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Towards bottom-up reconstitution of a functional FtsZ-based cell division machinery
Get PDFSynthetic biology aims at the understanding of living organisms through an engineering perspective, with the goal of improving or creating new biological systems. The prospect of building a synthetic cell focuses on producing life from basic elements by combining synthetic and/or organic cellular components in a bottom-up manner. To create a synthetic cell, the minimal functions of life are required and cell-free synthetic biology offers a suitable framework for understanding biological processes outside the inherently noisy environment of cells. A synthetic cell is expected to exhibit characteristics of a living cell, such as fundamental metabolism, proliferation, and communication. The bottom-up approach utilizes a wide range of in vitro tools/technologies such as biomimetic membranes, protein reconstitution, cell-free expression reactions, and microfluidics. As tools, they enable the thorough characterization of functional modules such as metabolism, replication, and cell division. The ultimate goal is to integrate these modules to construct a predictable, customizable, and controllable entity.
Among the functional modules of living organisms, cell division stands out as a hallmark feature. The machinery of division has evolved into a highly organized set of proteins with the aim of accurately splitting a mother cell into two daughter cells, while preserving the genetic information and cellular integrity. In the case of bacteria, and more concretely Escherichia coli, cell division is mediated by the divisome, a contractile ring consisting of a multiprotein complex that precisely assembles at midcell. At the center of this machinery is the essential FtsZ protein, which is able to polymerize and form the FtsZ-ring. This ring is key to the process, serving as a scaffold for the divisome and driving the division process. However, the molecular details of how the ring is functionally assembled, stabilized, and positioned are still not well understood. Therefore, the aim of this thesis is to develop and expand the knowledge about the molecular mechanism of the FtsZ-ring assembly and its function as a potential primary component in the minimal division machinery of synthetic cells.
To this end, and following a bottom-up approach, we conducted assays based on the in vitro reconstitution of FtsZ in cellular mimic environments using lipid vesicles. This allows the characterization of FtsZ’s behavior and functionalities in environments that are similar to a potential synthetic cell. Firstly, we designed a microfluidic device to deform lipid vesicles into bacterial rod-shaped compartments to analyze the effect of different geometries and membrane tension on FtsZ. We found that FtsZ filaments align with the shorter axis of the rod-shaped vesicles and reorganize into cone-like structures when the membrane tension is lowered, causing membrane deformations. This suggests that there is a geometry and tension-dependent mechanism in the assembly of FtsZ structures on membranes. Secondly, we designed an in vitro reconstitution assay based on soft lipid tubes pulled from FtsZ-decorated vesicles using optical tweezers. We observed the transformation of lipid tubes into 3D spring-like structures, where the GTPase activity of FtsZ drives spring compression likely through torsional stress. This allowed us to gain mechanistic insights into the molecular dynamics behind the force generated by FtsZ filaments. Thirdly, we studied the spatiotemporal localization of the division ring by co-reconstituting FtsZ inside lipid vesicles with the MinCDE system, which is involved in positioning the divisome in vivo, and FtsA, the natural tether of FtsZ to the membrane. We achieved the assembly, placement, and onset of constriction of a minimal division ring inside lipid vesicles using two different approaches: purified components or cell-free expression of the MinCDE, FtsA, and FtsZ proteins. This represents a significant advance towards the in vitro reconstitution of functional modules in a synthetic cell and expands our understanding of the molecular mechanism underlying the spatiotemporal organization of the FtsZ-ring. Lastly, we employed biochemical studies combined with cryo-ET visualization to characterize the stabilization of the division ring and the crosslinking of FtsZ filaments by ZapD, a protein known as one of the stabilizers of the divisome. We observed the formation of toroidal structures in solution that are assembled by short FtsZ filaments connected by ZapD and have bacterial size. Their characterization in 3D brings valuable structural information about the FtsZ-ring and its functional stabilization, which is important for its further reconstitution in minimal systems.
In conclusion, this thesis provides important insights into the molecular dynamics of the central protein of division in E. coli and most bacteria, addressing its activity on the membrane, mechanism of force constriction, spatiotemporal localization and stabilization of the FtsZ-ring. Furthermore, we demonstrate significant advancements towards the implementation of FtsZ-based division systems in minimal synthetic cells using a bottom-up approach
Improving persistence of anti-CD19 CAR T cells via overexpression of the IL-21 receptor
Get PDFAdoptive transfer of anti-CD19 chimeric antigen receptor (CAR) T cells has revolutionized the therapy of relapsed/refractory B-cell precursor ALL. High initial remission rates are nevertheless hampered by a lack of long-term persistence due to T cell exhaustion. Sustained proliferation and persistence of less differentiated T cell populations can counteract the effects of exhausted immune cells and confer a superior anti-tumor response to immunotherapy. Of particular interest in this regard is the interaction between interleukin-21, a common gamma chain receptor cytokine, and its receptor complex, IL-21R, which consists of the specific alpha chain and the common gamma chain (γC). The ligand/receptor interplay leads to improved proliferation and the generation of long-lived memory CD8+ T cells. This in vitro study characterizes the role of IL-21R expression in the setting of CAR T cells with the aim of improving long-term persistence of T cell-based immunotherapy.
