Naturwissenschaftliche Fakultät

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  • Doctoral thesis
    Open Access
    Analysis of immune cell migration in three dimensional environments
    (2024) Czerwinski, Tina Linda
    The immune system plays a key role in recognising and combating pathogens and tumour cells in the body. To achieve their function, immune cells constantly patrol a connective tissue of densely packed mesenchymal cells and extracellular matrix fibres with pores smaller than the cells, at high migration speeds of 5 - 10 μm/min. Immune cell migration has been extensively studied over the past decades, both in vitro using flat, two dimensional (2D) substrates and three dimensional (3D) matrices, and more recently in living animals using intra-vital imaging techniques. The 3D migratory ability of immune cells is commonly quantified by the migration speed and the directional persistence of cell trajectories. In this thesis, I demonstrate that the motile cell fraction is a vastly more sensitive indicator for the motility of immune cells in health, under pathological conditions and under different treatments, whereas migration speed and directional persistence remain surprisingly stable. The motile fraction indicates the fraction of cells in a population that are moving freely in a 3D matrix. Moreover, the remaining non-motile fraction is still viable and may only be temporarily unable to move. Hence, the motile fraction increases with measurement time. Taken together, this implies in a rather fundamental way that immune cells appear to either migrate at full speed, or they stop moving. In this thesis, I present new methods to accurately quantify the motile fraction of immune cell populations in different 3D matrices. Since the standard error of average values for the measured single cell parameters such as cell speed, persistence and motile fraction scales with the square root of the number of observed cells, an accurate estimate of the motile fraction requires live tracking of a large number of individual cells. Therefore, I developed a high-throughput 3D migration assay to track 1000 to 3000 individual cells per experiment, facilitating the detection of even very small changes in cell behaviour. With this tool in hand, I investigated and uncovered some of the fundamental laws that govern the ability of immune cells to infiltrate dense tissue. To mimic the process of immune cell migration through the connective tissue of different organs, I used biopolymer fibre networks as in vitro models, composed of collagen with tunable mechanical and structural properties, with pore sizes ranging from 3.5 μm to 5.7 μm and stiffness values (shear modulus) ranging from 100 Pa to 1200 Pa. To investigate the underlying cause of the sensitivity of the motile fraction in response to small changes in culture conditions or the microenvironment, I combined the 3D migration assay with high-speed 3D traction force microscopy (TFM). Current understanding suggests that immune cells infiltrate tissue using a so-called amoeboid migration mode, characterised by weak matrix adhesions and low traction forces. While my migration and traction force data confirm that immune cells (B cells, T cells, NK cells, neutrophils and monocytes) predominantly migrate in an amoeboid fashion, these data also demonstrate that the amoeboid migration mode is only used when the cells can move freely through matrix pores. However, when the cells encounter a narrow pore, they avoid getting stuck by switching to a highly contractile, mesenchymal-like mode of migration, which helps to pull the cell body through the narrow pore. Surprisingly, the magnitude of traction forces that immune cells are able to generate can reach values seen previously only for mesenchymal cells such as fibroblasts and cancer cells after endothelial-to-mesenchymal transition (EMT). Accordingly, the motile fraction appears to reflect the ability of an immune cell population to overcome the steric hindrance in a mechanically challenging environment. In support of this hypothesis, I demonstrate that NK92 cells, an NK cell line, as well as ex vivo expanded NK cells and primary NK cells, neutrophils, monocytes, T cells and B cells, all exert short bursts of contractile forces during 3D migration. These short contractile bursts occur predominantly after a cell slowed down on its way through the matrix and is in danger of getting stuck. After increasing their traction forces, cells tend to speed up. Decreasing cellular force generation by inhibiting actomyosin contraction decreases the motile fraction, while increasing cellular force generation by inhibiting microtubule polymerisation increases the motile fraction. I also show that the transmission of force to the extracellular matrix by immune cells is integrin-dependent, further highlighting the similarity of this mode of migration to mesenchymal cell migration. The motile fraction during 3D migration thus provides an indirect but surprisingly accurate and specific readout for the ability of immune cells to switch to a contractile mode of migration to overcome steric hindrance. With these novel insights and methods in hand, I next turned my attention to the development of improved expansion protocols that not only activate immune cells, but also enhance immune cell motility. Regulatory T cells are a prime target for ex vivo expansion because of the low number of these cells circulating in the peripheral blood. My data show that a clinical expansion protocol strongly increases the motility of expanded regulatory T cells by increasing the motile fraction, while cell speed and directional persistence remain nearly unchanged before versus after expansion. This finding strongly supports my earlier findings that a universal property of immune cells is that they are able to achieve the same cell speed and directional persistence under different conditions, but the number of cells that get stuck in the matrix varies significantly. Hence, the motile fraction is a clinically highly relevant and crucial parameter for optimising expansion protocols. Finally, I addressed the question of why current immune cell therapies fail in patients with solid tumours. I measured the motile fraction of NK cells derived from peripheral blood monocytes using a Good Manufacturing Practice (GMP) compliant NK cell expansion protocol. These protocols are used in a clinical context to generate a large, highly activated NK cell population from a patient’s blood sample, with the aim of reinjecting these expanded cells into the patient to enhance the immune response against blood-borne cancers or solid tumours. At the end of the expansion period, the cell population is cryopreserved to bridge the time required for biosafety testing. I found that after cryopreservation, the cytotoxicity (i.e. the killing ability of these cells) as well as the cell speed and directional persistence of motile NK cells is almost unchanged, while the motile fraction is decreased 6-fold. This finding may help explain the persistent failure of NK cell therapy in patients with solid tumours. In summary, the key finding of my thesis is that the default amoeboid migration mode of immune cells can be interrupted by a switch to a highly contractile, mesenchymal-like migration mode when the cell encounters a local microenvironment with a high degree of steric hindrance. This switch helps the cell to avoid getting stuck in the matrix. The ability to overcome the steric hindrance of the matrix can be readily estimated by the motile fraction in 3D migration assays. Together, these insights may be helpful for clinical applications including expansion protocols and novel treatments that target immune cell function.
  • Doctoral thesis
    Open Access
    A Primal-Dual Augmented Lagrangian Method for the Solution of Convex Semidefinite Programming Problems
    (2024) Kavand, Arefeh
    This thesis introduces a Penalty/Barrier Multiplier (PBM) approach within the augmented Lagrangian framework, to address convex semidefinite programming problems characterized by linear objective functions and linear matrix inequality (LMI) constraints. At the core of this work is a comprehensive convergence analysis carried out for this method. In the first part of this thesis, we provide an exploration of preliminaries, including crucial definitions, assumptions, such as the Slater's constraint qualification applied to both our primal and dual SDP problems, as well as the fundamental concepts related to exact penalty functions. Building upon these essential foundations, we introduce the PBM Augmented Lagrangian (PBM-AL) algorithm, which incorporates a specific matrix penalty function. Our analysis uses the established equivalence between the PBM-AL algorithm and the dual Proximal-Point Algorithm (PPA), where the produced multiplier in PBM-AL coincides with the variables of the dual PPA. This alignment is achieved by expressing the proximal term as a non-quadratic distance-like function linked to the Fenchel conjugate of the primal penalty function. This equivalence serves as the basis for our dual convergence analysis for PBM-AL, under the assumption that the sequence of the penalty parameters is a decreasing sequence approaching zero. Subsequently, we delve into the details of the primal convergence analysis. In our ongoing exploration, we are enhancing the stability of the PBM-AL algorithm by integrating a concept for Lagrange multiplier damping. This adjustment is accompanied by a thorough convergence analysis to ensure the preservation of convergence properties in this modified context. We introduce the Fenchel primal and dual optimization pair, which are the unconstrained minimization of the augmented Lagrangian function and the dual proximal-point maximization problem. Two pivotal concepts of weak and strong Fenchel duality emerge from this framework, playing a crucial role in shaping subsequent analyses. Utilizing the principles of Fenchel duality, we establish specific theoretical stopping criteria to enable the avoidance of obtaining an accurate solution for the sub-problems. It is noteworthy that in this scenario, we assume the algorithm initiates with a fully feasible multiplier, thus earning its designation as a theoretical algorithm. Subsequently, we present the convergence analysis of both the dual multiplier sequence generated by this algorithm and its primal counterpart. In our experiments, achieving full precision in solving the Newton-type systems arising within the PBM-AL algorithm, is often so expensive. This led us to explore the possibilities where the algorithm starts without a fully feasible dual variable. To investigate convergence, we introduced an $\ell_1$ penalty function, penalizing violations from equality constraints. Establishing a lower bound for the associated penalty parameter ensures its exactness. Introducing implementable stopping criteria, based on Fenchel duality, we analyze both dual and primal convergence in this setting as well. In order to generate multipliers that meet the so-called early stopping criteria is not possible within the original PBM-AL algorithm. This realization inspired us to introducing the Primal-Dual Augmented Lagrangian method (PDAL). Using this approach, we formulate a hybrid PDAL algorithm, which employs a direct solver to handle the primal-dual Newton system and integrates a hybrid primal-dual update strategy. Motivated by the necessity for iterative solvers to tackle large-scale SDP problems, we delve into the complexity associated with both the PBM-AL and PDAL approaches. Assuming a low-rank assumption for our dual variables, we introduce a practical preconditioner along with a Preconditioned Conjugate Gradient (PCG) into our hybrid PDAL algorithm. To demonstrate the efficiency of our proposed algorithms, the thesis presents numerical results for three distinct problem classes. We evaluate the performance of our hybrid PDAL algorithm by employing direct solvers to handle the Newton system and applying our derived practical stopping criteria. For large-scale problems, we employ iterative solvers with and without a preconditioner, and we assess the hybrid PDAL-PCG algorithm with some heuristic stopping criteria.
