Biologische Hochschulschriften (Goethe-Universität)
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Institute
Tissue size regulation is critical for the normal functioning of the organ as well as to prevent unwanted pathogenesis such as cancer. The Hippo signaling pathway is well known for its robust regulation of tissue growth by the negative regulation of its nuclear effectors YAP1 and WWTR1. In this study, I have described the role of Yap1/Wwtr1 in zebrafish development, with a primary emphasis on the cardiovascular system.
I have generated zebrafish yap1 and wwtr1 mutants by CRISPR/CAS9. The mutant alleles are likely to be nonfunctional due to a premature stop codon and they show evidence of nonsense-mediated decay. Given that Yap1 and Wwtr1 are closely related proteins and have overlapping functions, I am given the opportunity to perform combinatorial analysis of the mutations on zebrafish development. Together with molecular probing tools, high-throughput sequencing and high-resolution imaging, I showed that
1. Double yap1;wwtr1 mutants exhibit severe posterior elongation phenotype, but somitogenesis appears to proceed as usual.
2. Yap1 and Wwtr1 may play an important role in PCV development and secondary angiogenic sprouting. However, key experiments will be needed to elucidate the direct role of Yap1 and Wwtr1 on these processes.
3. wwtr1-/- larvae hearts have a reduction in trabeculation, but in mosaic WT hearts, mutant cardiomyocytes prefer to populate the trabecular layer. My studies revealed that the mutant compact wall could not support trabeculation, which explains the hypotrabeculation phenotype of wwtr1-/- hearts. Additionally, Wwtr1 is required for myocardial Notch activity and can inhibit compact wall cardiomyocytes from entering the trabecular layer.
In summary, the Hippo signaling pathway, through Yap1/Wwtr1 has important regulatory functions in growth control. My work has revealed a surprising role for Yap1/Wwtr1 in tissue morphogenesis such as posterior tail morphogenesis and specific developmental processes of the cardiovascular system. It will be of interest to elucidate the regulation of Yap1/Wwtr1 in individual cells that translates into the complex cellular behaviors that drives morphogenesis.
Gepulste dipolare EPR-Spektroskopie ist eine wertvolle Methode, um Abstände von 1.5 bis 10 nm zwischen zwei Spinmarkern zu messen. Diese Information kann für Strukturbestimmungen hilfreich sein, wo traditionelle Methoden wie Kristallstrukturanalyse und NMR nicht angewendet werden können. Zusätzlich ist es möglich, Änderungen in Konformation und Flexibilität zu verfolgen. Für diese Studien haben sich stabile Nitroxidradikale als Spinmarker etabliert. Diese werden spezifisch durch die site-directed spin labelling Methode (SDSL) kovalent an das zu untersuchende Biomolekül gebunden. In den letzten Jahren wurden weitere Spinmarker für Abstandsbestimmungen mittels EPR-Spektroskopie entwickelt. Besonders interessant sind Triarylmethylradikale (im Folgenden abgekürzt als Trityl) und paramagnetische Metallzentren.
Im Vergleich zu Nitroxidradikalen hat das Tritylradikal einige Vorteile: Eine höhere Stabilität in einer reduzierenden Umgebung wie im Inneren von Zellen, längere Elektronenspin-Relaxationszeiten bei Raumtemperatur und ein schmaleres EPR-Spektrum. Deswegen ist dieses organische Radikal ein alternativer Spinmarker, der besonders gut für die Forschung von Biomolekülen in einer nativen Umgebung unter physiologischen Bedingungen geeignet ist. Auch paramagnetische Metallzentren sind weniger reduktionsempfindlich als Nitroxidradikale. Zusätzlich sind diese Spinmarker interessant in biologischen Fragestellungen. Zum Beispiel besitzen zahlreiche Enzyme paramagnetische Manganzentren als Cofaktoren. Zudem kann Magnesium, ein wesentlicher Cofaktor in Enzymen, Nukleinsäuren und Nukleotid-Bindungsdomänen der G- und Membranproteine, oft durch das paramagnetische Mangan ersetzt werden. Um Abstandsmessungen an Biomolekülen, die nur ein Metallzentrum besitzen, durchzuführen, können zusätzliche Spinmarker in Form eines Nitroxid-, Tritylradikals oder eines anderen paramagnetischen Metallkomplexes mithilfe der SDSL-Methode kovalent gebunden werden.
Nitroxidradikale, Tritylradikale und Metallzentren haben deutlich unterschiedliche EPR-spektroskopische Eigenschaften, welche oft als orthogonale Spinmarker bezeichnet werden. Solche Spinmarker sind nützlich für die Untersuchung von verschiedenen Untereinheiten bei makromolekularen Komplexen. Somit können die intramolekularen Abstände innerhalb einer Untereinheit sowie intermolekularen Abstände zwischen den unterschiedlichen Untereinheiten mit nur einer einzigen Probe bestimmt werden. Zusätzlich können die orthogonalen Marker sehr effektiv genutzt werden, um Metallzentren in Biomolekülen mithilfe der Trilateration-Strategie genau zu lokalisieren.
Die hier vorliegende Doktorarbeit beschäftigt sich mit der Nutzung dieser neuen Spinmarker für Abstandsmessungen. Solche Spinmarker sind noch kaum erforscht, obwohl sie für biologische Anwendungen eine große Rolle spielen könnten.
Das erste Ziel dieser Doktorarbeit war eine Studie über Tritylradikale mithilfe der dipolaren EPR-Spektroskopie. Zu diesem Zweck wurden sowohl double quantum coherence (DQC) und single frequency technique for refocussing dipolar couplings (SIFTER) Experimente als auch Hochfrequenz pulsed electron electron double resonance (PELDOR) Experimente mit einem Trityl-Modellsystem durchgeführt. Dabei wurden die Besonderheiten der unterschiedlichen dipolaren Spektroskopiemethoden mit diesem Spinmarker untersucht, um die Empfindlichkeit und Robustheit für die Abstandsmessungen zu optimieren.
Das zweite Ziel war eine Studie über den Einfluss der Hochspin-Multiplizität des Mangans auf die Abstandsbestimmungen. Für diesen Zweck wurde zuerst ein Modellsystem mit einem orthogonalen Mn2+ Ion und Nitroxidradikal mithilfe der PELDOR-Spektroskopie untersucht. Anschließend wurde ein weiteres Modellsystem mit zwei Mn2+-Ionen untersucht, um PELDOR und relaxation-induced dipolar modulation enhancement (RIDME) Experimente bezüglich ihrer Empfindlichkeit und Robustheit sowie Genauigkeit der Datenanalyse zu optimieren.
Das Trityl-Modellsystem wurde in der Arbeitsgruppe von Prof. Sigurdsson synthetisiert. Die EPR Messungen wurden bei zwei verschiedenen Mikrowellenfrequenzen (34 und 180 GHz) durchgeführt. Es wurde gezeigt, dass die Auswahl der optimalen Methode von den EPR-spektroskopischen Eigenschaften des Systems bei den jeweiligen Mikrowellenfrequenzen abhängig ist. Das EPR-Spektrum des Trityls ist bei 34 GHz so schmal, dass das ganze Spektrum von einem üblichen Mikrowellenpuls angeregt werden kann. In diesem Fall sind die DQC und SIFTER Experimente am besten geeignet. Der mit diesen Methoden bestimmte Abstand von 4.9 nm ist in guter Übereinstimmung mit Werten aus der Literatur. Es wurde festgestellt, dass die SIFTER Messung eine höhere Empfindlichkeit als DQC besitzt, da das Signal-zu-Rausch Verhältnis um den Faktor vier größer ist. Außerdem ist die SIFTER-Methode experimentell weniger anspruchsvoll, da ein deutlich kürzerer Phasenzyklus für die Mikrowellenpulse benötigt wird. ...
Research in cell and developmental biology requires the application of three-dimensional model systems that reproduce the natural environment of cells. Processes in developmental biology are therefore studied in entire systems like insects or plants. In cell biology, three-dimensional cell cultures (e.g. spheroids or organoids) model the physiology and pathology of cells, tissues or organs. In all systems, the cellular neighborhood and interactions, but also physicochemical influences, are realistically presented. The production and handling of these model systems is rather simple and allows for reproducible characterization.
Confocal and light sheet-based fluorescence microscopy (LSFM) enable the observation of these systems while maintaining their three-dimensional integrity. LSFM is applicable to imaging live samples at high spatio-temporal resolution over long periods of time. The quality of the acquired datasets enables the extraction of quantitative features about morphology, functionality and dynamics in the context of the complete system. This approach is referred to as image-based systems biology. Exploiting the potential of the generated datasets requires an image analysis pipeline for data management, visualization and the retrieval of biologically meaningful values.
The goal of this thesis was to identify, develop and optimize modules of the image analysis pipeline. The modules cover data management and reduction, visualization, reconstruction of multiview image datasets, the segmentation and tracking of cell nuclei and the extraction of quantitative features. The modules were developed in an application-driven manner to test and ensure their applicability to real datasets from three-dimensional fluorescence microscopy. The underlying datasets were taken from research projects in developmental biology in insects and plants, as well as from cell biology.
The datasets acquired in fluorescence microscopy are typically complex and require common image processing steps in order to manage, visualize, and analyze the datasets. The first module accomplishes automatic structuring of large image datasets, reduces the data amount by image cropping and compression and computes maximum projection images along different spatial directions. The second module corrects for intensity variations in the generated maximum projection images that occur as a function of time. The program was published as a part of an article in Nature Protocols. Another developed module named BugCube provides a web-based platform to visualize and share the processed image datasets.
In LSFM, samples can be rotated in-between two acquisitions enabling the generation of multiview image datasets. Prior to my work, Frederic Strobl and Alexander Ross acquired the complete embryogenesis of the red flour beetle, Tribolium castaneum, and the field cricket, Gryllus bimaculatus, with LSFM. I evaluated a plugin for the software FIJI as a module for the reconstruction of such datasets. The plugin was optimized for automation and efficiency. We obtained the first high quality three-dimensional reconstructions of Tribolium and Gryllus datasets.
Optical clearing increases the penetration depth into samples, thus providing endpoint images of entire three-dimensional objects with cellular detail. This work contributes a quantitative characterization module that was applied to endpoint images of optically cleared spheroids. A program for the generation of ground truth datasets was developed in order to evaluate the cell nuclei segmentation performance. The program was part of a paper that was published in BMC Bioinformatics. Using the program, I could show that the cell nuclei segmentation is robust and accurate. Approaches from computational topology and graph theory complete the segmentation of cell nuclei. Thus, the developed module provides a comprehensive quantitative characterization of spheroids on the level of the individual cell, the cell neighborhood and the whole cell aggregate. The module was employed in four applications to analyze the influence of different stress conditions on the morphology and cellular arrangement of cells in spheroids. The module was accepted for publication in Scientific Reports along with the results for one application. The cell nuclei segmentation further provided a data source for simulation models that used correlation functions to identify structural zones in spheroids. These results were published in Royal Society Interface.
