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Institute
- Theodor-Boveri-Institut für Biowissenschaften (93)
- Graduate School of Life Sciences (51)
- Physikalisches Institut (42)
- Medizinische Klinik und Poliklinik II (31)
- Institut für Psychologie (29)
- Institut für Anorganische Chemie (27)
- Institut für Organische Chemie (27)
- Neurologische Klinik und Poliklinik (25)
- Rudolf-Virchow-Zentrum (21)
- Institut für Geographie und Geologie (20)
Schriftenreihe
Sonstige beteiligte Institutionen
- Johns Hopkins School of Medicine (2)
- VolkswagenStiftung (2)
- Bio-Imaging Center Würzburg (1)
- CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - the development agency of the Brazilian Federal Government (1)
- Center for Nanosystems Chemistry (CNC), Universität Würzburg (1)
- DAAD - Deutscher Akademischer Austauschdienst (1)
- Department of Hematology and Oncology, Sana Hospital Hof, Hof, Germany (1)
- Department of Laboratory Medicine and Medicine Huddinge, Karolinska Institutet and University Hospital, Stockholm, Sweden (1)
- Department of Medicine A, University Hospital of Münster, Münster, Germany (1)
- Ernst Strüngmann Institute for Neuroscience in Cooperation with Max Planck Society (ESI) (1)
ResearcherID
- B-4606-2017 (1)
The indepth metabolic profiling of the crude extracts of two African Ancistrocladus species viz. A. likoko from Central Africa and A. abbreviatus from West Africa, resulted in a total of 87 alkaloids among them 54 new ones. All of the compounds were intensely elucidated by 1D and 2D NMR, HRESIMS, as well as chemical and chiroptical techniques.
Among the newly discovered compounds are quinoid naphthylisoquinolines with an ortho-diketone in the naphthalene portion, nor-naphthylisoquinoline alkaloid lacking the always present methyl group at C-1, seco-(ring cleaved) naphthylisoquinolines, and a newly discovered class of natural products called the naphthylisoindolinones.
Some of the compounds displayed strong antitumoral activities against human pancreatic cancer cells and leukemia cells in-vitro.
This work deals with a class of nonlinear dynamical systems exhibiting both continuous and discrete dynamics, which is called as hybrid dynamical system.
We provide a broader framework of generalized hybrid dynamical systems allowing us to handle issues on modeling, stability and interconnections.
Various sufficient stability conditions are proposed by extensions of direct Lyapunov method.
We also explicitly show Lyapunov formulations of the nonlinear small-gain theorems for interconnected input-to-state stable hybrid dynamical systems.
Applications on modeling and stability of hybrid dynamical systems are given by effective strategies of vaccination programs to control a spread of disease in epidemic systems.
In Tissue Engineering, scaffolds composed of natural polymers often show a distinct lack in stability. The natural polymer gelatin is highly fragile under physiological conditions, nevertheless displaying a broad variety of favorable properties. The aim of this study was to fabricate electrospun gelatin nanofibers, in situ functionalized and stabilized during the spinning process with highly reactive star polymer NCO-sP(EO-stat-PO) (“sPEG”). A spinning protocol for homogenous, non-beaded, 500 to 1000 nm thick nanofibers from different ratios of gelatin and sPEG was successfully established. Fibers were subsequently characterized and tested with SEM imaging, tensile tests, water incubation, FTIR, EDX, and cell culture. It was shown that adding sPEG during the spinning process leads to an increase in visible fiber crosslinking, mechanical stability, and stability in water. The nanofibers were further shown to be biocompatible in cell culture with RAW 264.7 macrophages.
G protein-coupled receptor research looks out for new technologies to elucidate the complex
processes of receptor activation, function and downstream signaling with spatiotemporal
resolution, preferably in living cells and organisms. A thriving approach consists in making use
of the unsurpassed properties of light, including its high precision in space and time, noninvasiveness
and high degree of orthogonality regarding biological processes. This is realized
by the incorporation of molecular photoswitches, which are able to effectively respond to light,
such as azobenzene, into the structure of a ligand of a given receptor. The muscarinic
acetylcholine receptors belong to class A GPCRs and have received special attention in this
regard due to their role as a prototypic pharmacological system and their therapeutic potential.
They mediate the excitatory and inhibitory effects of the neurotransmitter acetylcholine and
thus regulate diverse important biological processes, especially many neurological functions in
our brain.
