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This thesis contains three theoretical works about certain aspects of the interplay of electronic correlations and topology in the Hubbard model.
In the first part of this thesis, the applicability of elementary band representations (EBRs) to diagnose interacting topological phases, that are protected by spatial symmetries and time-reversal-symmetry, in terms of their single-particle Matsubara Green’s functions is investigated. EBRs for the Matsubara Green’s function in the zero-temperature limit can be defined via the topological Hamiltonian. It is found that the Green’s function EBR classification can only change by (i) a gap closing in the spectral function at zero frequency, (ii) the Green’s function becoming singular i.e. having a zero eigenvalue at zero frequency or (iii) the Green’s function breaking a protecting symmetry. As an example, the use of the EBRs for Matsubara Green’s functions is demonstrated on the Su-Schriefer-Heeger model with exact diagonalization.
In the second part the Two-Particle Self-Consistent approach (TPSC) is extended to include spin-orbit coupling (SOC). Time-reversal symmetry, that is preserved in the presence of SOC, is used to derive new TPSC self-consistency equations including SOC. SOC breaks spin rotation symmetry which leads to a coupling of spin and charge channel. The local and constant TPSC vertex then consists of three spin vertices and one charge vertex. As a test case to study the interplay of Hubbard interaction and SOC, the Kane-Mele-Hubbard model is studied. The antiferromagnetic spin fluctuations are the leading instability which confirms that the Kane-Mele-Hubbard model is an XY antiferromagnet at zero temperature. Mixed spin-charge fluctuations are found to be small. Moreover, it is found that the transversal spin vertices are more strongly renormalized than the longitudinal spin vertex, SOC leads to a decrease of antiferromagnetic spin fluctuations and the self-energy shows dispersion and sharp features in momentum space close to the phase transition.
In the third part TPSC with SOC is used to calculate the spin Hall conductivity in the Kane-Mele-Hubbard model at finite temperature. The spin Hall conductivity is calculated once using the conductivity bubble and once including vertex corrections. Vertex corrections for the spin Hall conductivity within TPSC corresponds to the analogues of the Maki-Thompson contributions which physically correspond to the excitation and reabsorption of a spin, a charge or a mixed spin-charge excitation by an electron. At all temperatures, the vertex corrections show a large contribution in the vicinity of the phase transition to the XY antiferromagnet where antiferromagnetic spin fluctuations are large. It is found that vertex corrections are crucial to recover the quantized value of −2e^2/h in the zero-temperature limit. Further, at non-zero temperature, increasing the Hubbard interaction leads to a decrease of the spin Hall conductivity. The results indicate that scattering of electrons off antiferromagnetic spin fluctuations renormalize the band gap. Decreasing the gap can be interpreted as an effective increase of temperature leading to a decrease of the spin Hall conductivity.
Trait-dependent effects of biotic and abiotic filters on plant regeneration in Southern Ecuador
(2024)
Tropical forests have always fascinated scientists due to their unique biodiversity. However, our understanding of ecological processes shaping the complexity of tropical rainforests is still relatively poor. Plant regeneration is one of the processes that remain understudied in the tropics although this is a key process defining the structure, diversity and assembly of tropical plant communities. In my dissertation, I combine experimental, observational and trait-based approaches to identify processes shaping the assembly of seedling communities and compare associations between environmental conditions and plant traits across plant life stages. By working along a steep environmental gradient in the tropical mountains of Southern Ecuador, I was able to investigate how processes of plant regeneration vary in response to biotic and abiotic factors in tropical montane forests.
My dissertation comprises three complementary chapters, each addressing an individual research question. First, I studied how trait composition in plant communities varies in relation to the broad- and local-scale environmental conditions and across the plant life cycle. I measured key traits reflecting different ecological strategies of plants that correspond to three stages of the plant life cycle (i.e., adult trees, seed rain and recruiting seedlings). I worked on 81 subplots along an elevational gradient covering a large climatic gradient at three different elevations (1000, 2000 and 3000 m a.s.l.). In addition, I measured soil and light conditions at the local spatial scale within each subplot. My findings show that the trait composition of leaves, seeds and seedlings changed similarly across the elevational gradient, but that the different life stages responded differently to the local gradients in soil nutrients and light availability. Consequently, my findings highlight that trait-environment associations in plant communities differ between large and small spatial scales and across plant life stages.
Second, I investigated how seed size affects seedling recruitment in natural forests and in pastures in relation to abiotic and biotic factors. I set up a seed sowing experiment in both habitat types and sowed over 8,000 seeds belonging to seven tree species differing in seed size. I found that large-seeded species had higher proportions of recruitment in the forests compared to small-seeded species. However, small-seeded species tended to recruit better in pastures compared to large-seeded species. I showed that high surface temperature was the main driver of differences in seedling recruitment between habitats, because it limited seedling recruitment of large-seeded species. The results from this experiment show that pasture restoration requires seed addition of large-seeded species and active protection of recruiting seedlings in order to mitigate harmful conditions associated with high temperatures in deforested areas.
Third, I examined the associations between seedling beta-diversity and different abiotic and biotic factors between and within elevations. I applied beta-diversity partitioning to obtain two components of beta-diversity: species turnover and species richness differences. I associated these components of beta-diversity with biotic pressures by herbivores and fungal pathogens and environmental heterogeneity in light and soil conditions. I found that species turnover in seedling communities was positively associated with the dissimilarity in biotic pressures within elevations and with environmental heterogeneity between elevations. Further, I found that species richness differences increased primarily with increasing environmental heterogeneity within elevations. My findings show that the associations between beta-diversity of seedling communities and abiotic and biotic factors are scale-dependent, most likely due to differences in species sorting in response to biotic pressures and species coexistence in response to environmental heterogeneity.
My dissertation reveals that studying processes of community assembly at different plant life stages and spatial scales can yield new insights into patterns and processes of plant regeneration in tropical forests. I investigated how community assembly processes are governed by abiotic and biotic filtering across and within elevations. I also experimentally explored how the process of seedling recruitment depends on seed size-dependent interactions, and verified how these effects are associated with abiotic and biotic filtering. Identifying such processes is crucial to inform predictive models of environmental change on plant regeneration and successful forest restoration. Further exploration of plant functional traits and their associations with local-scale environmental conditions could effectively support local conservation efforts needed to enhance forest cover in the future and halt the accelerating loss of biodiversity.
This thesis develops a naturalist theory of phenomenal consciousness. In a first step, it is argued on phenomenological grounds that consciousness is a representational state and that explaining consciousness requires a study of the brain’s representational capacities. In a second step, Bayesian cognitive science and predictive processing are introduced as the most promising attempts to understand mental representation to date. Finally, in a third step, the thesis argues that the so-called “hard problem of consciousness” can be resolved if one adopts a form of metaphysical anti-realism that can be motivated in terms of core principles of Bayesian cognitive science.
Thomas Bowrey, who was an employee of the British colonial government, visited the Malay-speaking region at the end of the 17th century and published a dictionary of Malay (1701) which consists of 12,683 headwords. It is one of the oldest and largest collections of data on this language, which was the first language of the people he came into contact with while travelling through the Malay Peninsula, spending most of his time in harbours along its west coast. Malay, which was spoken in the various trading centres of this area (e.g. Penang, Malacca), had long previously begun to develop into a form of lingua franca during Bowrey’s stay there due to the fact that traders, especially those from Arabic countries (beginning in the 12th century), China (from the 15th century onwards), Portugal (since 1511), the Netherlands (since 1641), and less so from England, came into contact with Malays speaking their local dialects in the various trading posts in Malaya and probably began to become acquainted with the trade-language variant. Thus, Bowrey must have observed and recorded elements of both.
The data he collected is not limited to Malay variants spoken in coastal areas, but includes material from dialects which he encountered during his travels throughout the Malay Peninsula, though without, however, describing the locations in which he took notes on the lexicon and clauses. Not all of his material was written into manuscript form during his stay in Southeast Asia. A large part of his notes taken in situ were prepared for publication during his long journey home. His notes, which were used to print his dictionary, are in part kept in British libraries. Most of the material accessible to the public was studied during the preparation of this thesis.
Earlier works on this dictionary are quite limited in scope. They deal with very specific aspects such as the meanings of headwords found between the letters A and C (Rahim Aman, 1997 & 1998), and the work of Nor Azizah, who deals with the lexical change found in Bowrey’s dictionary between D and F, and syntactic and sociolinguistic aspects (Mashudi Kader, 2009), and collective nouns by Tarmizi Hasrah (2010). This study will discuss Bowrey’s dictionary as a whole in order to describe its contribution to our knowledge of linguistic and non-linguistic facts in 17th century Malaya. Besides analysing Malay synchronically, this thesis also deals with historical-comparative questions and asks whether Bowrey contributes to our knowledge of the changes to the Malay language between the 17th and 21st centuries.