  • Doctoral thesis
    Open Access
    Die Rolle von Siglecs in CLL und SLE
    (2024) Röder, Bettina
    CD22 und Siglec-G (bzw. das menschliche Ortholog Siglec-10) sind Mitglieder der Proteinfamilie der Siglecs (Sialic acid binding immunoglobulin-like lectins) und werden hauptsächlich auf B-Zellen exprimiert. Beide fungieren als inhibitorische Co-Rezeptoren des B-Zell-Rezeptors (BZR), indem sie die Calcium Mobilisierung dämpfen. Während die Funktion von CD22 auf konventionellen B2-Zellen zu beobachten ist, beschränkt sich die Wirkung von Siglec-G auf B1a-Zellen. Die Erkrankung der chronisch lymphatischen Leukämie (CLL) im Menschen zeichnet sich durch die klonale Expansion von CD5+ B-Zellen aus. Darüber hinaus ist bekannt, dass die BZR-Signaltransduktion wichtig für die Entstehung und die Aufrechterhaltung der Erkrankung ist. Siglec-G-defiziente Mäuse weisen durch den Verlust des inhibitorischen Rezeptors neben einer höheren BZR-vermittelten Calcium Antwort auch eine vergrößerte CD5+ B1a-Zellpopulation auf. Im Gegensatz dazu ergab die Untersuchung der kürzlich generierten Siglec-G überexprimierenden Mäusen eine signifikante Reduktion von B1a- Zellen in der Milz und im Peritoneum. Daher wurde im Rahmen dieser Arbeit der Einfluss der Expression von Siglec-G auf die CLL-Entwicklung im EμTCL1 Mausmodell analysiert. EμTCL1 transgene Mäuse entwickeln eine CLL-ähnliche Erkrankung, die den Verlauf der unmutierten CLL im Menschen simuliert. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der Verlust von Siglec-G zu einem früheren Ausbruch und einem schwereren Verlauf der CLL-ähnlichen Erkrankung im Mausmodell führt. Dies zeigte sich in einer früheren und stärkeren Expansion von CLL-ähnlichen Zellen (B220low CD5+) im Blut und weiteren Organen wie der Milz oder der Leber in TCL1 x Siglecg-/- Mäusen. Außerdem konnte ein verändertes BZR-Repertoire in diesen Mäusen festgestellt werden. Hierbei wurde ein früheres Auftreten von monoklonalen leukämischen Klonen und ein geringerer Anteil von Phosphorylcholin- und Phosphatidylcholin-bindender Zellen beobachtet. Schließlich führte der Verlust von Siglec-G zu einer kürzeren Überlebensspanne der Versuchstiere. Im Gegensatz dazu konnte durch die etwa fünffache Überexpression von Siglec-G auf B-Zellen die Expansion von CLL-ähnlichen Zellen in diesem Mausmodell deutlich verzögert und die somit die CLL-Entwicklung unterdrückt werden. Dies zeigt sich auch in einem Überlebensvorteil dieser Tiere im Vergleich zu TCL1 transgenen Mäusen. Interessanterweise konnte weiterhin eine Herunterregulation von Siglec-10, dem humanen Ortholog von Siglec-G, auf der Oberfläche humaner CLL-Zellen nachgewiesen werden. Diese Ergebnisse zeigen, dass Siglec-G eine entscheidende Rolle bei der Krankheitsentwicklung von CLL in Mäusen spielt, und legen nahe, dass ein ähnlicher Mechanismus für Siglec-10 bei CLL im Menschen existieren könnte. Im nächsten Schritt wurde untersucht, ob die Deletion von CD22, einem weiteren inhibitorischen Rezeptor auf B-Zellen, einen Einfluss auf die CLL-Entwicklung im Mausmodell hat. Der Verlust von CD22 führt zu ähnlich wie bei Siglec-G-defizienten Mäusen zwar zu einer höheren BZR-Signaltransduktion, aber nicht zu einer vergrößerten CD5+ B1a-Zellpopulation. Außerdem ist bekannt, dass die Expression von CD22 auf CLL- Zellen herunterreguliert ist. Dennoch konnten in TCL1 x CD22-/- Mäusen, trotz der höheren BZR-vermittelten Calcium-Mobilisierung in B-Zellen, keine wesentlichen Unterschiede in der CLL-Entwicklung im Blut nachgewiesen werden. Im Alter von 36 Wochen konnte eine signifikante Reduktion von Leukozyten und Lymphozyten durch hämatologische Untersuchungen festgestellt werden. Darüber hinaus konnte eine Tendenz zu weniger CLL- ähnlichen Zellen in den analysierten Organen beobachtet werden, die sich aber im weiteren Verlauf der Zeit, mit Ausnahme des Knochenmarks, wieder ausgleicht. Zusammengenommen scheint die verstärkte BZR-Signalweiterleitung durch den Verlust der inhibitorischen Co- Rezeptoren CD22 bzw. Siglec-G nicht allein ausschlaggebend für den drastischen Einfluss auf den Krankheitsverlauf zu sein, sondern zusätzlich die durch die Expression von Siglec-G regulierte Größe der CD5+ Vorläuferpopulation. Für ein funktionierendes Immunsystem ist die Unterscheidung zwischen Selbst- und Fremd- Antigenen essenziell. B-Zellen spielen bei der Entwicklung von Autoimmunerkrankungen vor allem durch ihre Fähigkeit zur Produktion von Autoantikörpern eine bedeutende Rolle. Für die auf B-Zellen vorkommenden inhibitorischen Co-Rezeptoren CD22 und Siglec-G, konnte eine wichtige Beteiligung in der Aufrechterhaltung der B-Zell Toleranz nachgewiesen werden. Im zweiten Teil dieser Arbeit wurde der Einfluss der B-Zell-spezifischen Überexpression von CD22 und Siglec-G auf die Entwicklung einer Autoimmunerkrankung untersucht. Die Überexpression von Siglec-G führt im Alter bereits zu einer Reduktion von Keimzentrums-B- Zellen, Plasmazellen und Follikulären T-Helferzellen (Tfh-Zellen). Durch die Injektion von Pristan eine Lupus-ähnliche Erkrankung in diesen Mäusen ausgelöst. In beiden Siglec- überexprimierenden Mauslinen wurde die Entwicklung von Pristan-induziertem Lupus unterdrückt. Diese Mäuse zeigten eine reduzierte Bildung von antinukleären Autoantikörpern (ANA), keine Anzeichen von Proteinurie und weniger Keimzentrums-B-Zellen, Plasmazellenund Tfh-Zellen. Darüber hinaus war dieser Effekt in Siglec-G überexprimierenden Mäusen deutlicher ausgeprägt als durch die Überexpression von CD22. Zusammengefasst konnte in den untersuchten Lupus-ähnlichen Krankheitsmodellen ein präventiver Effekt vor allem durch die Überexpression von Siglec-G aber auch von CD22 nachgewiesen werden.