In this work, the application of photopharmacological tool compounds to muscarinic receptors
is presented, consisting of pharmacophores extended with azobenzene as light-responsive
motif. Making use of the dualsteric concept, such photochromic ligands can be designed to bind
concomitantly to the orthosteric and allosteric binding site of the receptor, which is
demonstrated for BQCAAI (M1) and PAI (M2) and may lead to subtype- and functionalselective
photoswitchable ligands, suitable for further ex vivo and in vivo studies.
Moreover, photoswitchable ligands based on the synthetic agonist iperoxo were investigated
extensively with regard to their photochemical behavior and pharmacological profile, outlining
the advantages and challenges of using red-shifted molecular photoswitches, such as tetraortho-
fluoro azobenzene. For the first time on a GPCR it was examined, which impact the
different substitution pattern has on both the binding and the activity on the M1 receptor. Results
show that substituted azobenzenes in photopharmacological compounds (F4-photoiperoxo and
F4-iper-azo-iper) not just represent analogs with other photophysical properties but can exhibit
a considerably different biological profile that has to be investigated carefully.
The achievements gained in this study can give important new insights into the binding mode
and time course of activation processes, enabling precise spatial and temporal resolution of the
complex signaling pathway of muscarinic receptors. Due to their role as exemplary model
system, these findings may be useful for the investigation into other therapeutically relevant
GPCRs.
Since its first experimental implementation in 2005, single-molecule localization microscopy (SMLM) emerged as a versatile and powerful imaging tool for biological structures with nanometer resolution. By now, SMLM has compiled an extensive track-record of novel insights in sub- and inter- cellular organization.\\
Moreover, since all SMLM techniques rely on the analysis of emission patterns from isolated fluorophores, they inherently allocate molecular information $per$ $definitionem$.\\
Consequently, SMLM transitioned from its origin as pure high-resolution imaging instrument towards quantitative microscopy, where the key information medium is no longer the highly resolved image itself, but the raw localization data set.\\
The work presented in this thesis is part of the ongoing effort to translate those $per$ $se$ molecular information gained by SMLM imaging to insights into the structural organization of the targeted protein or even beyond. Although largely consistent in their objectives, the general distinction between global or segmentation clustering approaches on one side and particle averaging or meta-analyses techniques on the other is usually made.\\
During the course of my thesis, I designed, implemented and employed numerous quantitative approaches with varying degrees of complexity and fields of application.\\ \\
In my first major project, I analyzed the localization distribution of the integral protein gp210 of the nuclear pore complex (NPC) with an iterative \textit{k}-means algorithm. Relating the distinct localization statistics of separated gp210 domains to isolated fluorescent signals led, among others, to the conclusion that the anchoring ring of the NPC consists of 8 homo-dimers of gp210.\\
This is of particular significance, both because it answered a decades long standing question about the nature of the gp210 ring and it showcased the possibility to gain structural information well beyond the resolution capabilities of SMLM by crafty quantification approaches.\\ \\
The second major project reported comprises an extensive study of the synaptonemal complex (SNC) and linked cohesin complexes. Here, I employed a multi-level meta-analysis of the localization sets of various SNC proteins to facilitate the compilation of a novel model of the molecular organization of the major SNC components with so far unmatched extend and detail with isotropic three-dimensional resolution.\\
In a second venture, the two murine cohesin components SMC3 and STAG3 connected to the SNC were analyzed. Applying an adapted algorithm, considering the disperse nature of cohesins, led to the realization that there is an apparent polarization of those cohesin complexes in the SNC, as well as a possible sub-structure of STAG3 beyond the resolution capabilities of SMLM.\\ \\
Other minor projects connected to localization quantification included the study of plasma membrane glycans regarding their overall localization distribution and particular homogeneity as well as the investigation of two flotillin proteins in the membrane of bacteria, forming clusters of distinct shapes and sizes.\\ \\
Finally, a novel approach to three-dimensional SMLM is presented, employing the precise quantification of single molecule emitter intensities. This method, named TRABI, relies on the principles of aperture photometry which were improved for SMLM.\\
With TRABI it was shown, that widely used Gaussian fitting based localization software underestimates photon counts significantly. This mismatch was utilized as a $z$-dependent parameter, enabling the conversion of 2D SMLM data to a virtual 3D space. Furthermore it was demonstrated, that TRABI can be combined beneficially with a multi-plane detection scheme, resulting in superior performance regarding axial localization precision and resolution.\\
Additionally, TRABI has been subsequently employed to photometrically characterize a novel dye for SMLM, revealing superior photo-physical properties at the single-molecule level.\\
Following the conclusion of this thesis, the TRABI method and its applications remains subject of diverse ongoing research.
γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.
This dissertation deals with composite-based methods for structural equation models with latent variables and their enhancement. It comprises five chapters. Besides a brief introduction in the first chapter, the remaining chapters consisting of four essays cover the results of my PhD studies.Two of the essays have already been published in an international journal.
