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Charakterisierung der Pumpenversorgung bei ambulant palliativmedizinisch versorgten Patienten im Rahmen der SAPV der LMU München
Get PDFContribution of the K63-deubiquitinase Cylindromatosis (CYLD) to neuronal cell death after focal cerebral ischemia
Get PDFEvaluation of BMP2-loaded hybrid 3D collagen scaffolds: proliferation and osteogenic differentiation capacity of human mesenchymal stem cells
Get PDFMaturation of human induced pluripotent stem cell based myocardium by biomechanical stimulation of three-dimensional tissue cultures
Get PDFGeometric flows and the swampland
Get PDFAfter an introductory chapter on the quantum supersymmetric string, in which particular attention will be devoted to the techniques via which phenomenologically viable models can be obtained from the ultraviolet microscopic degrees of freedom, and a brief review of the swampland program, the technical tools required to deal with geometric flows will be outlined. The evolution of a broad family of scalar and metric bubble solutions under Perelman's combined flow will be then discussed, together with their asymptotic behaviour. Thereafter, the geometric flow equations associated to a generalised version of Perelman's entropy function will be derived and employed in defining the action-induced flow associated to a given theory for a scalar field and a dynamical metric. The problem of preserving Einstein field equations along the corresponding moduli space trajectories will be cured by allowing a supplementary energy-momentum tensor term to appear along the flow. In a particular example, such contribution will be shown to precisely reproduce the infinite tower of states with exponentially dropping masses postulated by the distance conjecture.Nach einer Einführung in den Superstring, in der besonders auf die Methoden eingegangen wird, mit welchen man aus mikroskopischen Freiheitsgraden im ultravioletten Bereich phänomenologisch brauchbare Modelle erhalten kann und einem kurzen Überblick über das Swampland-Programm werden die mathematischen Methoden vorgestellt, die für die Beschreibung von geometrischem Fluss notwendig sind. Danach wird die Entwicklung einer breitgefächerten Familie von skalaren und metrischen Blasenlösungen unter Perelmans kombiniertem Fluss, zusammen mit deren asymptotischen Verhalten diskutiert. Anschließend werden die geometrischen Flussgleichungen, die im Zusammenhang mit einer verallgemeinerten Version der Perelman-Entropiefunktion stehen, hergeleitet und zur Definition des von der Wirkung induzierten Flusses verwendet. Dieser kann mit einer bestimmten Theorie für ein skalares Feld und eine dynamische Metrik in Verbindung gebracht werden. Es wird ein zusätzlicher Energie-Impuls-Tensor eingeführt, so dass während des geometrischen Flusses die Einstein’schen Feldgleichungen entlang der entsprechenden Trajektorie im Modulraum unverändert bleiben. In einem speziellen Beispiel wird gezeigt, dass ein solcher Beitrag einen Turm aus unendlich vielen Zuständen mit exponentiell abfallenden Massen, wie er von der Abstandsvermutung postuliert wird, exakt reproduziert
Differential effects of acute and chronic hydrocortisone treatment on pyroptosis in vitro
Get PDFPyroptosis is a mode of inflammatory programmed cell death triggered by Path-ogen Associated Molecular Patterns (PAMP) of pathological microbes and intracellular components from damaged cells in order to attract immune cells and to prevent spread of infection. Pyroptosis occurs mostly in macrophages but can be also found in other immune cells and epithelial cells. Upon PAMP-stimulation the pro-inflammatory NF-κB signaling pathway is activated, which is followed by inflammasome assembly and cleavage of effector caspase-1. The main features of pyroptosis are cell lysis mediated by gasdermin D pores, and release of the pro-inflammatory cytokines IL-1β and IL-18 into the extracellular space.