First, primary T cells with altered expression of the IL-21R alpha chain were examined. A CRISPR/Cas9 genomic knock-out of the receptor had no detrimental effects on T cell functionality, whereas retroviral overexpression of the IL-21R led to slower expansion, a less differentiated phenotype and reduced secretion of pro-inflammatory cytokines in CD8+ cells, even after stimulation with IL-21. Co-expression of the common γC played no additional role in the IL-21/IL-21R interaction in primary T cells.
To examine this effect in a CAR setting, first- and second-generation bicistronic CAR constructs overexpressing the IL-21R alpha chain were compared to conventional CAR T cells. Specific anti-CD19 functionality was observed in all CAR constructs, with direct cytotoxicity and proliferation comparable after stimulation with CD19+ target cells. Interestingly, INF-γ secretion was consistently lower in IL-21R overexpressing CAR T cells, while addition of IL-21 partially reversed this trend. Further experiments focused on the effect of IL-21/IL-21R interaction in a second-generation CAR construct with a 4-1BB co-stimulatory domain. Not surprisingly, CAR T cells with a CRISPR/Cas9 genomic knock-out of IL-21R performed similarly to conventional CAR T cells. In contrast, IL-21R overexpressing CAR T cells demonstrated signs of improved persistence, as they retained a high proliferative capacity despite the rapid increase in effector functions after addition of IL-21 to the co-culture. No signs of early T cell dysfunction were detected, since no marked rise in expression of the co-inhibitory markers TIM-3 and PD-1 was observed. Moreover, the interplay between IL-21 and its receptor in the setting of IL-21R overexpressing CAR T cells led to the increased secretion of pro-inflammatory cytokines as well as IL-10. In a final experiment, IL-21 was overexpressed in CD4+ and IL-21R in CD8+ CAR T cells with the aim of mimicking the physiologic interaction between the two T cell populations. This novel approach led to superior functionality when compared to standard-of-care CAR T cells: the highest IFN-y Secretion was observed when IL-21 was overexpressed in CD4+ CAR T cells while IL21R was expressed in CD8+ CAR T cells.
These promising results underscore the in vitro benefits to T cell functionality after interaction between IL-21 and its receptor, which may improve the long-term persistence of CAR T cell-based immunotherapy. Combining IL-21 producing CD4+ with IL-21R overexpressing CD8+ CAR T cells can help further examine the in vivo persistence of IL-21 receptor overexpressing CAR T cells
Prävention einer beatmungsassoziierten Pneumonie bei Langzeitbeatmung am Universitätsklinikum der Ludwig-Maximilian-Universität München
Get PDFInvestigation on the bioactivities of natural products with the focus on phyllobilins, a novel substance class of bioactive phytochemicals
Get PDFSpin polarization dynamics in perovskite nanocrystals
Get PDFSeit dem kürzlichen Aufkommen von kolloidalen Blei-Halogenid Perowskit Nanokristallen (LHP NCs) begeistern sie die Branche durch ihre faszinierenden optischen Eigenschaften. Sie besitzen daher großes Potenzial optische Anwendungen wie Strahler, Solarenergiekollektoren und Spintronik zu reformieren. CsPbI3, eine anorganische Verbindung unter LHPs, zeigt besonders ausgeprägte Spin-Bahn-Kopplung, was zu signifikanter Feinstrukturspaltung und folglich zu lediglich zweifach entarteten Valenz- (VB) und Leitungsbändern (CB) führt. Hierdurch bestehen perfekte Bedingungen für maximal effiziente optische Spinausrichtung (oS), wobei Ladungsträger durch zirkular polarisierte Strahlung in bekannte VB- und CB-Zustände angeregt werden. Im Gegensatz dazu stehen konventionelle II-VI und III-V Halbleiter, worin sich dieselben auf mehrere VB- und CB-Zustände aufteilen, was die erreichbare oS um 50% gegenüber LHPs verringert. Das Potenzial der enormen Induktion von zirkularem Dichroismus in CsPbI3 und dessen Vielzahl an faszinierenden optischen Eigenschaften macht diese Verbindung zu einem Modellsystem für fundamentale Spinforschung. Das Wissen über Spindynamiken von Ladungsträgern ist ausschlaggebend für ein grundlegendes Verständnis elektronischer Prozesse in dieser Materialklasse.