In order to answer the research questions, this study not only relies on the dictionary in its entirety, but also on the notes found in British libraries as well as other material on early Malay, such as the Pigafetta list (1523), Houtman (1598–1603), and the Wilkinson dictionary (1901) as a complement to Bowrey’s dictionary; at the same time, the Malay Concordance Project (online), the SEAlang Project (online), Kamus Besar Bahasa Indonesia (online), and Kamus Dewan Edisi Keempat (2007) will represent modern Malay. It should be borne in mind that in contrast to the Thomas Bowrey dictionary (TBD), Kamus Dewan Edisi Keempat (KDE4) does not hold information on colloquial forms of Malay, many of which reflect features of lingua franca Malay. This study is divided into two different branches, namely the consideration of synchronic aspects and historical comparative aspects.
Finally, this study concludes that the Malay language in Thomas Bowrey’s dictionary is heavily influenced by both external and internal factors prevalent to the 17th century. The Malay language recorded in the Thomas Bowrey dictionary is very similar to modern Malay. The similarities between the Malay language of the 17th century and the Malay language of today are considerable, even though there are, of course, still some notable variances.
A powerful technique to distinguish the enantiomers of a chiral molecule is the Coulomb Explosion Imaging (CEI). This technique allows us to determine the handedness of a single molecule. In CEI, the molecule becomes charged by losing many electrons in a very short period of time by interacting with the light. The repulsion forces between the positive charged particles of the molecule leads the molecule to break into parts-fragments. By measuring the three vector momentum of (at least) four fragments, the handedness observable can be determined. In this thesis, CEI is induced by absorption of a single high energy photon, which creates an inner-shell hole (K shell) of the molecule. The subsequent cascade of Auger decays lead to fragmentation. We decided to work with the formic acid molecule in this thesis. Two different experiments were conducted. The first experiment focused on exciting electrons to different energy states, while the second experiment focused on extracting directly a photoelectron to the continuum and measure the angular distribution of the photoelectron in the molecular frame. The primary goal was to search for chiral signal in a pure achiral planar molecule under the previous electron processes. The results of these findings were further implemented to two more molecules.
While high-quality climate reconstructions of some past warm periods in the Cenozoic era now exist, the geological processes responsible for driving the observed longterm changes in atmospheric CO2 are not sufficiently well understood. The long-term change in atmospheric CO2 across the Cenozoic has been proposed to be driven by processes such as terrestrial weathering, organic carbon production and burial, reverse weathering, and volcanic degassing. One way of constraining the relative importance of the various driving forces proposed so far is to better understand the degree to which ocean chemistry has changed because the chemistry of seawater responds to geologic processes that drive atmospheric CO2. In addition, knowledge of the concentration of the major elements in seawater is crucial for accurately applying proxies such as those based on the boron isotopic composition and Mg/Ca of marine carbonates (a proxy for palaeo pH/CO2 and palaeotemperature, respectively). Previously reported records of seawater composition are primarily derived from fluid inclusions in marine evaporites; however, the results are sparse due to the limited availability of such deposits. In this thesis, changes in the Eocene seawater chemistry were reconstructed using trace element (elements/Ca) and isotopic (δ26Mg) proxies in a Larger Benthic Foraminifera (LBFs), i.e., Nummulites sp., to constrain the driving processes of long-term changes in seawater chemistry.
To achieve the objective of this thesis, first, a measurement protocol was established using LA-ICPMS to measure the K/Ca ratio simultaneously with other element/calcium ratios, which is challenging due to the interference of ArH+ on K+. Utilising this newly established measurement protocol, laboratory-cultured Operculina ammonoides grown at different seawater calcium concentrations ([Ca2+]), repeated at different temperatures, as well as modern O. ammonoides collected from different regions exhibiting a range of seawater parameters, were investigated. A significant correlation was observed between K/Casw and K/CaLBF, allowing K/CaLBF to potentially be used as a proxy for seawater major ion reconstructions. In addition, modern O. ammonoides demonstrated no significant influence of most seawater parameters (temperature, salinity, pH, or [CO32-]) on K/CaLBF. Modern O.
ammonoides were also assessed for their Mg isotopic composition (δ26Mg), revealing no significant effect of temperature or salinity on δ26MgLBF. Furthermore, the Mg isotopic fractionation in O. ammonoides was found to be close to that of inorganic calcite, indicating minimal vital effects in these large benthic foraminifera.
Operculina ammonoides is the nearest living relative of the abundant Eocene genus Nummulites, enabling the reconstruction of seawater chemistry using the calibration based on O. ammonoides. The trace elemental/calcium proxies, including Na/Ca, K/Ca, and Mg/Ca, as well as the δ26Mg proxy, were investigated in Eocene Nummulites. The result showed that during the Eocene, [Ca2+]sw was 1.6-2 times higher, while [K+]sw was ~2 times lower than the modern seawater composition. Furthermore, [Mg2+]sw decreased from the early Eocene (54.3− +9 7..69 mmol kg-1 at ~55 Ma) to Late Eocene (37.8− +4 4..3 4 mmol kg-1 at ~31 Ma), followed by
an increase toward modern seawater [Mg]. In contrast, the variability in δ26Mgsw values remained within a narrow range of ~0.3 ‰ throughout the Cenozoic. The reconstructed [Ca2+]sw agrees with the suggestion that Cenozoic seawater chemistry changes can be explained via a change in the seafloor spreading rate. When combined with existing records, the observed minimal change in δ26Mgsw with an increase in [Mg2+]sw suggests an additional possible role of a decrease in the formation of authigenic clay minerals coincident with the Cenozoic decline in deep ocean temperature, which is also supported by the increase in the [K+]sw reconstructed here for the first time. This finding highlights that the reduction in seafloor-spreading rate and decline in reverse weathering during the Cenozoic era has played a significant role in the evolution of seawater chemistry, emphasizing the importance of these processes in driving long-term changes in the carbon cycle.
During my initial days here in Frankfurt, in October 2020 amidst the pandemic crisis, all my notes revolved around three articles by Bolthausen and Kistler, which now form the starting point of this work.
The ones introduced by Bolthausen and Kistler are abstract mean field spin glass models, reminiscent of Derrida’s Generalized Random Energy Model (GREM), which generalize the GREM while remaining rigorously solvable through large deviations methods and within a classical Boltzmann-Gibbs formalism. This allows to establish, by means of a second moment method, the associated free energy at the thermodynamic limit as an orthodox, infinite-dimensional, Boltzmann-Gibbs variational principle.
Dual Parisi formulas for the limiting free energy associated with these Hamiltonians hold, and are revealed to be the finite-dimensional (”collapsed”) versions of the classical, infinite-dimensional Boltzmann-Gibbs principles.
In the 2nd chapter of this thesis, we uncover the hidden yet essential connection between real-world spin glasses, like the Sherrington-Kirkpatrick (SK) model and the random energy models. The crucial missing element is that of TAP-free energies: integrating it with the framework introduced by Bolthausen and Kistler results in a correction to the Parisi formula for the free energy, which brings it much, much closer to the ”true” Parisi solution for the SK-model. In other words, we can identify the principles that transform the classical Boltzmann-Gibbs maximization into the unorthodox (and puzzling) Parisi minimization.
This arguably stands as the primary achievement of this work.
ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. They play a role in many physiological processes but are also the reason for antibiotic resistance of microbes and multi drug resistance in cancer, and their dysfunction can lead to serious diseases. Transport is achieved through an ATP-driven closure of the two nucleotide binding sites (NBSs) which induces a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the connected transmembrane domains (TMDs). In contrast to this forward transition, the reverse transition (OF-to-IF) that involves Mg2+-dependent ATP hydrolysis and release is less understood. This is particularly relevant for heterodimeric ABC transporters with asymmetric NBSs. These transporters possess an ATPase active consensus NBS (c-NBS) and a degenerate NBS (d-NBS) with little or no ATPase activity.
Crucial details regarding function and mechanism of the transport cycle remain elusive.
Here, these open questions were addressed using pulse electron-electron double resonance (PELDOR or DEER) spectroscopy of the heterodimeric ABC exporter TmrAB.
To better understand the transport cycle, the underlying kinetics of the conformational transitions need to be elucidated. By introducing paramagnetic nitroxide (NO) spin probes at key positions of TmrAB and employing time-resolved PELDOR spectroscopy, the forward transition could be followed over time and the rate constants for the conformational transition at the TMDs and NBSs were characterized.