The widely used synthetical glucocorticoid (GC) hydrocortisone (HC) is a steroidal drug to limit inflammation. Physiologically, GCs are secreted from the adrenal cortex upon stress and act as immune suppressants and anti-inflammatory hormones by inhibiting pro-inflammatory signaling pathways (e.g. NF-κB). However, it was shown that HC also has the potential to upregulate pro-inflammatory genes such as TLRs (Toll like receptors) and NLRP3, which indicates that HC can have both positive and negative effects on inflammatory immune responses. The impact of HC on pyroptosis in macrophages was not addressed by now.
In the present work, the effects of incrementing HC concentrations on induc-tion and execution of pyroptosis were investigated in THP-1 macrophagelike cells. Accordingly, cells were pretreated either with 0.1 μg/ml HC to mimic ba-sal human cortisol levels or with 0.2 and 0.4 μg/ml to mimic chronically increased concentration levels. Pyroptosis was induced by LPS (lipopolysaccha-ride, 5 ng/ml) and ATP (adenosine triphosphate, 5 mM). Subsequent treatment with HC after pyroptosis induction was scheduled as an acute concentration increase from 0.1 µg/ml to 0.2 or 0.4 µg/ml, which corresponds to patients HC treatment upon severe inflammation or constant moderately (0.2 µg/ml) or high-ly (0.4 µg/ml) elevated HC levels, resembling stress condition or patients otherwise pretreated with HC, repectively.
Results derived from these investigations have shown, that the acute HC ex-posure did not change LPS- and ATP-mediated cell lysis compared to physiological control, however chronically elevated HC dose-dependently attenuated cell lysis. Interestingly, after acute exposure, NLRP3 and caspase-1 activation as well as extracellular IL-1β levels were dose-dependently augmented. However, NF-κB signaling pathway activation and IL-1β mRNA levels were unal-tered under these conditions. Chronic exposure suppressd the NF-κB signaling pathway, protein expression and their activation.
This study described that the effects of HC on pyroptosis in macrophages as well as on their release of the proinflammatory cytokine IL-1β are differently affected in dependence of an acute or chronic HC exposure. Moreover, the data promotes potential clinical studies to evaluate the impact of IL-1β as a possible inflammation and pharmacological biomarker in daily clinical routine.Die Pyroptose ist eine Form des entzündungsverursachenden programmierten Zelltodes, der durch PAMPs (Pathogen Associated Molecular Patterns / Patho-gen-assoziierte molekulare Muster) von pathologischen Mikroben und intrazel-lulären Komponenten von geschädigten Zellen ausgelöst wird. Dadurch sol-len Immunzellen angelockt und die Ausbreitung einer Infektion verhindert werden. Pyroptose findet hauptsächlich in Makrophagen statt, kann aber auch in anderen Immunzellen und Epithelzellen auftreten. Nach einer PAMP-Stimulation wird der proinflammatorische NF-κB-Signalweg aktiviert, was die Assemblierung des Inflammasoms und die Spaltung der Effektor-Caspase-1 zur Folge hat. Die Hauptmerkmale der Pyroptose sind eine durch Gasdermin D-Poren vermittelte Zelllyse und die Freisetzung der entzündungsfördernden Zytokine IL-1β und IL-18 in den extrazellulären Raum.
Das weitläufig verabreichte synthetische Glucocortikoid (GC) Hydrocortison (HC) ist ein steroidales Medikament zur Behandlung von schweren Entzün-dungen. Physiologisch werden GCs bei Stress aus der Nebennierenrinde se-zerniert und wirken als Immunsuppressivum und entzündungshemmendes Hormon, indem sie entzündungsfördernde Signalwege (z. B. NF-κB) hemmen. Für HC wurde jedoch gezeigt, dass es auch das Potenzial hat, entzündungs-fördernde Gene wie TLRs (Toll-like-Rezeptoren) und NLRP3 hochzuregulie-ren, was darauf hindeutet, dass HC sowohl positive als auch negative Auswir-kungen auf die Entzündungsreaktion haben kann. Der Einfluss von HC auf die Pyroptose in Makrophagen wurde bisher nicht adressiert.