The final part of this work presents a module for cell tracking and lineage reconstruction. In collaboration with Dr. Alexis Maizel, Dr. Jens Fangerau and Dr. Daniel von Wangenheim, I developed a module to track the positions of all cells involved in lateral root formation in Arabidopsis thaliana and used the extracted positions for extensive data analysis. We reconstructed the cell lineages and established the first atlas of all founder cells that contribute to the formation. The analysis of the retrieved data allowed us to study conserved and individual patterns in lateral root formation. The atlas and parts of the analysis presented in this thesis were published in Current Biology.
In this thesis, I developed modules for an image analysis pipeline in three-dimensional fluorescence microscopy and applied them in interdisciplinary research projects. The modules enabled the organization, processing, visualization and analysis of the datasets. The perspective of the image analysis pipeline is not restricted to image-based systems biology. With ongoing development of the image analysis pipeline, it can also be a valuable tool for medical diagnostics or industrial high-throughput approaches.
The cardiovascular system (CVS) consists of heart and blood vessels, forming a close circulatory loop. All tissues depend on the nutrients and molecular oxygen (O2) delivered by the blood. Therefore, it is not surprising that the CVS is one of the first working systems and the heart is the first functional organ in the forming embryo (Baldwin 1996). The building blocks of blood vessels are endothelial cells (ECs), which form the endothelium, a specialized epithelium that defines the luminal surface of the vessels (Pugsley and Tabrizchi 2000). The process of blood vessel development comprises several steps. The first events occurring are the formation of new vessels de novo to constitute the primary vascular loop known as vasculogenesis. During vasculogenesis the vascular precursors, known as angioblasts, migrate and coalesce to form the axial vessels. Subsequently, the main vessels undergo a specification step where they acquire either arterial or venous identity. As the embryo increases in size, the main vascular loop needs to increase in complexity. In order to reach all the different parts of the developing organs, new blood vessels are formed from pre-existing ones, a phenomenon known as angiogenesis (Gore et al. 2012).
Mature blood cells have a short lifespan. Therefore, hematopoietic stem cells (HSCs) are required throughout lifetime to constantly form new blood cells in a process called hematopoiesis. Interestingly, endothelial and immune cells development have been shown to converge at different points during their development, one of which is developmental hematopoiesis. During embryogenesis, definitive hematopoiesis occurs in a tissue called hemogenic endothelium (HE), a specialized subset of ECs at the ventral wall of the dorsal aorta (DA). HE acquires hematopoietic potentials and gives rise to HSCs, through a process known as endothelial-to-hematopoietic transition (EHT). During EHT, these specialized ECs extrude from DA and colonize the so-called aorta-gonadmesonephros (AGM) region, forming the native HSCs (Paik and Zon 2010).
As vascular development requires different steps, the molecular pathways involved are many. The Notch signaling pathway has been demonstrated to be one of the main players in vascular development. Among other functions, Notch signaling has been shown to be important during EHT. In the murine model, Runx1, a master regulator of HSC formation, has been shown to be transcriptionally regulated by NOTCH1 through GATA2 activation. This observation was later corroborated by knockdown studies for notch1a and notch1b in zebrafish (Butko, Pouget, and Traver 2016). Another essential pathway for vascular development is the HIF pathway. Hif-1α, Hif-1β and Hif-2α mouse mutants show severe vascular defects that result in early embryonic lethality (Simon and Keith 2008), which hinders a deep analysis of the phenotypes incurring in the mutant embryos. In addition, deletion of Hif-1α specifically in myeloid cells showed abnormalities in the motility, invasiveness, and adhesion of macrophages (Cramer et al. 2003). Intriguingly, Hif-1α deletion in vascular endothelial cadherin-expressing cells led to a significant but partial reduction of HSC number, suggesting that other players may be involved in this pathway (Imanirad et al. 2014).
Zebrafish embryos have been shown to be tolerant to hypoxia at very early stages of development (Padilla and Roth 2001). Also, zebrafish embryos develop externally and this allows to finely manipulate the environment where they grow (Lieschke and Currie 2007). These features make zebrafish an ideal model to investigate how hypoxia and Hif transcription factors affect vertebrate vascular development. In this study, I will examine the impact of hypoxia on zebrafish vascular development. Specifically, I will dissect the role of hif-1α in macrophage-EC interactions during vascular development and repair. Moreover, I show redundant functions for hif-1α and hif-2α in HSC development upstream of Notch signaling.
Structural biology often employs a combination of experimental and computational approaches to unravel the structure-function paradigm of biological macromolecules. This thesis aims to approach this combination by the application of Pulsed Electron-Electron Double Resonance (PELDOR/DEER) spectroscopy and structural modelling. In this respect, PELDOR spectroscopy in combination with site-directed spin labelling (SDSL) of proteins is frequently used to gain distance restraints in the range from 1.8 to 8 nm. The inter-spin distance and the flexibility of the spin labelled protein domains are encoded in the oscillation and the dampening of the PELDOR signal. The intrinsic flexibility of the commonly used MTSSL (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) spin label itself can be an obstacle for structural modelling if the flexibility of the label is large compared to the flexibility of the protein domains. In this thesis the investigation of two multi-domain proteins by the 4-pulse PELDOR sequence is presented. At first, the N-terminal polypeptide transport-associated (POTRA) domains of anaOmp85, a rigid three domain protein, giving well-defined PELDOR distance restraints, is investigated. The experimental restraints are used for structure refinement of the X-ray structure and reveal a strong impact of the intrinsic flexibility of MTSSL on the accuracy of structural refinement. The second example, K48-linked diubiquitin, is a highly flexible multi-domain protein on which the flexibility of MTSSL is of minor impact on structural modelling. In this case, the distance restraints are utilized to determine conformational ensembles. Due to the high intrinsic flexibility already characterizing diubiquitin the recently developed 7-pulse Carr-Purcell (CP) PELDOR sequence was applied to investigate longer ubiquitin chains. This sequence enables to measure dipolar oscillations with an extended time window, allowing a good separation between inter- and intramolecular contributions even for long distance and broad conformational distributions, thereby providing an increased accuracy of the obtained distance distributions.
The fruit fly Drosophila melanogaster is one of the most important biological model organisms, but only the comparative approach with closely related species provides insights into the evolutionary diversification of insects. Of particular interest is the live imaging of fluorophores in developing embryos. It provides data for the analysis and comparison of the threedimensional morphogenesis as a function of time. However, for all species apart from Drosophila, for example the red flour beetle Tribolium castaneum, essentially no established standard operation procedures are available and the pool of data and resources is sparse. The goal of my PhD project was to address these limitations. I was able to accomplish the following milestones:
- Development of the hemisphere and cobweb mounting methods for the non-invasive imaging of Tribolium embryos in light sheet-based fluorescence microscopes and characterization of most crucial embryogenetic events.
- Comprehensive documentation of methods as protocols that describe (i) beetle rearing in the laboratory, (ii) preparation of embryos, (ii) calibration of light sheet-based fluorescence microscopes, (iv) recording over several days, (v) embryo retrieval as a quality control as well as (vi) data processing.
- Adaption of the methods to record and analyze embryonic morphogenesis of the Mediterranean fruit fly Ceratitis capitata and the two-spotted cricket Gryllus bimaculatus as well as integration of the data into an evolutionary context.
- Further development of the hemisphere method to allow the bead-based / landmark-based registration and fusion of three-dimensional images acquired along multiple directions to compensate the shadowing effect.
- Development of the BugCube, a web-based computer program that allows to share image data, which was recorded by using light sheet-based fluorescence microscopy, with colleagues.
- Invention and experimental proof-of-principle of the (i) AGameOfClones vector concept that creates homozygous transgenic insect lines systematically. Additionally, partial proof-of-principle of the (ii) AClashOfStrings vector concept that creates double homozygous transgenic insect lines systematically, as well as preliminary evaluation of the (iii) AStormOfRecords vector concept that creates triple homozygous transgenic insect lines systematically.
- Creation and performance screening of more than fifty transgenic Tribolium lines for the long-term imaging of embryogenesis in fluorescence microscopes, including the first Lifeact and histone subunit-based lines.
My primary results contribute significantly to the advanced fluorescence imaging approaches of insect species beyond Drosophila. The image data can be used to compare different strategies of embryonic morphogenesis and thus to interpret the respective phylogenetic context. My technological developments extend the methodological arsenal for insect model organisms considerably.
Within my perspective, I emphasize the importance of non-invasive long-term fluorescence live imaging to establish speciesspecific morphogenetic standards, discuss the feasibly of a morphologic ontology on the cellular level, suggest the ‘nested linearly decreasing phylogenetic relationship’ approach for evolutionary developmental biology, propose the live imaging of species hybrids to investigate speciation and finally outline how light sheet-based fluorescence microscopy contributes to the transition from on-demand to systematic data acquisition in developmental biology.
During my PhD project, I wrote a total of ten manuscripts, six of which were already published in peer-reviewed scientific journals. Additionally, I supervised four Master and two Bachelor projects whose scientific questions were inspired by the topic of my PhD work.
Inhibition of midbrain dopamine (DA) neurons codes for negative reward prediction errors, and causally affects conditioning learning. DA neurons located in the ventral tegmental area (VTA) display two-fold longer rebound delays from hyperpolarizing inhibition in comparison to those in the substantia nigra (SN). This difference has been linked to the slow inactivation of Kv4.3-mediated A-type currents (IA). One known suppressor of Kv4.3 inactivation is a splice variant of potassium channel interacting protein 4 (KChIP4), KChIP4a, which has a unique potassium channel inactivation suppressor domain (KISD) that is coded within exon 3 of the KChIP4 gene. Previous ex vivo experiments from our lab showed that the constitutive knockout of KChIP4 (KChIP4 KO) removes the slow inactivation of IA in VTA DA neurons, with marginal effects on SN DA neurons. KChIP4 KO also increased firing pauses in response to phasic hyperpolarization in these neurons. Here I show, using extracellular recordings combined with juxtacellular labeling in anesthetized mice, that KChIP4 KO also selectively changes the number and duration spontaneous firing pauses by VTA DA neurons in vivo. Pauses were quantified with two different statistical methods, including one developed in house. No other firing parameter was affected, including mean frequency and bursting, and the activity of SN DA neurons was untouched, suggesting that KChIP4 gene products have a highly specific effect on VTA DA neuron responses to inhibitory input.
