agents. Previous structural studies have captured kasugamycin, edeine, and GE81112 bound to 30S subunits, but these structures were not determined in the context of active translation initiation complexes, and their resolution was limited (3.3-13 Å). Additionally, inconsistencies between these structures and associated biochemical data have left their mechanisms unclear. In this study, using cryo-electron microscopy (cryo-EM), we resolved the structures of kasugamycin, edeine, and GE81112 bound to 30S initiation complexes at resolutions between 2.0 and 2.9 Å. These findings reveal different binding sites for edeine and GE81112 compared to previous crystallographic studies. The high-resolution structures align with prior biochemical data, offering new insights into the molecular interactions of these inhibitors. We found that kasugamycin and edeine trap the pre-initiation 30S complex before the initiator tRNA decodes the mRNA, while GE81112 traps the 30S initiation complex after tRNA accommodation. Despite binding to a conserved site on the 30S subunit, these inhibitors block distinct steps in translation initiation due to their diverse chemical structures.
Additionally, we explored the inhibition mechanism of streptomycin, traditionally known as an elongation inhibitor, during translation initiation. Cryo-EM revealed that streptomycin disrupts interactions between initiation factors IF1 and IF3, leading to absence of IF3 from 30S initiation complexes and potentially accelerating the formation of non-productive 70S initiation complexes.
In conclusion, this body of work advances our understanding of bacterial RQC mechanisms, especially in Gram-negative bacteria. It also sheds light on how key inhibitors target translation initiation, providing insights that could be useful in combating antibiotic resistance
Die Gattung Amorphophallus Blume (Araceae): phylogenetische Untersuchungen und evolutionäre Muster
No full textThe genus Amorphophallus (ca. 237 species) is one of the largest genera of the Araceae and morphologically very diverse. The high species number and the high morphological variation make the understanding of the evolutionary history of the genus challenging. Therefore, in Publ.1, a new molecular phylogenetic analysis using nuclear (ITS1) and plastid (rbcL and matK) sequences based on 157 species is conducted and the resulting phylogenetic tree is used to delimit four subgenera. Moreover, several morphological and biochemical characters, some of which are related to mimicry, are explored in the phylogenetic context, demonstrating the congruence between molecular and some morphological and biochemical traits. However, several species complexes are difficult to resolve. Subsequently, the species boundaries are explored in Publ. 2 through the creation of artificial hybrids. As for the traits related to mimicry, at least two types of mimicry are encountered in several Amorphophallus species. One is a unique type of defensive colouration, petiolar lichen mimicry, providing the fleshy petiole the look of an old woody stem. Lichen mimicry in Amorphophallus has been previously described; however, in a few species only. Therefore, in Publ. 3, defensive colouration is explored in 138 Amorphophallus species, with an emphasis on lichen-like patterns. Mimicry of specific lichen types is identified in 69% of the investigated Amorphophallus species and the results are discussed in the context of the phylogenetic analysis. Deceit flowers, more precisely oviposition-site mimicry, is the second type of mimicry. The inflorescences mimic substrates, usually decomposing organic matter, which are used by Coleoptera and Diptera for feeding or breeding. Amorphophallus species are assumed to have specialised plant-pollinator interactions, involving specific pollinators, which in turn have contributed to the species richness of the genus. However, the available information about Amorphophallus pollinators is scarce; moreover, several reports are anecdotal. Therefore, the observations on visitors and pollinators in Amorphophallus are compiled, reviewed and discussed and the specificity of the plant-pollinator interaction is explored in Publ. 4. The key element of oviposition-site mimicry are the scent compounds. In previous investigations, the scent compounds of 92 Amorphophallus species have been identified and categorised to explore the evolution of floral odours in Amorphophallus. However, only few distinct evolutionary trends could be identified. One possible cause that has not been previously discussed, is intraspecific odour polymorphism. Consequently, the emitted scent compounds in Amorphophallus and the subjective odour classifications are reviewed and screened for odour polymorphism in Publ. 5. Significant odour polymorphism is identifiable in some Amorphophallus species, underlining the necessity for more investigations assessing the intraspecific variation of emitted scent compounds. Publ. 6 addresses thermogenesis, a floral temperature increase assumed to enhance scent volatilization during anthesis. The floral temperature of 80 Amorphophallus species has been measured and the resulting temperature curves have been used to explore and discuss the impact of thermogenesis on the evolution of the genus. The temperature curves show an unprecedented variation within the genus; moreover, the functionality of thermogenesis remains contentious, calling for further investigations. Lastly, using 36 Amorphophallus species, a phylogenomic study is conducted in Publ. 7, investigating the interrelationship between the main clades and providing a timeline for the evolution of the genus. For the first time, a phylogenetic hypothesis is presented that resolves the interrelationship between the African and the Asian clades. In a final chapter, the morphological variation is discussed in regard to the molecular phylogeny and evolutionary constraints. Moreover, further aspects of defensive colouration, odour polymorphism, thermogenesis and the plant-pollinator interaction are discussed
Adaptor proteins form an interaction network during clathrin-mediated endocytosis
No full textClathrin-mediated endocytosis is a process in eukaryotes that is highly conserved across this domain of life, and is essential for several functions such as receptor internalisation and nutrition. During clathrin-mediated endocytosis, selective assemblies of protein complexes form in specific regions of the membrane, ending in the formation of clathrin-coated vesicles (CCV). Controlling these assemblies are regulatory protein-protein interactions, involving proteins arriving at all time points of the Clathrin-coated pit (CCP) maturation. This includes the mid-coat adaptor protein Sla2, which connects the plasma membrane and the actin cytoskeleton, which is the primary force generator during membrane invagination.
