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Targeting the major pro-inflammatory interleukin-6-type cytokine receptor gp130 by antagonistic single domain antibodies
No full textIntroduction: Although Interleukin (IL)-6-type cytokine signaling is critical for maintaining the body's homeostasis, aberrant signaling has been observed in numerous diseases including autoimmunity and cancer. Currently, all approved biologics that inhibit IL-6-type cytokines specifically target the key pro-inflammatory mediator IL-6 or its receptor (IL6R). Historically, direct inhibition of glycoprotein 130 (gp130)—the shared transmembrane receptor for IL-6-type cytokines—was avoided due to concerns that broad suppression might cause more harm than benefit. However, this view is being reconsidered in light of the clinical success of Janus kinase (JAK) inhibitors, which broadly disrupt cytokine signaling, including pathways mediated by gp130.
Methods: Here we developed four single domain antibodies (sdAb), consisting out of a camelid-derived nanobody and a human Fc-fragment, and characterized them by direct protein interaction analysis, epitope binding, epitope binning, as well as inhibition of cytokine-induced stimulation and proliferation of appropriate Ba/F3 cell lines and trans-migration in HT-29 cells.
Results: The four sdAb-Fc constructs GP01-, GP11- GP13- and GP20-Fc bind directly to gp130 in the cytokine binding module (CBM) and largely inhibit IL-6-type cytokine signaling by interfering with the high-affinity binding site of IL-6, IL-11, CLCF1, CT1, CNTF, OSM and LIF. Furthermore, we functionally demonstrate the inhibitory effect of the selected nanobodies in cell-based transmigration assays of the human colorectal cancer cell line HT-29.
Discussion: In summary, our study has identified and characterized four novel inhibitory high-affinity gp130 nanobodies with potential for use in cytokine-dependent autoimmunity or cancer therapy
Towards Understanding and Reducing the Protein Retention on Microfluidic Paper Substrates
No full textTransferring point-of-care (POC) analytics onto microfluidic paper enables pump-free water transport at low cost and good availability of the biogenic cellulosic substrate. Although a number of interesting novel demonstrators for such paper-devices have been reported to date, a number of challenges still exist which limit a successful transfer to market-ready devices. A strong limitation to this respect is the (unspecific) adsorption of protein analytes to the paper fibers during the lateral flow assay. This interaction may significantly reduce the amount of analyte that reaches the detection (or reaction) zone of the microfluidic paper-based analytical device (µPAD), thereby reducing its overall sensitivity. Here we introduce a novel approach on reducing the non-specific adsorption of proteins to lab-made paper sheets, for the use in µPADs. By photolithographic cross-linking of benzophenone functionalized poly-(oligo-ethylene glycol methacrylate) (POEGMA) and poly-dimethyl acrylamide (PDMAA), cotton-linters fibers in lab-formed additive free paper sheets were modified with a surrounding thin hydrogel layer. This, as we show in tests similar to lateral flow assays, significantly reduces unspecific binding of model proteins. Furthermore, by evaporating the transport fluid during the microfluidic run at the end of the paper strip through local heating, model proteins can almost quantitatively be accumulated in that zone. The possibility of complete, almost quantitative protein transport in a µPAD opens up new opportunities to significantly improve the signal-to-noise ratio of paper based lateral flow assays
Entwicklung von Polyaspartaten als photoresponsive Orientierungsmedien zur induzierten Reorientierung eines Analyten
No full textDie vorliegende Dissertation beschreibt die Synthese und Untersuchung von Polyaspartarten mit ortho-flourierten Azobenzolen (FAB) zur Anwendung als photoresponsive Orientierungsmedien für die NMR-basierten Strukturaufklärung. Ein integraler Bestandteil der aktuellen Forschung befasst sich mit der Induktion mehrerer (unterschiedlicher) Orientierungen der zu untersuchenden Analyten innerhalb der gleichen Probe. Eine Reorientierung des Analyten kann beispielsweise durch die Verwendung stimuliresponsiver Orientierungsmedien erzielt werden, wobei Licht als Stimulus eine besondere Bedeutung zukommt, da dieser nicht-invasiv ist und sowohl Orts- als auch Zeit-aufgelöst gezielt angewendet werden kann. Für die im Rahmen dieser Arbeit synthetisierten Polymere werden die photoresponsiven Eigenschaften sowohl in isotroper Lösung als auch im flüssigkristallinen Zustand mit verschiedenen analytischen Methoden untersucht. Dabei wird vor allem der Einfluss des Isomerisierungszustandes der inkorporierten FAB-Einheiten auf die Sekundärstruktur des Polymers und die Orientierung des Modellanalyten im flüssigkristallinen Medium untersucht.