The temperature dependence of these rate constants was further analyzed to determine for the first time the activation energy of conformational changes in a large membrane protein. For TMD opening and c-NBS dimerization, values of 75 ± 27 kJ/mol and 56 ± 3 kJ/mol, respectively were found. These values agree with reported activation energies of peptide transport and peptide dissociation in other ABC transporters, suggesting that the forward transition may be the rate-limiting step for substrate translocation.
The functional relevance of asymmetric NBSs is so far not well understood. By combining Mg2+-to-Mn2+ substitution with Mn2+-NO and NO-NO PELDOR spectroscopy, the binding of ATP-Mn2+, the conformation of the NBSs, and the conformation of the TMDs could be simultaneously monitored for the first time. These results reveal an asymmetric post-hydrolytic state. Time-resolved investigation showed that ATP hydrolysis at the active c-NBS triggers the reverse transition, whereas opening of the impaired d-NBS regulates the return to the IF conformation.
Neurodevelopmental psychiatric disorders (NPDs) like attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia, affect millions of people worldwide. Despite recent progress in NPD research, much remains to be discovered about their underpinnings, therapeutic targets, effects of biological sex and age. Risk factors influencing brain development and signalling include prenatal inflammation and genetic variation. This dissertation aimed to build upon these findings by combining behavioural, molecular, and neuromorphological investigations in mouse models of such risk factors, i.e. maternal immune activation (MIA), neuron-specific overexpression (OE) of the cytoplasmatic isoforms of the RNA-binding protein RBFOX1, and neuronal deletion of the small Ras GTPase DIRAS2.
Maternal infections during pregnancy pose an increased risk for NPDs in the offspring. While viral-like MIA has been previously established elsewhere, this study was the first in our institution to implement the model. I validated NPD-relevant deficits in anxiety- and depression-like behaviours, as well as dose- and sex-specific social deficits in mouse offspring following MIA in early gestation. Proteomic analyses in embryonic and adult hippocampal (HPC) synaptoneurosomes highlighted novel and known targets affected by MIA. Analysis of the embryonic dataset implicated neurodevelopmental disruptions of the lipid, polysaccharide, and glycoprotein metabolism, important for proper membrane function, signalling, and myelination, for NPD-pertinent sequelae. In adulthood, the observed changes encompassed transmembrane trafficking and intracellular signalling, apoptosis, and cytoskeletal organisation pathways. Importantly, 50 proteins altered by MIA in embryonic and adult HPC were enriched in the NPD-relevant synaptic vesicle cycle. A persistently upregulated protein cluster formed a functional network involved in presynaptic signalling and proteins downregulated in embryos but upregulated in adults by MIA were correlated with observed social deficits. 49/50 genes encoding these proteins were significantly associated with NPD- and comorbidity-relevant traits in human phenome-wise association study data for psychiatric phenotypes. These findings highlight NPD-relevant targets for future study and early intervention in at-risk individuals. MIA-evoked changes in the neuroarchitecture of the NPD-relevant HPC and prefrontal cortex (PFC) of male and female mice highlighted sex- and region-specific alterations in dendritic and spine morphology, possibly underlining behavioural phenotypes.
To further investigate genetic risk factors of NPDs, I performed a study based on the implications of RBFOX1’s pleiotropic role in neuropsychiatric disorders and previous preclinical findings. Cytoplasmatic OE of RBFOX1, which affects the stability and translation of thousands of targets, was used to disseminate its role in morphology and behaviour. RBFOX1 OE affected dendritic length and branching in the male PFC and led to spine alterations in both PFC and HPC. Due to previously observed ASD-like endophenotypes in our Rbfox1 KO mice and the importance of gene × environment effects on NPD susceptibility, I probed the interaction of cytoplasmatic OE and a low-dose MIA on offspring. Both RBFOX1 OE alone and with MIA led to increased offspring loss during the perinatal period. Preliminary data suggested that RBFOX1 OE × MIA might increase anxiety- and anhedonia-like behaviours. Morphological changes in the adult male OE HPC and PFC suggested increased spine density and reduced dendritic complexity. A small post-mortem study in human dorsolateral PFC of older adults did not reveal significant effects of a common risk variant on RBFOX1 abundance.
To expand upon NPD genetic risks, I evaluated the effects of a homo- (KO) or heterozygous (HET) Diras2 deletion in a novel, neuron-specific mouse model. DIRAS2’s function is largely unknown, but it has been associated with ADHD in humans and neurodevelopment in vitro. In adult mice, there were subtle sex-specific effects on behaviour, i.e. more pronounced NPD-relevant deficits in males, in keeping with human data. KO mice had subtly improved cognitive performance, while HET mice exhibited behaviours in line with core ADHD symptoms, e.g. earning difficulties (females), response inhibition deficits and hyperactivity (males), suggesting Diras2 dose-sensitivity and sex-specificity. The morphological findings revealed multiple aberrations in dendritic and spine morphology in the adult PFC, HPC, and amygdala of HET males. KOs changes in spine and dendritic morphology were exclusively in the PFC and largely opposite to those in HETs and NPD-like phenotypes. Region- and genotype-specific expression changes in Diras2 and Diras1 were observed in six relevant brain regions of adult HET and KO females, also revealing differences in the survival and morphology regulator mTOR, which might underlie observed differences.
In conclusion, the effects of MIA and partial Diras2 knockdown resembled each other in core, NPD-associated behavioural and morphological phenotypes, while cytoplasmatic RBFOX1 OE and full Diras2 KO differed from those. My findings suggest complex dose- and sex-dependent relationships between these prenatal and genetic interventions, whose NPD-relevant influences might converge onto neurodevelopmental molecular pathways. An assessment of such putative overlap, based on available data from the MIA proteomic analyses of embryonic and adult HPC, suggested the three models might be linked via downstream targets, interactions, and upstream regulators. Future studies should disseminate both distinct and shared aspects of MIA, RBFOX1, and DIRAS2 relevant to NPDs and build upon these findings.
Molecular concepts for pandemic viruses : membrane fusion assays and targeting of reservoir cells
(2024)
In den letzten Jahren haben verschiedene pandemische Viren zu beträchtlichen Krankheits- und Todesfällen geführt. Um dieser ständigen Bedrohung entgegenzuwirken, ist es wichtig diagnostische Testsysteme und Therapien anzupassen oder neu zu etablieren. Diese Arbeit konzentriert sich auf die pandemischen Viren SARS-CoV-2 und HIV.
Der Zelleintritt von SARS-CoV-2 wird durch das Spike-Protein (S) ausgelöst, welches die Fusion der Virushülle mit der zellulären Membran bewirkt. Erste Studien haben gezeigt, dass das S-Protein eine hohe Fusionsaktivität aufweist. Aus diesem Grund sollten in dieser Arbeit neue Fusionstests etabliert werden, um potenzielle Inhibitoren der Zellfusion zu evaluieren. Im ersten Teil dieser Thesis wird die Etablierung von quantitativen Tests zur Evaluierung der Zell-Zell und Partikel-Zell Fusionsaktivität, welche durch S bewirkt wird, demonstriert.
Trotz jahrelanger Forschung können HIV-Patienten nicht geheilt werden und Virusinfektionen treten weiterhin weltweit auf. Das größte Problem bei der Entwicklung eines Heilmittels ist die frühe Bildung von Reservoirzellen während einer Infektion. Um diese Reservoirzellen zu identifizieren, wurde der Oberflächenmarker CD32a vorgeschlagen. Die Nutzung von Cas9-Nukleasen zur Inaktivierung von HIV ist in vitro erfolgreich, aber der effiziente Transfer in Reservoirzellen bleibt weiterhin herausfordernd. Im zweiten Teil dieser Thesis werden Rezeptor-gerichtete Adeno-assoziierte Vektoren (AAVs) für die HIV-Gentherapie präsentiert, die CD4 und CD32a für den Zelleintritt nutzen.