Following up on this result, I developed a new mouse line (KChIP4 Ex3d) where the KISD-coding exon 3 of KChIP4 is selectively excised by cre-recombinase expressed under the dopamine transporter (DAT) promoter, therefore disrupting the expression of KChIP4a only in midbrain DA neurons. I show that these mice have a highly selective behavioral phenotype, displaying a drastic acceleration in extinction learning, but no changes in acquisition learning, in comparison to control littermates. Computational fitting of the behavioral data with a modified Rescorla-Wagner model confirmed that this phenotype is congruent with a selective increase in learning from negative prediction errors. KChIP4 Ex3d also had normal open field exploration, novel object preference, hole board exploration and spontaneous alternation in a plus maze, indicating that exploratory drive, responses to novelty, anxiety, locomotion and working memory were not affected by the genetic manipulation. Furthermore semi-quantitative IHC revealed that KChIP4 Ex3d mice have increased Kv4.3 expression in TH+ neurons, suggesting that the absence of KChIP4a increases the binding of other KChIP variants, which known to increase surface expression of Kv4 channels.
Furthermore, in the course of my experimental study I identified that the most used mouse line where cre-recombinase is expressed under the DAT promoter (DAT-cre KI) has a different behavioral phenotype during conditioning in relation to WT littermate controls. These animals displayed increased responding during the initial trials of acquisition and delayed response latency extinction, consistent with an increase in motivation, which is in line with a decrease in DAT function.
I propose a working model where the disruption of KChIP4a expression in DA neurons leads to an increase in binding of other KChIP variants to Kv4.3 subunits, promoting their increased surface expression and increasing IA current density; this then increases firing pauses in response to synaptic inhibition, which in behaving animals translates to an increase in negative prediction error-based learning.
Introduction:
The evolutionary patterns of symbiotic organisms are inferred using cophylogenetic methods. Congruent phylogenies indicate cospeciation or host-switches to closely-related hosts, whereas incongruent topologies indicate independent speciation. Recent studies suggest that coordinated speciation is a rare event, and may not occur even in the highly specialized associations. The cospeciation hypothesis was mainly tested for free-living mutualistic associations, such as plant-pollinator interactions, and host-parasitic systems but was rarely tested on obligate, mutualistic associations involving intimate physiological interactions. Symbionts with lower partner selectivity may not experience coordinated speciation due to frequent switching of partners. On the other hand, symbionts with high partner selectivity may influence each other’s evolution owing to the highly interdependent lifestyles. Symbiont association patterns are also influenced by habitat and it has been proposed that symbiotic interactions are stronger in warm regions as compared to cooler regions (also referred as latitudinal gradient of biotic specialization). This hypothesis however, has recently been challenged and it has been suggested that a gradient of biotic specialization may not exist at all. Reliable species concepts are a prerequisite for understanding the association and evolutionary patterns of symbiotic organisms. The species concepts of many groups traditionally relied on the morphological species concept, which may not be adequate for distinguishing species due to the: i) homoplasious nature of morphological characters, an due to the inability to distinguish cryptic species. Thus phylogenetic species concept along with coalescent-based species delimitation approaches, which utilize molecular data for inferring species boundaries have been used widely for resolving taxonomic relationships. Lichens are obligatory symbiotic associations consisting of a fungal partner (mycobiont) and one or more photosynthetic partners, algae, and/or cyanobacteria (photobionts). I used the lichen forming fungal genus Protoparmelia as my study system, which consists of ~25-30 previously described species inhabiting different habitats, from the arctic to the tropics. This makes Protoparmelia an ideal system to explore the association and evolutionary patterns across different macrohabitats.
Objectives:
The objectives of this thesis were to 1. Elucidate the phylogenetic position of Protoparmelia within Lecanorales, and infer the monophyly of Protoparmelia; 2. Understand species diversity within Protoparmelia s.str. using coalescent-based species delimitation approaches; and 3. To identify the Trebouxia species associated with Protoparmelia using phylogenetic and species delimitation approaches and to infer the association and cophylogenetic patterns Protoparmelia and Trebouxia in different macrohabitats.
Results and discussion:
Chapter 1: Taxonomic position of Protoparmelia
In the first part of this study I explored the taxonomic position of Protoparmelia within the order Lecanorales. Overall this study included 54 taxa from four families, sequenced at five loci (178 sequences). I found Protoparmelia to be polyphyletic and sister to Parmeliaceae.
Chapter 2: Multilocus phylogeny and species delimitation of Protoparmelia spp.
In this part of the study, I identified and delimited the Protoparmelia species forming a monophyletic clade sister to Parmeliaceae i.e., Protoparmelia sensu stricto group, based on the multilocus phylogeny and coalescent-based species delimitation approaches. I included 18 previously described and three unidentified Protoparmelia species, which represents ~70% of the total described species, and 73 other taxa, sequenced at six loci. I found that the sensu stricto group comprised of 25 supported clades instead of 12 previously described Protoparmelia species. I tested the speciation probabilities of these 25 clades using species delimitation softwares BP&P and spedeSTEM. I found nine previously unrecognized lineages in Protoparmelia and I propose the presence of at least 23 species for Protoparmelia s.str., in contrast to the 12 described species included in the study.
Chapter 3: Association and cophylogenetic patterns of Protoparmelia and its symbiotic partner Trebouxia
...
The human brain is one of the most complex biological systems. More than 100 billion neurons build networks that control basic body functions and highly coordinated movements, enable us to express emotions, feelings and thoughts and to store memories over years and even throughout life time. Ultimately, “We are who we are because of what we learn and what we remember” (Kandel 2006). Under pathological conditions, the brain function is challenged. Most if not all neurological diseases have in common that they are either triggered and/or accompanied by inflammatory processes of brain tissue, referred to as neuroinflammation. Such inflammatory processes directly affect an elementary neural mechanism relevant for learning and memory: synaptic plasticity. Indeed, neurons are highly dynamic structures and able to respond to specific stimuli with morphological, functional and molecular adaptations that modify the strength and number of neuronal contact sides (synapses). Hence, the main motivation of this thesis was to identify the neural targets through which inflammation affects brain function and synaptic plasticity in particular. The principles of synaptic plasticity have been studied intensively in the hippocampus, an anatomical structure localized within the temporal lobes that is essential for the consolidation of memories and spatial navigation. Synaptic plasticity is coordinated by complex interactions of thousands of molecules and proteins. Among those proteins, synaptopodin (SP) is localized at a strategic position within excitatory synapses and has been shown to be fundamentally involved in the regulation of synaptic plasticity.
To induce neuroinflammation and to study its effects on SP as well as synaptic plasticity, the classic model of lipopolysaccharide (LPS) was applied. This thesis discloses that inflammatory processes impair the ability of neurons to express hippocampal synaptic plasticity in vivo, which is accompanied by a downregulation of SP-mRNA and protein level in the mouse hippocampus, indicating that SP is one of the cellular targets through which inflammatory signaling pathways affect synaptic plasticity and hence neural function. To learn more about the cellular and molecular mechanisms, an in vitro LPS model was established using entorhino-hippocampal organotypic slice cultures (OTCs).
While confirming the major effect of LPS on SP, this thesis furthermore shows that neuroinflammation crucially involves the cytokine TNFα to transduce its effects on SP, and that microglial cells are the main source of TNFα production under inflammatory conditions. In an attempt to learn more about the mechanisms that are affected under conditions of neuroinflammation effects of retinoic acid (RA), a vitamin A derivate were tested. This is mainly because SP as well as RA have been shown to modulate synaptic plasticity through the accumulation of glutamate receptors at the postsynaptic site: SP via the association with the actincytoskeleton as well as intracellular calcium stores, and RA directly via the modulation of local protein synthesis within dendrites. Indeed, in slice cultures exposed to RA, hippocampal SP cluster size is upregulated, both in vitro and in vivo. Intriguingly, a lack of SP prevents RA-induced synaptic strengthening of hippocampal dentate granule cells in OTCs. This suggests a direct contribution of SP in RA-dependent synaptic plasticity. Interestingly, co-immunoprecipitation of SP-mRNA together with the RA-receptor alpha (RARα) further implies that RA directly controls synaptic plasticity via regulation of SP-protein expression. It is therefore interesting to speculate that RA may increase SP expression or prevent its reduction and thus alterations in synaptic plasticity under conditions of neuroinflammation. Taken together, this thesis identifies SP as an important neuronal target of TNFα-mediated alterations in synaptic plasticity. Moreover, the work on RA indicates that SP affects the ability of neurons to express synaptic plasticity by modulating/mediating local protein synthesis. Since neuroinflammatory processes are an elementary concomitant feature and/or cause of neurological diseases, I am confident that future work on the effects of inflammatory processes on brain function may provide the perspective in devising new therapeutic strategies for the treatment of neuropathologies such as Alzheimer’s disease, multiple sclerosis, epilepsy or stroke, by targeting SP expression and SP-mediated synaptic plasticity.
Deciphering the ecological functions of fungal root endophytes based on their natural occurrence
(2017)
Plants are colonized by a large diversity of fungi, some residing on the surface and others penetrating the plant tissues, the latter referred to as fungal endophytes (endon Gr., within; phyton, plant; de Bary 1879). Despite the saprotrophic potential of fungal endophytes, they are not found to cause visible disease symptoms to the host. Plants are colonized simultaneously by various fungal species, which form rich and diverse endophytic assemblages. Although it is hypothesized that fungal endophytes contribute to the fitness of their hosts and to the functioning of ecosystems, the ecological function of fungal endophytic assemblages remains cryptic. The aims of this doctoral thesis are to gain insight to the ecological functions of root fungal endophytes, by deciphering their roles in ecosystems based on their natural occurrence and the structure of their assemblages. The thesis focuses on studying the diversity and structure of the endophytic mycobiome within roots of two annual and widespread plant hosts Microthlapsi perfoliatum and M. erraticum (Brassicaceae) in several locations across northern Mediterranean and central Europe. The thesis is composed by six Chapters, with a primary focus on Chapter 1, 2 and 3.
Chapter 1 (Glynou et al., 2016) aimed at characterizing the diversity of fungal endophytes in roots at a continental scale and at assessing the factors affecting the structure of endophytic assemblages with the use of cultivation-based methods. For that, root samples were collected from 52 plant populations, along with a collection of soil, bioclimatic, geographic and host data. Cultivation of surface-sterilized root samples on culture media and isolation of fungal colonies in pure culture generated 1,998 fungal colonies. Grouping of sequences into Operational Taxonomic Units (OTUs), based on the 97% similarity of the isolates’ rDNA Internal Transcribed Spacer (ITS) sequence, generated in total 296 OTUs, representing taxa mostly within the phylum Ascomycota with a minor representation of Basidiomycota. Endophytic assemblages were mostly correlated with variation in bioclimatic conditions. Interestingly, despite the large diversity revealed, the assemblages were dominated by only six OTUs related to the orders Hypocreales, Pleosporales and Helotiales, which had a widespread distribution across populations but with some following patterns of ecological preferences.