Zur Erzeugung verschiedener photoinduzierter Orientierungen eines Analyten innerhalb der gleichen anisotropen Probe werden hierbei unterschiedliche Strategien verfolgt. Hierzu werden Homopolyaspartate mit unterschiedlichen FAB-Motiven in der Polymerseitenkette synthetisiert (Projekt A), was die resultierende photoresponsiven Sekundärstruktur beeinflusst. Projekt B beschäftigt sich mit dem gezielten Design eines photoinduzierten Helix-Helix-Übergangs (Helixinversion) durch die Synthese von Copolyaspartaten. Die Kombination und Zusammensetzung unterschiedlicher (FAB-)Strukturmotive in der Seitenkette, die die Präferenz besitzen die gleiche oder entgegengesetzte Helixgängigkeit einzunehmen, ermöglichen unterschiedliche photoinduzierte Konformationsübergänge der Copolyaspartate. Durch eine Belichtung der anisotropen Proben können unterschiedliche flüssigkristalline Zustände und unterschiedliche Orientierungen eines Modellanalyten induziert werden. Der Unterschied der photoinduzierten Orientierungen innerhalb einer Probe (u. a. Photodifferenzierung) wird ermittelt. Zur weiteren Destabilisierung der Helixinversionsbarierre erfolgt die Synthese von FAB-basierten Terpolymeren mit achiralen Einheiten (Aib) als dritte Komponente. In Projekt D wird die Synthese von Polyaspartaten mit FAB-Gruppen im Polymerrückgrat und ihr photoresponsives Verhalten beschrieben
Ankommen in der Sekundarstufe I: Von der Vorbereitungs- zur Regelklasse
No full textDer Beitrag untersucht die Umsetzung eines Förderkonzepts an bayerischen Realschulen, das auf die Integration neu zugewanderter Schülerinnen und Schüler abzielt (Bayerisches Realschulnetz (BRN) 2023). Im Fokus stehen die Auswahlkriterien für die Teilnahme an der Fördermaßnahme sowie die Ausgestaltung verschiedener Beschulungsmodelle. Anhand der qualitativen Auswertung von Gruppeninterviews mit Lehrkräften und Schülerinnen und Schülern wird aufgezeigt, dass das Konzept durch seine Flexibilität und Offenheit eine erfolgreiche Integration von Neuzugewanderten fördert. Dabei erweisen sich differenzierte Aufnahmekriterien und die Kombination aus Sprachförderung und stufenweiser Eingliederung in Regelklassen als entscheidend für den Lernerfolg
Acoustic indices predict recovery of tropical bird communities for taxonomic and functional composition
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Zur statistischen Analyse überparametrisierter tiefer neuronaler Netze trainiert durch Gradientenabstieg
No full textDer Erfolg des Deep Learnings ist unübersehbar, insbesondere bei großen neuronalen Netzen wie ChatGPT-3 mit 175 Mrd. und BERT-Large mit 340 Mio. Parametern. Dies zeigt den Trend zur Überparametrisierung, bei der die Anzahl der Parameter die der Trainingsdaten übersteigt. Klassische Theorien erwarten hier eine Überanpassung und damit eine schlechte Generalisierung auf neuen Daten. Dennoch liefern solche Netze oft sehr gute Ergebnisse.
Diese Arbeit untersucht überparametrisierte neuronale Netze, die durch den Gradientenabstieg trainiert werden, im Rahmen der nichtparametrischen Regression, insbesondere in Bezug auf universelle Konsistenz und Konvergenzraten. Dabei zeigt sich, dass die gängige Überanpassungshypothese in diesem Kontext nicht zutrifft.