  • Doctoral thesis
    Open Access
    On the edge of active systems: The theory of bead-spring microswimmers
    (2024) Ziegler, Sebastian
    Microswimmers are autonomous active agents, able to self-propel in viscous fluids. As an example of active systems, microswimmers continuously dissipate energy and thus are not subject to the restrictions of thermal equilibrium. While the emerging complex single-swimmer and collective behaviors have been studied for various systems in experiments, numerically or theoretically, the physical principles governing locomotion in the viscous regime are not yet fully understood. In particular, the impact of a swimmer's elasticity and the resulting altering of the swimming stroke on the swimmer dynamics is not explored in detail and requires further investigation. This thesis, which builds on six peer-reviewed publications [P1 - P6], focuses on bead-spring swimmers consisting of spherical beads connected by harmonic springs and driven by bead forces. In contrast to previous microswimmer models with a prescribed swimming stroke, bead-spring swimmers are able to adapt their stroke in dependence on their environment, which is an important ability of also biological microswimmers. Since the associated equations of motion for bead-spring swimmers are non-linear in the positions of the beads, we have developed a versatile perturbative approach to calculate the swimmer behavior [P1]. It compares very well to numerical results and presents a refinement of previous analytical results in the literature. Inspired by experiments with driving forces prescribed in the laboratory reference frame, we study the behavior of the triangular bead-spring swimmer under external driving forces. This swimmer undergoes translations as well as rotations and relaxes towards one of six stable orientations, which are equidistantly interspersed with unstable steady states [P1]. The stable and unstable states are associated with opposite swimming directions and a puller- and pusher-like dipolar time-averaged flow field, respectively. Employing the perturbative approach, it is generalized that the magnitude of the time-averaged flow field is proportional to the swimming velocity. Even more generally, we show that this relation can be proven for any swimmer with only two internal degrees of freedom being excited [P4]. In the publications [P2, P3], the frequency-dependent swimming velocity of the triangular bead-spring swimmer is compared to that of the magnetocapillary triangular swimmer studied in Lattice-Boltzmann simulations. Both the bead-spring swimmer and the magnetocapillary swimmer in Lattice-Boltzmann simulations show an optimum in the propulsion speed with respect to the frequency of the applied forces or the magnetic field, respectively. However, the optima are associated with different time scales in both systems. In the bead-spring system, inertia is generally neglected, and the time scale associated with maximal velocity emerges from the interplay of viscous drag and spring forces. In the case of the magnetocapillary swimmer, however, the time scales associated with the optimal swimming velocity of the magnetocapillary swimmer are shown to depend strongly on the beads' inertia [P2, P3]. The study of bead-spring swimmers is extended to the collective behavior of two linear three-sphere bead-spring swimmers in publication [P4]. As the main result, two such swimmers benefit mutually in a large fraction of the parameter space due to effects resulting from the time-averaged flow field each swimmer produces. This so-called passive interaction is complemented by active interaction effects, which result from the interplay of the swimmer's own activity with the time-dependent flow fields produced by nearby swimmers. We show that active interactions are particularly relevant for swimmers' rotations due to interactions with other swimmers. The rotations are responsible for the complex long-term trajectories of two interacting swimmers, which can be understood from the instantaneous rotations experienced by the swimmers [P4]. The results for two interacting swimmers in our framework, which uses an analytical expression for the hydrodynamics, are compared to simulations of the bead-spring swimmer using a coupled Lattice-Boltzmann and immersed boundary method [P5]. While a good agreement between both methods is observed for a single swimmer, there is a qualitative mismatch on which of two interacting collinear swimmers moves faster. Preliminary results suggest that this mismatch can be resolved by assuming a time delay associated with flow propagation, known to exist in Lattice-Boltzmann simulations. Studies on single swimmers are eventually extended to the behavior of bead-spring swimmers in viscosity gradients as a common environment of motile bacteria and other microorganisms. To this end, a framework to calculate the mobility matrix for spherical particles in asymptotically small viscosity gradients, based on the Lorentz reciprocal theorem and a reflection method, is presented in [P6]. It is demonstrated that a linear viscosity gradient gives rise to interactions between the particles that do not decay with distance as long as the separation between the particles is smaller than the size of the viscosity gradient by orders of magnitude. While previous studies typically considered infinitely large viscosity gradients, we show that the relative positioning of the particles within the finite-size linear gradients substantially affects their interaction behavior. With these results at hand, the behavior of the triangular bead-spring swimmer is studied within asymptotically small linear viscosity gradients. For an equilateral swimmer with equal beads, the swimmer generally aligns either parallel or anti-parallel to the viscosity gradient. Whether the swimmer eventually swims towards higher viscosity, i.e., behaves viscophilic, or lower viscosity, i.e., behaves viscophobic, depends strongly on the swimmer's parameters. However, the swimmer behaves generally viscophilic and viscophobic in the limits of very stiff and very soft springs, respectively. By exploiting the self-developed versatile perturbative framework for bead-spring swimmers, this thesis significantly advances the understanding of microswimming by linking flow field and swimming velocity as well as providing the relation between stroke-based and elastic force-based swimmers. Furthermore, the emergence of the collective behavior from single-swimmer properties is further elucidated, as well as the impact of swimmer rigidity on its behavior in a viscosity gradient. Beyond the fundamental results obtained, the methods presented in this thesis lay the foundations for future studies. Especially by allowing for a swimmer's response to external stimuli, our approach is well-suited to study, e.g., swimming algae cells or artificial magnetocapillary swimmers on a liquid interface, which will be addressed in the future. Furthermore, the swimmer's responsiveness is a vital ingredient in the design of smart microscopic devices for, e.g., medical applications, and our results might be helpful in their development.
  • Doctoral thesis
    Open Access
    Epitaktische Kobaltoxidfilme auf Au(111): Wachstum, Eigenschaften und Adsorption organischer Moleküle
    (2024) Ammon, Maximilian Michael
    Molekulare Selbstanordnungen auf Oberflächen sind für die Grundlagenforschung faszinierend und potenziell technologisch nutzbar. Vielversprechend ist dabei die Funktionalisierung von Oberflächen mit Porphyrinen, die für metallische Substrate weitgehend verstanden ist. Für Anwendungen wie z.B. die Photovoltaik ist es jedoch notwendig, die Moleküle elektrisch vom Substrat zu entkoppeln. Dies kann unter anderem durch die Verwendung isolierender oder halbleitender Substrate wie Metalloxide erreicht werden. Ein mögliches und relevantes Metalloxid ist Kobaltoxid, das sich in chemischer Hinsicht durch die Variabilität der Oxidationszahlen der Metallionen auszeichnet. Die bekannten Volumenkristalle CoO (Kochsalzstruktur) und Co3O4 (Spinellstruktur) sind Halbleiter. Diese Arbeit beschreibt das epitaktische Wachstum von Kobaltoxid auf der Au(111) Oberfläche, das hauptsächlich mit Rastertunnelmikroskopie und Beugung langsamer Elektronen untersucht wurde. Durch sorgfältige Kontrolle der Präparationsparameter konnte erstmals das epitaktische Wachstum von CoO mit Wurtzitstruktur, CoO mit Kochsalzstruktur und Co3O4 mit Spinellstruktur konsistent beschrieben und gegeneinander abgegrenzt werden. Anschließend wird auf die Adsorption von 5,10,15,20-Tetrakis(p-Cyanophenyl)Porphyrin auf der (100)-Oberfläche von Kochsalz-CoO eingegangen. Kochsalz-CoO(100) kann auf Au(111) als etwa 1 nm dicker, halbleitender Film gewachsen werden, der die Oberfläche nur partiell bedeckt. Dies ist ein ideales Modellsystem, um die Adsorption mit Rastertunnelmikroskopie zu untersuchen. Nach Adsorption bei Raumtemperatur bilden die Moleküle eine mit dem Substrat kommensurable Selbstanordnung, die durch ein Gleichgewicht zwischen Molekül-Molekül- und Molekül-Substrat-Wechselwirkung erklärt werden kann. Mittels Tunnelspektroskopie konnten zwei Molekülvarianten in der Selbstanordnung nachgewiesen werden, von denen die eine durch Metallierung mit Kobaltatomen aus dem Film entsteht.
  • Doctoral thesis
    Open Access
    Deciphering the role of the presenilins Psen1 and Psen2 in intestinal epithelial homeostasis and inflammation
    (2024) Erkert, Lena
    Intestinal epithelial cells (IECs) form a protective barrier against harmful pathogens present in the intestinal lumen, while acting as a communication platform between the microbiome in the lumen and adjacent immune cells in the lamina propria. Tight control of IEC differentiation, proliferation and death is therefore essential to ensure intestinal homeostasis. If the balance between these vitally important processes is disturbed, intestinal pathologies such as inflam-matory bowel diseases (IBD) can result. To date, the pathophysiology of IBD is incompletely understood and requires further investigation. However, several factors have been identified that contribute to disease pathology, including genetic and environmental factors, such as al-terations in the microbiome that induce the recruitment of mucosal immune cells to the in-flamed areas, ultimately leading to barrier defects. In this context, different human studies have already identified various risk genes associated with IBD. Preliminary analysis by our group of RNA sequencing data from inflamed tissue of mice with experimentally induced colitis identified, for the first time, deregulation of several molecules involved in the Alzheimer’s Dis-ease pathway, including the presenilin Psen1 and Psen2. Although presenilins are well studied molecules in the brain due to their association with Alzheimer's disease and their important function in the γ-secretase complex, their role in the gut remains largely unknown. Therefore, the aim of this work was to verify the hypothesis that presenilins play an important role during intestinal homeostasis and under inflammatory conditions. To this end, an IEC-specific Psen1 deletion in mice (Psen1ΔIEC) was established. The absence of Psen1 in IECs had no effect on cell differentiation, proliferation or death, which could be explained by a compensatory overex-pression of Psen2. Based on these findings, an inducible Psen1/Psen2 double knockout (Psen2-/- Psen1iΔIEC) mouse line was established. Mice lacking both presenilins in the intestinal epitheli-um rapidly and severely lost weight and developed spontaneous intestinal inflammation. In addition, Psen2-/- Psen1iΔIEC mice showed a breakdown of the intestinal barrier with bacterial translocation into the lamina propria. However, attempts to rescue the observed phenotype by antibiotic treatment to eliminate the microbiota improved the inflammatory signature but not the wasting disease. Most importantly, Psen2-/- Psen1iΔIEC mice showed a disturbance of IEC differentiation, with a significant upregulation of the number of secretory cells and an almost complete loss of absorptive enterocytes. This alteration in cell composition might be responsi-ble for the observed wasting disease, attributed to compromised nutrient uptake. Molecularly, the present thesis was able to demonstrate that the observed phenotype is mainly the result of a complete blockade of Notch signaling. In conclusion, this work has identified the prese-nilins Psen1 and Psen2 as important molecules in the maintenance of intestinal homeostasis. Their absence results in barrier breakdown with subsequent bacterial translocation, goblet cell hyperplasia and, most importantly, absorptive enterocyte hypoplasia and hence malnutrition.
