Die Konflikte in den Verhältnissen zwischen der Führung der organisierten deutschen Arbeiterbewegung und Marx/Engels im Zeitraum von 1847 bis 1852 bilden den Gegenstand der vorliegenden Dissertation. Den fundamentalen Konflikt zwischen der Arbeiterführung und Marx/Engels sieht die Arbeit in der Frage, ob Kommunisten und arbeitende Klassen in der Revolution von 1848/1849 zum Anhängsel des Bürgertums werden oder ob sie eine unabhängige und selbstständig handelnde Partei bleiben und für ihre eigenen Interessen und Bedürfnisse kämpfen müssten. Während die Arbeiterführung, wie Weitling, Gottschalk und Willich, sowohl gegen den Feudalismus als auch gegen die Bourgeoisie antrat und nicht für die Interessen der bürgerlichen Klasse, sondern für die Interessen der arbeitenden Klassen und Besitzlosen zu kämpfen bezweckte, versuchten Marx/Engels die Kommunisten zur Unterstützung der Bourgeoisie zu überreden. Dieser Konflikt war also der Ausgangspunkt aller Konflikte und spielte im andauernden Kampf zwischen der Arbeiterführung und Marx/Engels immer wieder die ausschlaggebende Rolle. Der intensive Kampf zwischen der Führung der deutschen Arbeiterbewegung und Marx/Engels führte dann schließlich im September 1850 zum Ausschluss von Marx/Engels aus dem Bund der Kommunisten. Die von Marx/Engels selbst aufgestellte Behauptung, sie hätten die Führung des Bundes der Gerechtigkeit für ihren „wissenschaftlichen“ Kommunismus gewonnen und so hätte eine Verschmelzung zwischen ihrer Theorie und der Arbeiterführung stattgefunden, widerlegt daher die vorliegende Dissertation grundlegend. Sie zeigt, dass die Theorie der Arbeiterführung, nämlich der revolutionäre Kommunismus im Zeitraum von 1847-1852, also in einem „Knotenpunkt“ der Geschichte, eine konkrete Macht unter den arbeitenden Klassen und Besitzlosen und gerade deswegen ein „Schreckgespenst“ für die herrschenden Klassen war, während der „wissenschaftliche“ Kommunismus von Marx/Engels lediglich ein Projekt war, das gegen den „wirklich existierenden Kommunismus“ der Zeit ins Feld geführt wurde, weil dieses Projekt die Bourgeoisie unter dem Deckmantel der Wissenschaft als „höchst revolutionär“ bezeichnet, ihre Herrschaft als historisch notwendig legitimiert und öffentlich ankündigt, gemeinsam mit ihr zu kämpfen.
Weniger anzeigenDiseases of the central nervous system (CNS) are a global health threat to both humans and animals with high mortality rates and frequent occurrence of convalescence, occasionally also including long-term sequela. Thus, the role of the different CNS cell types involved is at the forefront of comprehending the development, function, and diseases of the brain. The CNS cell type glia includes microglia and astrocytes, both are crucial players in the brain and are involved in pathogen defense, innate immune activation, and signaling. Their intimate crosstalk maintains the delicate balance between homeostasis and rapid detection of invading pathogens and has progressively gathered research attention. Therefore, the improvement of methods for the isolation, cultivation, and purification of distinct CNS-derived cell types at a high viability and purity is indispensable in order to study the individual contributions of cellular populations to the proper function of the brain. The objective of this study was to develop an in vitro protocol for the isolation, cultivation, and purification of highly viable and pure microglia and astrocytes from neonatal mice and subsequently investigate their cell type-specific effects as well as their contribution to the CNS immune response and cellular crosstalk. In the first step, a glial isolation protocol was developed by optimizing pre-existing techniques for the in vitro isolation and cultivation of microglia and astrocytes from whole neonatal murine brains. Microglial cell yields were maximized by stimulating with macrophage colony-stimulating factor (M-CSF) and applying multiple microglial harvests derived from the same mixed glial culture. Then, a purification protocol for magnetic-activated cell sorting (MACS®) was improved by using cultivated primary glial cell suspensions instead of directly sorting dissociated single cell suspensions. Thereby, microglia and astrocytes were successfully isolated, cultivated, and MACS-purified at a purity of 99-100%, as confirmed by fluorescence-activated cell sorting (FACS) analysis. Furthermore, glial cells generated with our novel protocol are highly viable (~100%) and show a preserved integrity, as demonstrated by field emission scanning electron microscopy (FESEM). Subsequently, non-purified and MACS-purified microglial and astrocyte monocultures were stimulated with different TLR ligands. Their pro- and anti-inflammatory response was determined by using Tumor necrosis factor (TNF) and Interleukin-10 (IL-10) enzyme-linked immunosorbent assays (ELISA). In fact, these experiments demonstrated a significantly weakened protein response in MACS-purified microglia and astrocytes in contrast to the non-purified glial monocultures, providing the first hint of a microglia-astrocyte crosstalk. In the next step, our hypothesis of an existing crosstalk was reinforced by stimulating TLR2-knockout (TLR2-KO) microglial and astrocyte monocultures with wild-type (WT) supernatants (SN) derived from the respective other glial cell type. Here, a significant TNF protein release by TLR2-KO astrocyte monocultures was observed, which were activated with microglial WT SN in response to Pam3CSK4, in comparison to the untreated control. Interestingly, transcriptome analysis using RNA sequencing (RNA-seq) unraveled a wide array of significantly up- and down-regulated genes in comparison to their untreated controls, which may be candidate mediators of the intimate molecular glial conversation. Finally, co-culture experiments with microglia and astrocytes coming from different genetic backgrounds were performed. Interestingly, a significant TNF protein release by Pam3CSK4-activated WT microglia co-cultured with TLR2-KO astrocytes was detected in comparison to the untreated control, confirming the results from the prior monoculture experiments. In this thesis, we describe a novel protocol to optimize in vitro isolation, cultivation, and purification of neonatal murine primary microglia and astrocytes. With our proposed method, we maximized microglial yields with the big advantage of sacrificing as few mice as possible following the principles of the 3Rs (replacement, reduction, and refinement). By using ~100% pure glial mono-/co-cultures derived from mice with different genotypes, we were able to analyze the microglia-astrocyte crosstalk on a cell-specific level. Moreover, unlike previous protocols, we showed that the immune response during the crosstalk underlies a TLR2/1-dependent mechanism. Consequently, we postulate our novel protocol to be a suitable and efficient method to investigate cell type-specific effects which contributes to immune modulation as well as cellular crosstalk in future approaches. Thereby, we further extended the possibilities to study glial cells in different experimental issues on brain function as well as CNS infections.