Zur Charakterisierung der Fusionsaktivität von SARS-CoV-2 wurden drei quantitative Fusionstests etabliert, welche Partikel- und Zell-Zell Fusionen berücksichtigen. Für den Partikel-Zell Fusionstest wurden lentivirale Vektoren (LV) verwendet, welche das S-Protein auf ihrer Oberfläche präsentierten. Die Transduktionseffizienz von S-LV erreichte auf Zellen, die den SARS-CoV-2 Rezeptor ACE2 exprimieren, ein Signal-zu-Hintergrund Verhältnis von über 2000. Durch die Präsentation von S auf leeren LV-Partikeln konnte die Fusion von benachbarten Zellen detektiert und quantifiziert werden („fusion-from-without“ (FFWO)). Für die Quantifizierung wurde ein Reporter-Komplementationstest etabliert. Hierbei wurden die Alpha- und Omega-Fragmente der β-Galaktosidase getrennt in zwei Zielzellpopulationen exprimiert, die beide ACE2 exprimierten. Durch die Zugabe von S-Partikeln kam es zur Fusion der Zielzellen und zur Komplementation der Alpha- und Omega-Fragmente. Die resultierende β-Galaktosidase-Aktivität konnte anschließend quantifiziert werden. Unter optimalen Versuchsbedingungen erreichte dieser Assay ein Signal-zu-Hintergrund Verhältnis von 2,7 Größenordnungen. Anschließend wurde der Komplementationstest für die Messung der Zell-Zell Fusion verwendet. In diesem Test exprimierten Effektorzellen S und das Alpha-Fragment, Zielzellen ACE2 und das Omega-Fragment. Obwohl die S-Expression auf den Effektorzellen sehr gering war, konnte dennoch eine signifikante Fusion nachgewiesen werden. Auch hier konnte unter optimalen Versuchsbedingungen ein hohes Signal-zu-Hintergrund Verhältnis von 2,9 Größenordnungen festgestellt werden. Nach der Etablierung der Testsysteme wurden S-spezifische Inhibitoren verwendet. Im Gegensatz zu Partikel-Zell-Fusionen wurde die Fusionsaktivität von S auf Zellen nur mäßig inhibiert. Dies deutet daraufhin, dass das Eindringen von Partikeln in Zellen wirksamer verhindert werden kann als die Ausbreitung durch Zell-Zell Fusionen.
Um AAVs spezifisch an HIV-Reservoirzellen zu binden, wurden CD4- und CD32a-spezifische DARPins („designed ankyrin repeat proteins“) in Rezeptor-verblindete AAVs eingebaut. Ebenso wurden beide DARPins gleichzeitig auf dem Kapsid präsentiert, um eine höhere Spezifität für doppelt-positive Zellen zu erreichen. Wenn diese Partikel einer Zellmischung aus CD4-, CD32a- und CD4/CD32a-exprimierenden Zellen zugesetzt wurden, transduzierten die bispezifischen Vektoren vorzugsweise doppelt-positive Zellen. Diese Präferenz war am höchsten in Zellkulturen, die stark unterrepräsentierte CD4/CD32a-exprimierende Zellen enthielten. Unter diesen Voraussetzungen erreichten bispezifische Vektoren eine bis zu 66-fach höhere Transduktionseffizienz auf CD4/CD32a-positive Zellen im Vergleich zu CD32a-exprimierenden Zellen. Darüber hinaus zeigten bispezifische AAV eine präferentielle Bindung und Transduktion von isolierten Primärzellen und Zellen in Vollblut. Selbst nach systemischer Injektion in humanisierte Mäuse wurden doppelt-positive Zellen effizienter von bispezifischen als von monospezifischen AAVs transduziert. Schließlich zeigten die generierten Vektoren, welche die Cas9 Nuklease transferierten, eine effiziente Inhibition der HIV-Replikation.
Inorganic phosphate is one of the most abundant and essential nutrients in living organisms. It plays an indispensable role in energy metabolism and serves as a building block for major cellular components such as the backbones of DNA and RNA, headgroups of phospholipids and in posttranslational modifcations of many proteins. Disturbances in cellular phosphate homeostasis have a detrimental effect on the viability of cells. There- fore, both the import and export of phosphate is strictly regulated in eukaryotic cells. In the eukaryotic model organism Saccharomyces cerevisiae, the uptake of phosphate is carried out either by transporters with high affinity or by transporters with low affinity, depending on the cytosolic phosphate concentration. While structures are available for homologues of the high-affinity transporters, no structures of low-affinity transporters have been solved so far. Interestingly, only the low-affinity transporters have a regulatory SPX domain, which is found in various proteins involved in phosphate homeostasis.
In this work, structures of Pho90 from Saccharomyces cerevisiae, a low-affinity phosphate transporter, were solved by cryo-EM, providing insights into its transport mechanism. The dimeric structure resembles the structures of proteins of the divalent anion symporter superfamily (DASS) and of mammalian transporters of the solute carrier 13 (SLC13) family. The transmembrane domain of each protomer consists of 13 helical elements and can be subdivided into scaffold and transport domains. The structure of ScPho90 in the presence of phosphate shows the phosphate binding site within the transporter domain in an outward-open conformation with a bound phosphate ion and two sodium ions. In the absence of phosphate, an asymmetric dimer structure was determined, with one protomer adopting an inward-open conformation. While the dimer contact and the scaffold domain are identical in both conformations, the transport domain is rotated by about 30° and shifted by 11 Å towards the cytoplasmic side, leading to the accessibility of the binding pocket from the cytoplasm. Based on these findings and by comparison with known structures, a phosphate transport mechanism is proposed in the present work that involves substrate binding on the extracellular side, conformational change by a rigid-body motion of the transport domain, in an "elevator-like" motion, and substrate release into the cytoplasm. The regulatory SPX domain is not well resolved in the ScPho90 structures, so that no direct conclusions were drawn about its regulatory mechanism. The findings provide new insights into the function and mechanism of eukaryotic low-affinity phosphate transporters.
While eukaryotic cells express various phosphate import proteins, most eukaryotes have only a single highly conserved and essential phosphate exporter. These exporters show no sequence homology to other transporters of known structure, but also possess a regulatory SPX domain. In this work, the structural basis for eukaryotic phosphate export is investigated by elucidating the structures of the homologous phosphate exporters Syg1 from Saccharomyces cerevisiae and Xpr1 from Homo sapiens, using cryo-EM. The structures of ScSyg1 and HsXpr1 show a conserved homodimeric structure and the transmembrane part of each protomer consists of 10 TM helices. Helix TM1 establishes the dimer contact by means of a glycine zipper motif, which is a known oligomerization motif. Helices TM2-5 form a hydrophobic pocket that has density for a lipid molecule. Whether the lipid binding into the hydrophobic pocket has an allosteric effect on the phosphate export activity or only serves protein stabilization is not known. Helices TM5-10 form a six-helix bundle, which constitutes a putative phosphate translocation pathway in its center. This bundle is formed by the protein sequence annotated as EXS domain.
The respective phosphate translocation pathways of ScSyg1 and HsXpr1 show structural differences. While the translocation pathway in HsXpr1 is accessible from the cytoplasm, in ScSyg1 it is closed by a large loop of the SPX domain. Interestingly, this loop is not conserved in higher eukaryotes and is therefore not present in HsXpr1. Another difference are distinct conformations of helix TM9. In ScSyg1, TM9 adopts a kinked conformation, which results in the translocation pathway being open to the extracellular side. In contrast, TM9 adopts a straight conformation in HsXpr1, resulting in the placement of a highly conserved tryptophane residue in the middle of the translocation pathway. As a result, the translocation pathway in HsXpr1 is closed to the extracellular side.
Exploring strategies to improve the reverse beta-oxidation pathway in Saccharomyces cerevisiae
(2024)
Microbes are the most diverse living organisms on Earth, with various metabolic adaptations that allow them to live in different conditions and produce compounds with different chemical complexity. Microbial biotechnology exploits the metabolic diversity of microorganisms to manufacture products for different industries. Today, the chemical industry is a significant energy consumer and carbon dioxide emitter, with processes that harm natural ecosystems, like the extraction of medium-chain fatty acids (MCFAs). MCFAs are used as precursors for biofuels, volatile esters, surfactants, or polymers in materials with enhanced properties.
However, their current extraction process uses large, non-sustainable monocultures of coconut and palm trees. Therefore, the microbial production of MCFAs can help reduce the current environmental impact of obtaining these products and their derivatives.
In nature, fatty acids are mostly produced via fatty acid biosynthesis (FAB). However, the reverse β-oxidation (rBOX) is a more energy-efficient pathway compared to FAB. The rBOX pathway consists of four reactions, which result in the elongation of an acyl-CoA molecule by two carbon units from acetyl-CoA in each cycle. In this work we used Saccharomyces cerevisiae, an organism with a high tolerance towards toxic compounds, as the expression host of the rBOX pathway to produce MCFAs and medium-chain fatty alcohols (MCFOHs).
In the first part of this work, we expanded the length of the products from expressing the rBOX in the cytosol and increased the MCFAs titres. First, we deleted the major glycerol-3-phosphate dehydrogenase (GPD2). This resulted in a platform strain with significantly reduced glycerol fermentation and increased rBOX pathway activity, probably due to an increased availability of NADH. Then, we tested different combinations of rBOX enzymes to increase the length and titres of MCFA. Expressing the thiolase CnbktB and β-hydroxyacyl-CoA dehydrogenase CnpaaH1 from Cupriavidus necator, Cacrt from Clostridium acetobutylicum and the trans-enoyl-CoA reductase Tdter (Treponema denticola) resulted in hexanoic acid as the main product.