In dieser Arbeit wird die Dynamik von Ladungsträger-Spinrelaxation (LSR) und dessen zugrundeliegenden theoretischen Mechanismen mittels zeitaufgelöster differenzieller Transmissionsspektroskopie (DTS) beleuchtet. Dabei stellt sich heraus, dass die intrinsisch achiralen NCs beträchtlichen zirkularen Dichroismus kurz nach Anregung durch zirkular polarisierte Laserpulse aufweisen. Darauffolgende LSR gleicht das präparierte Spin-Ungleichgewicht aus. Energetischere optische Anregung bewirkt ein Abkühlen der Ladungsträger zur Bandlücke durch Phononenemission. Dabei entsteht eine große Nicht-Gleichgewichts-Phononenpopulation, welche den Wirkungsquerschnitt der Ladungsträger-Phononenstreuung vergrößert. Der Elliott-Yafet- (EY) Mechanismus, den ich der maßgeblichen LSR in CsPbI3 zuordne, besagt, dass LSR durch Ladungsträger-Phononenstreuung erfolgt. Die gemessene Ensemble-Spinpolarisation veringert sich dementsprechend erheblich mit erhöhter LSR während des Abkühlvorgangs der Ladungsträger. Temperaturabhängige DTS offenbart, dass die LSR-Geschwindigkeit bei Raumtemperatur derer bei kryogenen Temperaturen um eine Größenordnung übersteigt. Entsprechende Raten enthüllen einen klaren und adequaten Zusammenhang jeweils zur Phononenbesetzung und EY-Funktionalität. Der Entzug von Elektronen aus den CsPbI3 NCs durch Beimischung eines Elektron-Absorbermoleküls erlaubt die fast ausschließliche Beobachtung der Loch-Spinrelaxation, welche sich als langsamer, als die der Elektronen erweist.Recently, colloidal lead halide perovskite nanocrystals (LHP NCs) have emerged and impress the community with their intriguing optical properties ever since. They demonstrate great potential to reform optical applications such as light emitting devices, solar energy harvesting and spintronics. Among LHPs, the all-inorganic compound CsPbI3 exhibits particularly strong spin-orbit coupling, leading to significant fine structure splitting, which makes both, valence (VB) and conduction band (CB) only two-fold degenerate. This renders perfect conditions for maximally efficient optical orientation, whereupon charge carriers are excited into precisely known VB and CB states by circularly polarized radiation. This is in contrast to conventional II-VI and III-V semiconductors, where circularly photoexcited charge carriers are distributed among multiple VB and CB states, dropping their maximally achievable optical orientation by as much as 50% compared to LHPs. The potential of photoinducing tremendous circular dichroism into CsPbI3 NCs through optical orientation, in combination with their multiplicity of intriguing optical properties, make them a model system for fundamental spin studies. The knowledge about the spin dynamics of charge carriers is crucial for a profound comprehension of electronic processes in this material class.
In this thesis, charge carrier spin polarization dynamics and underlying theoretical mechanisms are elucidated in colloidal CsPbI3 NCs by employing time-resolved differential transmission spectroscopy (DTS). Thereby, the intrinsically achiral NCs are found to exhibit considerable circular dichroism shortly after excitation with a circularly polarized laser pulse. Subsequent charge carrier spin relaxation equilibrates the prepared spin imbalance. Elevated photoexcitation energy causes charge carriers to cool down to the band gap via phonon emission. Thereby, a large non-equilibrium phonon population develops, increasing the carrier-phonon scattering cross-section. The Elliott-Yafet (EY) mechanism, which I assign to govern spin relaxation in CsPbI3 NCs, predicts that spin relaxation is a consequence of carrier-phonon scattering. Accordingly, the investigated ensemble spin polarization is measured to diminish significantly in the process of carrier cooling at an increased spin relaxation rate. Temperature-dependent DTS reveals that room temperature spin relaxation dynamics are one order of magnitude faster than at cryogenic temperatures. The corresponding rates reveal a clear and adequate correlation to phonon occupation and EY functionality, respectively. The removal of electrons from the CsPbI3 NCs through admixture with an electron scavenger molecule permits the almost exclusive investigation of hole spin relaxation, which is revealed to occur slower compared to that of electrons
The mass composition of massive early-type galaxies
Get PDFEs ist anzunehmen, dass die Vielfalt der Galaxien im lokalen Universum aus sukzessiven Generationen von Galaxienverschmelzungen hervorgegangen ist. Massereiche Ellipsen stehen dabei an der Spitze der Hierarchie der Galaxienverschmelzungen. Außerdem bergen sie die größten supermassereichen Schwarzen Löcher.