Weniger anzeigenMit der Digitalisierung und dem Aufkommen partizipativer Plattformen haben sich die Bedingungen zur Herstellung medialer Sichtbarkeit verändert. Zugleich hat feministischer Aktivismus mit der Digitalisierung neue Formen angenommen. Feministische Hashtag-Kampagnen oder Initiativen wie Wikipedia-Schreibaktionen für Frauen verfolgen vor allem die Absicht, Sichtbarkeit und Anerkennung für Frauen und/oder Geschlechterungerechtigkeiten in der digitalen Öffentlichkeit einzufordern. In dieser Dissertation stelle ich die übergeordnete Frage: Welche Bedingungen und Folgen haben Sichtbarkeiten innerhalb von Netzwerköffentlichkeiten für feministischen Online-Aktivismus? Frühere Forschung zeigt, dass Sichtbarkeiten und aktivistische Initiativen auf partizipativen Plattformen durchzogen sind von Geschlechterungleichheiten und antifeministischer Gegenmobilisierung. Ungleichheiten in digitalen Öffentlichkeiten wurden bisher vor allem als Ergebnis ungleichen Zugangs zu und ungleicher Nutzung von Informations- und Kommunikationstechnologien erforscht. Zugang und Nutzung reichen aber nicht immer aus, um zu erklären, warum Geschlecht einen Einfluss darauf zu haben scheint, wer oder was tatsächlich öffentlich sichtbar wird. (Un-)Sichtbarkeiten auf partizipativen Plattformen verstehe ich dabei im Sinne des Netzwerk-Gatekeeping-Ansatzes als Ergebnis konnektiver Prozesse des (Un-)Sichtbarmachens und damit als emergente Netzwerkphänomene. Da diese Prozesse auf sozialen Interaktionen basieren, schlage ich theoretisch vor, Sichtbarkeit um Anerkennung und Missachtung als soziale, moralisch-normative Beziehungsformen zu erweitern. Entlang der zwei Dimensionen Aufmerksamkeit und Anerkennung lassen sich Sichtbarkeiten und ihre Folgen für feministischen Online-Aktivismus innerhalb partizipativer Plattformen systematisieren und untersuchen. In der ersten empirischen Studie untersuche ich Unterschiede in der Netzwerksichtbarkeit von verschiedenen Akteurstypen innerhalb des deutschsprachigen #MeToo-Protestes auf Twitter. Auch wenn sich überwiegend private Nutzer*innen beteiligten, erhielten vor allem die Accounts einiger Massenmedien und einzelner Journalist*innen große Sichtbarkeit. Außerdem zeigte sich eine starke Gegenmobilisierung und Abwertung von #MeToo durch antifeministische Akteur*innen, welche die Möglichkeiten zur strategischen Vernetzung intensiver nutzten als Unterstützer*innen des Protests. Die zweite Studie widmet sich der Möglichkeit zur (böswilligen) Beeinflussung solcher Twitter-Diskurse durch die Automatisierung von Accounts. Indem dieselben Twitteraccounts mit drei verschiedenen Erkennungsverfahren auf sogenannte Social Bots getestet werden, zeige und diskutiere ich die Grenzen der methodischen Möglichkeiten, Automatisierung zu erkennen und damit den Einfluss von Social Bots auf Diskurse zu bestimmen. Die dritte Studie untersucht, wie Nutzer*innen in der Wikipedia über die Löschung von Biografie-Artikeln diskutieren. Da hier explizit ausgehandelt und festgeschrieben wird, welches Wissen „relevant“ für die Öffentlichkeit ist, eignet sich das Beispiel gut, um den Zusammenhang zwischen Sichtbarkeit und Anerkennung zu untersuchen. Aus dem Ergebnis, dass Frauenbiografien häufiger als Männerbiografien infrage gestellt werden, folgere ich, dass Sichtbarkeit nicht immer nur ermächtigend wirkt, sondern Geschlechterunterschiede auch aufgrund mangelnder Anerkennung in Form von Kontrolle und Überwachung entstehen.
Weniger anzeigenValvular disease and successful therapy is dependent on a complex interplay of metabolic, hemodynamic / fluid dynamic, neuro‐hormonal and immunologic processes. In order to grasp the mechanism of a disease or evaluate novel therapies, biological systems are needed that are capable of mimicking this fascinating complexity. Due to the closely resembling anatomical landmarks of the valvular apparatus and ventricular cavities as well as intra‐ventricular and intra‐aortic fluid dynamic behavior of blood flow, farm pigs should be considered when there is a need for translational large animal model in the field of heart valve research. The use of 90‐100 kg pigs enables deployment of human grade devices as cardiac dimensions in this weight‐class of animals closely resembles one of adult patients. Further, clinical‐grade echocardiography imaging equipment with minimal adaptations of the application technique can be routinely used for TEE guidance of valvular procedures in experimental projects using this animal species. For navigationally challenging procedures, such as transseptal interventions on the mitral valve, porcine animal model could be humanized by relatively simple surgical intervention. Besides proving researchers with robust, human‐like platform for valvular implant testing and development, porcine models provide exceptional value in investigations of diseases where clinical studies are either impractical, time consuming or unethical. Such being studies of cardiac injury in poly‐trauma or precise hemodynamic effects of valvular regurgitation. In such studies, clinical grade medical equipment (such as standard CT and MRI machines and anesthesia equipment) can routinely be used and novel protocols developed and tested, which can then rapidly be adapted for human use, further closing the translation gap. Finally, experience gathered during preclinical testing and learning curve achieved with translational animal models could and should be used to provide practitioners with a solid starting point when novel valve therapies finally find their way to clinical use. Yet, a certain translational gap still remains.