Unser theoretischer Ansatz verbindet die drei zentralen Aspekte des Deep Learnings: Optimierung, Approximation und Generalisierung. Die Optimierung erfolgt, wie in der Praxis üblich, mittels Gradientenabstieg, wobei kein zusätzlicher Regularisierungsterm verwendet wird. Im Bereich der Approximation zeigen wir, dass geeignete Netzwerktopologien eine präzise Annäherung an komplexe Funktionen ermöglichen. Die Generalisierung beschreibt die Leistungsfähigkeit auf neuen Daten. Unsere Untersuchungen zeigen, dass überparametrisierte neuronale Netze trotz der großen Anzahl von Parametern in der Lage sind, zuverlässige Vorhersagen zu treffen.
Die theoretischen Ergebnisse dieser Arbeit verdeutlichen das Potenzial überparametrisierter neuronaler Netze in mehrfacher Hinsicht. So lässt sich mit der sigmoidalen Aktivierungsfunktion die Eigenschaft der universellen Konsistenz nachweisen. Für glatte Regressionsfunktionen können zudem nahezu optimale Konvergenzraten abgeleitet werden. Besonders bemerkenswert ist, dass sich für ReLU-Netze unter kompositionellen Annahmen sogar dimensionsunabhängige Raten erzielen lassen und damit der Fluch der Dimensionalität umgangen werden kann
3D‐Printing with Steel ‐ Additive Manufacturing Connections and Structures
No full textAutomated production is finding its way into fabrication of structural steel. One robot holds attachments (stiffeners, head plates, etc.) to a steel beam or column and another robot produces weld seams. However, welding robots can also be used for additive manufacturing (Wire + Arc Additive Manufacturing, WAAM). The wire electrode serves as printing material. The Institute for Steel Construction and Materials Mechanics in Darmstadt is investigating how typical connecting elements of steel construction can be printed directly on steel beams using Additive Manufacturing with arc welding and robots. Furthermore structural elements like nodal points are printed and even complete structures like columns and a little bridge have been manufactured additively already. The main focus is on determining suitable welding and process parameters. In addition, topology optimization is used to find good structures using a low amount of material. This is possible due to the free design prospects of 3D‐printing. This opens for novel design and production strategies
Regulation of homologous recombination sub-pathways at two-ended double-strand breaks
No full textDNA double strand breaks (DSBs) are a detrimental form of DNA damages that drives genome instability and cancer development. DSBs can spontaneously arise during replication or as a result of the exposure to exogenous agents such as ionizing radiation (IR). Accurate repair of DSBs is necessary to preserve the genome integrity for cell survival. Homologous recombination (HR) is an important mechanism of DSB repair that utilizes an intact homologous DNA sequence as a template to faithfully restore the genetic information. In the repair of two-ended DSBs, two sub-pathways of HR are involved: the double Holliday Junction (dHJ) and the synthesis-dependent strand annealing (SDSA) sub-pathways. Although SDSA is previously thought to be the predominant HR sub-pathway in somatic cells, it appears that a distinct dHJ sub-pathway that is solely processed by dHJ resolution is primarily used to repair IR-induced DSBs during G2 phase. The chromatin remodeler ATRX has been shown to promote the dHJ sub-pathway via the interaction with DNA polymerase processivity factor PCNA. SDSA only becomes the primary HR sub-pathway in cell lines naturally lacking functional ATRX like U2OS cells and is completely dependent on RECQ5 helicase, but the underlying mechanisms of how RECQ5 promotes SDSA during G2 phase is unclear. Although ATRX and RECQ5 have been shown to promote the dHJ and SDSA sub-pathway respectively, little is known about how somatic cells regulate the usage of distinct HR sub-pathways.
In this thesis, I show that RECQ5 ATPase/helicase activity and its PCNA-interacting motifs are required for SDSA in ATRX-null U2OS cells. Notably, the over-expression of ATRX with functional ATPase and PCNA-interacting motifs outcompetes the RECQ5-dependent SDSA in U2OS cells. In ATRX-positive HeLa cells, ATRX is dominant over RECQ5 for HR repair. I further reveal that the over-expression of RECQ5 lacking the ATPase/helicase activity, but not the PCNA interacting motifs, in HeLa cells results in an HR repair defect. These data suggest that the binding of RECQ5 ATPase mutant to PCNA blocks interaction between PCNA and ATRX during HR. Taken together, the results obtained in this thesis indicate that HR sub-pathway choice during G2 phase is defined by the interaction of ATRX and RECQ5 with PCNA.