In der vorliegenden Arbeit wurden die Auswirkungen einer Erhöhung der HC-Konzentrationen auf die Induktion und Ausführung der Pyroptose in THP-1-Makrophagen untersucht. Hierfür wurden die Zellen mit 0,1 μg/ml HC vorbe-handelt, um basale Cortisolspiegel nachzuahmen, oder mit 0,2 und 0,4 μg/ml für chronisch erhöhte Konzentrationsspiegel. Pyroptose wurde durch LPS (Lipopolysaccharid, 5 ng/ml) und ATP (Adenosintriphosphat, 5 mM) induziert. Die weitere Behandlung mit HC nach Pyroptoseinduktion erfolgte als eine akute Erhöhung von 0,1 µg/ml auf 0,2 bzw. 0,4 µg/ml, die Konzentrationen ei-ner klinischen Bedingung bei der Behandlung von schweren Infektion ent-sprechen bzw. bei mäßig (0,2 µg/ml) oder stark (0,4 µg/ml) gestressten oder mit HC vorbehandelten Patienten auftreten können.
Die Ergebnisse der Untersuchungen haben gezeigt, dass eine akute HC-Exposition die LPS- und ATP-vermittelte pyroptotische Zelllyse im Vergleich zur physiologischen Kontrolle nicht veränderte, jedoch eine chronische erhöh-te HC-Exposition die Zelllyse dosisabhängig verminderte. Interessanterweise waren nach akuter HC-Exposition die NLRP3- und Caspase-1-Aktivierung so-wie die extrazellulären IL-1β-Spiegel dosisabhängig erhöht. Die Aktivierung des NF-κB-Signalweges und die IL-1β-mRNA-Spiegel blieben jedoch unter diesen Bedingungen unverändert. Die chronische Exposition hingegen un-terdrückte den NF-κB-Signalweg, die Proteinexpression und deren Aktivie-rung. Die Ergebnisse dieser Untersuchungen zeigen, dass in Abhängigkeit einer akuten oder chronischen Exposition die Effekte von HC auf die Pyropto-se und die Ausschüttung des pro-inflammatorischen Zytokins IL-1β unter-schiedlich ausfallen. Sie legen zudem die Umsetzung klinischer Studien zur Bewertung der Auswirkungen von IL-1β als möglichen Entzündungs- und pharmakologischen Biomarker für den klinischen Alltag nahe
Mikrobiologische In-vitro-Untersuchung zur Plaqueakkumulation auf additiv gefertigten Polymeren für provisorischen und definitiven festsitzenden Zahnersatz
Get PDFStructural characterization of CD47 and STING in complex with the activating nanobody
Get PDFcGAS-STING pathway is one of the most important first line defense mechanisms in a human body. Its action begins when a dsDNA from a pathogen enters the cell. Upon its detection by cGAS, the small molecule ligand of STING, called 2’,3’-cGAMP, is synthesized. It binds to STING which resides in form of a dimer in endoplasmic reticulum. This butterfly-shaped molecule then closes its wings and rotates. Afterwards, the partner protein, TBK1 binds at its top and the oligomerization process begins. Such complex migrates to Golgi apparatus and due to several phosphorylation events it activates IRF3 which migrates to the nucleus and initiates the expression of type I interferons. These molecules trigger the further cascade of events that leads to inflammation and defense against the pathogen. This important physiological process can also lead to various disease states and thus is tightly regulated. Despite long-term research efforts, this complex pathway is still not fully understood.