Weniger anzeigenThe geodynamic processes that gave rise to the first cratons on Earth remain controversial. Whether the first preserved cratons formed through horizontal tectonic processes similar to those operating on Earth today or in a stagnant lid regime dominated by vertical stacking and reworking of crustal material continues to be debated. Central to the question of Eoarchean geodynamics is whether and to what extent material from Earth’s surface was recycled into the magmatic system and mantle. Horizontal tectonic processes similar to those active today would be expected to rework material including sulfur from the Earth’s surface into igneous rocks and the mantle. In contrast, significant reworking of sulfur from the crust into the mantle under proposed vertical tectonic regimes is not expected. Multiple sulfur isotopes provide a valuable window into early Earth processes. They can preserve a record of mass independent fractionation (MIF-S) that took place as a result of photolytic processes in the atmosphere prior to the Great Oxidation Event (GOE). This MIF-S was preserved in sediment and hydrothermal deposits that incorporated sulfur species fractionated in the atmosphere. Large MIF-S signatures are a unique feature of surface deposits formed prior to the GOE, and these signatures are robust enough to survive reworking into the magmatic system. The occurrence of MIF-S in igneous lithologies is therefore an indication that the rocks include material recycled from Earth’s surface in the Archean or before. In this dissertation, the results of multiple sulfur isotope analyses of igneous lithologies from the Eoarchean Itsaq Gneiss Complex (IGC) in southern West Greenland are reported, alongside petrographic observations and additional supporting analyses of the same. Investigated lithologies include tonalite-trondhjemite-granodiorites (TTGs), amphibolites, and peridotites from the IGC. The TTGs were subject to bulk multiple sulfur isotope and petrographic analysis. The majority of measured TTGs were found to contain MIF-S (positive Δ33S values up to +0.30‰), indicating incorporation of surface-derived sulfur dominated by sedimentary material. Elevated Δ36S up to +0.80‰ and δ34S up to +3.36‰ in IGC TTG samples point to additional incorporation of seawater sulfate. The surface-derived material is interpreted to have been incorporated into the TTGs in the context of modern-like arc accretion. Two IGC amphibolites with tholeiite-like compositions were concurrently analyzed for multiple sulfur isotopes. One of the two amphibolites was found to contain positive bulk Δ33S (+0.14‰), similar to other IGC amphibolites reported in the literature. iv These tholeiite-like amphibolites are interpreted to represent the source rocks of the TTGs and to have incorporated sediment-dominated sulfur during subduction in the Eoarchean. Peridotites found in ultramafic enclaves in the IGC were also investigated. These rocks have been well characterized by previous investigations and have been interpreted to represent the oldest remnants of obducted mantle. A mantle origin for these peridotites remains controversial, however, with some researchers arguing that they are ultramafic cumulates. The peridotites were found to contain positive bulk Δ33S values between +0.04‰ and +0.21‰, indicating that they incorporated sulfur dominated by sedimentary material. Trends emerge when plotting the Δ33S and δ34S values of the peridotites against trace and major element concentration data on these rocks from the literature, as well as Hf isotope data published in previous studies. The trends indicate that the peridotites incorporated sediment derived sulfur early in their history, prior to variable melt overprinting that delivered additional sulfur of seawater sulfate origin. The complex history of multiple overprinting events in the Eoarchean is interpreted to have taken place in a mantle wedge. The presence of MIF-S in peridotites originating in the mantle is a significant line of evidence in support of Eoarchean subduction. Additional in-situ sulfur and lead isotope analysis of sulfides in the studied peridotites was conducted by secondary ion mass spectrometry (SIMS), in conjunction with electron microscopy and electron microprobe analysis. The sulfides were predominantly pentlandite and pyrrhotite, typical mantle minerals, and their Δ33S values were consistent with bulk analyses. Amphibole crosscutting the sulfides demonstrates that the sulfides predate amphibolite facies metamorphism the peridotites experienced in the Neoarchean. Highly unradiogenic lead isotope results are consistent with Eoarchean origins followed by partial reequilibration during metamorphism. Because lead is a trace component of the sulfides and sulfur is a major component, this reequilibration is not expected to have influenced Δ33S values. The presence of MIF-S in a majority of studied samples and in representatives of all studied rock types points strongly in the direction of widespread Eoarchean crustal recycling in the IGC. This is consistent with interpretations of Eoarchean geodynamics that include horizontal processes in which plates override one another.
Weniger anzeigenIn this thesis we investigate sign mappings, which for a fixed rank r map subsets of {1,...,n} of size r to one of the two signs + and -, while avoiding sign patterns on induced substructures. Particular focus will be on signotopes and generalized signotopes which originate from pseudohyperplane arrangements and simple drawings. Using those combinatorial encodings for topological objects, we prove classic results in a more general setting.
We consider Levi's extension lemma for pseudoline arrangements and prove that it generalizes to signotopes of odd rank r. Levi showed in 1926 that every pseudoline arrangement can be extended by an additional pseudoline going through two prescribed points. A generalization to dimension 3 fails as Goodman and Pollack (1981) provided an example of pseudoplane arrangements and three prescribed points which is not extendable even though for hyperplane arrangements an extension through d points in dimension d is trivial. Later Richter-Gebert (1993) showed that even an extension through two prescribed points is not possible in dimension 3. We show that signotopes, a subclass of pseudohyperplane arrangements, admit an extension theorem for all even dimensions, that is if the rank r is odd. Moreover, we provide signotopes which are not extendable for rank 4, 6, 8, 10 and 12. Next, we focus on theorems from convex geometry such as Carathéodory's, Helly's and Kirchberger's theorem and study them in the more general setting of simple drawings of the complete graph. In particular we determine in which layer of the convexity hierarchy introduced by Arroyo et al. (2022) the statements hold, and in which layer there are counterexamples. The convexity hierarchy describes several layers between point sets in the plane and simple drawings using a generalized notion of convexity. For the proof of Kirchberger's theorem generalized signotopes, which encode the triangle orientations of simple drawings in the plane, played an essential role. Additionally to the mentioned theorems we introduce the notion of holes in the setting of simple drawings, which are classically considered in point sets. We show that convex drawings behave similarly to point sets in the sense that every sufficiently large convex drawing contains a 6-hole while there are arbitrarily large drawings without 7-holes. Moreover, we show that Rafla's conjecture (1988) is true for convex drawings. The conjecture states that every simple drawing of the complete graph admits a plane Hamiltonian cycle. The best known partial results are plane paths of length $\Omega(log(n)/ log log(n))$ (Suk, Zeng and Aichholzer et al. 2022) and plane matchings of size $\Omega(\sqrt{n})$ (Aichholzer et al. 2022). We investigate several variations and strengthenings of this conjecture. In particular we prove that every convex drawing admits a plane substructure consisting of a plane Hamiltonian cycle and additional n-2 additional edges.