  • Doctoral thesis
    Open Access
    Modeling, Analysis, and Simulation of Single Droplet Drying in an Acoustic Levitator
    (2024) Doß, Martin
    Acoustic levitation offers the possibility to perform single droplet drying experiments in a non-intrusive manner. More precisely, a standing ultrasound wave is used to counteract gravity and thus levitate the droplet in one of its pressure nodes. Even if acoustic levitation requires no physical contact, one has to be aware of its secondary effects on the drying process: The droplet is deformed by the acoustic radiation pressure and the acoustic streaming affects the heat and mass transfer. In this thesis, we model and simulate the full drying process of a single colloidal droplet in an acoustic levitator. Our mathematical model combines all relevant principles from acoustics, thermodynamics, colloidal rheology, and transport in porous media. The acoustic pressure amplitude is directly computed from the Helmholtz equation taking into account the realistic geometry of the levitation and drying chamber. The evolution of the free droplet surface is governed by the equilibrium of forces between surface tension, acoustic radiation pressure, and gravity. We solve the Stokes equations to derive the acoustic streaming from its limiting velocities at the droplet surface. The drying process of the colloidal droplet is modeled in two stages. During the first drying stage, the droplet contains enough water for the colloids to move freely within its liquid interior. Their local volume fraction is governed by a convection-diffusion equation whose nonlinear diffusivity diverges at random close packing. Once the colloids are randomly close packed, the liquid droplet turns into a wet particle which is regarded as a porous medium. This critical point of time initiates the second drying stage. We apply a distributed evaporation model to capture how the residual water evaporates from within the wet particle after the critical point of time. The evaporation rate is either derived from the thermodynamic equilibrium or non-equilibrium between the saturated and the actual vapor pressure. Taking into account the acoustic streaming, we fully resolve its convective impact on the heat and mass transfer within and around the levitated droplet. From a mathematical perspective, the first drying stage is modeled by a coupled ODE-PDE system for the droplet volume, the temperature distribution, and the vapor mass density. Its well-posedness is analyzed for a spherical pure water droplet under suitable simplifications. First of all, the celebrated d²-law is reproduced from our governing equations without convection. In particular, we derive an implicit formula for the temperature of a spherical droplet evaporating into stagnant air. In the presence of convection, our single droplet evaporation model is shown to be well-posed in the sense that it admits a unique maximal weak solution until the droplet has evaporated completely. Our proof relies on a contraction argument using Banach's fixed-point theorem. The nonlinear coupling arising from the evaporation rate is handled with the method of upper and lower solutions. Finally, we apply the finite element method to perform direct numerical simulations. Depending on the particular focus of our single droplet drying studies, the corresponding simulations are implemented either in FEniCS or COMSOL Multiphysics. The acoustic streaming turns out to have a significant impact on the heat and mass transfer during the first and the second drying stage. First of all, it makes the colloids accumulate at the equator of the levitated droplet. Moreover, the resulting convection in the gas phase accelerates the drying process before and after the critical point of time. Empirical data from the literature are used to validate our model and to determine its effective parameters. In particular, we identify the effective sound pressure level near the levitated droplet from the measured evolution of its equivalent radius. Motivated by pharmaceutical applications, we approximate protein molecules as colloids to simulate their enzymatic inactivation throughout the drying process of a single protein formulation droplet in an acoustic levitator. Reproducing activity measurements from the literature allows us to unravel the underlying denaturation kinetics. The dehydration of the protein molecules turns out to be primarily responsible for their denaturation right after the critical point of time.
  • Doctoral thesis
    Open Access
    Sterically Encumbering Tris(aryloxide)- and Cyclopentadienyl-Supported Uranium Complexes and Their Reactivity
    (2024) Löffler, Sascha
    The abundance of depleted uranium as a waste product of the enrichment process and the relatively high abundance of natural uranium makes it a considerable object of research. Any contribution of fundamental research in uranium (coordination) chemistry helps to understand the unique physicochemical properties and reactivity of the metal and its complexes. This lays the foundation for any beneficial use and application of the uranium complexes in the future. The present thesis consists of four chapters that correlate to four publications. Three chapters (3.2, 3.4, and 3.5) include uranium complexes stabilized by tris(aryloxide) ligands and their reactivity and interactions with the (small) molecules methylcyclohexane, methylene chloride, silver fluoride, water, nitrous oxide, and 9,10-diphenylanthracene-9,10-endoperoxide. Chapter 3.3 focusses on uranium complexes with cyclopentadienyl ligands and their molecular and electronic structures. In chapter 3.2, the intermolecular forces behind a uranium–alkane adduct formation are discussed. This study follows up on a publication in JACS 2003 that reports an alkane coordination to the uranium center of the complex [UIII((Ot-Bu,t-BuAr)3tacn)]. To gain insight into the forces behind the adduct formation, the adduct was resynthesized and subsequently analyzed and characterized by X-ray diffraction measurements at 6 K and a variety of spectroscopic methods. The experimental results served as a benchmark to adjust the theoretical methods and calculations that were used to describe the interactions between the uranium and the alkane. Chapter 3.3 includes bent and linear uranium pentabenzyl cyclopentadienyl complexes. It is shown that the electronic structure changes significantly when going from bent to linear. For this purpose, several bent and linear complexes were synthesized and thoroughly characterized by 1H NMR spectroscopy, CHN elemental analysis, UV/Vis/NIR spectroscopy, and SQUID magnetization measurements. The spectroscopic findings were supported and explained by theoretical studies. Chapter 3.4 deals with the activation of small molecules, such as methylene chloride, silver fluoride, water, and nitrous oxide. The primary focus is on the synthesis of a uranium(IV/V/VI) oxide and a uranium(IV/V) hydroxide series. All complexes are stabilized by the same cyclen-based tris(aryloxide) ligand. This allows for an unprecedented comparison of the oxide and hydroxide complexes, which aids drawing conclusions about the physicochemical properties of the uranium complexes. For this purpose, all compounds were extensively characterized by 1H NMR spectroscopy, CHN elemental analysis, UV/Vis/NIR spectroscopy, IR spectroscopy, cyclic voltammetry, and SQUID magnetization measurements. Chapter 3.5 describes a rare uranium-mediated peroxide activation of 9,10-diphenylanthracene-9,10-endoperoxide. The resulting uranium bis(alkoxide) complex was subsequently reduced to serve as a precursor for the formation of a uranyl complex via photochemical release of diphenyl anthracene. Theoretical calculations indicate that the synthesis of the uranyl complex proceeds through an elusive uranium cis-dioxo intermediate.
  • Doctoral thesis
    Open Access
    Generation of Kerr squeezed light and its application to interferometry
    (2024) Kalinin, Nikolay
    The quantum nature of light limits the precision with which some of its characteristics can be defined. A variety of nonclassical states exists which, in principle, allows to lower some of these limits. Squeezed light states are among the simplest of these nonclassical states, and yet they are very effective. In this thesis, we investigate the generation of squeezed states using the Kerr effect in fibers, and the application of these states to interferometry. In particular, we develop a novel setup for the generation of polarization-squeezed states which is very stable and does not require any active feedback loop. We investigate the range of possible parameters of this system to optimize squeezing, both experimentally and numerically. We suggest a way to apply Kerr squeezed states to interferometry, and implement it in the experiment. For the first time, we demonstrate that it is indeed possible to enhance the sensitivity of an interferometer beyond the shot noise limit with the use of such Kerr squeezed states.