Chapter 2 aimed at characterizing the uncultivable fraction of the root fungal endophytic diversity, which was not possible to capture in Chapter 1. High-throughput sequencing via the
Illumina Miseq platform was implemented in 43 of the 52 original populations and mostly in the same root samples. In comparison with the cultivation-based approach, the HTS managed to cover the overall diversity within samples. It revealed a large non-cultivated endophytic diversity but the same cultivable fungi dominated assemblages. Moreover, the endophytic diversity was grouped mostly within fungal orders with demonstrated ability to grow in culture and taxonomically related groups were found to have divergent ecological preferences.
The genetic identity of the most abundant OTUs was further investigated in Chapter 3 (Glynou et al., 2017), aiming to unravel genotypic variability, which was possibly overlooked due to the use of lTS, as a universal genetic marker, and could explain their high abundance and widespread distribution. Multi-locus gene sequencing and AFLP profiling for the five most abundant OTUs suggested a low within-OTU genetic variability and show that these fungi have ubiquitous distribution and are not limited by environmental conditions within the ecological ranges of the study. A selection of endophytes frequently isolated in Chapter 1 was functionally characterized in Chapter 4 (Kia et al., 2017) based on the isolates’ traits and interactions with plants. In Chapter 5 (Cheikh-Ali et al., 2015) fungal cultures of Exophiala sp. with differential colony structure where investigated for their production of secondary metabolites. Moreover, Chapter 6 (Maciá-Vicente et al., 2016) comprises the description of the new species Exophiala radicis based on morphological and molecular characteristics.
Compilation of all results shows that the fungal endophytic diversity in roots of Microthlaspi spp. is high but few widespread OTUs dominate the assemblages, and have unlimited dispersal ability. These fungi seem also to have a wide niche breadth and are not affected by environmental filtering. The findings indicate that the local environment but also processes of competitive exclusion determine the structure of endophytic assemblages. In addition, the fungal endophytes associated with Microthlapsi spp. likely have saprotrophic activity however the interactions with plants are likely context-dependent. Further research is needed to assess the biotic interactions among endophytes and their effect on the structure of fungal endophytic assemblages. Ultimately, the findings of this thesis are useful to shed light on the processes underlying the structure of endophytic assemblages. They also upraise the need to describe diversity by combining genetic, metabolic and physiological data, in order to disentangle the elusive ecological roles of the endophytic mycobiome.
Gallery forests (GFs) are floristically and functionally distinct forests along rivers and watercourses. The GFs of the West African savannas form thin stripes with a particular, species-rich flora differing significantly from the surrounding vegetation. Due to their relative isolation in the savannas and their dependence on rivers, GFs are particularly endangered by the expected global climate changes in the 21st century. Despite their high diversity, little is known about the ecology and biogeography of GFs in West Africa. Especially, their response to climate changes and their vegetation dynamics remain largely unknown. This thesis combines floristic, phylo-geographic and ecological data to investigate the biodiversity, historical and recent biogeography and conservation status of GF species in Burkina Faso (BFA) as a model for West Africa.
The process of urbanization is one of the major causes of the global loss of biodiversity; however, cities nowadays also have the potential to serve as new habitats for wildlife. The European rabbit (Oryctolagus cuniculus, L. 1758) is a typical example of a wildlife species that reaches stable population densities in cities. Due to intense plant and soil damages, German city authorities aim to control high rabbit densities through the application of a yearly hunting regime (e. g., in Munich, Berlin or Frankfurt am Main). In contrast, population densities of O. cuniculus are on decline in German rural areas, i. e., numbers of yearly hunting bags decreased. The aim of my doctoral thesis was to answer the following research questions: Do population densities of the European rabbit correlate with the intensity of urbanization in and around Frankfurt am Main and if so, which factors play a role in varying densities? How are burrow construction behaviors and group sizes, daytime activity patterns and anti-predator behaviors as well as communication behaviors of this mammal affected by urbanization?
In my first study, I focused on population dynamics across 17 different study sites in and around Frankfurt. As one of yet few studies, I invented an approach that quantified the intensity of urbanization (degree of urbanity) of each study site base on four variables: (1) intensity of anthropogenic disturbance per min and ha, (2) number of residents within a radius of 500 m, (3) proportion of artificial ground cover and (4) numbers of anthropogenic objects per ha. Spearman rank correlations confirmed that with increasing degree of urbanity also rabbit and burrow densities increased. The access to dense shrubs, bushes etc. as suitable sites for burrow construction is the most determining factor for rabbit abundances, and therefore I presumed different densities along the rural-to-urban gradient to be driven by shifts in the availability of thick vegetation.
In the second study, I calculated two indices that in both cases classified burrows to be either accumulated, evenly or randomly distributed within study sites. Additionally, in cooperation with local hunters the number of burrow entrances and animals that occupy the same burrow had been determined during the hunting season. With increasing degree of urbanity burrow distribution patterns shifted from accumulated in rural areas towards more evenly distributed within the city center of Frankfurt. This is a clear sign for an increasing access to sites suitable for burrow construction along the rural to-urban gradient. Additional Spearman rank correlations revealed that the external dimensions of burrows decreased (shorter distances between entrances) and that burrows became less complex (fewer entrances) along the rural-to-urban gradient. In accordance, the number of rabbits that commonly shared the same burrow system was highest within rural areas, whereas I found mainly pairs and single individuals within highly urbanized study sites.
In the last study I compared activity patterns, burrow use and percentages of anti-predator behaviors from one hour before sunrise until one hour after sunset of rural, suburban and urban rabbit groups. A linear mixed model (LMM) and Spearman rank correlations confirmed that rabbits located at urban and suburban sites spent more time outside their protective burrows compared to their rural conspecifics. At suburban sites, individuals invested the least amount of time in anti-predator behavior. Results of this third study gave evidence that suburban rabbit populations on one hand benefit from less predation pressure by natural predators in comparison to rural sites, whereas on the other hand are exposed to less intense disturbance by humans compared to urban study sites.
The last study focused on the effects that urbanization had on the latrine-based communication behavior of rabbits. As many other mammals, O. cuniculus exchange information via the deposition of excreta in latrines, and depending on the intended receiver(s), latrines are either formed in central areas for within-group communication or at territorial boundaries, e. g., for between-group communication. The relative importance of within- vs. between-group communication depends on, amongst other factors, population densities and group sizes which I proved both to shift along the considered rural-to-urban gradient. I determined latrine sizes, latrine densities and latrine utilization frequencies relative to their distance to the nearest burrow at 15 different study sites. Latrine densities and utilization frequencies increased with increasing distance from the burrow in suburban and urban populations whereas at rural sites, largest latrines and those containing the most fecal pellets were close to the burrow, suggesting that within-group communication prevailed.
To sum up, for the first time, I was able to relate shifts in the ecology and behavior of the European rabbit as adaptations to a gradual anthropogenic habitat alteration that are typical for “urban exploiters”. Especially the suburban habitat provides high landscape heterogeneity (“edge habitat“) which is essential for high and stable rabbit populations. Moreover, here, comparably low human disturbance and predation pressure are given in contrast to the agriculturally transformed, open landscapes which are nowadays typical for most rural areas in central Europe. I argue that this mainly leads to the observed behavioral changes along the rural-to-urban gradient. Future plans for rural land management actions should aim to increase refuge availability by generating networks of ecotones. This would also benefit species that depend on similar ecosystem structures as the European rabbit and are on decline in Germany.