Weniger anzeigenThe land-ocean interface is the most important source or sink for many trace elements in the ocean. Investigating the geochemical behaviour of beryllium (Be) isotopes, encompassing the cosmogenic radionuclide 10Be and the stable nuclide 9Be, and the transfer pathways of Be at this interface is imperative. First, knowing the mechanisms of Be transfer is a prerequisite for the application of the oceanic 10Be/9Be ratio as a sensitive proxy for past continental weathering. Second, understanding the transfer of Be will contribute to our current knowledge of marine biogeochemical cycles of particle-reactive trace metals in general. Third, it helps to expand the potential applications of the 10Be/9Be ratio in coastal marine environments. However, research in this field, particularly on the distribution of 10Be/9Be in coastal seawater, is still lacking due to the analytical challenges in accurately determining ultra-low concentrations of 9Be (at the level of pg/g) and 10Be (at the level of single atoms) in seawater. In this thesis, I propose a new, time-efficient procedure for the simultaneous preconcentration of the exceedingly low levels of 9Be and 10Be from (coastal) seawater based on the iron co-precipitation method. This new procedure contributes towards more time-efficient handling of samples, less risk of sample cross-contamination, and a more accurate 10Be/9Be ratio. I also evaluated the iron co-precipitation method with respect to: i) the impact of major matrix elements on the accuracy of obtained Be concentrations, ii) its extraction efficiency for pg/g-levels Be in the presence and absence of organic matter, and iii) the comparison with existing preconcentration methods. For the determination of 9Be and 10Be in open ocean seawater samples, a precision on the 10Be/9Be ratio of <5% can be attained using less than 3 L of seawater, whereas previously more than 20 L were used to obtain this precision. Even for coastal seawater with extremely low 10Be concentration of only 100 atoms/g, a maximum seawater amount of 10 L is sufficient. I applied this new procedure to measure dissolved 9Be and 10Be along the entire salinity gradient in two of the largest estuaries on Earth: The Changjiang and the Amazon River estuary. In addition, I determined the 10Be and 9Be concentrations in different chemically extractable fractions of the corresponding suspended and bottom sediments. The main objectives were i) to investigate terrigenous 9Be pathways into the ocean under a variety of hydro-chemical estuarine conditions; and ii) to fill the current knowledge gap of the distribution of 10Be/9Be at the land-ocean interface and explore its applications in reconstructing past continental denudation rates of the adjacent river catchments. In both estuaries, dissolved 9Be and 10Be display similar, non-conservative behaviour during estuarine mixing. The removal and release of dissolved 9Be in the Changjiang Estuary and its relationship with corresponding particulate 9Be distribution and hydrochemical data (e.g., dissolved oxygen) suggest three land-to-ocean pathways of 9Be through estuaries. These are: i) riverine dissolved input after coastal scavenging, ii) 9Be desorption from the suspended particulate matter (SPM), and iii) coastal benthic inputs which involve porewater diffusion and/or submarine groundwater discharge. Among these pathways, benthic input potentially presents the most important contributor to the marine 9Be budget and thus likely dominates the paleo-marine 10Be/9Be record. The concentration of dissolved oxygen in coastal bottom seawater may play a role in controlling the benthic 9Be flux through time. Despite the non-conservative behaviour of both dissolved 9Be and 10Be, the 10Be/9Be ratios of dissolved Be exhibit exponential increases along the salinity gradient, generally following the conservative water mixing lines, except where hypoxic bottom waters are affected by submarine groundwater discharge. In contrast, for sediment within the inner-shelf regions (approximately 150 km from the coast), a change in 10Be/9Be ratios of only within a factor of 2 is observed in authigenic (reactive) suspended and bottom sediment phases, indicating that a continent-derived 10Be/9Be signal is largely preserved in this zone. Beyond this zone where boundary currents prevail, reactive 10Be/9Be of outer-shelf/slope sediments markedly increase by a factor of 3 to 30. This increase is likely a result of water-sediment interactions through “boundary exchange” processes. Using the 10Be/9Be ratios of reactive phases of inner-shelf sediments, I calculated the denudation rates for the entire Changjiang and Amazon catchment and compared them with denudation rates derived from in situ cosmogenic beryllium isotopes. The good agreement (within a factor of 2) suggests that in large river-dominated shelves characterized by high sedimentation rates and weakened shoreward diffusion of seawater, the reactive 10Be/9Be ratio of coast-proximal sediments can be a direct recorder of terrigenous denudation of the adjacent river catchments. In summary, with this thesis I demonstrate that the 10Be/9Be ratio in the authigenic phase of open ocean sediments is mainly sensitive to the benthic 9Be flux, while the 10Be/9Be ratio in detrital continental inner-shelf sediments, less affected by “boundary exchange”, can faithfully record the denudation rate of the adjacent river catchments. Thus, both systems are suited to explore past changes of these processes.
Weniger anzeigenDue to advances in sequencing technologies, the amount of sequencing data is continuously increasing and has reached an amount that calls for new data management methods, to actually utilize the sequencing data. In the last years, a number of different indices haven been developed to simplify the data, thereby reducing the amount of space needed and enabling analysis on large collections of sequencing data. In this thesis, the index Needle will be introduced, which allows (semi-)quantitative analyses on large data sets and outperforms other existing solutions with regards to both space and speed. Needle, like other indices, is based on alignment-free methods because in this way the costly step of classical sequence analyses, the alignment, can be omitted. Alignment-free methods are based on short subsequences of the actual sequence data. There are multiple different methods to determine these subsequences and this thesis provides a detailed analysis and comparison to determine the best method for such indices. Moreover, the benchmarking application minions is introduced, which will make comparisons between these methods easier as adding future new methods is simple. Needle is capable of utilizing large collections of sequencing data and determining their gene expressions. Three analyses are performed, which act as a proof of concept for how Needle can be utilized for large collections of sequencing data. Therefore, Needle is applied in this thesis to find cancer signatures, a newly annotated mouse transcript and tissue specific differentially expressed genes for different large data sets. In summary, indices like Needle are needed to actually take advantage of the data wealth currently present in the biological and medical research field
Weniger anzeigenThe knowledge of drug metabolism is fundamental for scientific fields where a comprehensive understanding of steroid metabolism is of high relevance, including but not only limited to anti-doping studies, endocrinology, forensic toxicology, and safety assessment in drug development. This work concentrates on the metabolism of anabolic androgenic steroids (AAS) in both in vivo and in vitro models, with a specific focus on the AAS compounds testosterone (T), metandienone (MD), methyltestosterone (MT), clostebol (CLT), dehydrochloromethyltestosterone (DHCMT), and methylclostebol (CLMT) due to their structural similarities. It introduces new alternative models for studying AAS metabolism and provides valuable knowledge on metabolic properties based on chemical structural characteristics, offering the possibility for the enhancement of AAS detection. Firstly, in order to have a deep understanding of the A-ring reduction in MD, isolated enzyme assays (AKR1C2, AK1C3, ALR1C4, and AKR1D1) were performed. The results obtained substantiate the sequence of A-ring reduction in MD, as previously suggested in the literature [93, 136, 137], i.e., the 4,5-double bond is reduced first, then the 3 oxo group, and finally the 1,2-double bond. Moreover, it appears that AKR1C2, AKR1C3, and AKR1C4 exhibited varying stereoselectivity in catalyzing the 3-oxo reduction in 5α- or 5β-DHMD. AKR1C2 and AKR1C4 showed both 3α- and 3β-HSD activities, whereas AKR1C3 functioned as 3α-HSD only. The sequence of A-ring reduction provided valuable insights for the metabolic pathway analysis for subsequent in vitro studies in human skin cells. The metabolism of T, MT, CLT, and CLMT by keratinocytes and fibroblasts derived from human foreskins produced metabolites with partially or fully reduced A-ring. However, no metabolites of MD or DHCMT could be detected. The metabolite profiles suggest that 3α-HSD, 3β-HSD, and 5α-reductase activities play important roles in steroid metabolism by human keratinocytes and fibroblasts, whereas 17β HSD activity is weak. The stereochemistry of fully reduced metabolites (i.e., 3α,4α,5α-THCLT, 3β,4α,5α-THCLT, 3α,4α,5α-THCLMT, and 3β,4α,5α-THCLMT) of CLT and CLMT was newly identified and confirmed in this study. Differences in the chemical structures of compounds appear to affect A-ring reduction order and cellular metabolic capacities, especially the chlorine group at position 4. Keratinocytes appear to have a higher metabolic capability for compounds containing a chlorine substituent in position 4, whereas the opposite is true in fibroblasts. The medaka embryo model was used for the first time as an alternative model for in vivo studies of AAS. There were four metabolites detected after incubation of medaka embryos with MD, including 6βOH-MD and 5β-DHMD as well as tentatively assigned 18OH-MD and 16OH-MD. These metabolites have also been reported in previous human administration studies [136, 177, 180]. In comparison to the in vitro models, the medaka embryo model allows for simultaneous analysis of biotransformation and potential toxicity. Given the similar outcomes with human administration studies, medaka embryos may serve as an alternative model to identify potential metabolites for human biotransformation of doping-relevant compounds. Investigations on the metabolism of [13C3]-T and MT in the medaka embryo model show that the main metabolic reactions for both two substrates include hydrogenation of the 4,5-double bond and reduction of the 3-oxo function. Additionally, the oxidation of 17β-hydroxy group in [13C3]-T was also observed. The metabolites observed indicate the activities of steroid 5α-reductases, steroid 5β-reductases, 3α-HSD, 3β-HSD, and 17β-HSD in medaka embryos. 3β,5α- and 3α,5β-isomers were detected as the main fully reduced A-ring metabolites in both incubations. However, this study only showed preliminary results, further studies are necessary. In conclusion, appropriate in vivo and in vitro models were evaluated and used for metabolic studies of AAS. The findings presented in this thesis hold significant implications not only for doping control analysis, but also for other scientific areas where a comprehensive understanding of steroid metabolism is highly relevant, such as endocrinology, forensic toxicology, and safety assessment in drug development.