  • Doctoral thesis
    Open Access
    Crystalline organic thin films: from growth to applications in electronic devices
    (2024) Johnson, Manuel
    Functional organic electronic devices such as organic field-effect transistors (OFETs), or-ganic light-emitting devices, or organic photovoltaics represent a promising alternative to expensive Si-based circuitry. To raise their performance to a competitive level, a detailed understanding of electronic states and their dependence on film morphology is required. This thesis therefore investigates the growth dependence, properties and applications of organic semiconducting thin films employing complementary micro-spectroscopic tools taking ad-vantage of electron, X-ray and scanning probes. In the first part of this thesis, structure formation in a confined liquid surrounding using the so-called “floating-lens approach” – a relatively novel tool for organic thin films preparation from saturated solutions at the solvent-liquid interface – is explored. The detailed balance of intermolecular interactions, primarily π-π interactions and extended times for the molecular self-organization yield films of superior structural quality and domain sizes exceeding those of conventional preparation schemes. In this regard, a dialkylated benzothiophene-based compound (C8-BTBT-C8) is used as a model system to demonstrate the advantageous influ-ence of the close-to-equilibrium growth conditions at the solvent-water interface. Utilizing a combination of atomic force microscopy (AFM) and scanning transmission X-ray micros-copy (STXM), the high degree of molecular order and significantly decreased number of defects and domain boundaries in contrast to vacuum processed samples is illustrated. The molecular orientation derived from linear dichroism in near-edge X-ray absorption fine structure (NEXAFS) spectroscopy is in full agreement with electron diffraction tomography (EDT), which allows precise determination of the unit cell structure of the prepared films. The applicability of the floating-lens approach was extended to monoalkylated benzothio-phene derivative (C13-BTBT). Within these studies, well-defined thin films with close-to-upright standing molecules were obtained for this type of compounds. In particular, the ab-sence of molecular orientation contrast in STXM and the pronounced linear NEXAFS di-chroism, are indicative of the high structural quality and 2D extension of the crystalline do-mains. Detailed microscopic investigations by EDT reveal that the molecules arrange in a head-to-head bilayer packing motif. Used as active layer in simple top-contact/bottom-gate OFETs, excellent electrical properties (charge carrier mobility > 2cm2/Vs) are obtained as a result of the long-range 2D crystallinity. The influence of the process parameters, i.e., solvent, droplet volume and time for self-as-sembly on the structure formation is examined on the basis of α,ω-dihexylsexithiophene (α,ω-DH6T). Careful evaluation of differently prepared thin films reveals that aromatic sol-vents as well as sufficient time for self-assembly favor for the formation of long-range or-dered structures and the initial droplet volume can be used to control the final film thickness. Applying cutting-edge diffractive imaging, the structural parameters of bulk crystals are re-vealed already for monolayer films. In multilayer systems, we observe the azimuthal rota-tions of subsequent layers. Astonishingly, the sense of rotation between the first two layers determines the sense of rotation (“chirality”) for the ongoing growth into thicker films. This specific finding is indicative for a strong coupling of the layers via interactions of the termi-nal hexyl chains, which progresses through the intermediate sexithiophene unit. The exper-imental findings are supported by molecular dynamics simulations by D. Vivod (CCC Er-langen): As organic films with high structural definitions favor improved device performances, in-operando studies were considered. In this context, the effect of gate polarization in p-type self-assembled monolayer OFETs is investigated. By comparing NEXAFS spectra in differ-ent transistor modes (on- vs off-state and gate-bias dependences), spectral features are re-vealed for negative gate voltages, while no change is observed for positive gate voltages. These effects are attributed to field-induced charges which are accumulated at the OSC-insulator interface in the on-state of the device. The energetic levels within the band gap are indicative for polaronic states, which is in line with previous findings in optical spectroscopy studies. It is worth noting that the optimization of the probing volume in SAMFETs is indis-pensable for the detection of these polaronic states in conventional electron spectroscopy. The fabrication of a novel high-performance 2D hybrid phototransistor based on a mono-layer MoS2/BTBT-SAM heterojunction is furthermore demonstrated. Its electronic structure is studied using a toolbox of micro-spectroscopic techniques. AFM, Raman and ultraviolet-visible measurements are employed to confirm the successful transfer of MoS2 onto the BTBT-SAM and verify its monolayer nature and Kelvin probe force microscopy (KPFM) is used to gain detailed insights into the charge-transfer under illumination. The experiments reveal an efficient charge separation at the organic-inorganic heterointerface and a localiza-tion of positive charges underneath the MoS2 crystal, which leads to a strong photogating effect, explaining the exceptional high photoresponsivity (475 A/W) of the thus prepared devices. The last project refers to the fabrication of PEDOT:PSS/MWCNT composite electrodes as potential application in OFETs using micro-contact printing (μCP). A precise control of the contact pressure during the printing process is required to produce high-quality electrodes with well-defined interfaces. AFM, scanning and transmission electron microscopy (SEM/TEM) are employed to reveal insights into the micromorphology and internal inter-faces and electrical measurements are conducted to evaluate the performance of the organic electrodes. For BTBT-based OFET structures, we find improved electrical performances (contact resistance, charge carrier injection), for μCP-prepared contacts compared to OFETs with conventional vacuum processed gold contacts.
  • Doctoral thesis
    Open Access
    Perylene Bisimide Cyclophanes and Fullerene Derivatives as Supramolecular Architectures
    (2024) Solymosi, Iris

    The present work deals with perylene bisimide (PBI) based cyclophanes and fullerene derivatives as supramolecular systems. The majority of the molecules were synthesized and characterised, but most importantly the intra- and intermolecular interactions in the different assemblies were investigated, either between macrocyclic PBI building blocks or in complexes consisting of various host and guest molecules. For this purpose, PBI cyclophanes of different sizes were synthesised in a condensation reaction with malonyl dichloride and subsequently isolated (publication 1). The flexible malonate linkers caused the two-membered ring to exhibit different properties compared to the larger macrocycles containing three or four PBI units. The π-π stacking of the two-membered ring was intensively investigated by low-temperature NMR spectroscopy and it was found that the homochiral arrangement of the PBI units is preferred at low temperatures due to a solvent-dependent diastereoselectivity. Furthermore, the two-membered ring was functionalised with fullerene pentakisadducts, thereby modifying the macrocycle's external environment and further enhancing the π-π stacking within the cyclophane. Additionally, the photophysical properties of the cyclophanes and hybrid molecules were measured and compared with one another. After the extensive characterisation in solution, the π-stacking behaviour of the PBI units in the solid state was investigated (publication 2). A slip-stack arrangement of the PBI units could be observed. As a special feature, there are both intramolecular and intermolecular π-π interactions, which lead to the formation of a one-dimensional π-stacking, polymer-like structure. This particular solid state structure could not have been deduced from the properties in solution and from theoretical calculations.

    Moreover, the two-membered ring and a structurally similar open molecule structure with only one PBI unit were functionalised with pristine fullerene C60 and the respective target compounds subsequently characterised (publication 3). For the dumbbell-like molecule with two PBI units, the low-temperature NMR measurements revealed that there is a lower diastereoselectivity in favour of the homochiral arrangement of the PBI units than for the unsubstituted two-membered ring or the hybrid molecule with fullerene pentakisadducts as substituents, which were presented in publication 1. In addition, the influence of the bridging unit on the complexation of the fullerene substituents with [10]cycloparaphenylene ([10]CPP) was investigated in o-DCB. With the help of fluorescence as well as NMR spectroscopies and, especially, ITC titrations it could be shown that the single or double PBI bridge has no significant influence on the enthalpically driven bis-pseudorotaxane formation. However, in other derivatives the bridging unit demonstrated to play a crucial role on the supramolecular interaction. Thus, the complexation behaviour of four other dumbbell molecules with carbohydrate based (isosorbide or isomannide) bridging units and [10]CPP was also investigated (publication 4). Although the four dumbbell molecules have the same molecular weight and very similar electrochemical properties, the isosorbide derivatives are thermally more stable. In the ITC titrations with [10]CPP, characteristic 1:1 ([10]CPPdumbbell molecule) and 2:1 complexes (([10]CPP)2dumbbell molecule) could be detected for the furan-fused dumbbell molecules. In contrast, the fullerene derivatives with cyclopropane rings on the fullerene substituents also formed oligomer-like structures in addition to the complexes mentioned, which was assumed by NMR measurements.

    Besides the dumbbell molecules, the binding affinities and thermodynamic parameters of pristine C60 and C70 with [10]CPP were determined using ITC (publication 5). It was found for the first time that when the fullerenes were saturated with [10]CPP in o-DCB, not only the 1:1 complex ([10]CPPC60/70) but also the 2:1 complex (([10]CPP)2C60/70) was formed. The binding constants for the complexation of C60 and [10]CPP are between Ka = (2.5-3.8) · 105 M-1 and about an order of magnitude lower for C70 and [10]CPP. In addition to the 1:1 complexes, the 2:1 complexes could also be observed in the gas phase, which was further supported by theoretical calculations. Finally, the interaction of the endohedral fullerene [Li+@C60](PF6)- with [10]CPP was investigated by ITC titrations (publication 6). Unlike C60 and C70, the 2:1 complex could only be observed in the gas phase, but not in solution. However, the binding affinity in o-DCB is two orders of magnitude higher than for C60, most likely due to the charge distribution throughout the entire complex and additional charge-transfer interactions.

    These investigations help to gain a deeper understanding of the supramolecular interactions between perylene bisimide units in a predefined arrangement and the host-guest systems based on fullerene derivatives and [10]CPP.