Proteinen die ExHepatitis C ist eine entzündliche Erkrankung der Leber, die durch das Hepatitis-C-Virus (HCV) verursacht wird. Trotz vieler Bemühungen ist heutzutage immer noch keine prophylaktische Vakzinierung verfügbar. Neuartige Therapien versprechen eine hohe Heilungsrate, sind aber mit hohen Kosten verbunden. HCV induziert oxidativen Stress, welcher für das Auftreten und die Progression der Pathogenese eine zentrale Rolle spielt. Um zellulären Stress (z.B. durch ROS) entgegenzuwirken, haben Zellen cytoprotective und detoxifizierende Mechanismen entwickelt, die die zelluläre Homöostase aufrechterhalten. Dabei kontrolliert der redoxsensitive Transkriptionsfaktor Nrf2 als Heterodimer zusammen mit sMaf- pression von cytoprotective und ROS-detoxifizierenden Genen. Vorherige Studien haben gezeigt, dass HCV den Nrf2/ARE-Signalweg beeinträchtigt. Dabei induziert HCV eine Translokation der sMaf-Proteine aus dem Zellkern in das Cytoplasma, wo diese das virale Protein NS3 binden. Im Cytoplasma lokalisierte sMaf-Proteine verhindern dadurch eine Translokation von Nrf2 in den Zellkern. Folglich ist die Expression von Nrf2/ARE-abhängigen cytoprotective Genen inhibiert und intrazelluläre ROS-Spiegel dauerhaft erhöht. Ein weiterer zentraler cytoprotective Mechanismus ist die Autophagie. Sie dient der Aufrechterhaltung der zellulären Homöostase durch den Abbau von defekten Proteinen und Organellen. Des Weiteren ist bekannt, dass Autophagie nicht nur im Laufe von Nährstoffmangel induziert wird, sondern auch durch erhöhte Mengen an ROS. In sämtlichen Studien konnte beobachtet werden, dass Autophagie für die Aufrechterhaltung des viralen Lebenszyklus eine wesentliche Rolle spielt, da sie mit der Ausbildung des membranous web, der Translation, der Replikation und der Freisetzung des Virus interferiert. Ausgehend davon sollte in dieser Arbeit zunächst die Relevanz von HCV-induziertem oxidativen Stress, resultierend aus der Nrf2/ARE-Signalweginhibition, als möglicher Aktivator der Autophagie untersucht werden. Dabei wurde in HCV-positiven Zellen eine Akkumulation von LC3-II beobachtet, was auf eine Induktion der Autophagie schließen lässt. In Übereinstimmung damit wurde eine erhöhte Expression von Autophagie-Markerproteinen in HCV-infizierten PHHs detektiert. Im Laufe der Autophagie wird p62 abgebaut. Somit sollte eine Induktion der Autophagie in einer Verminderung der Menge an p62 resultieren. Nichtsdestotrotz ist eine Akkumulation von p62 in HCV-positiven Zellen nachzuweisen. Dies erscheint zunächst widersprüchlich. Aufgrund der Tatsache, dass die Expression der katalytischen Untereinheit des Proteasoms (PSMB5) Nrf2-abhängig ist, führt die beeinträchtigte Nrf2-Aktivität in HCV-positiven Zellen jedoch zu einer verringerten Aktivität des konstitutiven Proteasoms. Dieser Befund kann auch die erhöhte Halbwertzeit von p62 in HCV-positiven Zellen erklären. Kürzlich wurde ein Zusammenspiel des Nrf2/ARE-Signalwegs und der Autophagie beobachtet. Dabei kann Nrf2 nicht nur über den kanonischen Signalweg aktiviert werden, sondern auch durch eine direkte Interaktion des phosphorylierten Autophagie-Adaptorproteins p62 (pS[349] p62) mit Keap1. In HCV-positiven Zellen können nicht nur eine Zunahme der Gesamtmenge von p62 beobachtet werden, sondern auch erhöhte Mengen an pS[349] p62. Die Berechnung des Quotienten aus pS[349] p62 und p62 zeigt in etwa eine Verdopplung der Menge an pS[349] p62 , was auf eine vermehrte Phosphorylierung von p62 in HCV-positiven Zellen rückschließen lässt. Des Weiteren konnte beobachtet werden, dass erhöhte Mengen an ROS, wie sie auch in HCV-positiven Zellen vorkommen, Autophagie induzieren können, die durch eine Akkumulation von LC3-II und die Zunahme von LC3 Puncta charakterisiert ist. Auch eine Zunahme von pS[349] p62 konnte beobachtet werden. Ferner resultierte die Überexpression der phosphomimetischen Mutante (p62 [S351E]) in einer Akkumulation von LC3-II, was auf die Fähigkeit von pS[349] p62 rückschließen lässt, Autophagie zu induzieren. Eine Modulation der Autophagie mittels der Inhibitoren 3-Methyladenin und Bafilomycin führte zu einer inhibierten Freisetzung von infektiösen viralen Partikeln und unterstreicht damit, dass der Autophagie eine essentielle Bedeutung bei der Freisetzung viraler Partikel zukommt. Eine HCV-Infektion wird sowohl von erhöhten Mengen an ROS als auch von einer Induktion der Autophagie begleitet. Dementsprechend führte eine Verminderung des intrazellulären Radikalspiegels durch eine Inkubation mit den Radikalfängern PDTC und NAC zu geringeren Mengen an LC3-II und pS[349] p62. Dabei konnte auch eine Abnahme der freigesetzten infektiösen viralen Partikel beobachtet werden, was ein Zusammenspiel zwischen erhöhten Mengen an ROS, Induktion der Autophagie und Virusfreisetzung nahelegt. Vorschlag: Erhöhte Mengen an ROS werden durch eine Aktivierung des Nrf2/ARE-Signalwegs detoxifiziert und würden somit den zuvor beschriebenen viralen Mechanismus verhindern. HCV die Aktivierung Nrf2/ARE-regulierter Gene beeinträchtigt, wurde die Hypothese aufgestellt, dass in HCV-positiven Zellen dieser komplexe Mechanismus dazu dient, die Translokation des pS[349] p62-abhängig freigesetzte Nrf2 in den Zellkern zu verhindern. Das wiederum hat eine eingeschränkte Expression von Nrf2/ARE-abhängigen Genen und Detoxifizierung von ROS zur Folge. Um diese Hypothese experimentell zu untersuchen, wurden HCV-positive und negative Zellen cotransfiziert mit dem p62 Wildtyp (p62 [wt]), der p62 phosphomimetischen Mutante (p62 [S351E]) oder einem Kontrollplasmid in Kombination mit einem Reporterkonstrukt, welches die Nrf2-Aktivierung darstellt (OKD48). Während in HCV-negativen Zellen im Vergleich zum p62 [wt] eine Transfektion mit p62 [S351E] zu einer signifikanten Aktivierung des Nrf2-abhängigen Reportergens führt konnte dies in HCV-positiven Zellen nicht beobachtet werden. Zusammengenommen beschreiben diese Ergebnisse einen neuartigen Mechanismus wie HCV das Zusammenspiel zwischen dem Nrf2/ARE-Signalweg, erhöhten Mengen an ROS und Autophagie beeinflusst. Dabei übt HCV einen negativen Effekt auf den Nrf2/ARE-Signalweg aus, um dem pS[349] p62-abhängig freigesetzten Nrf2 zu entkommen. Folglich werden erhöhte Mengen an ROS aufrechterhalten, die eine Induktion der Autophagie ermöglichen, welche für die Freisetzung viraler Partikel essentiell ist.
The composition of cellular membranes is extremely complex and the mechanisms underlying their homeostasis are poorly understood. Organelles within a eukaryotic cell require a non-random distribution of membrane lipids and a tight regulation of the membrane lipid composition is a prerequisite for the maintenance of specific organellar functions. Physical membrane properties such as bilayer thickness, lipid packing density and surface charge are governed by the lipid composition and change gradually from the early to the late secretory pathway. As the endoplasmic reticulum (ER) is situated at the beginning of the cells secretory pathway, it has to accept and accommodate a great variety and quantity of secretory and transmembrane proteins, which enter the ER on their way to their final cellular destination. Secretory proteins can be translocated into the lumen of the ER co- or posttanslationally and membrane proteins are being inserted and released into the ER membrane. In the oxidative milieu of the ER-lumen, supported by a variety of chaperones, proteins can fold into their native form.
If the folding capacity of the ER-lumen is exceeded, an accumulation of mis- or unfolded proteins in the lumen of the ER occurs, consequently triggering the unfolded protein response (UPR). This highly conserved program activates a wide-spread transcriptional response to restore protein folding homeostasis. In fact, 7 – 8% of all genes in the yeast Saccharomyces cerevisiae (S. cerevisiae) are regulated by the UPR. The mechanism underlying the activation of the UPR by protein folding stress has been investigated thoroughly in the last decades and many of its mechanistic details have been elucidated. Recently, it became evident that aberrant lipid compositions of the ER membrane, collectively referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism of this membrane-activated UPR, however, remained unclear.
This study focuses on the UPR in S. cerevisiae and characterizes the inositol requiring enzyme 1 (Ire1) as the sole UPR sensor in S. cerevisiae. Active Ire1 forms oligomers and, collaboratively with the tRNA ligase Rlg1, splices immature mRNA of the transcription factor HAC1, which results in the synthesis of mature HAC1 mRNA and the production of the active Hac1 protein, which binds to UPR-elements in the nucleus and activates the expression of UPR target genes. Here, the combination of in vivo and in vitro experiments is being used, which is supplemented by molecular dynamics (MD) simulations performed by Roberto Covino and Gerhard Hummer (MPI for Biophysics, Frankfurt), aiming to identify the molecular mechanism of Ire1 activation by lipid bilayer stress. This study focuses on the analysis of the juxta- and transmembrane region of Ire1. Bioinformatic analyses revealed a putative ER-lumenal amphipathic helix (AH) N-terminally of and partially overlapping with the transmembrane helix (TMH). This predicted AH contains a large hydrophobic face, which inserts into the ER membrane, forcing the TMH into a tilted orientation within the membrane. The resulting unusual architecture of Ire1’s AH and TMH constitutes a unique structural element required for the activation of Ire1 by lipid bilayer stress.
To investigate the function of the AH in the physiological context, different variants of Ire1 were produced under the control of their endogenous promoter and from their endogenous locus. The functional role of the AH was tested, by disrupting its amphipathic character by the introduction of charged residues into the hydrophobic face of the AH. The role of a conserved negative residue between the TMH and the AH (E540 in S. cerevisiae) was tested by substituting it by a unipolar, polar, or positively charged residue. These variants were intensively characterized using a series of assays:
This thesis provides evidence that the AH is crucial for the function of Ire1: Mutant variants with a disrupted (F531R, V535R) or otherwise modified AH (E540A) exhibited a lower degree of oligomerization and failed to catalyze the splicing of the HAC1 mRNA as the Wildtype control. Likewise, the induction of PDI1, a target gene of the UPR, was greatly reduced in mutants with a disrupted or defective AH. These data revealed an important functional role of the AH for normal Ire1 function.
An in vitro system was established to analyze the membrane-mediated oligomerization of Ire1. This system enabled the isolated functional analysis of the AH and TMH during Ire1 activation by lipid bilayer stress. A fusion construct, coding for the maltose binding protein (MBP) from Escherichia coli (E. coli), N-terminally to the AH and TMH of Ire1 was produced. The heterologous production in E. coli, the purification and reconstitution of this minimal sensor of Ire1 in liposomes was established as part of this study. To analyze the oligomeric status of the minimal sensor in different lipid environments, continuous wave electron paramagnetic resonance (cwEPR) spectroscopic experiments were performed. These experiments revealed that the molecular packing density of the lipids had a significant influence of the oligomerization of the spin-labeled membrane sensor: increasing packing densities resulted in sensor oligomerization. The AH-disruptive F531R mutant, in which the amphipathic character of the AH was destroyed, showed no membrane-sensitive changes in its oligomerization status.
Thus, the activation of Ire1 by lipid bilayer stress is achieved by a membrane-based mechanism. According to the current model, the AH induces a local membrane compression by inserting its large hydrophobic face into the membrane. As membrane thickness and acyl chain order are interconnected, this compression simultaneously results in an increased local disordering of lipid acyl chains. Supporting MD simulations performed by Roberto Covino and Gerhard Hummer revealed that the bilayer compression is significantly more pronounced in a densely packed lipid environment, than in a lipid environment of lower lipid packing density. Hence, the energetic cost of the local compression increases with the packing density of the membrane, but is compensated for by the oligomerization of Ire1. This minimization of energetic cost induced by the membrane deformation of Ire1 forms the basis for the activation of Ire1 by lipid bilayer stress.
Characterizing the hologenome of Lasallia pustulata and tracing genomic footprints of lichenization
(2017)
The lichen symbiosis – consisting of fungal mycobionts and photoautotroph photobionts (green algae or cyanobacteria) – is globally successful. It covers an estimated 6% of the global surface with habitats ranging from deserts to the arctic. This success is reflected in the diversity of the mycobionts, with around 21% of all fungal species participating in lichen symbioses that can be facultative or obligate. Lichenization is furthermore evolutionary old, with fossil evidence for lichens reaching back 415 million years. For an individual fungal lineage, the Lecanoromycetes, the lichenization happened around 300 million years ago. This longstanding symbiotic relationship and the diversity of observed symbiotic dependency make them promising models to study the genomic consequences that follow the establishment of symbioses. Despite this, only little is known about the genomic effects of lichenization and extreme symbiotic dependency. To fill this gap we sequenced the hologenome of the lichen Lasallia pustulata, where the mycobiont could so far not been cultivated, suggesting that it might be more dependent on its symbionts.