The first essay considers an alternative way of construct modeling in structural equation modeling.While in social and behavioral sciences theoretical constructs are typically modeled as common factors, in other sciences the common factor model is an inadequate way construct modeling due to its assumptions. This essay introduces the confirmatory composite analysis (CCA) analogous to confirmatory factor analysis (CFA). In contrast to CFA, CCA models theoretical constructs as composites instead of common factors. Besides the theoretical presentation of CCA and its assumptions, a Monte Carlo simulation is conducted which demonstrates that misspecifications of the composite model can be detected by the introduced test for overall model fit.
The second essay rises the question of how parameter differences can be assessed in the framework of partial least squares path modeling. Since the standard errors of the estimated parameters have no analytical closed-form, the t- and F-test known from regression analysis cannot be directly used to test for parameter differences. However, bootstrapping provides a solution to this problem. It can be employed to construct confidence intervals for the estimated parameter differences, which can be used for making inferences about the parameter difference in the population. To guide practitioners, guidelines were developed and demonstrated by means of empirical examples.
The third essay answers the question of how ordinal categorical indicators can be dealt with in partial least squares path modeling. A new consistent estimator is developed which combines the polychoric correlation and partial least squares path modeling to appropriately deal with the qualitative character of ordinal categorical indicators. The new estimator named ordinal consistent partial least squares combines consistent partial least squares with ordinal partial least squares. Besides its derivation, a Monte Carlo simulation is conducted which shows that the new estimator performs well in finite samples. Moreover, for illustration, an empirical example is estimated by ordinal consistent partial least squares.
The last essay introduces a new consistent estimator for polynomial factor models.
Similarly to consistent partial least squares, weights are determined to build stand-ins for the latent variables, however a non-iterative approach is used.
A Monte Carlo simulation shows that the new estimator behaves well in finite samples.
Polygonum cuspidatum (Japanese knotweed, also known as Huzhang in Chinese), a plant that produces bioactive components such as stilbenes and quinones, has long been recognized as important in traditional Chinese herbal medicine. To better understand the biological features of this plant and to gain genetic insight into the biosynthesis of its natural products, we assembled a draft genome of P. cuspidatum using Illumina sequencing technology. The draft genome is ca. 2.56 Gb long, with 71.54% of the genome annotated as transposable elements. Integrated gene prediction suggested that the P. cuspidatum genome encodes 55,075 functional genes, including 6,776 gene families that are conserved in the five eudicot species examined and 2,386 that are unique to P. cuspidatum. Among the functional genes identified, 4,753 are predicted to encode transcription factors. We traced the gene duplication history of P. cuspidatum and determined that it has undergone two whole-genome duplication events about 65 and 6.6 million years ago. Roots are considered the primary medicinal tissue, and transcriptome analysis identified 2,173 genes that were expressed at higher levels in roots compared to aboveground tissues. Detailed phylogenetic analysis demonstrated expansion of the gene family encoding stilbene synthase and chalcone synthase enzymes in the phenylpropanoid metabolic pathway, which is associated with the biosynthesis of resveratrol, a pharmacologically important stilbene. Analysis of the draft genome identified 7 abscisic acid and water deficit stress-induced protein-coding genes and 14 cysteine-rich transmembrane module genes predicted to be involved in stress responses. The draft de novo genome assembly produced in this study represents a valuable resource for the molecular characterization of medicinal compounds in P. cuspidatum, the improvement of this important medicinal plant, and the exploration of its abiotic stress resistance.
The respiratory system is amongst the most important compartments in the human body. Due to its connection to the external environment, it is one of the most common portals of pathogen entry. Airborne pathogens like measles virus (MV) carried in liquid droplets exhaled from the infected individuals via a cough or sneeze enter the body from the upper respiratory tract and travel down to the lower respiratory tract and reach the alveoli. There, pathogens are captured by the resident dendritic cells (DCs) or macrophages and brought to the lymph node where immune responses or, as in case of MV, dissemination via the hematopoietic cell compartment are initiated. Basic mechanisms governing MV exit from the respiratory tract, especially virus transmission from infected immune cells to the epithelial cells have not been fully addressed before. Considering the importance of these factors in the viral spread, a complex close-to-in-vivo 3D human respiratory tract model was generated. This model was established using de-cellularized porcine intestine tissue as a biological scaffold and H358 cells as targets for infection. The scaffold was embedded with fibroblast cells, and later on, an endothelial cell layer seeded at the basolateral side. This provided an environment resembling the respiratory tract where MV infected DCs had to transmigrate through the collagen scaffold and transmit the virus to epithelial cells in a Nectin-4 dependent manner. For viral transmission, the access of infected DCs to the recipient epithelial cells is an essential prerequisite and therefore, this important factor which is reflected by cell migration was analyzed in this 3D system.