  • Doctoral thesis
    Open Access
    Dissection of astrocyte-mediated cerebral homeostasis in synucleinopathies
    (2024) Schneider, Yanni
    Multiple system atrophy (MSA) represents a rare atypical parkinsonian disorder characterized by a rapid and fatal course currently lacking causal therapies. Clinical features of MSA com- prise classical parkinsonism, cerebellar ataxia and pronounced autonomic failure occurring in the mean of the 6th decade of life. So far, the disease is classified as a sporadic disease with rare cases of familial MSA. Data concerning environmental factors causing the disease is still controversial. Dependent on the clinical manifestation, MSA is subdivided into a parkinsonian or a cerebellar type. Due to the low frequency of the disease and the fact that a definitive diagnosis is based on post mortem, animal models have been developed to mirror the molec- ular and motor phenotype of the disease. The MBP29-haSyn mouse model expresses human α-synuclein (aSyn) under the myelin basic protein (MBP) promotor specifically in oligodendro- cytes thus resembling MSA-related molecular pathology, such as glial cytoplasmic aSyn inclu- sions, severe demyelination, and microgliosis. Moreover, MBP29-haSyn mice display motor impairments, gait instability and a reduced lifespan. So far, comprehensive molecular profiling of astrocytes in MSA is lacking. Astrocytic expres- sion of glial fibrillary acidic protein (GFAP) was assessed as an indicator for astrocytic re- sponse to alterations in the microenvironment of MBP29-haSyn mice. Reactive astrogliosis was observed in the cortex and striatum on protein and RNA level analyzing expression levels of GFAP and vimentin. Moreover, focusing on cortex and striatum, an impaired capacity of upregulation of homeostasis-associated aquaporin-4, growth-associated protein 43, and glu- tamine synthetase was observed in the striatum of MBP29-haSyn-Syn mice compared to cor- tex. Additionally, expression of glutamate reuptake transporters was downregulated in the stri- atum of MBP29-haSyn mice, but not altered in the cortex. Based on these differences, a magnetic-activated cell sorting protocol for astrocyte isolation was adapted to ensure a high yield of pure astrocytes and bulk RNA sequencing was per- formed subsequently. Using these datasets, a transcriptomic landscape was generated high- lighting altered gene expression in astrocytes in the cortex and the striatum of MBP29-haSyn mice. Comprehensive analysis of both astrocyte populations revealed a dichotomous profile of astrocytes in the regions analyzed. Striatal astrocytes exhibit a pronounced immuno-active profile with upregulated pro-inflammatory transcripts involved in cytokine-cytokine interaction, toll-like receptor signaling, and phagocytosis. Conversely, analysis of the cortical astrocyte population demonstrated a decreased expression of transcripts associated with the inflamma- tory response, such as cytokine-cytokine interaction, and chemokine signaling. Since an increased presence of oligodendrocyte-associated transcripts was found upregulat-ed, the cellular components were further analyzed using gene set enrichment. Notably, the results indicated an enrichment of upregulated transcripts predominantly in the myelin sheath and the maintenance of axon implying a supportive role of astrocytes in the cortex of MBP29-haSyn mice. To examine the presence of oligodendroglial transcripts in astrocytes, in situ RNA hybridization was performed targeting Sry-related HMG-box 10 (SOX10) and myelin regulatory factor (MYRF) as prototypical oligodendroglial transcription factor. SOX10 and MYRF were identified in the proximity of astrocytic processes suggesting a potential oligoden-droglia-astroglia interaction in the cortex of MBP29-haSyn mice. Finally, reactive astrogliosis was investigated in human MSA-P patients post mortem. MSA-P patients display a strong reactive astrogliosis in the cortex, the striatum, and the substantia nigra. Moreover, expression of excitatory amino acid transporter 2 (EAAT2) was investigated expand the analysis of astrocytic characteristics. While no general decrease in expression of EAAT2 was detected, astrocytes in MSA-P patients show a re-distribution of EAAT2 from the branches towards the soma. The re-distribution of EAAT2 implies altered astrocyte functions in MSA-P patients, with EAAT2 as a potential target for a more detailed investigation of the glutamate metabolism in MSA. In conclusion, the findings of this thesis strongly demonstrate the heterogeneity of astrocytes within the CNS dependent on the region and the predominant pathology. The MBP29-haSyn mouse model provided a powerful tool to generate a transcriptomic landscape, thus highlight-ing the dynamic characteristic of astrocytes, displaying distinguished molecular profiles de-spite showing a similar upregulation pattern of GFAP. This work highlights the necessity of a comprehensive profiling of astrocytes to unravel the molecular complexity of this cell type to identify dysregulated pathways. Identification of potential interventional targets may be a promising approach to modulate the astrocytic phenotype in MSA-related pathology and thus slowing disease progression.
  • Doctoral thesis
    Open Access
    Energy Storage in Derivatized Norbornadiene/Quadricyclane as Molecular Solar Thermal System Investigated by XPS
    (2024) Hemauer, Felix
    The indispensable transition to renewable energy sources goes hand in hand with appropriate energy storage solutions. Molecular solar thermal (MOST) systems combine the utilization of solar power with the direct storage of the gained energy in a chemical manner. Upon irradiation, an energy-lean compound is converted into its energy-rich photoisomer, whereby the release of the stored energy can be catalytically triggered on demand. The molecule pair norbornadiene (NBD) and quadricyclane (QC) appears promising for MOST-based applications. By suitable derivatization of its molecular framework, the conversion and storage properties are optimized. In particular, the overlap of the absorption profile of NBD with the solar spectrum defines the overall efficiency and requires novel molecular design. Not only the photoconversion yield from NBD to QC is essential for the general applicability, but also the back reaction from QC to NBD must occur in a controlled and efficient way. In this thesis, several 2,3-disubstituted NBD derivatives and their corresponding QC isomers were surveyed on model catalyst surfaces (Pt, Ni, Au) by synchrotron radiation-based X-ray photoelectron spectroscopy (XPS). Specifically, the derivatization included cyano moieties, phenyl-ester substitution, and ester-substituted oxa-NBD/QC of various size. Investigations on the adsorption at low temperatures and subsequent temperature-programmed experiments allowed for information on the respective systems on the molecular level, which enabled the deduction of thermally induced surface reactions. The focus was set on the conditions and the extent of the energy-releasing cycloreversion reactions of the QC derivatives, by which the feasibility of the different molecule and catalyst combinations was assessed.
  • Doctoral thesis
    Open Access
    Myeloid ZEB1 in the gastrointestinal tumor and metastatic microenvironment
    (2024) Fuchs, Kathrin
    Cancer is one of the leading causes of morbidity worldwide. Colorectal (CRC) and pancreatic (PDAC) cancer will account for almost 20% of all expected cancer deaths in 2023. While cancer research mainly focused on the malignant tumor cells for many years, the critical impact of the tumor microenvironment (TME) on tumor progression has recently come into focus. In particular, tumor associated macrophages (TAMs) in the TME play a dual role in influencing clinical outcome of cancer patients, depending on their polarization towards tumor suppressive or supportive subtypes. The transcription factor ZEB1 conveys plasticity to numerous cell types, for example the epithelial-to-mesenchymal transition in tumor cells, which fosters malignant progression. Interestingly, ZEB1 is also present in stromal cells of TME. In this study, we therefore sought to investigate how plasticity factor ZEB1 can alter TAM polarization and functions and thereby affect gastrointestinal tumor development and metastatic colonization. Based on clinical data, we demonstrate the expression of ZEB1 in TAMs in primary tumors and metastases. Employment of a mouse model with conditional homozygous knockout of myeloid Zeb1 (ZEB1LysMDel) revealed no evident impact of myeloid ZEB1 on organ development, tissue and immune homeostasis in mice. In contrast, myeloid ZEB1 depletion resulted in increased tumor growth and tumorgenicity of subcutaneous syngeneic allografts of CRC cell line CMT 93. These results were partially reproduced in CRC cell line MC 38 and PDAC cell line KPCz661. Moreover, metastatic lung colonization of KPCz661 was also considerably enhanced in ZEB1LysMDel compared to control mice. Surprisingly, in vitro characterization of macrophages derived from ZEB1LysMDel mice did not reveal ZEB1 as a master regulator of macrophage polarization transcription, but rather as fine tuner of specific macrophage effectors. Remarkably, we provide evidence that the secretion of cytokines CCL2 and CCL22 by ZEB1 proficient macrophages may be responsible for macrophage-mediated chemotaxis of cytotoxic T cells into the TME. ZEB1 deficient macrophages were incapable of recruiting sufficient cytotoxic T cells into the TME, resulting in diminished tumor cell apoptosis and subsequently increased tumor or metastatic colony burden. Collectively, our data provide evidence for a novel and unanticipated tumor suppressive function of ZEB1 in TAMs by chemokine mediated cytotoxic T cell recruitment. This study reinforces the importance of the complex interactions in the TME and their influence on cancer biology.