As the poor culturability of lichen symbionts renders their genomes inaccessible to standard sequencing practices, we evaluated the extent to which different metagenome sequencing- and de novo assembly-strategies can be used to sequence and reconstruct the genomes of the individual symbionts. We find that the abundances of individual genomes present in the L. pustulata hologenome vary substantially, with the mycobiont being most abundant. Using in silico generated data sets and real Illumina sequencing data for L. pustulata we observe that the skewed abundances prevent a contiguous assembly of the underrepresented genomes when using only short-read sequencing. We conclude that short-read sequencing can offer first insights into lichen hologenomes. The fragmentation of the reconstructions hinders downstream analyses into the genomic consequences of lichenization though, as these are focused on identifying the gain and loss of genes.
We thus demonstrate a hybrid genome assembly strategy that is based on both short- and long-read sequencing. We show that this strategy is capable of creating highly contiguous genome reconstructions, not only for the L. pustulata mycobiont but also its photobiont Trebouxia sp., along with substantial amounts of the bacterial microbiome. A subsequent analysis of the microbiome of L. pustulata – performed over nine different samples collected in Germany and Italy – showed a stable taxonomic composition across the geographic range. We find that Acidobacteriaceae, which are known to thrive in nutrient poor habitats, are the dominant taxa. These would make them well adapted for the co-habitation with L. pustulata, which largely grows on rocks. Whether the Acidobacteriaceae are functionally involved in the lichen symbiosis is unclear so far.
As further comparative genomic studies rely on comprehensive genome annotations, we evaluate the completeness and fidelity of the gene annotations for the mycobiont L. pustulata as well as four further Lecanoromycetes. This reveals that un- and mis-annotated genes impact all evaluated genomes, with artificially joined genes and unannotated genes having the largest impact. In addition to these factors we find that the sequence composition – especially G/C-rich inverted repeats – lead to sequencing errors that interfere with the gene prediction. We minimize the effects of these artifacts through a rigorous curation.
Given the extremely sparse taxon sampling of available green alga genomes, we focus our search for the genomic footprints of lichenization on the mycobionts. We compare the genomes of the Lecanoromycetes to their closest relatives, the Eurotiomycetes and Dothideomycetes. This reveals that the last common ancestor of the Lecanoromycetes has lost around 10% of its genes after they split from the non-lichenized ancestor they share with the Eurotiomycetes. These losses are furthermore enriched, showing an excessive loss of genes involved with the degradation of polysaccharides. The loss of these genes fits a change from an ancestral saprotrophic lifestyle that depends on degrading complex plant matter, to the symbiotic lifestyle that relies on simpler nutrients provided by the photobionts. While the last common ancestor of the Lecanoromycetes additionally gained around 400 genes these could so far not be further characterized due to a lack of functionally annotated reference data.
As the mycobiont L. pustulata could so far not been grown in axenic culture, we initially expected to find an extensive genomic remodeling compared to the other mycobionts that easily grow in culture. We do not find evidence for this. Analyzing both the contraction of gene families and the loss of genes, we observe that L. pustulata and Umbilicaria muehlenbergii – its close relative that is easily grown in culture – share most of these. Furthermore, L. pustulata does not show an excessive loss of evolutionary old and well-conserved genes. These effects are mirrored on the functional level, as neither gene family contractions nor gene losses show a functional enrichment. This is partially due to the lack of functional reference data, analogous to the genes gained in the Lecanoromycetes, rendering their characterization hard. Thus, further studies on the genomic consequences of lichenization and differences in symbiotic dependence will have to be conducted, including larger taxon sets. This will be even more important for the photobionts, as the Chlorophyta are even more sparsely sampled today, hindering an effective functional and evolutionary study.
Rhabdomyosarcoma is the most common paediatric soft-tissue sarcoma, and for tumour recurrence, the prognosis is still unfavourable. The current standard therapy consisting of surgery, radiation and combined chemotherapy does not consider the specific biology of this tumour.
Histone deacetylases (HDACs) and the Lysine-specific demethylase-1 (LSD1) are two epigenetic modifiers which are both part of repressor complexes leading to transcriptional silencing of target genes. Whereas HDACs lead to deacetylation of several lysine-residues within the histone tail, LSD1 is specific for demethylation of H3K4me2 and H3K4me1, as well as in a different context for H3K9me2. Rhabdomyosarcoma is reported to harbour high levels of LSD1, but the functional relevance is yet unclear. HDAC inhibition proved to be effective as single agent treatment, however, the proximity of HDAC1/2 and LSD1 in repressor complexes at the DNA implies a suitable rationale for a combination therapy potentially leading to cooperative effects on target gene transcription. In this study, we aimed to evaluate the potential of a combined LSD1 and HDAC inhibition for cell death induction in rhabdomyosarcoma cell lines. Whereas LSD1 inhibitors failed to induce cell death on their own, the combined inhibition of HDACs and LSD1 resulted in highly synergistic cell death induction. This effect extended to several combinations of LSD1 and HDAC inhibitors as well as to four different rhabdomyosarcoma cell lines, two of embryonal and two of alveolar histology.
With the use of the HDAC inhibitor JNJ-26481585 and the reversible LSD1 inhibitor GSK690, we demonstrated that the cell death induced by the combination matches with the details of intrinsic mitochondrial apoptosis. JNJ-26481585/GSK690-induced cell death is partially caspase-dependent and leads to caspase cleavage, followed by substrate cleavage as shown for PARP, as well as loss of the mitochondrial membrane potential.
Furthermore, JNJ-26481585 and GSK690 acted together to transcriptionally upregulate the proapoptotic proteins NOXA, BIM and BMF, which resulted in respective changes on protein level for both cell lines. However, the antiapoptotic BCL-2 family proteins BCL-2, MCL-1 and BCL-xL displayed only minor changes in protein levels upon treatment with GSK690 and JNJ-26481585, which did not rely on transcriptional activity. Therefore, the increase in proapoptotic proteins induces a shift towards proapoptotic signalling at the mitochondrial membrane. This shift is functionally relevant since knockdown of a proapoptotic protein or overexpression of one of the antiapoptotic proteins BCL-2 and MCL-1, as well as a stabilized mutant MCL-1, can significantly protect from GSK690/JNJ-26481585-induced cell death.
Knockdown of the mitochondrial membrane protein BAK, which is directly guarding the mitochondrial membrane integrity, potently protected from GSK690/JNJ-26481585- induced cell death, directly linking the shift in the BCL-2 family proteins to the observed loss of mitochondrial membrane potential and the further downstream activation of caspases. Furthermore, treatment with JNJ-26481585 and GSK690 resulted in a cell cycle arrest in G2/M phase, indicating additional effects on the tumour cells beside apoptosis induction. Taken together, the combined inhibition of LSD1 and HDACs is a promising strategy for rhabdomyosarcoma treatment.
Surface water can contain a complex mixture of organic micropollutants (i.e. residues of pharmaceuticals or biocides). Conventional wastewater treatment plants (WWTPs) do not completely remove a broad range of anthropogenic chemicals and therefore represent a leading point source. To upgrade WWTPs, technical solutions based on oxidative and sorptive processes have been developed and successfully implemented. Acknowledging these substantial advances, this thesis focuses on another key topic and aims to investigate whether improved biological treatment processes likewise effectively remove anthropogenic micropollutants from wastewater. The work conducted on this topic was part of two European research projects (ATHENE, ENDETECH).
The ATHENE project aimed to go beyond the state-of-the-art by developing biological wastewater treatment processes that exploit the full potential of biodegradation. With the objective to explore the potential of complementary strictly anaerobic conditions within the biological wastewater treatment, combinations of aerobic and anaerobic treatments on site of a WWTP were implemented. Based on pre-experiments, two promising treatment combinations were selected for a more comprehensive evaluation. An aerobic treatment was paired with an anaerobic pre-treatment under iron-reducing conditions, and an activated sludge treatment was combined with an anaerobic post-treatment under substrate-limiting conditions. For the evaluation of these processes, an effect-based assessment was applied and combined with chemical data of 31 selected target organic micropollutants as well as ten metabolites. To assess the removal of endocrine disrupting chemicals (EDCs), yeast based reporter gene assays covering seven receptor-mediated mechanisms of action including (anti-)estrogenicity, (anti-) androgenicity, retinoid-like, and dioxin-like activity were conducted. Furthermore, the removal of unspecific toxicity (Microtox assay) and oxidative stress response as a marker for reactive toxicity (AREc32 assay) were analyzed to cover micropollutants acting via a non-specific mechanism of action. Moreover, to assess toxicity of the whole effluent in vivo, standardized in vivo bioassays with four aquatic model species (Desmodesmus subspicatus, Daphnia magna, Lumbriculus variegatus, Potamopyrgus antipodarum) were performed.
The combination of aerobic and anaerobic treatments resulted in a low additional removal of the selected target organic micropollutants (by 14-17%). In contrast, the removal of endocrine and dioxin-like activities (by 17-75%) and non-specific in vitro toxicities (by 27-60%) was significantly enhanced. Compared to technical solutions (i.e. ozonation), the combination with an anaerobic pre-treatment under iron-reducing conditions was likewise effective in removing the estrogenic activity as well as the unspecific toxicity, whereas anti-androgenic activity and dioxin-like activity were less effectively removed. Exposure to effluents of the conventional activated sludge treatment did not induce adverse in vivo effects in the investigated aquatic model species. Accordingly, no further improvement in water quality could be observed. In conclusion, the combination of aerobic and anaerobic treatment processes significantly enhanced the removal of specific and non-specific in vitro toxicities. Thus, an optimization of the biological wastewater treatment can lead to a substantially improved detoxification. These capacities of a treatment technology can only be uncovered by complementary effect-based measurements.
The global objective of the ENDETECH project was to develop a biotechnological solution to eliminate recalcitrant pharmaceuticals in wastewater direct from sites, where high loads are expected (i.e. hospitals). For this purpose, laccase, an enzyme mainly found in wood decaying fungi, was immobilized on ceramic membranes for application in bioreactors. In a proof of principle experiment, the performance of immobilized laccase in removing a mixture of 38 antibiotics without and in combination with a natural mediator (syringaldehyde; SYR) was investigated. For the evaluation of the enzymatic membrane bioreactors, chemical data on the elimination of the selected target antibiotics was combined with the outcomes of two in vitro bioassays. Growth inhibition tests with an antibiotic sensitive Bacillus subtilis strain were conducted to assess the residual antibiotic activity of the effluents, and Microtox assays were performed to detect a potential formation of toxic by-products.