The enhanced motility of specifically MV-infected DCs in the 3D models was observed, which occurred independently of factors released from the other cell types in the models. Enhanced motility of infected DCs in 3D collagen matrices suggested infection-induced cytoskeletal remodeling, as also verified by detection of cytoskeletal polarization, uropod formation. This enforced migration was sensitive to ROCK inhibition revealing that MV infection induces an amoeboid migration mode in DCs. In support of this, the formation of podosome structures and filopodia, as well as their activity, were reduced in infected DCs and retained in their uninfected siblings. Differential migration modes of uninfected and infected DCs did not cause differential maturation, which was found to be identical for both populations. As an underlying mechanism driving this enforced migration, the role of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) was studied in MV-exposed cultures. It was shown in this thesis that MV-infection increased S1P production, and this was identified as a contributing factor as inhibition sphingosine kinase activity abolished enforced migration of MV-infected DCs. These findings revealed that MV infection induces a fast push-and-squeeze amoeboid mode of migration, which is supported by SphK/S1P axis. However, this push-and-squeeze amoeboid migration mode did not prevent the transendothelial migration of MV-infected DCs.
Altogether, this 3D system has been proven to be a suitable model to study specific parameters of mechanisms involved in infections in an in vivo-like conditions.
Herein described are the isolation, structural elucidation, and biological evaluation of highly thrilling monomeric and dimeric new naphthylisoquinoline alkaloids from A. ealaensis. The separation, chiral resolution, and characterization of a series of stereoisomeric 2,3-dihydrobenzofuran neolignans are also reported. The analytical and phytochemical analysis on two Congolese antimalarial herbal drugs is part of the last chapter of the results. In this last case, major concerns on widely used Congolese herbal drugs are discussed.
In addition to bradykinesia and tremor, patients with Parkinson’s disease (PD) are known to exhibit non-motor symptoms such as apathy and hypomimia but also impulsivity in response to dopaminergic replacement therapy. Moreover, a plethora of studies observe differences in electrocortical and autonomic responses to both visual and acoustic affective stimuli in PD subjects compared to healthy controls. This suggests that the basal ganglia (BG), as well as the hyperdirect pathway and BG thalamocortical circuits, are involved in affective processing. Recent studies have shown valence and dopamine-dependent changes in synchronization in the subthalamic nucleus (STN) in PD patients during affective tasks. This thesis investigates the role of dopamine, valence, and laterality in STN electrophysiology by analyzing event-related potentials (ERP), synchronization, and inter-hemispheric STN connectivity. STN recordings were obtained from PD patients with chronically implanted electrodes for deep brain stimulation during a passive affective picture presentation task. The STN exhibited valence-dependent ERP latencies and lateralized ‘high beta’ (28–40 Hz) event-related desynchronization. This thesis also examines the role of dopamine, valence, and laterality on STN functional connectivity with the anterior cingulate cortex (ACC) and the amygdala. The activity of these limbic structures was reconstructed using simultaneously recorded electroencephalographic signals. While the STN was found to establish early coupling with both structures, STN-ACC coupling in the ‘alpha’ range (7–11 Hz) and uncoupling in the ‘low beta’ range (14–21 Hz) were lateralized. Lateralization was also observed at the level of synchrony in both reconstructed sources and for ACC ERP amplitude, whereas dopamine modulated ERP latency in the amygdala. These results may deepen our current understanding of the STN as a limbic node within larger emotional-motor networks in the brain.
Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity
(2019)
Adaptation to alterations in nutrient availability ensures the survival of organisms. In vertebrates, adipocytes play a decisive role in this process due to their ability to store large amounts of excess nutrients and release them in times of food deprivation. In todays western world, a rather unlimited excess of nutrients leads to high-caloric food consumption in humans. Nutrient overload together with a decreased energy dissipation result in obesity as well as associated diseases such as insulin resistance, diabetes, and liver steatosis. Obesity causes a hormonal imbalance, which in combination with altered nutrient levels can aberrantly activate G-protein coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D (PKD) 1 is a DAG effector integrating multiple hormonal and nutritional inputs. Nevertheless, its physiological role in adipocytes has not been investigated so far. In this thesis, evidence is provided that the deletion of PKD1 in adipocytes suppresses lipogenesis as well as the accumulation of triglycerides. Furthermore, PKD1 depletion results in increased mitochondrial biogenesis as well as decoupling activity. Moreover, PKD1 deletion promotes the expression of the β3-adrenergic receptor (ADRB3) in a CCAAT/enhancer-binding protein (C/EBP)-α and δ-dependent manner. This results in elevated expression levels of beige markers in adipocytes in the presence of a β-agonist. Contrarily, adipocytes expressing a constitutive active form of PKD1 present a reversed phenotype. Additionally, PKD1 regulates adipocyte metabolism in an AMP-activated protein kinase (AMPK)-dependent manner by suppressing its activity through phosphorylation of AMPK α1/α2 subunits. Thus, PKD1 deletion results in an enhanced activity of the AMPK complex. Consistent with the in vitro findings, mice lacking PKD1 in adipocytes demonstrate a resistance to high-fat diet-induced obesity due to an elevated energy expenditure caused by trans-differentiation of white into beige adipocytes. Moreover, deletion of PKD1 in murine adipocytes improves systemic insulin sensitivity and ameliorates liver steatosis. Finally, PKD1 levels positively correlate with HOMA-IR as well as insulin levels in human subjects. Furthermore, inhibition of PKD1 in human adipocytes leads to metabolic alterations, which are comparable to the alterations seen in their murine counterparts. Taken together, these data demonstrate that PKD1 suppresses energy dissipation, drives lipogenesis, and adiposity. Therefore, increased energy dissipation induced by several complementary mechanisms upon PKD1 deletion might represent an attractive strategy to treat obesity and its related complications.
Doping plays a decisive role for the functionality of semiconductor-based (opto-)electronic
devices. Hence, the technological utilization of semiconductors necessitates control and a
fundamental understanding of the doping process. However, for low-dimensional systems like
carbon nanotubes, neither concentration nor distribution of charge carriers is currently well known.
The research presented in this thesis investigated the doping of semiconducting carbon nanotubes by spectroscopic methods. Samples of highly purified, intrinsic (6,5) single-wall carbon nanotubes were fabricated using polymer stabilization.
Chapter 4 showed that both electro- and redox chemical $p$-doping lead to identical bleaching,
blueshift, broadening and asymmetry of the S$_1$ exciton absorption band. The similar spectral changes induced by both doping schemes suggest that optical spectra can not be used to infer what process was used for doping. Perhaps more importantly, it also indicates that the distribution of charges and the character of the charge transfer states does not depend on the method by which doping was achieved.
The detailed analysis of the doping-induced spectral changes in chapter 5 suggests that surplus charges are distributed inhomogeneously. The hypothesis of carrier localization is consistent with the high sensitivity of the S$_1$ exciton photoluminescence to additional charge carriers and with the stretched-exponential decay of the exciton population following ultrafast excitation.
Both aspects are in good agreement with diffusion-limited contact quenching of excitons
at localized charges. Moreover, localized charges act – similar to structural defects – as
perturbations to the bandstructure as evidenced by a doping-induced increase of the D-band
antiresonance in the mid-infrared spectrum.
Quantum mechanical model calculations also suggest that counterions play a crucial role in
carrier localization. Counterion adsorption at the nanotube surface is thus believed to induce charge traps of more than 100 meV depth with a carrier localization length on the order of 3 - 4 nm. The doping-induced bleach of interband absorption is accompanied by an absorption increase in the IR region below 600 meV. The observed shift of the IR peak position indicates a continuous transition from localized to rather delocalized charge carriers. This transition is caused by the increase of the overlap of charge carrier wavefunctions at higher charge densities and was modeled by classical Monte-Carlo simulations of intraband absorption.
Chapter 6 discussed the spectroscopy of heavily (degenerately) doped nanotubes, which are
characterized by a Drude-response of free-carrier intraband absorption in the optical conductivity spectrum. In the NIR spectral region, the S$_1$ exciton and X$+^_1$ trion absorption is replaced by a nearly 1 eV broad and constant absorption signal, the so-called H-band. The linear and transient absorption spectra of heavily doped nanotubes suggest that the H-band can be attributed to free-carrier interband transitions.
Chapter 7 dealt with the quantification of charge carrier densities by linear absorption spectroscopy.
A particularly good measure of the carrier density is the S$_1$ exciton bleach. For a
bleach below about 50 %, the carrier density is proportional to the bleach. At higher doping
levels, deviations from the linear behavior were observed. For doping levels exceeding a
fully bleached S$_1$ band, the determination of the normalized oscillator strength f$\text{1st}$ over the
whole first subband region (trion, exciton, free e-h pairs) is recommended for quantification of carrier densities. Based on the nanotube density of states, the carrier density $n$ can be estimated using $n = 0.74\,\text{nm}^{−1} \cdot (1 − f_\text{1st})$.