Weniger anzeigenZiel dieser Studie war es, das Applanationstonometer TonoPen Avia Vet für die Tierarten Hund, Katze und Kaninchen mit Hilfe eines Manometers zu kalibrieren. Anhand der Ergebnisse sollte ein Korrekturfaktor bestimmt werden, mit dem der tonometrisch gemessene intraokulare Druck (IOD) in den manometrischen IOD umgerechnet werden kann. Im zweiten Teil der Studie sollten Referenzwerte für den mit dem TonoPen Avia Vet gemessenen IOD für Hunde, Katzen und Kaninchen sowie Referenzwerte für den mit dem TonoVet gemessenen IOD für Kaninchen bestimmt werden. Ein möglicher Einfluss von Alter, Gewicht und Geschlecht auf den IOD sollte untersucht werden. In der Kalibrierungsstudie war ein positiv linearer Zusammenhang zwischen den beiden Messmethoden erkennbar. Der TonoPen Avia Vet unterschätzt bei steigendem Druck zunehmend den manometrisch gemessenen IOD. Die errechneten Korrekturfaktoren lassen eine Umrechnung des tonometrisch gemessenen IOD in den manometrischen IOD zu: Bei Hunden, Katzen und Kaninchen wird der mit dem TonoPen Avia Vet gemessene IOD mit dem Faktor 1,5 multipliziert, um den entsprechenden manometrischen IOD zu berechen. Anhand von Messungen des IOD bei augengesunden Tieren wurden Referenzwerte bestimmt. Untersucht wurden insgesamt 94 Hunde (188 Augen), 64 Katzen (128 Augen) und 122 Kaninchen (244 Augen). Die Referenzwerte wurden anhand des 2,5%- und des 97,5%-Perzentils bestimmt und betrugen für das TonoPen Avia Vet für Hunde 9-18 mmHg, für Katzen 9-20 mmHg und für Kaninchen 6-16 mmHg. Die Referenzwerte betrugen für den mit dem TonoVet gemessenen IOD bei Kaninchen 7-17 mmHg. Das Alter hatte bei allen drei Tierarten einen signifikanten Einfluss auf den IOD. Bei steigendem Alter sinkt der IOD zunehmend ab. Es gab keinen signifikanten Einfluss des Geschlechts oder des Körpergewichts auf den IOD. Insgesamt war der TonoPen Avia Vet und der TonoVet einfach anwendbar, die Messungen waren schnell durchführbar und wurden von den Tieren gut toleriert.
Weniger anzeigenEnzymes are indispensable proteins for all types of organisms, capable of fulfilling various functions by catalyzing diverse chemical reactions. This makes them important tools not only for fundamental research in biochemistry but also for applied research in pharmacology and organic chemistry. However, the recombinant production of enzymes is not trivial, and conventional isolations and expressions from cells are often unsuitable for many applications due to decreased enzyme activity. Cell-free protein synthesis can simplify the synthesis of some enzymes that were previously difficult to produce. Representatives of various enzyme classes were synthesized and subsequently analyzed. Modifications to the established synthesis platforms based on translationally active CHO and Sf21 lysates were made. These modifications allowed for the production of a soluble enzyme, a representative of the hydrolases, GH78 from Xylaria polymorpha. Kinetic analyses after synthesis optimization confirmed the kinetic similarity to the naturally synthesized enzyme. In particular, temperature and synthesis condition adjustments, as well as DNA template modifications, were found to increase enzyme activity. Efforts to adapt a cell-free synthesis platform for the production of further active fungal enzymes, mainly comprising unspecific peroxygenases in active CHO lysates have not yet yielded successful results. However, progress has been made in laying the foundation for future advancements, including the generation of templates, successful synthesis, and the establishment of activity assays. To provide a mammalian enzyme alternative to the unspecific peroxygenases, difficult-to-express monooxygenases comprising members of the cytochrome P450 enzyme family (CYP), the membrane-bound enzymes CYP1A2, CYP2B6, and CYP3A4 from Homo Sapiens, were produced in a eukaryotic translationally active cell lysate. By genetically modifying the CHO cells used for lysate production, an increased amount of CPR, the coenzyme of CYPs, was integrated into the endogenous membrane vesicles of the lysate, thus increasing the activity of the cell-free produced CYPs. Further adaptations of the synthesis, such as synthesis temperature and supplementation with heme, led to further increases in CYP activity in the endogenous membrane vesicles. This allowed for exemplary screening experiments with known CYP substrates.