Expressing Cncrt2 (C. necator) or YlECH (Y. lipolytica) as enoyl-CoA hydratases resulted in octanoic acid as the main product. Then, we integrated the octanoic (Cncrt2 or YlECH) and the hexanoic acid (Cacrt)-producing variants in the genome of the platform strain and we achieved titers of ≈75 mg/L (hexanoic acid) and ≈ 60 mg/L (octanoic acid) when growing these strains in a complex, highly buffered medium. These are the highest titers of octanoic and hexanoic acid obtained in S. cerevisiae with the rBOX. Additionally, we deleted TES1 and FAA2 to prevent competition for butyryl-CoA and degradation of the produced fatty acids, respectively.
However, these deletions did not improve MCFA titers. In addition, we tested two dual acyl-CoA reductase/alcohol dehydrogenases (ACR/ADH), CaadhE2 from C. acetobutylicum and the putative ACR/ADH EceutE from Escherichia coli, in an octanoyl-CoA-producing strain to produce MCFOH. As a result, we produced 1-hexanol and 1-octanol for the first time in S. cerevisiae with these two enzymes. Nonetheless, the titres were low (<10 mg/L and <2 mg/L, respectively), and four-carbon 1-butanol was the main product in both cases (>80 mg/L). This showed the preference of these two enzymes for butyryl-CoA.
In the second part of this work, we expressed the rBOX in the mitochondria of S. cerevisiae to benefit from the high levels of acetyl-CoA and the reducing environment in that organelle. First, in an adh-deficient strain, we mutated MTH1, a transcription factor regulating the expression of hexose transporters, and deleted GPD2. This resulted in a strain with a reduced Crabtree effect and, therefore, an increased carbon flux to the mitochondria. We partially validated the increased flux to the mitochondria by expressing the ethanol-acetyltransferase EAT1 from Kluyveromyces marxianus in this organelle. This resulted in a higher isoamyl acetate production in the MTH1-mutant strain. Isoamyl acetate is synthesised by Eat1 from acetyl-CoA and isoamyl alcohol, a product of the metabolism of amino acids in the mitochondria. Then, we targeted different butyryl-CoA-producing rBOX variants to the mitochondria, and we used the production of 1-butanol and butyric acid as a proof-of-concept. The strong expression of all the enzymes was toxic for the cell, and the highest butyric acid titres (≈ 50 mg/L) in the mitochondria from the rBOX were obtained from the weak expression of the pathway. The highest 1-butanol titers (≈ 5 mg/L) were obtained with the downregulation of the mitochondrial NADH-oxidase NDI1. However, this downregulation led to a non-desirable petite phenotype.
In summary, we produced hexanoic and octanoic acid for the first time in S. cerevisiae using the rBOX and achieved the highest reported titers of hexanoic and octanoic acid so far using this pathway in S. cerevisiae. In addition, we successfully compartmentalised the rBOX in the mitochondria. However, competing reactions, some of them essential for the viability of the cell, limit the use of this organelle for the rBOX.
In dieser Dissertation werden die Erfahrungen mit verschiedenen Präparationsmethoden für CH-Kavitäten beschrieben, um die Leistung der Kavitäten nach der Herstellung weiter zu steigern. Die Leistung wird anhand von zwei wichtigen HF-Parametern bewertet:
dem elektrischen Feld Ea und der intrinsischen Güte Q0. Im Gegensatz zu normalleitenden (NC) Kavitäten kann die intrinsische Güte von supraleitenden (SC) Kavitäten mit zunehmendem elektrischem Feld erheblich variieren. Das optimale Ergebnis für die Kavitätenpräparation ist die Erhöhung des maximalen elektrischen Feldes unter Beibehaltung eines höheren Q0 über die gesamte Feldspanne. Da Q0 umgekehrt proportional zu den Kavitätsverlusten ist, reduziert eine Erhöhung des Qualitätsfaktors die Kryoverluste für den Betrieb bei gegebenem Feldniveau. Die Entwicklung der Kavitätenperformanz im Verlauf dieser Arbeit dargestellt.
Die meisten SC-Kavitäten sind elliptische Strukturen, welche bei hoher Geschwindigkeit und Tastrate angewendet werden. Die Präparationsmethoden wurden daher überwiegend auf diese Strukturen angewandt und optimiert. Diese Arbeit konzentriert sich auf die Umsetzung der zuverlässigsten und vielversprechendsten Oberflächenbehandlungen mittels des ersten vom IAP entwickelten SC 360MHz CH-Prototyps. Diese Kavität wies nach 11 Jahren Lagerung eine verminderte Leistung auf, welche mit Röntgenstrahlung bei bereits niedrigen elektrischen Feldern einherging. Dies deutet auf eine unbeabsichtigte Belüftung mit normaler Luft hin, durch die Partikel eingeführt wurden, die als verstärkende Quellen von Elektronen fungierten. Außerdem musste der Leistungskoppler aufgrund einer starken Überkopplung neu ausgelegt werden.
Die Kavität wurde für 48 Stunden bei 120◦ C mittels Heizbändern in der Experimentierhalle des IAP’s ausgeheizt, was zu einer Verbesserung des Qualitätsfaktors bei niedrigen Werten und zu einer Verkürzung der für die Konditionierung von Multipacting-Barrieren erforderlichen Zeit führte. Allerdings wurde durch diese Behandlung das maximale erreichbare elektrische Feld weiter verringert. Die Verbesserung der Güte ist auf das Ausgasen der Kohlenwasserstoffe während des Backvorgangs zurückzuführen. Die negative Auswirkung auf das maximale elektrische Feld ist weniger auf das Backen selbst zurückzuführen als auf den Transport der Kavität und die verwendeten Vakuumkomponenten, die in der Versuchshalle gelagert sind.
Die beobachtete Leistungseinschränkung lässt sich hauptsächlich durch Partikel im Inneren des Resonators erklären, da Feldemission bei niedrigen Feldstärken auftrat. Eine Hochdruckspülung mit ultrareinem Wasser (HPR) ist das Standardverfahren, um nach Behandlungen, bei denen das Risiko einer Oberflächenkontamination besteht, eine hohe Reinheit der inneren Oberflächen zu erreichen. Die HPR wurde in Zusammenarbeit mit dem Helmholtz-Institut-Mainz und der Gesellschaft für Schwerionenforschung geplant und durchgeführt. Der Resonator zeigte bereits während der Messung der Q-E-Kurve eine Zunahme der transmittierten Leistung bei konstanter Vorwärtsleistung, was vor der HPR nicht der Fall war. Bei der CW-HF-Konditionierung zeigte die Kavität den höchsten Gradienten bei einem deutlich schwächeren Q-Abfall bei hohen Feldstärken.
Sowohl bei der Messung von 2008 als auch bei der beschriebenen Messung wurde die Kavität mit einer HPR-Behandlung fertiggestellt, aber für die HPR-Behandlung bei HIM in Mainz wurden einige Anpassungen vorbereitet. Der CH Prototyp verfügt über keine zusätzlichen Spülports und wurde daher mit zwei verschiedenen Düsen mit unterschiedlichen Sprühwinkeln gespült, um die erreichbaren inneren Resonatorflächen zu maximieren. Die Verwendung mehrerer Sprühwinkel könnte auch für CH-Kavitäten mit Spülöffnungen von Vorteil sein und sollte für zukünftige HPR-Anwendungen in Betracht gezogen werden.
Die Heliumbehandlung wurde am CH-Prototyp 2,5 Stunden lang durchgeführt und lieferte vielversprechende Ergebnisse in Bezug auf die Güte und die Gradientenoptimierung.