Previously, one of the technical challenges in the study of HR sub-pathways was the lack of human cell lines that specifically use SDSA or the dHJ sub-pathway, respectively. Given that U2OS cells exclusively use SDSA during G2 phase, I use this opportunity to conduct a candidate-based siRNA screen to look for SDSA-promoting helicases in response to IR. I identify RECQ1 helicase as a crucial factor for SDSA in G2 cells, in addition to RECQ5. Importantly, RECQ1 ATPase/helicase activity and DNA strand annealing activity are essential for SDSA. RECQ1 appears to be involved in the downstream strand annealing steps during SDSA. Besides a direct role in HR, I uncover an unexplored function of RECQ1 in regulating the DSB repair pathway choice in G2 phase. It has been previously shown that the core HR factor BRCA2 acts in concert with RAD52 during G2 phase to restrict the usage of DNA polymerase θ (POLθ)-mediated end-joining until mitosis. Here, I provide evidence that, in G2-phase U2OS cells, RAD52 also prevents the premature usage of an alternative DSB repair pathway that is independent of POLθ. The presence of RECQ1 in G2 phase may counteract the role of RAD52 by specifically removing RAD52 and promoting the usage of RECQ1-mediated SDSA over the POLθ-independent repair in U2OS cells. The pilot experiment suggests that HELQ is essential for this POLθ-independent repair pathway which is normally inhibited by RECQ1 and RAD52 during G2 phase.
Dynamic processing of D-loops is believed to be critical for HR sub-pathway usage. Importantly, our group has recently identified that RAD54 and its paralog RAD54B, which are both implicated in promoting D-loop formation and stabilization, facilitate the dHJ and SDSA sub-pathways, respectively. Presumably, the alteration of D-loop stability could affect the HR sub-pathway choice. Recent in vitro studies showed that the regulation of D-loop disruption by the topoisomerase TOP3A could funnel HR towards distinct sub-pathway. I therefore test if the loss of TOP3A alters the HR sub-pathway usage in vivo. Interestingly, the depletion of TOP3A in U2OS cells rescues the RECQ1-/RECQ5-/RAD54B-dependent repair defect demonstrating that the loss of TOP3A in U2OS cells bypasses the canonical SDSA. Surprisingly, the repair is not channeled to dHJ but to an alternative HR pathway that requires RAD54. I also investigate whether the loss of TOP3A affects the HR sub-pathway choice in HeLa cells that pre-dominantly use dHJ sub-pathway. I confirm that the ATRX-deficient HeLa cells exhibited an HR repair defect and strongly reduced the usage of IR-induced dHJ sub-pathway. Surprisingly, the loss of TOP3A in ATRX-deficient HeLa cells rescues this repair defects and re-allows the usage of dHJ sub-pathway. The occurrence of ATRX-independent dHJ sub-pathway in the absence of TOP3A illustrates an antagonistic relationship between ATRX and TOP3A in controlling the dHJ sub-pathway.
Despite the processing of dHJ intermediates being well defined, it remains poorly understood how cells promote the formation of these recombination intermediates. I discover RAD18 as a new dHJ-specific factor in response to IR. I present evidence that RAD18 facilitates the dHJ sub-pathway and maintains chromosomal stability in HeLa cells. The loss of RAD18 does not affect the IR-induced HR repair in G2-phase U2OS cells that exclusively use SDSA. Whether RAD18 is involved in supporting dHJ formation or processing of dHJs remains to be defined.
Collectively, this study contributed to our understandings of how human cells regulate the HR sub-pathway usage for repairing two-ended DSBs, with implications of the importance of PCNA interaction and D-loop disruption in manipulating the HR sub-pathway choice. The discovery and characterization of additional HR sub-pathway factors will further provide insights into the mechanisms of distinct HR sub-pathways and the crosstalk between HR and other DSB repair pathways