Weniger anzeigenDie degenerativen Augenerkrankungen, wie die diabetische Retinopathie, das Glaukom oder die Retinitis pigmentosa sind Gegenstand der Forschung, sodass immer mehr Studien zu möglichen Therapien vorliegen. Der neurotrophe Faktor BDNF ist ein vielversprechender Wachstumsfaktor, der seine Wirkung über den Tyrosinkinaserezeptor B (TrkB) entfaltet und der viele kritische Funktionen im Zentralnervensystem (ZNS) erfüllt. Die vorliegende Arbeit beabsichtigt einerseits durch den Einsatz eines bereits publizierten Aptamers den neuroprotektiven Effekt von BDNF zu imitieren sowie andererseits die unerwünschten Nebeneffekte zu vermeiden. Zunächst erfolgte dazu Ausschließung der Bedenklichkeit des Aptamers sowohl an retinalen Zellen als auch in Retinaexplantaten. In einem weiteren Schritt wurde die Verweildauer sowie die Bindungseffizienz des TrkB-Aptamers an primären dissoziierten retinalen Zellen sowie an Retinaexplantaten bestätigt. Ebenfalls wurde nicht nur nachgewiesen, dass das TrkB-Aptamer den BDNF-Signalweg aktiviert, sondern auch, dass es spezifisch bindet. Nachdem die Spezifität des TrkB-Aptamers bestätigt wurde, erfolgte eine Langzeitinkubation über mehrere Tage. Daraus resultierte, dass bei der langen Kultivierung mit dem TrkB-Aptamer ein neuroprotektiver Effekt entsteht. Anschließend wurde der neuroprotektive Effekt des TrkB-Aptamers in einem retinalen bereits etablierten Kobaltchlorid (CoCl2) Schädigungsmodell bestätigt.Mit der Gewinnung der neuen Forschungsergebnisse besteht die Hoffnung, einen wesentlichen Beitrag zur Entwicklung neuer therapeutischen Möglichkeiten bei den degenerativen Retinaerkrankungen geleistet zu haben.
Weniger anzeigenHCC is a leading cause of cancer related mortality globally and most patients are not diagnosed with this devastating disease until advanced stage. Systemic treatment represents the standard of care for these patients, which, until recently, was only able to provide a modest survival benefit. Recently, the introduction of immunotherapy and more specifically checkpoint inhibitors targeting the PD-1/PD-L1 axis have markedly improved outcomes. As a caveat, however, severe heterogeneity in terms of the survival benefit has been observed. While 15-20% of patients respond to single-agent anti-PD1 treatment and have an outstanding outcome, the majority will exhibit either stable disease or progressive disease where the benefit is severely mitigated. Evidence from other cancer types suggests that tumors with an inflamed microenvironment with heavy effector cell infiltration and an intact antigen-presentation machinery may be more amenable to checkpoint inhibition. The present work incorporates clinical and translational aspects aimed at laying groundwork for the introduction of precision oncology in this space. First, it defines factors driving immunogenicity and immune exclusion in HCC, developing an immune-based molecular tumor classification that characterizes immunogenic and non-immunogenic subclasses through integrative multi-omics analysis. Second, it defines molecular markers predictive of both response and resistance to anti-PD1 using transcriptomic analysis of tumor samples from patients undergoing treatment. Third, through metaanalysis it characterizes the impact of the underlying liver disease on outcomes after immunotherapy for HCC. Increasingly checkpoint inhibitors are tested in earlier disease stages to reduce high recurrence rates after resection. To this end, ensuring a patients availability for adjuvant treatment is contingent on fast recovery after surgery and high quality safety outcomes. The final aspects of this work include a detailed analysis of how laparoscopic liver surgery has facilitated this prerequisite and defines risk factors for adverse outcomes. Together the incorporated reports contribute to our grasp of immunotherapy in the HCC field and provide informative markers that may guide clinical decision making.
Weniger anzeigenWhy are some populist challenges more successful than others? Theorizing that a successful challenge occurs from a combination of demand, opportunity, and activation, I examine these conditions in the Visegrad Countries (Czechia, Hungary, Poland, and Slovakia) and how the challenger party in each country utilized these conditions to stage their challenges. Using a mix of public opinion surveys, secondary literature, and qualitative textual analysis, I find that parties that use identity-based appeals (e.g. to religion or nation) are better able to establish hegemonic electoral positions and delegitimize their opponents than parties that either coincidentally or by design fail to do so. This study contributes a review of populist challenges in post- communist Central Europe and a qualitative textual analysis of party literature that is largely unavailable in other languages. In considering why some challenges result in monolithic state capture (e.g. Poland and Hungary) while others founder (e.g. Czechia and Slovakia), we gain a broader picture of how parties instrumentalize identities and values, and a clearer picture of the structural weaknesses in liberal democratic regimes that facilitate democratic erosion and breakdown.
Weniger anzeigenIn this thesis we investigated set systems on geometric point data in Euclidean space from a macroscopic (first two chapters) and microscopic (last chapter) perspective.
In Chapter A we described cloud sets, which are set systems build on points which give rise to simplicial complexes as control structures. The preferred instance of such structure is a simplicial surface (making the cloud complex a cloud surface), so that the system of clouds mimics the properties of a collection of subsets we find in an atlas describing a 2-manifold. We discussed the generation of cloud surfaces on one hand starting with a simplicial surface, generating points, and placing them in clouds. On the other hand we focused on the reverse direction, obtaining the clouds from a point set which led to topolgical questions on coverings. In practice we used the cloud surfaces' underlying simplicial surface as a skeleton to perform point motions in Euclidean space.
In Chapter B we derived a second set system which is dual to the one in the first chapter under certain conditions. For the resulting complex we conjectured them to be surface-like, i.e. having combinatorial properties so that each point has at least an abstract vicinity which is perceivable to be 2-dimensional. For their construction we presented a k-means clustering approach for point sets coupled with an application of generating a simplified polygonal surface out of the segmentation also reconstructing polygonal faces which are not necessarily simply connected.
In Chapter C we partitioned the microscopic investigation inside a cloud in two. First we set up a large scale evaluation to determine a neighborhood around a point accounting as an approximation of a 2-dimensional vicinity using an energy model on feature classification entities built from eigenvalues obtained by a weighted point distribution analysis. The evaluation explores the parameter spaces of the weighting function and combinatorial neighborhood sizes of the point distributions. Secondly we proposed the flatness model as a feature classification entity favoring point distributions which appear to be 2-dimensional and not curve-like or volumetric. This continuous model then is incorporated into a point set denoising application to evaluate its potency using less parameters and preserving geometric features without the necessity of point normal information.
Weniger anzeigenAdulte, multipotente mesenchymale Stammzellen (MSCs) stellen nicht nur für die regenerative Medizin, sondern auch für die Etablierung von in-vitro-Modellen einen wichtigen Forschungsschwerpunkt dar. MSCs aus Fettgewebe (ASCs) eignen sich hierfür besonders und haben das Potential, in verschiedene Zelltypen zu differenzieren, darunter in Kardiomyozyten-ähnliche Zellen (CLCs), wie in Studien bei Menschen, Kaninchen und Nagern beschrieben wurde.