Während dieses Prozesses wurde die emittierte Röntgenstrahlung in Richtung am Arbeitsplatzs gemessen und zeigte starke zeitabhängige Fluktuationen. Dies deutete auf die Beseitigung von Partikeln hin und wurde anschließend durch einen Anstieg des elektrischen Feldes von 8,4 auf 8,7 MV/m bestätigt. Eine unerwartete Auswirkung wurde bei der Q-Steigung im mittleren bis hohen Feld festgestellt, bei der der Qualitätsfaktor im Vergleich zum HF-konditionierten Fall eine Erhöhung von 5% oberhalb von 2MV/m aufwies. Dieser systematische Anstieg wurde für diesen Beschleuniger vor der Behandlung bisher nicht beobachtet. Stickstoffgedopte Kavitäten zeigen ein ähnliches Verhalten, bei dem Wechselwirkungen innerhalb der Oxidschicht mit Änderungen der Qualitätsfaktoren korreliert sind. Da Helium ein nicht reaktives Element ist, sind mögliche Erklärungen für diesen Effekt der Sputterprozess und die Einlagerung von Helium innerhalb der Oberfläche. Eine Serie von Heliumbehandlungen ist geplant, um ein optimiertes und sicheres Rezept für CH-Kavitäten zu finden. Die Q-E-Messung nach der Abkühlung und vor der Behandlung wird auch zeigen, ob der Leistungsgewinn durch ein Aufwärmen auf Raumtemperatur beeinträchtigt wird.
Die in dieser Arbeit skizzierte Behandlungssequenz wird für CH-Kavitäten dringend empfohlen. Das Ausheizen hat sich bei der Verringerung des Multipactings and der Güteabnahme bei hohen Feldern als wirksam erwiesen und bleibt von der anschließenden HPR unbeeinflusst. In dieser Arbeit wurden keine negativen Auswirkungen der HPR auf das Multipactingverhalten festgestellt. Anschließend wird eine CW-HF-Konditionierung durchgeführt, bis keine weitere Leistungszunahme der Kavität mehr zu verzeichnen ist.
Wenn die Kavität immer noch durch Feldemission begrenzt ist, sollte eine Wiederholung der HPR-Behandlung in Betracht gezogen werden, da bei sorgfältiger Durchführung der HPR keine der bisherig gefertigten CH-Kavitäten hierdurch begrenzt war. Es ist auch anzumerken, dass die Heliumbehandlung nur an der 360MHz CH-Kavität durchgeführt wurde, als diese eine geringe Strahlung durch Feldemission aufwies. Das Risiko des Heliumprocessing an CH-Kavitäten unter starker Feldemission ist unbekannt. Es ist zu erwarten, dass die Elektronenströme und damit die Ionenbeschusslawinen zunehmen und ein größeres Risiko für die Beschädigung von der Komponenten darstellen. Nach dem derzeitigen Kenntnisstand sollte die Heliumbehandlung nur für gut vorbereitete Kavitäten mit minimaler Feldemission in Betracht gezogen werden.
The strong force is one of the four fundamental interactions, and the theory of it is called Quantum Chromodynamics (QCD). A many-body system of strongly interacting particles (QCD matter) can exist in different phases depending on temperature (T) and baryonic chemical potential (µB). The phases and transitions between them can be visualized as µB−T phase diagram. Extraction of the properties of the QCD matter, such as compressibility, viscosity and various susceptibilities, and its Equation of State (EoS) is an important aspect of the QCD matter study. In the region of near-zero baryonic chemical potential and low temperatures the QCD matter degrees of freedom are hadrons, in which quarks and gluons are confined, while at higher temperatures partonic (quarks and gluons) degrees of freedom dominate. This partonic (deconfined) state is called quark-gluon plasma (QGP) and is intensively studied at CERN and BNL. According to lattice QCD calculations at µB=0 the transition to QGP is smooth (cross-over) and takes place at T≈156 MeV. The region of the QCD phase diagram, where matter is compressed to densities of a few times normal nuclear density (µB of several hundreds MeV), is not accessible for the current lattice QCD calculations, and is a subject of intensive research. Some phenomenological models predict a first order phase transition between hadronic and partonic phases in the region of T≲100 MeV and µB≳500 MeV. Search for signs of a possible phase transition and a critical point or clarifying whether the smooth cross-over is continuing in this region are the main goals of the near future explorations of the QCD phase diagram.
In the laboratory a scan of the QCD phase diagram can be performed via heavy-ion collisions. The region of the QCD phase diagram at T≳150 MeV and µB≈0 is accessible in collisions at LHC energies (√sNN of several TeV), while the region of T≲100 MeV and µB≳500 MeV can be studied with collisions at √sNN of a few GeV. The QCD matter created in the overlap region of colliding nuclei (fireball) is rapidly expanding during the collision evolution. In the fireball there are strong temperature and pressure gradients, extreme electromagnetic fields and an exchange of angular momentum and spin between the system constituents. These effects result in various collective phenomena. Pressure gradients and the scattering of particles, together with the initial spatial anisotropy of the density distribution in the fireball, form an anisotropic flow - a momentum (azimuthal) anisotropy in the emission of produced particles. The correlation of particle spin with the angular momentum of colliding nuclei leads to a global polarization of particles. A strong initial magnetic field in the fireball results in a charge dependence and particle-antiparticle difference of flow and polarization.
Anisotropic flow is quantified by the coefficients vₙ from a Fourier decomposition of the azimuthal angle distribution of emitted particles relative to the reaction plane spanned by beam axis and impact parameter direction. The first harmonic coefficient v₁ quantifies the directed flow - preferential particle emission either along or opposite to the impact parameter direction. The v₁ is driven by pressure gradients in the fireball and thus probes the compressibility of the QCD matter. The change of the sign of v₁ at √sNN of several GeV is attributed to a softening of the EoS during the expansion, and thus can be an evidence of the first order phase transition. The global polarization coefficient PH is an average value of the hyperon’s spin projection on the direction of the angular momentum of the colliding system. It probes the dynamics of the QCD matter, such as vorticity, and can shed light on the mechanism of orbital momentum transfer into the spin of produced particles.
In collisions at √sNN of several GeV, which probe the region of the QCD phase diagram at T≲100 MeV and µB≳500 MeV, hadron production is dominated by u and d quarks. Hadrons with strange quarks are produced near the threshold, what makes their yields and dynamics sensitive to the density of the fireball. Thus measurement of flow and polarization, in particular of (multi-)strange particles, provides experimental constraints on the EoS, that allows to extract transport coefficients of the QCD matter from comparison of data with theoretical model calculations of heavy-ion collisions.
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Chronische Entzündungen und die daraus resultierenden Morbiditäten gehören zu den häufigsten Ursachen für einen frühen Tod beim Menschen. Einer der Hauptfaktoren für die Verschlechterung des Gesundheitszustands bei Patienten mit chronischen-entzündlichen Erkrankungen ist die pathologische Infiltration von Leukozyten in gesundes Gewebe, die zu Gewebeschäden und dem Fortschreiten der Krankheit führt. Das vaskuläre Endothel, das die Innenseite der Blutgefäße auskleidet, spielt eine entscheidende Rolle bei der Entzündungsreaktion, da es als Schnittstelle für die Interaktion mit Leukozyten fungiert, um die Extravasation von Leukozyten aus dem Blutstrom in das Gewebe zu ermöglichen. Die Adhäsion von Leukozyten an die Zellen des Endothels wird dabei hauptsächlich durch die von Zytokinen ausgelösten pro-inflammatorischen NFκB- und AP-1-Signalkaskaden ermöglicht, die die Hochregulierung der wichtigsten endothelialen Adhäsionsmoleküle – ICAM-1, VCAM-1 und E-Selektin – bewirken. Eine Klasse von Wirkstoffen, die für ihre entzündungshemmenden Eigenschaften und ihren Nutzen bei der Behandlung chronischer Entzündungskrankheiten bekannt sind, sind die Mikrotubuli-bindenden-Substanzen (microtubule-targeting-agents; MTAs), die nachweislich auch den Entzündungszustand in den Zellen des Endothels und die Leukozyten-Adhäsionskaskade beeinflussen können. MTAs lassen sich in Mikrotubuli-Destabilisatoren, die eine Depolymerisation des Mikrotubuli-Zytoskeletts bewirken, und Mikrotubuli-Stabilisatoren, die die Depolymerisation der Mikrotubuli verhindern, unterteilen. Die zugrundeliegenden biomolekularen Vorgänge und Wirkungen, die die MTAs auf die Zellen des Gefäßendothels haben, und wie sie die Adhäsionskaskade der Leukozyten beeinflussen, sind jedoch weitgehend unbekannt.
Ziel dieser Studie war es, die Auswirkungen des neuartigen Mikrotubuli-Destabilisators Prätubulysin, eines Vorläufers der Tubulysine, die ursprünglich in Stämmen des Myxobakteriums Angiococcus disciformis entdeckt wurden, auf die entzündlichen Prozesse zu untersuchen, die die Leukozyten-adhäsion in TNF-aktivierten primären Endothelzellen aus der menschlichen Nabelschnurvene (HUVECs) ermöglichen. Zusätzlich wurden auch die Auswirkungen der bereits klinisch etablierten Mikrotubuli-Destabilisatoren Colchicin und Vincristin sowie des Mikrotubuli-Stabilisators Paclitaxel untersucht.