The treatment by laccase without SYR did not reduce the load of antibiotics significantly. In contrast, in combination with a SYR concentration of 10 µmol L-1, 26 out of 38 antibiotics were removed by >50% after 24 h treatment. Moreover, increasing the SYR concentration to 1000 µmol L-1 resulted in a further improvement of the antibiotic removal. 32 out of 38 antibiotics were removed by over 50%, whereby 17 were almost completely eliminated (>90%). However, the treatment with laccase in combination with SYR resulted in a time-dependent increase of unspecific toxicity. While SYR alone did not affect B. subtilis, the combination of laccase with SYR led to a strong time-dependent growth inhibition up to 100%. Similar to that, a time-dependent increase of unspecific toxicity in the Microtox assay was observed. In conclusion, the laccase-mediator process successfully degrades a broad spectrum of antibiotics and thus represents a promising technology to treat wastewater from sites, where high loads are expected. However, further research is required to reduce the formation of unspecific toxicity before an implementation of this technology can be considered.
Pulsed electron-electron double resonance (PELDOR), also called Double Electron-Electron Resonance, (DEER) is a pulsed EPR technique that can provide structural information of biomolecules, such as proteins or nucleic acids, complementary to other structure determination methods by measuring long distances (from 1.5 up to 10 nm) between two paramagnetic labels. Incorporation of the rigid Ç-label pairwise into DNA or RNA molecules enables the determination not only of the distance but also of the mutual orientation between the two Ç-labels by multi-frequency orientation-selective PELDOR data (X-, Q- and G-band frequencies). Thus, information about the orientation of secondary structure elements of nucleic acids can be revealed and used as additional angular information for structure determination. Since Ç does not have motion independent from the helix where it resides, the conformational flexibility of the nucleic acid molecule can be directly determined. This thesis demonstrates the advancement of PELDOR spectroscopy, beyond its original scope of distance measurements, to determine the mutual orientation between two rigid spin labels towards the characterization of the conformational space sampled by highly flexible nucleic acid molecules. Applications of the methodology are shown on two systems: a three-way junction, namely a cocaine aptamer in its bound-state, and a two-way junction, namely a bent DNA.
More in detail, the conformational changes of the cocaine aptamer upon cocaine binding were investigated by analysis of the distance distributions. The cocaine-bound and the unbound states could be differentiated by their conformational flexibility, which decreases in the presence of the ligand. Moreover, the obtained distance distributions revealed a small change in the mean distance between the two spin labels upon cocaine binding. This indicates a ligand-induced conformational change, which presumably originates at the junction where cocaine is known to bind. The investigation of the relative orientation between the two spin-labeled helices of the aptamer revealed further structural insights into the conformational dynamics of the cocaine-bound state. The angular information from the orientation-selective PELDOR data and the a priori knowledge about the secondary structure of the aptamer were helpful in obtaining a molecular model describing its global folding and flexibility. In spite of a large flexible aptamer, the kink angle between the Ç-labeled helices was found to be rather well-defined.
As for the bent DNA molecule, a two-step protocol was proposed to investigate the conformational flexibility. In the first step, a database with all the possible conformers was created, using available restraints from NMR and distance restraints derived from PELDOR. In a second step, a weighted ensemble of these conformers fitting the multi-frequency PELDOR data was built. The uniqueness of the obtained structural ensemble was checked by validation against an independent PELDOR data set recorded at a higher magnetic field strength. In addition, the kink and twist angle pairs were determined and the resulting structural ensemble was compared with the conformational space deduced both from FRET experiments and from the structure determined by the NMR restraints alone.
Overall, this thesis underlines the potential of using PELDOR spectroscopy combined with rigid spin labels in the context of structure determination of nucleic acids in order to determine the relative orientation between two helices, the conformational flexibility and the conformational changes of nucleic acid molecules upon ligand binding.
Der Gyrus dentatus ist eine anatomische Region im Hippocampus und besitzt die einzigartige Fähigkeit auch im adulten Gehirn lebenslang neue Nervenzellen zu generieren. Dieser Prozess wird als adulte Neurogenese bezeichnet, stellt eine besondere Form struktureller Plastizität dar und es wurde gezeigt, dass adult neugebildete Körnerzellen im Gyrus dentatus essentiell am Prozess des hippocampalen Lernens und der Gedächtnisausbildung beteiligt sind. Es wird vermutet, dass neue Körnerzellen aufgrund ihrer charakteristischen Eigenschaften verstärkt auf neue Informationsmuster reagieren können und darauf spezialisiert sind Muster, die eine hohe Ähnlichkeit zueinander haben zu separieren und diese Unterschiede zu kodieren. Obwohl bereits eine Vielzahl von wissenschaftlichen Studien zum Verständnis der Entwicklung und Funktion adult neugebildeter Körnerzellen beitragen konnte, bestehen immer noch Unklarheiten darin, wie sich diese neuen Nervenzellen strukturell entwickeln, wann es zu einer funktionellen Integration kommt und wie diese beiden Prozesse miteinander zusammenhängen. In den vorliegenden Arbeiten wurde die strukturelle Entwicklung und synaptische Integration adult neugebildeter Körnerzellen in das bestehende hippocampale Netzwerk der Ratte und Maus unter in vivo Bedingungen untersucht. Zur Beantwortung dieser Fragen wurden Methoden aus der Anatomie, Histologie und in vivo Elektrophysiologie kombiniert. Der Nachweis neuer Körnerzellen erfolgte entweder durch immunhistologische Färbungen gegen spezifische Marker für unreife und reife Körnerzellen, Markierungen mit Bromdesoxyuridin oder retro- bzw. adenovirale intrazerebrale Injektionen und Expression von GFP. Es wurde eine in vivo Stimulation des Tractus perforans in der anästhesierten Ratte zur Langzeitpotenzierung der Körnerzellsynapsen und anschließend eine immunhistologische Analyse der Expression von synaptischen Aktivitäts- und Plastizitätsmarkern in neugebildeten und reifen Körnerzellen nach der Stimulation durchgeführt. Zusätzlich wurden detaillierte drei-dimensionale Rekonstruktion dendritischer Bäume erstellt und dendritische Dornenfortsätze an retroviral markierten Zellen analysiert.
Die vorliegenden Daten belegen den generellen Verlauf der Entwicklung neugeborener Körnerzellen in zwei unterschiedliche Phasen: eine frühe dendritische Reifung und eine späte funktionelle und synaptische Integration. Neugeborene Körnerzellen zeigten ein rasches dendritisches Auswachsen, dass innerhalb der ersten drei bis vier Wochen abgeschlossen war. Während dieses Wachstumsprozesses passieren Dendriten nacheinander die Körnerzellschicht und anschließend die innere, mittlere und äußere Molekularschicht. Dadurch sind sie innerhalb ihrer morphologischen Entwicklungsphasen anatomisch auf spezifische präsynaptische Partner limitiert. In der wissenschaftlichen Literatur wird eine transiente kritische Phase beschrieben, in der neugeborene Körnerzellen eine starke Plastizität und sensitivere synaptische Erregbarkeit aufweisen. Obwohl die vorliegenden Resultate keine direkten Hinweise auf eine stärkere bzw. sensitivere Plastizität neugeborener Körnerzellen liefern, konnte eine Phase zwischen vier und fünf Wochen identifiziert werden, in der neue Körnerzellen einen sprunghaften Anstieg in ihrer Fähigkeit zur Expression synaptischer Aktivitätsmarker (z.B. Arc und c-fos) und Ausbildung struktureller Plastizität (Dendriten und Dornenfortsätze) zeigten. Die präsentierten Resultate machen deutlich, dass Dornenfortsätze neuer Körnerzellen nach elf Wochen eine vergleichbare Dichte, Größenverteilung und Plastizität aufzeigen, die vergleichbar mit denen vorhandener Körnerzellen sind. Die Fähigkeit zur dendritischen Plastizität nach synaptischer Aktivierung zeigten jedoch nur neugeborene Körnerzellen zwischen der vierten und fünften Woche. Diese Ergebnisse implizieren, dass die Integration neugebildeter Körnerzellen kontinuierlich verläuft und obwohl die vorliegenden Daten die Existenz einer dendritischen Plastizität und einen sprunghaften Anstieg synaptischer Plastizität in der vierten und fünften Woche belegen, wurden keine weiteren Hinweise auf eine transiente kritische Phase gefunden. Des Weiteren zeigten dendritische Bäume von gereiften adult neugeborenen und reifen Körnerzellen Unterschiede, die daraufhin deuten, dass neue Körnerzellen eine eigene Subpopulation darstellen.
In the dentate gyrus (DG) of the mammalian hippocampus, neurogenesis continues to take place throughout an organism’s life. Adult neurogenesis includes proliferation and differentiation of neural stem cells into dentate granule cells (GCs) that mature and integrate into the existing cellular network. This thesis work presents a novel approach that enables longitudinal examination of living postnatally generated GCs in their endogenous niche by using retroviral (RV) labeling in organotypic entorhino-hippocampal slice cultures (OTCs). Older GCs were fluorescence-labeled with an adeno-associated virus controlled by the synapsin 1 promoter (AAV-Syn). The combination of time-lapse imaging and 3-D reconstruction of newborn developing GCs and older, more mature GCs enabled comparative analyses of dendritic growth and cellular dynamics as well as investigations of spine formation and the establishment of synaptic contacts.