Das Szenario der hierarchischen Verschmelzungen kann viele der beobachteten Eigenschaften von Ellipsen erklären. Dennoch bleibt die genaue Zusammensetzung der Massen in diesen Galaxien schleierhaft. Die Massenfunktion lokaler schwarzer Löcher, und insbesondere ihr oberes Ende, sind nicht bekannt. Auch wissen wir nicht, welcher Anteil der Gesamtmasse einer Galaxie den Sternen und welcher der dunklen Materie zuzuschreiben ist, da es hier stets eine unbekannte Fraktion an stellaren Objekten gibt, welche Masse zur Galaxie beitragen, aber kaum oder gar kein Licht. Auf der einen Seite gibt es eine unbekannte Anzahl an lichtschwachen Zwergsternen, und auf der anderen Seite einen unbekannten Bruchteil an Sternen, der zu Relikten kollabiert ist. Die ursprüngliche massen funktion (UMF) der Sterne umfasst diese Information. Verschiedene Studien der UMF haben eine andere UFM in massiven Ellipsen als in weniger massereichen Galaxien wie unserer Milchstraße ermittelt. Doch meistens produzieren verschiedene Methoden widersprüchliche Resultate für dieselben Galaxien.
Auf der Messung nicht-parametrischer Sichtliniengeschwindigkeitsverteilungen (SGV) basierende dynamische Modelle können genützt werden, um Galaxienmassen zu messen und in einzelne Komponenten zu zerlegen. In dieser Dissertation messe ich die nicht-parametrischen SGV von 9 + 1 Ellipsen bis zur Fluchtgeschwindigkeit des jeweiligen Potentials mit unserem Code WINGIFT. Darauf basierend konstruiere ich für acht der Galaxien Schwarzschild Orbit-Modelle.
Dabei präsentiere ich hier die Entdeckung eines von nur vier bisher dynamisch gemessenen Schwarzen Löchern mit M_BH > 10^10 M⊙, sowie zwei empirische Relationen zwischen M_BH und der zentralen Flächenhelligkeit, sowie zentralen Oberflächendichte massiver Ellipsen.
Mit diesen Relationen lässt sich das obere Ende der Massenfunktion lokaler schwarzer Löcher in der Zukunft gezielt erforschen. Für sieben der Galaxien präsentiere ich dynamische Evidenz für interne Gradienten der UMF.
Solche intrinsischen Gradienten der UMF könnten die Diskrepanzen bisheriger auf verschiedenen Methoden basierenden Messungen der UMF lösen.
Die gefundenen Gradienten suggerieren, dass sich in den Zentren von Ellipsen sehr kompakte Regionen vorfinden ≲ 1 kpc, deren stellare Populationen einen höheren Anteil an entweder lichtschwachen Zwergsternen oder Relikten vorweisen als es für Populationen im Rest des Universums der Fall ist.It is thought that most galaxies in the local universe are the outcome of several generations of hierarchical mergers of progenitor galaxies. Massive early-type galaxies (ETGs) occupy the top ranks of this hierarchy. They also harbour the biggest supermassive black holes (SMBHS) in the local universe.
The merger framework can explain many of the observed properties of different kinds of ETGs. However, the exact mass compositions of these objects remains elusive: For once, the local SMBH mass function is poorly understood and barely sampled at the high mass end. We also do not know how much galaxy mass is contributed by stars and how
much by dark matter, because an unknown fraction of stars are low-luminosity dwarf stars, and another unknown fraction of more massive stars have turned into remnants – both of these contribute a significant amount of mass to galaxies, but little or no light. The stellar
initial mass function (IMF) underlying the stellar population(s) of a galaxy encompasses this information. Different studies, using different methods have claimed that the IMF in massive ETGs is different from that of less massive galaxies like the Milky Way. But these results have thus far remained overwhelmingly contradictory on the level of individual galaxies.
Accurate measurements of non-parametric line-of-sight velocity distributions (LOSVDs) in ETGs can be analysed with Schwarzschild orbit models to produce precise galaxy mass decompositions. In this thesis, I measure the full non-parametric shape of LOSVDs all the way to the escape velocity of each galaxy’s gravitation potential for a total of 9 +
1 massive ETGs using our kinematic fitting code WINGFIT. For eight of the galaxies I construct Schwarzschild models based on these kinematics. I present the discovery of one of so far only four SMBHs more massive than 10^10 M⊙ with direct dynamical detections, and two new SMBH-host scaling relations between MBH and the central surface brightness, as well as surface mass-density of massive galaxies. In the future, these empirical relations can be used for a targeted sampling of the high mass end of the local SMBH mass function. For seven of the ETGs, I present dynamical evidence for internal radial gradients of the IMF. Such gradients can potentially explain the contradictions between previous IMF measurements from different methods. These measurements suggest that the centers of ETGs contain very spatially concentrated regions (r ≲ 1 kpc) of stellar populations with an enhanced fraction of either low-luminosity dwarfs or remnants relative to stellar
populations in the rest of the universe