Ziel der Untersuchungen war es, (1) die mittels zwei Isolierungsverfahren aus unterschiedlichen Fettgewebelokalisationen gewonnenen MSCs hinsichtlich ihrer in-vitro-Charakteristika zu vergleichen. Im zweiten Teil der Studie (2) sollte das kardiomyogene Differenzierungspotential der Zellen erstmals beim Pferd in vitro untersucht werden, um das Grundlagenwissen in Bezug auf die Etablierung eines pferdespezifischen in-vitro-Kardiomyozytenmodells zu erweitern.
In die in-vitro-Studie wurden n = 16 Pferde einbezogen, von denen post mortem MSCs aus abdominalem (abd), retrobulbärem (rb) und subkutanem (sc) Fettgewebe mittels des Explantat-Verfahrens (ASCs-EXP) und nach Kollagenaseverdau (ASCs-SVF) gewonnen wurden. Nach Evaluierung des Isolierungserfolgs (n = 16), des Proliferationspotentials (n = 3) sowie des für das Zellwachstum am besten geeigneten Serumsupplements (n = 3) wurden die Zellen auf ihre Charakteristika als MSCs überprüft, wozu die Fähigkeit zur Plastikadhärenz, die Untersuchung des tripotenten Differenzierungspotentials über 7, 14 bzw. 21 Tage (n = 6) und das spezifische Oberflächenmarkerprofil (n = 5) zählten. Es folgte die Untersuchung des kardiomyogenen Differenzierungspotentials von abd-ASCs-SVF (n = 5) mithilfe des Induktionsfaktors 5-Azacytidin (5-AZA) sowie der Faktoren Activin A (Act A), knochenmorphogenetisches Protein-4 (BMP-4) und Dickkopf-1 (DKK-1). Eine Untersuchung zellmorphologischer Veränderungen sowie Analyse der Expression kardialer und pluripotenzassoziierter Marker sowie eines Muskelmarkers mittels SYBR Green RT-qPCR wurden an Tag 0 (T0) und 3 Wochen nach Induktion (T3) angeschlossen.
Plastikadhärente, Fibroblasten-ähnliche ASCs-EXP und ASCs-SVF konnten aus abd-, rb- und sc-Fettgewebe gewonnen werden. Während sich abd- und rb-ASCs hinsichtlich des Isolierungserfolgs annähernd gleich verhielten, wiesen sc-ASCs einen signifikant niedrigeren Isolierungserfolg auf. In Bezug auf die Zellproliferation wurden keine signifikanten Unterschiede zwischen den Lokalisationen und Isolationsmethoden festgestellt, jedoch ein signifikanter Effekt des Serumsupplements. Das höchste adipogene Differenzierungspotential wiesen abd-ASCs-EXP gegenüber rb- und sc-ASCs-EXP an Tag 7 und abd-ASCs-SVF gegenüber abd-ASCs-EXP an Tag 14 auf. Das osteogene Differenzierungspotential erwies sich an Tag 14 zwischen ASCs der verschiedenen Lokalisationen und Isolierungsverfahren annähernd gleich. An Tag 21 wiesen abd-ASCs-EXP gegenüber abd-ASCs-SVF und rb-ASCs-EXP ein höheres osteogenes Differenzierungspotential auf. Das chondrogene Differenzierungspotential war bei allen Zellen unabhängig von der Gewebequelle und Isolationsmethode vergleichbar. Die untersuchten Zellen waren positiv für CD29, CD44, CD90 sowie negativ für CD34 und CD45 und exprimierten die pluripotenzassoziierten Marker OCT4/POUF5, MYC und DNMT3B. Eine kardiomyogene Differenzierung konnte weder mithilfe von 5-AZA noch mithilfe unterschiedlicher Konzentrationen von Act A, BMP-4 und DKK-1 nachgewiesen werden. Es wurden weder zellmorphologische Veränderungen, eine spontane Schlagaktivität noch eine Aufregulierung der kardialen Marker NKX2-5, GATA4, TNNI3, MYH6, MYH7 sowie des Muskelmarkers MYF6 zum Zeitpunkt T3 im Vergleich zu den Positivkontrollen mittels SYBR Green RT-qPCR nachgewiesen.
Die vorliegende Studie vergleicht die mittels der beiden Isolierungsverfahren aus den drei Fettgewebelokalisationen isolierten equinen MSCs in Bezug auf ihre Charakteristika in vitro, wobei eine Isolierung equiner rb-ASCs zuvor noch nicht beschrieben wurde. Auch wenn eine kardiomyogene Differenzierung von MSCs bei verschiedenen Spezies in vitro nachgewiesen wurde, lagen hierzu bisher keine Studien zum Pferd vor. In der vorliegenden Studie wird erstmals der Effekt des in der Literatur am häufigsten eingesetzten kardiomyogenen Induktors 5-AZA sowie der an der embryonalen Kardiomyogenese beteiligten Faktoren Act A, BMP-4 und DKK-1 untersucht.
Es konnte gezeigt werden, dass equine ASCs-EXP und ASCs-SVF aus allen untersuchten Fettgewebelokalisationen gewonnen und aufgrund ihrer Charakteristika eindeutig als MSCs identifiziert werden konnten. Die Zellen wiesen ein hohes Proliferations- und tripotentes Differenzierungspotential auf. Eine kardiomyogene Differenzierung equiner abd-ASCs-SVF konnte allerdings unter Einsatz der gewählten Induktionsfaktoren und Versuchsbedingungen nicht erreicht werden. Die vorliegende Studie trägt dazu bei, das Grundlagenwissen über die in-vitro-Charakteristika equiner ASCs zu erweitern. Weitere Vergleichsstudien, in denen der Einfluss der Isolationsmethode auf equine MSCs verschiedener Herkünfte überprüft wird, sind anzuraten. In Bezug auf die kardiomyogenen Differenzierungsversuche ist der Einsatz pluripotenter Stammzellen anstelle der multipotenten MSCs in Erwägung zu ziehen, da diese ein höheres Differenzierungspotential aufweisen.
Weniger anzeigenColorectal cancer, a highly heterogeneous cancer, continues to be a leading cause of mortality worldwide. While the 5-year survival rates for patients with Stage I and II are high, there has been little or no improvement of survival for patients with metastases. To make matters worse, 20% of the patients already present with metastasis at the time of diagnosis. Therefore, early detection of patients who are at high risk of developing metastases using biomarkers is key to improving patient survival.
Metastasis-associated in colon cancer 1 (MACC1) is one such biomarker that has been directly linked to metastasis development, reduced survival, and worse overall outcomes. In addition to identifying high-risk patients, MACC1 is biologically linked to tumor and metastasis development. Specifically, the MACC1 structure contains diverse domains and several tyrosine sites capable of versatile interactions. Therefore, the aim of the first part of the project was to study the role of tyrosine sites close to the N-terminus of MACC1. Employing computational tyrosine phosphorylation prediction tools, site Tyr379 and SRC kinase as one of the promising kinases responsible for its phosphorylation were identified. Preliminary examination reveals an association between MACC1 and SRC.