Das entzündungshemmende Potenzial von Prätubulysin wurde daher zunächst in vivo in einem Imiquimod-induzierten psoriasiformen Dermatitis-Mausmodell getestet, wobei sich zeigte, dass Prätubulysin den Entzündungszustand deutlich verringert. Um zu beweisen, dass der entzündungshemmende Effekt mit einer verringerten Interaktion von Leukozyten mit dem Endothel zusammenhängt, wurde die Wirkung von Prätubulysin in vivo mittels Intravitalmikroskopie des TNF-aktivierten Kremaster-Muskels der Maus untersucht. Dabei zeigte sich, dass die Behandlung mit Prätubulysin zu einer signifikant verringerten Adhäsion von Leukozyten an die Zellen des Gefäßendothels führte. Die verringerte Adhäsion von Leukozyten an Endothelzellen wurde auch in der in vitro Umgebung bestätigt, indem die Adhäsion von Leukozyten unter Flussbedingungen getestet wurde. Mittels Durchflusszytometrie, Western-Blot-Analyse, sowie qRT-PCR-Analyse der jeweiligen mRNA-Level konnte gezeigt werden, dass die verringerten Leukozyten-Interaktionen auf der verringerten Expression der Zelladhäsionsmoleküle ICAM-1 und VCAM-1 sowie teilweise von E-Selektin nach Behandlung mit Prätubulysin, Vincristin und Colchicin beruhen, wobei Paclitaxel keine signifikanten hemmenden Auswirkungen hatte. Weitere Untersuchungen des Einflusses von Prätubulysin auf die NFκB- und AP-1-Signalübertragung zeigten, dass diese intrazellulären Signalkaskaden durch Prätubulysin nicht behindert werden, wobei NFκB und AP-1 weitgehend in den Promotoren der Zelladhäsionsmoleküle angereichert waren, wie durch Chromatin-Immunpräzipitation nachgewiesen wurde. Darüber hinaus induzierte die Behandlung mit Prätubulysin die Aktivität der NFκB-induzierenden Kinase IKK und führte zu einem signifikanten Anstieg der Aktivität der AP-1 Upstream-Kinase JNK, wie eine Western Blot Analyse ergab. Die Prüfung der Transkriptionsaktivität von NFκB und AP-1 in Reportergen Assays zeigte, dass insbesondere die Mikrotubuli-Destabilisatoren die Promotoraktivität dieser Transkriptionsfaktoren in einer konzentrationsabhängigen Weise verringerten. Weitere Tests zur Abhängigkeit der durch Prätubulysin induzierten Hemmung der Zelladhäsionsmoleküle von der Aktivität der JNK zeigten, dass die Hemmung empfindlich auf die Aktivität dieser Kinase reagiert. Es konnte gezeigt werden, dass die Inhibition der Aktivität der JNK die Expression der Zelladhäsionsmoleküle durch die Behandlung mit Prätubulysin auf mRNA und Proteinebene wiederherstellt. Mit Hilfe der Chromatin-Immunpräzipitation konnte weiterhin gezeigt werden, dass die Behandlung mit Prätubulysin zunächst die Assoziation des Bromodomänen-enthaltenden Proteins 4 mit den Promotoren/Genen von ICAM-1 und VCAM-1 erhöhte, aber zu einem behandlungszeitabhängigen Rückgang der Anreicherung führte. Darüber hinaus wurde durch die Behandlung mit Prätubulysin auch der Abbau dieses Proteins leicht erhöht. Durch den Einsatz eines JNK Inhibitors konnte gezeigt werden, dass die Verdrängung des Bromodomänen-enthaltenden Proteins 4 von icam-1 und vcam-1, sowie der erhöhte Abbau dieses Faktors auch von der Aktivität der JNK abhängig sind. Die Verdrängung des Bromodomänen-enthaltenden Proteins 4 induzierte auch das Vorhandensein von repressiven Chromatinmarkierungen in den Genen von ICAM-1 und VCAM-1. Die Prüfung der Anreicherung der RNA-Polymerase II an den Promotoren/Genen von ICAM-1 und VCAM-1 zeigte jedoch auch eine behandlungszeitabhängige differentielle Anreicherung dieser Polymerase, wobei die Anreicherung nach kurzen Behandlungszeiten reduziert war, sich nach mittleren Behandlungszeiten erholte und nach längeren Behandlungszeiten wieder stark reduziert war. Die anschließende Prüfung der Bedeutung des Bromodomänen-enthaltenden Proteins 4 für die Expression von ICAM-1 und VCAM-1 durch Knock-down-Experimente ergab, dass das vcam-1 Gen durch Knock-down dieses Proteins unterdrückt, das icam-1 Gen jedoch induziert wird. Dies deutet auf das Vorhandensein zusätzlicher Faktoren hin, die auch auf die Aktivität der JNK reagieren und neben dem Bromodomänen-enthaltenden Proteins 4 die Transkriptionsverlängerung des icam-1 Gens bewirken.
Inflammation is a regulated reaction of the body to control a threat such as infection or injury. An efficient resolution of inflammation is critical to prevent the development of chronic inflammation and to restore tissue homeostasis. Macrophages (Mf) play a crucial role in the onset, but also in the resolution of inflammation, because they phagocytose and eliminate pathogens and tissue debris. Efficient efferocytosis, i.e. the engulfment of apoptotic cells, represents an important trigger for the onset of the resolution response and contributes to the pro-resolving reprogramming of Mf. Despite the importance of post- transcriptional modes of regulation during the resolution phase and translational control as a key node modulating gene expression in immune cells, relevant translational alterations remain largely elusive.
In the present study, I aimed to identify translationally regulated targets in inflammatory primary murine Mf upon resolution-promoting efferocytosis. To this end, I used total RNA-sequencing as well as de novo proteomics analyses to determine global transcriptional and translational changes. Sequencing data confirmed that efferocytosis induced a pro-resolution signature in inflammatory Mf and pointed towards translational regulation because the related integrated stress response was enriched upon efferocytosis. While changes of gene expression between efferocytic and non-efferocytic Mf appeared rather small at the transcriptional level, I observed considerable differences at the level of de novo synthesized proteins. This finding suggests a regulation at the level of translation. Furthermore, the tight connection between translational and metabolic changes was confirmed by enriched metabolism-associated terms of targets upregulated by efferocytosis at both RNA and de novo protein level. Interestingly, analysis of translationally regulated targets in response to inflammatory stimulation showed reduced translation for most targets, with only little impact of efferocytosis. Among those targets, I identified pro-resolving matrix metallopeptidase 12 (Mmp12) as a novel candidate, which showed translational repression during early inflammation and translational increase during the resolution phase. Noteworthy, a first indicator for a potential translation regulatory component of Mmp12 were the extremely high mRNA levels and not overly high de novo protein levels. Validation experiments recapitulated a slight elevation of Mmp12 mRNA expression and a significant downregulation of MMP12 intracellular protein levels in inflammatory Mf, as observed in the RNA-seq and de novo proteomics datasets. To investigate whether the discrepancy in mRNA and protein expression were due to changes in translation, I applied polysomal fractionation analysis to determine the translational status of Mmp12. Inflammatory Mf displayed a significantly lower relative Mmp12 mRNA abundance in the late polysomes compared to naïve Mf, suggesting reduced translational efficiency upon inflammatory stimulation. Consequently, extracellular MMP12 levels in the supernatant of inflammatory Mf decreased, although with a slight delay.
The functional impact of attenuated Mmp12 translation upon inflammatory stimulation was assessed in migration assays. While siRNA-mediated knockdown of Mmp12 did not alter Mf migration on uncoated plates, it increased migration 3-fold on matrigel/elastin-coated plates. Importantly, the increase in migrated distance driven by siMmp12 could be lowered by the addition of exogenous recombinant MMP12 protein. In line with reduced Mmp12 translation and MMP12 protein in inflammatory Mf, I observed a significant increase in cell migration on matrigel/elastin-coated plates, while it remained unaltered on uncoated plates. Consequently, Mf elastase MMP12 degrades elastin, thereby cell migration along elastin fibers is diminished. In inflammatory Mf, Mmp12 is translationally downregulated, thereby enhancing the migratory capacity.
In summary, the present study identifies a substantial contribution of translational regulation in the course of inflammation shown by high changes between inflammatory naïve and efferocytic Mf at the de novo proteomic level. Specifically, I was able to determine the translational regulation of pro-resolving Mmp12, which is repressed during early inflammation and recovers during the resolution phase. Functionally, translational control of MMP12 emerged as a strategy to alter the migratory properties of Mf, enabling enhanced, matrix- dependent migration of Mf during the early inflammatory phase, while restricting migration during the resolution phase.