In the last part of this thesis (chapter 8), the time-resolved spectroelectrochemistry was
extended to systems beyond photostable carbon nanotube films. The integration of a flowelectrolysis cell into the transient absorption spectrometer allows the investigation of in-situ electrochemically generated but photounstable molecules due to a continuous exchange of sample volume. First time-resolved experiments were successfully performed using the dye
methylene blue and its electrochemically reduced form leucomethylene blue.
A compound with a boron-boron triple bond is shown to undergo stepwise hydroboration reactions with catecholborane to yield an unsymmetrical hydro(boryl)diborene and a 2,3-dihydrotetraborane. Abstraction of H– from the latter compound produces an unusual cationic, planar tetraborane with a hydrogen atom bridging the central B2 moiety. Spectroscopic and crystallographic data and DFT calculations support a ‘protonated diborene’ structure for this compound, which can also be accessed via direct protonation of the corresponding diborene.
Bacterial meningitis occurs when blood-borne bacteria are able to penetrate highly specialized brain endothelial cells (BECs) and gain access to the meninges. Neisseria meningitidis (Nm) is a human-exclusive pathogen for which suitable in vitro models are severely lacking. Until recently, modeling BEC-Nm interactions has been almost exclusively limited to immortalized human cells that lack proper BEC phenotypes. Specifically, these in vitro models lack barrier properties, and continuous tight junctions. Alternatively, humanized mice have been used, but these must rely on known interactions and have limited translatability. This motivates the need to establish novel human-based in vitro BEC models that have barrier phenotypes to research Nm-BEC interactions. Recently, a human induced pluripotent stem cell (iPSC) model of BECs has been developed that possesses superior BEC phenotypes and closely mimics the in vivo blood vessels present at the blood-meningeal barrier.
Here, iPSC-BECs were tested as a novel cellular model to study Nm-host pathogen interactions, with focus on host responses to Nm infection. Two wild type strains and three mutant strains of Nm were used to confirm that these followed similar phenotypes to previously described models. Importantly, the recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, at distinct time points of infection, and the secretion of IFN γ and RANTES by iPSC-BECs. Nm was directly observed to disrupt tight junction proteins ZO-1, Occludin, and Claudin-5 at late time points of infection, which became frayed and/or discontinuous upon infection. This destruction is preceded by, and might be dependent on, SNAI1 activation (a transcriptional repressor of tight junction proteins). In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability was observed at late infection time points. Notably, bacterial transmigration correlated with junctional disruption, indicating that the paracellular route contributes for bacterial crossing of BECs. Finally, RNA-Sequencing (RNA-Seq) of sorted, infected iPSC-BECs was established through the use of fluorescence-activated cell sorting (FACS) techniques following infection. This allowed the detection of expression data of Nm-responsive host genes not previously described thus far to play a role during meningitidis.
In conclusion, here the utility of iPSC-BECs in vitro to study Nm infection could be demonstrated. This is the first BEC in vitro model to express all major BEC tight junctions and to display high barrier potential. Altogether, here this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes and suggests that the paracellular route contributes to Nm traversal of BECs.
Social attention is a ubiquitous, but also enigmatic and sometimes elusive phenomenon.
We direct our gaze at other human beings to see what they are doing
and to guess their intentions, but we may also absorb social events en passant as
they unfold in the corner of the eye. We use our gaze as a discrete communication
channel, sometimes conveying pieces of information which would be difficult
to explicate, but we may also find ourselves avoiding eye-contact with others in
moments when self-disclosure is fear-laden. We experience our gaze as the most
genuine expression of our will, but research also suggests considerable levels of
predictability and automaticity in our gaze behavior. The phenomenon’s complexity
has hindered researchers from developing a unified framework which can
conclusively accommodate all of its aspects, or from even agreeing on the most
promising research methodologies.
The present work follows a multi-methods approach, taking on several aspects
of the phenomenon from various directions. Participants in study 1 viewed dynamic
social scenes on a computer screen. Here, low-level physical saliency (i.e.
color, contrast, or motion) and human heads both attracted gaze to a similar extent,
providing a comparison of two vastly different classes of gaze predictors in
direct juxtaposition. In study 2, participants with varying degrees of social anxiety
walked in a public train station while their eye movements were tracked. With
increasing levels of social anxiety, participants showed a relative avoidance of gaze
at near compared to distant people. When replicating the experiment in a laboratory
situation with a matched participant group, social anxiety did not modulate
gaze behavior, fueling the debate around appropriate experimental designs in the
field. Study 3 employed virtual reality (VR) to investigate social gaze in a complex
and immersive, but still highly controlled situation. In this situation, participants
exhibited a gaze behavior which may be more typical for real-life compared to laboratory situations as they avoided gaze contact with a virtual conspecific unless
she gazed at them. This study provided important insights into gaze behavior in
virtual social situations, helping to better estimate the possible benefits of this
new research approach. Throughout all three experiments, participants showed
consistent inter-individual differences in their gaze behavior. However, the present
work could not resolve if these differences are linked to psychologically meaningful
traits or if they instead have an epiphenomenal character.