Despite extensive evidence describing the functional diversity of MACC1, little is known about the structural features and self-association property of MACC1. To address this gap in knowledge, the goal of the second part of this project was to systematically evaluate the structural properties of MACC1 and the self-association capability of MACC1. Using AlphaFold2, the structures of MACC1 and MACC1 dimer were revealed. Val212, Ileu214, and Cys216 present in the ZU5 domain of MACC1 were found to be critical for dimerization. The knowledge gained from the AI prediction was transferred to set up a bioluminescence resonance energy transfer (BRET) assay to analyze MACC1 dimerization and the effect of mutation on dimerization. In addition to validating the presence of MACC1 dimer in living cells, the BRET assay confirmed reduced MACC1 self-association when the above residues were mutated. Ultimately, the impact of these mutations on MACC1 signaling and metastasis properties was verified using an in vitro metastasis assay.
In summary, these results shed new light on the MACC1 structural characteristics particularly the presence of MACC1 homodimer, and reveal the residues important for dimerization, thus providing a framework for future development of intervention strategies.
Weniger anzeigenThe purpose of this research is to advance the understanding of beautification practices in urban China by analyzing those beauty ideals and those forms of cosmetic surgery that are distinctive of the Chinese landscape. The focus is on female beauty not only because women are the main consumers of beautification in China, but also because the female body has been a ground for expressing social change throughout history. This study adopts an ethnographic approach, including observation and person-centered interviews in Hangzhou and Shanghai in 2017 and 2018. These two cities are located in the Yangtze River Delta, a highly developed and urbanized area in Eastern China, which may be representative of urban China due to a certain uniformity in beauty trends in the country. The findings of this research revealed that individualization, success and naturalness each highlight a feature of women’s pursuit of beauty. Within a sociological institutional environment, the concept of habitus interacts with the notion of constructive power over bodies in order to analyze the social significance of beautification trends. Women in urban China negotiate their social value (also) through their looks, between market logics and traditional norms. I argue that the beauty that these women pursue represents the modern woman of highly developed urban areas and position my argument in a larger discourse of desire for success, related to quality and the overall social engineering of neoliberal China. The individualization of society has enabled women to pursue beautification and encouraged them to win the social competition. This quest for happiness relates to an obsession with success that pervades the lives of Chinese urbanities, reflecting on aesthetic ideals. A fair and pure skin, an oval shape, a pointed chin, a straight high-bridged nose, and large double-lidded eyes are the ideal face features that symbolize success in a sense that transcends one’s social class and one’s material achievements. The treatments that aim to obtain the ideal face belong to weizheng or micro cosmetic surgery, a distinctive category of cosmetic surgery: naturalness characterizes both the outcome and the process of weizheng, distancing it from conventional cosmetic surgeries and pushing the boundaries of natural beauty toward a broader meaning. Studying the way female beauty is interpreted and negotiated, mainly by the post-1980 generation, can help us better understand the development of Chinese urban society.
Weniger anzeigenClass switch recombination (CSR) is a somatic recombination reaction occurring in mature B lymphocytes, that involves programmed double-strand break (DSB) formation and repair at immunoglobulin heavy chain locus (Igh). CSR is essential for adaptive immunity since it diversifies the effector functions of antibody responses. Many key players, including components of the DNA damage response and DSB repair, transcriptional regulators, long non-coding RNAs and chromatin remodelers, have been reported to contribute to the different phases of this complex process. However, how all these DNA-RNA-protein interactions are established and evolve to support the repair dynamics of CSR remains elusive. In order to bridge this knowledge gap, I have established a primary B cell model system that couples locus-specific chromatin purification and proximity labelling to high-resolution mass-spectrometry to identify the factors that selectively bind within the Igh locus to support DSB processing and repair during CSR. 53BP1 is a DNA damage response factor that protects DSB ends from nucleolytic processing to promote their repair via the nonhomologous end-joining (NHEJ) pathway. Because of this activity, 53BP1 is not only crucial for CSR, but also facilitates toxic mis-repair (toxic-NHEJ) of DSBs in BRCA1-deficient cells. Phosphorylation of 53BP1 on its N-terminus by the DSB repair kinase ataxia telangiectasia mutated (ATM) is essential for DNA end-protection, and, as a consequence, for both these physiological (CSR) and pathological 53BP1 (toxic-NHEJ) repair events. Through a SILAC-based mass-spectrometry approach, we identified acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) as a potential interactor of 53BP1 under unphosphorylated conditions. I tested the hypothesis that ANP32B might interact with 53BP1 and negatively regulate it during DSB repair, which on further assessment I confirmed is dispensable for repair-associated activity. Rap1-interacting factor 1 (RIF1) is a multi-functional protein that, together with 53BP1, acts as a pro-NHEJ protein because of its ability to mediate DSB end-protection. As a consequence, RIF1 promotes both CSR in mature B cells and toxic-NHEJ reactions on BRCA1-deficient backgrounds. However, the post-translational regulation of RIF1 DNA end-protection function has not yet been elucidated. To this end, I implemented label-free mass-spectrometry in primary B lymphocytes to define potential phosphorylation events supporting RIF1 DNA end-protection activity. I identified a serine residue (S2138) that is phosphorylated upon DSB induction; however, this post-translational modification was dispensable for regulating RIF1 function during DSB repair and CSR. Altogether, CSR repair components, including 53BP1 and RIF1, define a crucial balance between immunity and lymphomagenesis in B cells. Therefore, identifying novel factors and post-translational modifications involved during CSR break repair will expand our understanding of the mechanisms preserving genome stability during antibody diversification in mature B cells.
Weniger anzeigenThis dissertation comprises original experimental work exploring the intermediate stages of the perception-action loop in the somatosensory domain. The stages correspond to the maintenance of working memory (WM) content, the goal-directed manipulation of the content, and the formation of memory-based decisions. Concurrently, the thesis addresses ongoing debates in cognitive neuroscience, specifically, the localization of the respective WM- and decision-making content. For both debates, the central issue rests with the extent of influence that experimental design has on the localization of the resulting representations. By taking advantage of modifications of experimental paradigms, advanced whole-brain data analysis techniques, and the extensive literature in the somatosensory domain, the dissertation provides evidence in favour of the distributed representation of WM content. The distribution of WM representations is not limited to either frontal or sensory regions. Indeed, the fronto-parietal network - specifically the intraparietal sulcus, inferior frontal gyrus, and the premotor cortex - is necessary for the successful performance of the intermediate stages of the perception-action loop. Therefore, the maintenance of WM content, the manipulation and maintenance of the resulting content, and the computation of the decision variable, all take place in a network consisting primarily of frontal and parietal regions with the specific distribution of WM content depending on the experimental paradigm.
Weniger anzeigen