Research on the human and animal microbiome has become increasingly important in recent years. It is now widely accepted the gut microbiome is of crucial importance to health, as it is involved in a large number of physiological processes. The term ‘microbiome’ refers to the all living microorganisms including their genes and metabolites in a defined environment, while the specific composition of microorganisms consisting of bacteria, archaea and protozoa is referred to as the ‘microbiota’ (Lane-Petter, 1962; Lederberg and McCray, 2001).
In recent years, research has focused on various of these communities in the soil (Fierer, 2017), water (Sunagawa et al., 2015), air (Leung et al., 2014) and especially in the human gut. However, this topic is also becoming increasingly relevant for the conservation of endangered species. In the face of global mass extinctions and the listing of over 42,000 animal species as ‘critically endangered’, conservation breeding programmes are more important than ever (Díaz et al., 2019; IUCN, 2022). The responsibility for these tasks lies with zoological institutions, which are dedicated to animal conservation and the continuous monitoring of animal welfare. Microbiome research offers a non-invasive method to support species conservation. By analysing faecal samples, microbial markers can be identified that provide important information about the health status and reproductive cycle of animals (Weingrill et al., 2004; Antwis et al., 2019). Zoological facilities also provide an ideal research environment for comparing individuals from different habitats. In addition, all necessary metadata such as age, sex, kinship or medical treatment are documented and can be used for the analysis.
This is the starting point for this thesis. In order to identify such microbial markers, it is necessary to understand the microbiome of a variety of animal species. The first aim is therefore to characterise the faecal microbiota of 31 mammalian species, focusing on herbivores and carnivores. It could be shown that they differ significantly in terms of both microbial diversity and microbiota composition. Herbivorous species express a very diverse microbial composition, consisting mainly of cellulose-degrading taxa of the families Fibrobacteraceae or Spirochaetaceae. In contrast, the microbiota of carnivorous species is less diverse and is dominated by protein-degrading Fusobacteriaceae and Clostridiaceae. In addition, this thesis proves that the microbiota of herbivorous species is highly consistent, whereas the microbiota of carnivorous species is highly variable. The results of this study provide important insights for the sampling scheme of future projects. Especially when analysing carnivorous species, single samples are not sufficient to capture the full variability of the microbiome.
These results lead to the question of whether this variability can be explained by daily fluctuations in the individual microbiome and whether this can be used to distinguish between species or individuals. Using individual longitudinal data and a combined approach of clustering algorithms and dynamic time warping, it is shown that such a distinction is possible at the species and individual level. This was confirmed for both a carnivorous (Panthera tigris) and a herbivorous (Connochaetes taurinus) species. These results confirm the influence of the host individual on the faecal microbiota, in addition to the often described influence of diet (Ley et al., 2008a; Kartzinel et al., 2019).
Based on the knowledge gained from these studies, a methodology has been developed that will enable the conservation of species in the field to be supported by microbiome research in the future. The focus here lays on the identification of host-specific metadata based on the faecal microbiota. The developed regression model is able to distinguish between carnivorous, herbivorous and omnivorous hosts with up to 99% accuracy. In addition, a more accurate phylogenetic classification of the family (Canidae, Felidae, Ursidae, Herpestidae) can be made for carnivorous hosts. For herbivorous hosts, the model can predict the respective digestive system with up to 100% accuracy, distinguishing between ruminants, hindgut fermenters and a simple digestive system. The acquisition of host-specific metadata from an unknown faecal sample is an important step towards establishing microbiome research in species conservation. Field studies in particular will benefit from such new methods. Usually, costly microsatellite analysis and high-quality host DNA are required to obtain host-specific information from faecal samples. The newly developed method offers a less costly and labour-intensive alternative to conventional techniques and opens up a more accessible field for microbiome research in the field.
Fluorescence microscopy has significantly impacted our understanding of cell biology. The extension of diffraction-unlimited super-resolution microscopy opened an observation window that allows for the scrutiny of cellular organization at a molecular level. The non-invasive nature of visible light in super-resolution microscopy methods renders them suitable for observations in living cells and organisms. Building upon these advancements, a promising synergy between super-resolution fluorescence microscopy and deep learning becomes evident, extending the capabilities of the imaging methods. Tasks such as image modality translation, restoration, single-molecule fitting, virtual labeling, spectral demixing, and molecular counting, are enabled with high precision. The techniques explored in this thesis address three critical facets in advanced microscopy, namely the reduction in image acquisition time, saving photon budget during measurement, and increasing the multiplexing capability. Furthermore, descriptors of protein distributions and their motion on cell membranes were developed.
Autophagy is an important degradation pathway mediating the engulfment of cellular material (cargo) into autophagosomes followed by degradation in autophagosomes.
Different stress stimuli, e.g. nutrient deprivation, oxidative stress or organelle damage, engage autophagy to maintain cellular homeostasis, recycle nutrients or remove damaged cell organelles. Autophagy not only degrades bulk cytoplasmic material but also selective autophagic cargo, for example lysosomes (lysophagy), mitochondria (mitophagy), ER (ER-phagy), lipid droplets (lipophagy), protein aggregates (aggrephagy) or pathogens (xenophagy). Selective autophagy pathways are regulated by selective autophagy receptors which bind to ubiquitinated cargo proteins and link them to LC3 on the autophagosomal membrane.
Ubiquitination is an essential post-translational modification controlling different cellular processes such as proteasomal and lysosomal degradation or innate immune signaling.
M1-linked (linear) poly-Ubiquitin (poly-Ub) chains are exclusively assembled by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the M1 poly-Ub-specific OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN). In addition to key functions in innate immune signaling and nuclear factor-κB (NF-κB) activation, M1 ubiquitination is also implicated in the regulation of autophagy.
LUBAC and OTULIN control autophagy initiation and maturation and the autophagic clearance of invading bacteria via xenophagy. However, additional functions of LUBAC- and OTULIN-regulated M1 ubiquitination in autophagy are largely unknown and it also remains unexplored if LUBAC and OTULIN control other selective autophagy pathways in addition to xenophagy. This study aimed to unravel the role of LUBAC- and OTULIN-controlled M1 ubiquitination in bulk and selective autophagy in more detail.
In this study, characterization of OTULIN-depleted MZ-54 glioblastoma (GBM) cells revealed that OTULIN deficiency results in enhanced LC3 lipidation in response to autophagy induction and upon blockade of late stage autophagy with Bafilomycin A1 (BafA1). Furthermore, electron microscopy analysis showed that OTULIN-deficient cells have an increased number of degradative compartments (DGCs), confirming enhanced autophagy activity upon loss of OTULIN. APEX2-based autophagosome content profiling identified various OTULIN-dependent autophagy cargo proteins. Among these were the autophagy receptor TAX1BP1 which regulates different forms of selective autophagy (e.g. lysophagy, aggrephagy) and the glycan-binding protein galectin-3 which serves key functions in lysophagy, suggesting a role of OTULIN and M1 poly-Ub in the regulation of aggrephagy and lysophagy.
Abstract 2
To study aggrephagy, protein aggregation was induced with puromycin which causes premature termination of translation and accumulation of defective ribosomal products (DRiPs). Loss of OTULIN increased the number of M1 poly-Ub-positive foci and insoluble proteins and reduced the levels of soluble TAX1BP1 and p62 in response to puromycin-induced proteotoxic stress.
Intriguingly, upon induction of lysosomal membrane permeabilization (LMP) with the lysosomotropic drug L-Leucyl-L-Leucine methyl ester (LLOMe), M1 poly-Ub strongly accumulated at damaged lysosomes and colocalized with TAX1BP1- and galectin-3-positive puncta. M1 poly-Ub-modified lysosomes formed a platform for NF-κB essential modulator (NEMO) and inhibitor of κB (IκB) kinase (IKK) complex recruitment and local NF-κB activation in a K63 poly-Ub- and OTULIN-dependent manner. Furthermore, inhibition of lysosomal degradation enhanced LLOMe-induced cell death, suggesting pro-survival functions of lysophagy following LMP. Enrichment of M1 poly-Ub at damaged lysosomes was also observed in human dopaminergic neurons and in primary mouse embryonic cortical neurons, confirming the importance of M1 poly-Ub in the response to lysosomal damage.
Together, these results identify OTULIN as a negative regulator of autophagy induction and the autophagic flux and reveal OTULIN-dependent autophagy cargo proteins.
Furthermore, this study uncovers novel and important roles of M1 poly-Ub in the response to lysosomal damage and local NF-κB activation at damaged lysosomes.