  • Doctoral thesis
    Open Access
    Interplay between membrane fluctuations and the kinetics of a membrane-anchored receptor
    (2024) Janeš, Josip Augustin
    The main topic of this dissertation is the interplay between a bendable thin sheet (membrane) and its highly localized pinning. The most general formulation of the problem allows for the reversible breaking and formation of the membrane pinning, with the kinetic rates depending on the dynamics of the membrane. In order to break the problem into more manageable parts, we first investigate the effect of a permanent pinning on the static and dynamic properties of the pinned membrane, such as its shape and fluctuations. After that we explore how are the kinetic rates for the pinning breaking and formation affected by membrane fluctuations. A paradigmatic example that motivates investigation of such problems is a biological membrane anchoring receptors which interact with environmental ligands. The receptor-ligand ”lock-key” interactions are effective only when receptors and their corresponding ligands are in close proximity for a certain amount of time, which in turn depends on the fluctuations of the receptor-anchoring membrane. On the other hand, the formed ligand-receptor bond pins the membrane locally, affecting membrane dynamics and consequently the rate for bond rupture. Of course, the described problem of a bendable sheet locally pinned by a stochastic pinning is more general than the biological case and can be applied to many different systems. However, we will frame our investigations mostly through the biological perspective. Along the way we will build on the corresponding literature which already offers many useful attempts at a general descriptive model. In the General Introduction, we discuss the theoretical frameworks that define the problems we aim to solve. We first review the continuum Canham-Helfrich model of the membrane energetics [1, 2], which describes the membrane by only two coarsegrained parameters; bending rigidity and tension. We then describe the extensions of the model that account for the non-specific interactions of the membrane with the environment, and introduce the Hamiltonian that accounts for the local, specific interactions. After that, we present the Langevin formalism used for modeling membrane dynamics in a hydrodynamic surrounding [3], together with the obtainable analytic solutions for the case of non-specific interactions. Finally, we consider the available models for the receptor-ligand interactions in situations in which at least one of the interacting molecules is anchored to a fluctuating membrane. We start with the frequently used phenomenological models [4, 5] and end with the coarse-grained kinetic rates that account for the effect of membrane fluctuations [6]. Publication P1 treats the classical Canham-Helfrich model of the membrane extended by two additional terms; one representing the non-specific interactions with the environment, and the other representing a localized pinning of the membrane modeling specific receptor-ligand interaction. We assume that the pinning is permanent and immobile and explore its consequences on the thermal equilibrium properties of the membrane, such as its mean shape, spatial two-point correlation function and shape fluctuations. Interestingly, we find that the correlation function of a pinned membrane is proportional to the free-membrane correlation, and consequently that the correlation length of a pinned membrane does not depend on the pinning properties. However, we find a non-monotonous dependence of the correlation length on the membrane tension, indicating that the effect of the pinning cannot be accounted for by a constant effective tension. We explore the corresponding tension regimes of the correlation length and show its universally exponential decay. We use these results to gain insight into the membrane-mediated interactions between two pinnings and find that interactions are present even in the absence of the mean shape deformation, solely due to the effect of thermal fluctuations of the membrane. Publication P2 extends the treatment of the pinned membrane by exploring the membrane dynamics in the context of the Langevin formalism accounting for the hydrodynamic effects of the surrounding solution. We resolve the membrane dynamics by analytically calculating the Green’s function of the differential equation defining the problem. We use this result to focus on the case of thermal forces and calculate the power spectral density (PSD) of a thermally agitated pinned membrane in a hydrodynamic surrounding. We validate the correctness of the analytical result with explicit numerical simulations of the membrane dynamics. We then propose several experimental protocols that can use the derived theoretical results for the extraction of system parameters from the measurements of membrane spatio-temporal dynamics. In publication P3 we tackle the problem of the reversible ligand-receptor (LR) interactions, with the aim to account for the effect of membrane fluctuations on these interactions by developing effective LR reaction rates. First, we give a firstprinciple derivation of the otherwise phenomenologically introduced rates with familiar Bell-Dembo properties [4, 5]. We then assume that the receptor is anchored on a fluctuating membrane and account for the effect of membrane fluctuations by calculating the expected (un)binding rates with respect to the time-independent probability distribution of membrane fluctuations. Based on the fluctuation measurements in non-activated and activated human macrophages and red blood cells, we construct a general model that captures both Gaussian and non-Gaussian fluctuations exhibited by both cell types. Thus, the introduced fluctuation model, which is a convolution of Gaussian and exponential distributions, enables us to model the effect of active fluctuations in a cell-type-independent way. Finally, we show that even the calculated non-Gaussian LR rates have a Bell-Dembo structure in the biologically relevant regime in which the receptor is much stiffer than the membrane. This result emphasizes the robustness of the Bell-Dembo assumptions and gives a framework that unifies the treatment of the effective LR rates under passive and active membrane fluctuations. Work in this dissertation builds a theoretical framework for the investigation of the interplay between membrane fluctuations and the kinetics and affinity of the membrane-anchored receptors. The framework is based on a few simple principles and as such offers well-defined metrics for testing the validity of the corresponding models. Furthermore, it offers a way to bridge the gap between the models of passive and active fluctuations, as well as between different fluctuation sources in the active case, thus tying separate models into a single, coherent picture. All of this should facilitate analytical modeling of complex systems that were out of reach until now, as well as increase the spatio-temporal scales on which the systems can be effectively simulated
  • Doctoral thesis
    Open Access
    Transport phenomena on the nanoscale: from isotropic systems to extreme confinement
    (2024-04-29) Baer, Andreas
    Nanoscale systems, including particles or macromolecules as well as fluids confined to nanochannels and liquid films, are fascinating as they feature a transition between macroscopic continuum hydrodynamics and the intrinsically discrete molecular scale. A wealth of new phenomena arises due to this cross over as the separation of time or length scales between the different components (liquid – confinement – particle) is often not satisfied. The present thesis tackles this transition region focusing on diffusive transport in a series of six peer-reviewed articles [P1] to [P6]. Molecular dynamics (MD) simulations are applied as primary method, that is a unique tool allowing to resolve molecular details and sampling statistical averages on the mesoscopic scale. The thesis starts with addressing the validity of the Stokes-Einstein-Sutherland (SES) equation [P1]. It captures a fundamental relation between the diffusion coefficient of a particle or molecule, its hydrodynamic radius and the surrounding fluid viscosity. The derivation of the SES equation at equilibrium assumes a continuous description of the fluid and a separation of time and length scales: the particle is required to be large and heavy compared to solvent molecules such that the momentum of the particle changes slowly compared to molecular time scales of the fluid. With these conditions violated for a nanoparticle, a breakdown at the nanoscale was often proposed and the latter even confirmed by several MD studies. Contrastingly, most experiments including those of our collaborators confirmed its validity for particles down to 1 nm in diameter. This discrepancy is tackled in the thesis by extensive MD simulations of the C60 buckminsterfullerene diffusing in toluene. This system clearly violates crucial conditions underlying the SES equation, yet the law is restored in simulations in the framework of the linear response to a constant drag force. This explains the success of the experiments that typically rely on the analysis of particle sedimentation when applying the SES equation. Notably, consistent with the Knudsen number, small deviations from perfect stick boundary conditions at the particle interface are required to obtain uniform results in experiments and simulations. The study of bulk systems is extended to understand diffusion in confined liquids. Here the ordering of the solvent at the interface with the solid or the vapour phase may occur on similar length scales as the characteristic length scale of the confinement and the size of the diffusing object. Similarly, the characteristic time scales of diffusion life times of structural fluctuations become comparable to those of interactions of molecules with interfaces and other molecules. The anisotropy due to confinement also requires separate handling of the directions parallel and orthogonal to the confining walls when analysing transport properties. These issues are first tackled in MD simulations of the solvent phase within solid pores or thin films in [P2] to [P4]. Using the analysis tools developed in the PULS group it is possible to show that significant anisotropic oscillations of transport coefficients may take place due to effective interactions with the interfaces. Understanding the behaviour of confined solvents is a prerequisite for the investigation of diffusive transport of nanoparticles as solutes in such confined systems [P5]. Using the fullerenes C60 and C70 in toluene filled alumina pores as model systems, it is shown that an effective diffusion coefficient can be well estimated by measuring the diffusivity in the centre of a pore and at the interface, as well as the transition rates between these two regions. These rates are estimated from the potential of mean force (PMF) of the particle with the solid. This approach is of particular relevance for understanding separation techniques including chromatography, as the direct relation between the effective transport coefficients in the pore and the particle retention time is established. With equilibrium transport properties at the nanoscale extensively analysed, the attention finally shifts towards non-equilibrium systems, specifically addressing the viscosity of water in electric fields [P6]. Due to their dipole moment, water molecules couple to such fields, altering its intrinsic structure as well as relaxation processes in an anisotropic manner. In the extensive analysis of relaxations of thermal excitations and changes in the first and second hydration shell it is possible to study the competition between order imposed by the intrinsic tetrahedral structure and order imposed by the field. It is furthermore possible to assign different modes to time dependent viscosity, each relating to different molecular relaxation processes, and ultimately explain the anisotropic response of the system. The transport phenomena of nanoscale systems tackled in the present thesis demand further research to be conducted in this area. The theoretical foundations of the SES equation are recapped and brought into the context of experiments and simulations, providing a solid framework for studying the diffusion of small dispersed nanoparticles. Relaxing the stick boundary condition demands an in-depth analysis covering various systems and particle sizes to allow for an a priori estimate of the boundary condition, e.g. from the Knudsen number. Furthermore, the study on the transport of dispersions through narrow pores paves the way for establishing a unified view of the relationship between interactions and transport properties of such systems in general. For systems with polar components, the investigations of water in an electric field furthermore provide valuable insights, into how the transport phenomena are altered in the presence of these fields, most relevant for water-filled nanopores. Therefore, the tools and concepts developed in this work shall find applications well beyond the systems studied herein.