Functional analysis of polarization and podosome formation of murine and human megakaryocytes
(2019)
In mammals, blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MK) that extend polarized cell protrusions (proplateles) into BM sinusoids. Proplatelet formation (PPF) requires substantial cytoskeletal rearrangements that have been shown to involve the formation of podosomes, filamentous actin (F-actin) and integrin-rich structures. However, the exact molecular mechanisms regulating MK podosome formation, polarization and migration within the BM are poorly defined. According to current knowledge obtained from studies with other cell types, these processes are regulated by Rho GTPase proteins like RhoA and Cdc42.
In this thesis, polarization and podosome formation were investigated in MKs from genetically modified mice, as well as the cell lines K562 and Meg01 by pharmacological modulation of signaling pathways.
The first part of this thesis describes establishment of the basic assays for investigation of MK polarization. Initial data on polarization of the MK-like erythroleukemia cell line K562 revealed first insights into actin and tubulin dynamics of wild type (WT) and RhoA knock-out (RhoA-/-) K562 cells. Phorbol 12-myristate 13-acetate (PMA)-induction of K562 cells led to the expected MK-receptor upregulation but also RhoA depletion and altered polarization patterns.
The second part of this thesis focuses on podosome formation of MKs. RhoA is shown to be dispensable for podosome formation. Cdc42 is revealed as an important, but not essential regulator of MK spreading and podosome formation. Studies of signaling pathways of podosome formation reveal the importance of the tyrosine kinases Src, Syk, as well as glycoprotein (GP)VI in MK spreading and podosome formation.
This thesis provides novel insights into the mechanisms underlying polarization and podosome formation of MKs and reveals new, important information about cytoskeletal dynamics of MKs and potentially also platelets.
Protein kinase A (PKA) is the main effector of cyclic-adenosine monophosphate (cAMP) and plays an important role in steroidogenesis and proliferation of adrenal cells. In a previous study we found two mutations (L206R, 199_200insW) in the main catalytic subunit of protein kinase A (PKA C) to be responsible for cortisol-producing adrenocortical adenomas (CPAs). These mutations interfere with the formation of a stable holoenzyme, thus causing constitutive PKA activation. More recently, we identified additional mutations affecting PKA C in CPAs associated with overt Cushing syndrome: S213R+insIILR, 200_201insV, W197R, d244 248+E249Q, E32V.
This study reports a functional characterization of those PKA Cmutations linked to CPAs of Cushing’s patients. All analyzed mutations except for E32V showed a reduced interaction with at least one tested regulatory (R) subunit. Interestingly the results of the activity differed among the mutants and between the assays employed. For three mutants (L206R, 199_200insW, S213R+insIILR), the results showed enhanced translocation to the nucleus. This was also observed in CRISPR/Cas9 generated PRKACA L206R mutated HEK293T cells. The enhanced nuclear translocation of this mutants could be due to the lack of R subunit binding, but also other mechanisms could be at play. Additionally, I used an algorithm, which predicted an effect of the mutation on substrate specificity for four mutants (L206R, 199_200insW, 200_201insV, d244 248+E249Q). This was proven using phosphoproteomics for three mutants (L206R, 200_201insV, d244 248+E249Q). In PRKACA L206R mutated CPAs this change in substrate specificity also caused hyperphosphorylation of H1.4 on serine 36, which has been reported to be implicated in mitosis. Due to these observations, I hypothesized, that there are several mechanisms of action of PRKACA mutations leading to increased cortisol secretion and cell proliferation in adrenal cells: interference with the formation of a stable holoenzyme, altered subcellular localization and a change in substrate specificity. My data indicate that some PKA C mutants might act via just one, others by a combination of these mechanisms. Altogether, these findings indicate that several mechanisms contribute to the development of CPAs caused by PRKACA mutations. Moreover, these findings provide a highly illustrative example of how alterations in a protein kinase can cause a human disease.
The Myb-MuvB (MMB) multiprotein complex is a master regulator of cell cycle-dependent gene expression. Target genes of MMB are expressed at elevated levels in several different cancer types and are included in the chromosomal instability (CIN) signature of lung, brain, and breast tumors.