Here, I present the STING-activating nanobody which can be a tool to explore this protein’s mysteries. The nanobody was generated using alpaca immunization and found to significantly increase expression of CXCL10 - one of the interferon-stimulated genes, and to cause IRF3 migration to the nucleus. The first insight on how it happens was provided by the crystal structure of the nanobody with a soluble domain of STING. The wings of STING do not close during this interaction, which resembles the ways in which bacterial cyclic dinucleotides activate this protein. For now we may only speculate on how exactly the nanobody activates STING and further structural work is required to explain it. Examples of potential ways would be: release of STING C-terminal tail, positive or negative influence on interaction with partner proteins and triggering an oligomerization.
CD47, the marker of self, is a membrane protein present on the surface of every human cell, making it a “friend” of the host immune system. “Foes”, e.g. cancer cells, often use its power to evade detection and thrive. Such observations lead to development of more and more successful therapies based on antibodies that bind to CD47 and prevent its interaction with SIRPα on macrophages, priming cells for destruction. However, the problem is that no one fully determined how actually CD47 works.
Here, I present my multiple structural attempts to do that, using cryo-EM. I used multiple powerful tools, like nanodiscs, detergents, affinity binders to facilitate structure determination. Despite that, a high-enough resolution was not obtained, most probably because of too high inherent flexibility of this challenging target. Even recently published, crystal apo structure did not provide enough information to explain how this molecule perform its function. To decipher its mysteries multiple more structures of CD47 with its known and to-be-known partner proteins are needed
Identification of the novel antitumor targets of Lagunamide A and Neocarzilin A
Get PDFNatural products are a rich source of bioactive compounds and have traditionally contributed to the discovery and exploration of prospective drug development targets. Novel targets, particularly for cancer, are desperately needed to study cancer biology and develop precision therapeutics. Inspired by the promising anticancer activities of the marine cyanobacterial product Lagunamide A (Lag A) and the streptomyces carzinostaticusin product Neocarzilin A (NCA), we performed in depth studies on their modes of action.
For Lagunamide A, a biologically active natural compound, the cellular target is yet unidentified. With an affinity-based approach (ABPP), we failed to identify a target protein. In contrast, by using thermal proteome profiling, we found three proteins EYA3, SMC1A and NDC80 as putative targets. For validation of the proposed targets, genetic depletion of EYA3 showed only limited impact on antagonizing the anti-proliferative and pro-apoptotic effect of Lag A. However, EYA3 turned out to be important for DNA damage repair since incubation with Lag A sensitized cancer cells to doxorubicin treatment. Thus, this work not only shows that Lag A might serve as a chemosensitizing agent in the tumor context, but also pinpoints that depending on the target proteins, different target identification techniques are required.
Besides the chemoresistance machinery, cell proliferation is another hotspot in the field of cancer research. Our previous study showed that the natural compound NCA exhibited potent anti-migratory and anti-proliferative effects on cancer cells. We have previously identified VAT-1 as a putative anti-migratory target of NCA. However, the target and mechanism for the anti-proliferative effect of NCA were unknown to date. Affinity-based protein profiling (ABPP) based on an NCA-derived probe, and subsequent mass spectrometry were employed to identify additional potential target proteins of NCA. For validation of the proposed targets, we used genetic knockdown (with siRNA) or knockout (with CRISPR/Cas9) and over-expression approaches. By applying ABPP, Bone Marrow Stromal Antigen 2 (BST-2) was identified as it was highly enriched by binding to an NCA-derived probe. Binding of BST-2 to the probe was outcompeted by NCA itself, proposing BST-2 as a target of NCA. Silencing or knockout of BST-2 decreased the anti-proliferative action of NCA against HeLa cells, while overexpression or reconstitution of BST-2 increased sensitivity of cells towards NCA, thereby supporting the hypothesis of BST-2 as an anti-proliferative target of NCA. In-depth mechanistic studies revealed that NCA promoted BST-2 degradation via the lysosomal pathway and influenced the EGFR signaling pathway via disturbing lipid rafts, which in turn regulated cell proliferation. In conclusion, we identify BST-2 as an innovative target of NCA, and NCA as an interesting lead structure for inhibiting cancer cell proliferation