  • Doctoral thesis
    Open Access
    Impacts of supraglacial lakes and snowmelt on glacier velocity on the example of the Baltoro Glacier in Pakistan
    (2024) Wendleder, Anna
    Millions of people live along the Indus River in Pakistan and are dependent on the meltwater from the glaciers in the Karakoram. Glacial meltwater also mainly controls the glacier dynamics which is a key information on glacier evolution in a changing climate. Timing and amount of meltwater influences the easonal evolution and the variation of glacier dynamic. Though, the glaciers in the Karakoram are not yet been sufficiently researched to better understand their glacier dynamics and their drivers. In addition, most of the glaciers are covered with an extensive debris cover and thus react more complex to climate changes than glaciers without debris. The focus of this work is to better understand the dynamics of Baltoro Glacier in Pakistan and hydrological drainage within the glacier. The long-term time series of glacier velocity fields derived from multi-mission Synthetic Aperutre Radar (SAR) data with high temporal and spatial resolution enable the monitoring of (intra-)seasonal and annual glacier dynamics. In combination with time series of supraglacial lakes derived from Earth observation data and precipitation and temperature from reanalysis and satellite-based data complex relationships were found between winter precipitation, summer melt, supraglacial lakes, crevasses and glacier acceleration. The high winter precipitation is associated with an acceleration of the glaciers in spring, while the heavy precipitation in spring leads to an increase in supraglacial lakes. The higher temperatures during the early melting season also influenced the formation of lakes and thus the increase in meltwater in the glacier. The mapping of supraglacial lakes is based on an annual resolution and little is known about their seasonal behaviour. The multi-temporal and multi-sensor summer time series made it possible to determine the characteristic filling and discharge periods, the lake area change over the years, and how the seasonal development varies over the years. The supraglacial lakes filled between mid-April to mid-June and drained between mid-June to mid-September and expanded faster than they contracted. A tendency towards the formation of larger lakes (>0.04 km2 ) over time is visible. The combination of the dense and high-temporal time series of supraglacial lakes with the glacier surface velocity, snowmelt, runoff, precipitation, and temperature derived of earth observation and reanalysis data enables to analyze influence of supraglacial lakes and snowmelt and the drivers of glacier velocity. The prolonged period of positive air temperatures in spring affected both snowmelt and supraglacial lake formation. Snow and ice melt had the greatest influence on the spring acceleration and the high glacier velocities in summer. The drainage of the supraglacial lakes caused the glacier acceleration in fall. The influence of melting and drainage of supraglacial lakes on glacier dynamics is therefore surprisingly large, with the former also leading to efficient drainage. Despite the insulation provided by the debris cover on the main branch, the Baltoro glacier is sensitive to temperature rise, leading to additional ice loss.
  • Doctoral thesis
    Open Access
    Grundlegende Untersuchungen zur Entwicklung Bioorthogonaler Reaktionssysteme auf Basis von Olefinmetathesereaktionen
    (2024) Benkert, Tobias
    Carrying out artificial chemical reactions in living cells is one of the greatest challenges in modern chemistry. To date, there are only a few reactions that are able to fulfil the strict constraints of a bioorthogonal reaction. In this work, fundamental studies on a bioorthogonal reaction system based on olefin metathesis have been investigated. Several synthetic routes to different systems were investigated and carried out. Particular attention was paid to the preparation of piperidine-labelled systems for future targeted use in cell lysosomes. Three different approaches were studied in detail and led to the conclusion that the detailed investigation of suitable fluorescent dyes prior to their direct synthesis is a more promising strategy, as no surprising discrepancies arise between the existing and the required properties of the systems for use in cells.
  • Doctoral thesis
    Open Access
    Signaling circuits involved in the selection of high-affinity antigen-specific B cells in the germinal center
    (2024) Kuria, Timothy Chege
    The germinal center (GC) serves as the site for somatic hypermutation, affinity maturation, and the selection of high-affinity B cells. Two selection models have been proposed to take place in the GC: a death-limited model and a birth-limited model. In the simplified death-limited model, low-affinity GC B cells undergo apoptosis, while high-affinity B cells do not. In the more pragmatic birth-limited model, selection signals are mediated through increased upregulation of metabolic factors in high-affinity cells, promoting faster proliferation compared to low-affinity GC B cells. Regardless of the selection model, high-affinity B cells consistently outcompete low-affinity B cells in the GC. However, the signaling circuits that mediate this selection are not yet fully understood. An adoptive experimental approach utilizing a 4-hydroxy-3-nitrophenyl (NP) antibody mouse model (B1-8 mouse model) coupled to a non-responsive NP recipient mouse model was employed. Donor high and low-affinity B cells were competitively transferred into the recipient mice. On days 6 and 9 of the GC response, recipient mice were sacrificed, and the GC response was analyzed. RNA was also extracted from the donor B cells, followed by bulk RNA sequencing and differential gene expression analysis to identify potential genes mediating selection in the GC. High-affinity B cells consistently outcompeted low-affinity B cells, even with a starting ratio of high-affinity to low-affinity B cells of 1:138. Differential expression analysis of low-affinity and high-affinity GC B cells revealed a panel of genes, including the B cell co-receptor, CD72. In multiple follow-up experiments, high-affinity B cells downregulated the expression of CD72 not only at the transcriptome level but also at the protein level. CD72 is expressed on all B cells except in antibody-secreting cells, and the downregulation of CD72 likely serves to prepare high-affinity GC B cells for differentiation into antibody-secreting cells. Additionally, CD72 knock-out B cells were outcompeted by wild-type B cells owing to the lack of CD72-CD100 mediated activation and proliferation. In this thesis, novel genes were identified, including the B cell co-receptor CD72, which may play a role in mediating the GC selection process and differentiation into antibody-secreting cells. We have gained meaningful insights through the possible identification of a new GC selection signaling pathway that CD72 mediates.
  • Doctoral thesis
    Open Access
    Methods to monitor and improve cell viability and behaviour during and after bioprinting
    (2024) Fischer, Lena
    The ultimate goal of biofabrication is to create biologically functional constructs that mimic human tissues and organs in structure and complexity. To achieve this, living cells immersed in biomaterials are printed into three-dimensional (3-D) tissue-constructs using methods such as laser-assisted, inkjet or extrusion-based bioprinting. Due to many advantages, extrusion-based bioprinting is the most commonly used method. However, it inevitably subjects cells to shear stress due to the application of hydrostatic pressure to pass cells through a thin printing needle. This shear stress can cause damage to the cellular plasma membrane and possibly intracellular organelles, which can impair cell survival or functional performance. Despite this inherent problem of extrusion bioprinting, methods to monitor shear stress-induced plasma membrane damages, and to decrease the vulnerability of cells to shear stress are not well established. In this work, a fluorescence-based assay using the styryl dye FM 1-43 was developed to visualize and quantify bioprinting-induced plasma membrane damages. FM 1-43 is incorporated into the outer lipid layer of the plasma membrane without crossing it and becomes fluorescent upon insertion. Following plasma membrane damage, e.g. through shear stress, the dye enters the cell and additionally stains intracellular membrane systems (e.g. mitochondria), resulting in an increased fluorescence intensity. Using FM 1-43 in confocal microscopy and microfluidic setups, it could be confirmed that the fluorescence intensity increases with applied shear stress and resulting cell deformation. Moreover, it could be shown in this work that bioprinting at high pneumatic pressure (6 bar) leads to cellular plasma membrane damages and impaired cell survival. Calcium ions play a fundamental and well-described role in the immediate repair of plasma membrane lesions. Based on this, it was shown in this work that supplementing the bioink with 1.9 mM CaCl2 improved cell survival during bioprinting in all tested bioinks (alginate, GelMA) and cell types (NIH/3T3, A125, MDA-MB-231, hiPSC, HUVEC/Tert2). Importantly, stiffer cells (hiPSC, HUVEC/Tert2) underwent less cell deformation than softer cells (NIH/3T3, A125, MDA-MB-231) at similar shear stress, resulting in less plasma membrane damage and cell death. This result strongly suggests that it is the shear stress-induced cell deformation, rather than the shear stress itself, that results in cell damage and death. In addition to providing tools for visualizing and improving the immediate effects of bioprinting on cell survival, methods for long-term monitoring of cell behavior were developed in this work. Lentiviral genetic reporter constructs for different cellular processes (proliferation, morphology and apoptosis) were generated and stably integrated into different cell types. Notably, bioprinting experiments using NIH/3T3-FUCCI proliferation reporter cells revealed that the application of high shear stress (6 bar) did not impair the proliferation of surviving cells. Moreover, NIH/3T3-tdTomato-Farnesyl morphology reporter cells exhibited increased spreading and elongation in biomaterials containing adhesion motifs (collagen) compared to non-adhesive bioinks (alginate), emphasizing the relevance of efÏcient adhesive functionalization of bioinks. As demonstrated by those experiments, genetically encoded reporter for cell behaviour can be used for the optimization of printing processes as well as the characterization of bioinks, and for live monitoring of the same cells over a longer period of time.