This doctoral thesis showed that the complete loss of the MMB core subunit LIN9 leads to strong proliferation defects and nuclear abnormalities in primary lung adenocarcinoma cells. Transcriptome profiling and genome-wide DNA-binding analyses of MMB in lung adenocarcinoma cells revealed that MMB drives the expression of genes linked to cell cycle progression, mitosis, and chromosome segregation by direct binding to promoters of these genes. Unexpectedly, a previously unknown overlap between MMB-dependent genes and several signatures of YAP-regulated genes was identified. YAP is a transcriptional co-activator acting downstream of the Hippo signaling pathway, which is deregulated in many tumor types. Here, MMB and YAP were found to physically interact and co-regulate a set of mitotic and cytokinetic target genes, which are important in cancer. Furthermore, the activation of mitotic genes and the induction of entry into mitosis by YAP were strongly dependent on MMB. By ChIP-seq and 4C-seq, the genome-wide binding of MMB upon YAP overexpression was analyzed and long-range chromatin interaction sites of selected MMB target gene promoters were identified. Strikingly, YAP strongly promoted chromatin-association of B-MYB through binding to distal enhancer elements that interact with MMB-regulated promoters through chromatin looping.
Together, the findings of this thesis provide a so far unknown molecular mechanism by which YAP and MMB cooperate to regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.
In recent years many discoveries have been made that reveal a close relation between quantum information and geometry in the context of the AdS/CFT correspondence. In this duality between a conformal quantum field theory (CFT) and a theory of gravity on Anti-de Sitter spaces (AdS) quantum information quantities in CFT are associated with geometric objects in AdS. Subject of this thesis is the examination of this intriguing property of AdS/CFT. We study two central elements of quantum information: subregion complexity -- which is a measure for the effort required to construct a given reduced state -- and the modular Hamiltonian -- which is given by the logarithm of a considered reduced state.
While a clear definition for subregion complexity in terms of unitary gates exists for discrete systems, a rigorous formulation for quantum field theories is not known.
In AdS/CFT, subregion complexity is proposed to be related to certain codimension one regions on the AdS side.
The main focus of this thesis lies on the examination of such candidates for gravitational duals of subregion complexity.
We introduce the concept of \textit{topological complexity}, which considers subregion complexity to be given by the integral over the Ricci scalar of codimension one regions in AdS. The Gauss-Bonnet theorem provides very general expressions for the topological complexity of CFT\(_2\) states dual to global AdS\(_3\), BTZ black holes and conical defects. In particular, our calculations show that the topology of the considered codimension one bulk region plays an essential role for topological complexity.
Moreover, we study holographic subregion complexity (HSRC), which associates the volume of a particular codimension one bulk region with subregion complexity. We derive an explicit field theory expression for the HSRC of vacuum states. The formulation of HSRC in terms of field theory quantities may allow to investigate whether this bulk object indeed provides a concept of subregion complexity on the CFT side. In particular, if this turns out to be the case, our expression for HSRC may be seen as a field theory definition of subregion complexity. We extend our expression to states dual to BTZ black holes and conical defects.
A further focus of this thesis is the modular Hamiltonian of a family of states \(\rho_\lambda\) depending on a continuous parameter \(\lambda\). Here \(\lambda\) may be associated with the energy density or the temperature, for instance.
The importance of the modular Hamiltonian for quantum information is due to its contribution to relative entropy -- one of the very few objects in quantum information with a rigorous definition for quantum field theories.
The first order contribution in \(\tilde{\lambda}=\lambda-\lambda_0\) of the modular Hamiltonian to the relative entropy between \(\rho_\lambda\) and a reference state \(\rho_{\lambda_0}\) is provided by the first law of entanglement. We study under which circumstances higher order contributions in \(\tilde{\lambda}\) are to be expected.
We show that for states reduced to two entangling regions \(A\), \(B\) the modular Hamiltonian of at least one of these regions is expected to provide higher order contributions in \(\tilde{\lambda}\) to the relative entropy if \(A\) and \(B\) saturate the Araki-Lieb inequality. The statement of the Araki-Lieb inequality is that the difference between the entanglement entropies of \(A\) and \(B\) is always smaller or equal to the entanglement entropy of the union of \(A\) and \(B\).
Regions for which this inequality is saturated are referred to as entanglement plateaux. In AdS/CFT the relation between geometry and quantum information provides many examples for entanglement plateaux. We apply our result to several of them, including large intervals for states dual to BTZ black holes and annuli for states dual to black brane geometries.