The work presented in this thesis presents an interaction network with Sla2 as the central node, and the interacting proteins Clathrin Light Chain (CLC), Sla1, and Pan1 as the primary focus of the biophysically characterised interactions. Sla2, the Fungi homolog, is compared to Hip1R, homolog from Metazoa. The central region of Sla2 is shown to have an additional interaction with CLC not found in Hip1R. This region is also responsible for the regulation of Pan1. Sequence conservation in comparison to Hip1R highlighted the specificity of this Fungi specific interaction with Pan1 and Sla1. The non-conserved region is dominant over the shared region with Hip1R in vivo. The structure, determined by electron cryogenic-microscopy, of the C-terminal region of Sla2 is presented to contextualise these results as the domains detailed here are regulated by the interaction with CLC. The use of AlphaFold3 enabled several key functions of this research: initial model production for structure refinement, protein protein complex prediction, and folded domain annotation for Sla1. This last function narrowed down the most likely interaction site between Sla2 and Sla1 through to the third SH3 domain instead of the other folded domain, which was determined to be a PH domain, which are characterised by the ability to bind phosphatidylinositol.
The central region of Sla2 has been shown to also interact with one of the initiator proteins of endocytosis, Ede1. Ede1 phase separates both in vitro and in vivo and we show that Sla2 can be recruited to phase separated droplets. In addition, Sla2 can recruit Clathrin Light Chain and Clathrin Heavy Chain into its own phase separated droplet.
These findings fill in details of the regulatory interactions in the endocytic pit, with biophysical and biochemical data, fluorescence microscopy, structural determination by two different methods, AI assisted protein-protein interaction modelling, and in vivo determination of the interaction network surrounding Sla2.Die Clathrin-vermittelte Endozytose ist ein hochkonservierter Prozess in Eukaryoten und essenziell für verschiedene Funktionen wie die Internalisierung von Rezeptoren und die Nährstoffaufnahme. Während der Clathrin-vermittelten Endozytose bilden sich selektive Protein-Komplexe in spezifischen Bereichen der Membran, was schließlich zur Bildung Clathrin-beschichteter Vesikel (CCV) führt. Die Kontrolle dieser Assemblierungen erfolgt durch regulatorische Protein-Protein-Interaktionen, an denen Proteine beteiligt sind, die zu verschiedenen Zeitpunkten während der Reifung der Clathrin-beschichteten Grube (CCP) eintreffen. Dazu gehört das Mid-Coat-Adapterprotein Sla2, das die Plasmamembran mit dem Aktin-Zytoskelett verbindet, welches die primäre treibende Kraft bei der Membraneinstülpung darstellt.
Die in dieser Arbeit vorgestellte Forschung beschreibt ein Interaktionsnetzwerk mit Sla2 als zentralem Knoten und den interagierenden Proteinen Clathrin-Leichtkette (CLC), Sla1 und Pan1 als Hauptfokus der biophysikalisch charakterisierten Interaktionen. Sla2, das homologe Protein in Pilzen, wird mit Hip1R, dem Homolog aus Metazoa, verglichen. Es zeigt sich, dass die zentrale Region von Sla2 eine zusätzliche Interaktion mit CLC eingeht, die in Hip1R nicht vorhanden ist. Diese Region ist zudem für die Regulation von Pan1 verantwortlich. Der Vergleich der Sequenzkonservierung mit Hip1R unterstreicht die Spezifität dieser pilzspezifischen Interaktion mit Pan1 und Sla1. In vivo zeigt sich, dass die nicht-konservierte Region eine dominante Rolle gegenüber der mit Hip1R geteilten Region spielt.
Die mittels kryogener Elektronenmikroskopie bestimmte Struktur der C-terminalen Region von Sla2 wird vorgestellt, um diese Ergebnisse in einen strukturellen Kontext zu setzen, da die hier beschriebenen Domänen durch die Interaktion mit CLC reguliert werden. Der Einsatz von AlphaFold3 ermöglichte mehrere zentrale Aspekte dieser Forschung: die Erstellung eines Ausgangsmodells für die Strukturoptimierung, die Vorhersage von Protein-Protein-Komplexen sowie die Annotation gefalteter Domänen in Sla1. Letzteres erlaubte es, die wahrscheinlichste Interaktionsstelle zwischen Sla2 und Sla1 auf die dritte SH3-Domäne einzugrenzen, anstatt auf eine andere gefaltete Domäne, die als PH-Domäne identifiziert wurde – eine Domänenklasse, die für ihre Fähigkeit zur Bindung von Phosphatidylinositol bekannt ist.
Zudem wurde gezeigt, dass die zentrale Region von Sla2 mit einem der Initiatorproteine der Endozytose, Ede1, interagiert. Ede1 bildet sowohl in vitro als auch in vivo Phasenseparationen, und wir zeigen, dass Sla2 in diese phasenseparierten Tropfen rekrutiert werden kann. Darüber hinaus kann Sla2 sowohl die Clathrin-Leichtkette als auch die Clathrin Schwerkette in seine eigenen phasenseparierten Tropfen einbinden.
Diese Erkenntnisse liefern detaillierte Einblicke in die regulatorischen Interaktionen innerhalb der endozytischen Grube. Sie basieren auf biophysikalischen und biochemischen Daten, Fluoreszenzmikroskopie, struktureller Bestimmung durch zwei verschiedene Methoden, KI-gestütztem Protein-Protein-Interaktionsmodellieren sowie der in vivo-Analyse des Interaktionsnetzwerks um Sla2