Postnatal neurogenesis was studied in the mouse and rat DG in vivo by analysis of the distribution of chemical neuronal maturation markers doublecortin (DCX) and calbindin in combination with the GC marker Prox1 between P7 and P42. The marker expression patterns at different time points indicated that the number of mature GCs increased gradually over time and that young, immature GCs were added to the inner layers of the granule cell layer (GCL), as is the case in the adult brain. The most substantial shift in GC maturation took place between P7 and P14, though GCs in the rat DG matured faster (i.e. by ~5 days) than GCs in the mouse. Immunocytochemical in vitro analysis in OTCs at DIV 7, 14, and 28 exhibited a distribution of marker expression over time that was comparable to in vivo, though the number of DCX-expressing GCs was low at DIV 28, indicating a considerable decrease in neurogenesis rate over time in the OTC. Nevertheless, RV-labeling of newborn GCs at DIV 0 yielded successful visualization and enabled time-lapse imaging of complete developing GCs up to 4 weeks after mitosis. During the second week of development, newborn GCs exhibited a high level of structural dynamics, including extension and retraction of dendritic segments. In the third week, newborn GCs displayed high dendritic complexity which was followed by pronounced dendritic pruning. Finally, a phase of structural stabilization and local refinement could be observed during the fourth week. Older AAV-Syn-labeled GCs did not exhibit such dynamic structural remodeling. Anterograde tracing of entorhinal projection fibers using the biotinylated dextran amine Mini Ruby showed innervation of the outer molecular layer (OML) by entorhinal axons at early time points, i.e. DIV 8 when newborn GCs started to extend dendrites into the ML, as well as at DIV 20 when RV-labeled GCs exhibited elaborate dendritic trees with processes in the OML intermingling with entorhinal fibers. This shows that newborn GCs in the OTC grow into an area of existing entorhinal axon terminals, which is highly similar to the situation in the adult brain. Hence, the results show that postnatal neurogenesis can be studied effectively in the OTC system as a model of adult neurogenesis. The first appearance of spine-like protrusions in newborn GCs was observed two weeks post RV injection. Ultrastructural electron-microscopic images revealed that spines established synaptic contacts with axonal boutons. These findings suggest that newborn GCs are successfully integrated into the existing cellular circuitry in the OTC system. The high level of structural flexibility found in this study might be a necessary requisite of new neurons for successful dendritic maturation and functional integration into a neuronal network. Thus, live imaging of postnatally born GCs in the OTC appears as a useful novel approach to elucidate the mechanisms that affect cellular dynamics of neurogenesis.
Savannas provide essential ecosystem services for human well-being in West Africa. Thus, ecosystem change not only directly affects biodiversity but also human livelihoods. Human land use considerably shaped these savanna ecosystems for millennia, particularly agriculture, livestock grazing, logging and the collection of non-timber forest products (NTFPs). NTFPs are wild plant products and comprise all organic matter from herbaceous plants, shrubs, and trees (excluding timber). Current increasing land use pressure through fast demographic changes is widely esteemed as a severe threat for savanna biodiversity and the socio-economy of rural communities. In consideration of the pivotal role of NTFP species for biodiversity and livelihoods, it is important to evaluate the effect of increasing land use change on savanna vegetation and on its provisioning service for human well-being. Thus, the major aim of this thesis is to investigate the impacts of land use intensification on vegetation composition, diversity and function and its consequences for provisioning ecosystem services (NTFPs) and human well-being in a West African savanna.
The research for this study was conducted in the North Sudanian vegetation zone of south-eastern Burkina Faso, where population growth exceeds the nationwide trend. Generally, Burkina Faso belongs to the worldwide poorest countries, where nearly one quarter of the population suffers from malnutrition (FAO 2014). The integration of NTFPs and particularly wild food species into rural household economies is, thus, an important measure in the national combat against poverty and food insecurity (FAO 2014). Against this background, I focus on vegetation changes, the economic importance of NTFPs as well as the decrease and substitution of wild food species in this study.
Vegetation resurveys of different vegetation types since the early 1990s showed that land use change led to more pronounced changes in the herbaceous than in the woody vegetation layer. Most woody vegetation types stayed stable in species composition and richness, even though some highly useful tree species (Vitellaria paradoxa, Parkia biglobosa) declined in some woody vegetation types. In contrast, in most herbaceous vegetation types species richness increased and species composition considerably changed. This change might be explained by a general ruderalisation process through a pronounced increase of wide-ranging herbaceous species. However, in spite of a general species increase in the herbaceous layer, a decrease of preferred herbaceous fodder species was found. Thus, the decline of useful species in both layers is alarming. Herbaceous vegetation types also showed more pronounced changes in plant functional trait characteristics in comparison to woody vegetation types. However, an increase of smaller plant species and species with a high diaspore terminal velocity (VTerm) was found in both vegetation layers. Since these two trait responses are generally related to grazing and browsing, the strong increase of livestock herds is likely to be responsible for the detected vegetation changes.
In addition to the vegetation study, interviews showed that all useful food species were widely considered to decline. The two economically most important tree species, the shea tree (Vitellaria paradoxa) and the locust bean tree (Parkia biglobosa) that contribute with 70% to wild food income, were considered among the most declining species of all cited wild food species. On this matter, local perceptions of species decline and results from field observations are in accordance. However, a wide range of cited substitutes indicated a great knowledge on alternative plant species in the area. Most wild food species are, however, substituted by other highly valued wild food species. Although our results suggest that rural communities are able to cope with the decrease or absence of wild food species, growing decline of one species would concurrently increase the pressure on other native food species. Therefore, the need to counteract the decrease of highly useful wild food species should be of high priority in management measures. In general, I showed that NTFPs are an essential component in rural households, since it contributed with 45 % to total household income. Significant differences in NTFP dependency between the two investigated villages and across the three main ethnic groups were detected, reflecting different traditional uses and harvesting practices. In general, it was shown that poorer households depend more on NTFP income than wealthier households. Against the background of this study, management strategies for agroforestry systems and poverty alleviation should consider local differences, and ethnicity-dependent NTFP-use patterns.
Overall, the combination of field studies on temporal and functional vegetation change with socio-economic and ethno-botanic interviews increases the knowledge on qualitative and quantitative vegetation changes and on the consequences for rural populations. This thesis gives a thorough insight into decreasing trends of economically valued plant species and thus gives evidence on the consequences of vegetation changes for ecosystem services of West African savanna ecosystems. Further, different NTFP-dependencies and use preferences according to socio-economic and cultural variables, such as ethnicity, present a valuable basis for specific decision-making and should be considered in management plans.
Alzheimer’s disease is a chronic neurodegenerative disease that causes problems with memory, thinking and behavior. The pathophysiological hallmarks of AD are extracellular senile plaques and intracellular neurofibrillary tangles. Amyloid plaques mainly contain the amyloid-β (Aβ) peptide, which appears as a cleavage product of the APP. APP is a type I transmembrane protein with a large extracellular domain and a short cytoplasmic tail. It is expressed in variety of tissues e.g. in neuronal tissue (brain, spinal cord, retina), and non-neuronal tissues (kidney, lung, pancreas, prostate gland, and thyroid gland) (Dawkins and Small, 2014). APP has been studied because of its link to AD, however, its role in normal brain function is poorly understood. APP is processed by two different pathways, amyloidogenic pathway and non-amyloidogenic pathway. In physiological condition, the majority of APP is processed via the non-amyloidogenic, thus leading to the generation of the secreted N-terminal APP processing product sAPPα. sAPPα is formed due to the cleavage of APP by α-secretase. In previous studies, our group has shown that sAPPα produce potent neuroprotective effect by altering gene expression, as well as by antagonizing several different types of neurotoxic stress stimuli (Copanaki et al., 2010; Kögel et al., 2003, 2005; Milosch et al., 2014). Several studies have shown that protein degradation is reduced in AD (Hong et al., 2014; Lipinski et al., 2010) but the role of APP and its cleavage products in protein degradation is still unknown. This thesis discusses about the physiological functions of APP in neuroprotection and protein homeostasis.
In the first part of the thesis (Section 4.1 - 4.4), the neuroprotective properties of yeast derived sAPPα and E1 (N-terminal domain of sAPPα) were investigated under serum and glucose deprivation conditions. In previous work, it was shown that recombinant sAPPα evoked a significant decrease in serum deprivation triggered cell death in human SH-SY5Y neuroblastoma cells and mouse embryonic fibroblast MEF cells. It was also observed that sAPPα induces the phosphorylation of Akt which leads to neuroprotection (Milosch et al., 2014). This study investigated whether this neuroprotection is associated with altered expression of downstream intracellular Akt targets such as FoxO, Bim, Bcl-xL and Mcl-1 under stress conditions. Here it was shown that sAPPα prevents activation and nuclear translocation of FoxO. FoxO act as a transcription factor for different proapoptotic genes such as Bim. It was also observed that Bim protein and mRNA expression was significantly reduced with sAPPα and E1 treatment. The expression of antiapoptotic protiens such as Bcl-xL and Mcl-1 were also examined and it was observed that sAPPα and E1 increases expression of both these proteins. Furthermore, it was previously demonstrated that uncleaved holo-APP functionally cooperates with sAPPα to activate Akt and provide neuroprotection (Milosch et al., 2014). Therefore, to investigate the function of the APP in sAPPα regulated Akt downstream proteins expressions, MEF APP KO cells were used. E1 and sAPPα only showed neuroprotective modulatory effect on these Akt downstream targets in MEF wt cells, but not in APP KO cells. In addition, sAPPα also showed neuroprotection in primary wt hippocampal neurons under trophic factor deprivation. Cellular fractionation experiments were also done to determine the role of sAPPα in cytochrome c release from mitochondria. It was observed that sAPPα treatment can inhibit mitochondrial cytochrome c release in wt MEF cells.
The second part of the thesis (Section 4.5 - 4.9) discusses about the role of sAPPα in protein homeostasis. It was observed that sAPPα prevents proteotoxic stress induced BAG3 protein expression in SH-SY5Y and MEF cells. This was also observed in mRNA levels which indicate a transcriptional regulation. Furthermore, treatment with sAPPα was also shown to decrease aggresomes formation. Aggresomes are perinuclear aggregates which are formed due to accumulation of damaged and misfolded proteins and BAG3 plays important role in their formation and the transport of degradation prone proteins into these structures. The analysis of proteasomal activity showed a reduced accumulation of proteasomal substrate d2 by sAPPα under proteasomal stress. In proteasomal activity assay, sAPPα was shown to increase the degradation of proteasomal substrate SUC-LLVY-AMC and the fluorigenic signal was measured spectrophotometrically. The sAPPβ fragment which is generated via the amyloidogenic pathway was also examined for its role in BAG3 expression and proteasomal degradation. sAPPβ, which has almost similar structure as sAPPα, only 17 amino acids at the C-terminus is missing, was failed to modulate BAG3 expression and proteostasis. This indicates that these biological effects are highly specific for sAPPα.
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The focus of this research was to understand the molecular mechanism that lies behind the insertion of tail-anchored membrane proteins into the ER membrane of yeast cells. State-of-art instruments such as LILBID, and Cryo-EM, combined with the introduction of direct electron detectors, were used to analyze the proteins that capture tail-anchored proteins near the ER membrane and help their releases from a chaperone, an ATPase named Get3. Get3 escorts TA proteins to the ER membrane, where both Get3 and the TA proteins interact sequentially to Get3 membrane bound receptors Get1 and Get2. Get1 and Get2 are homologs of mammalian WRB and CAML.
The native host was used to separately produce Get1, Get2, and the Get2/Get1 single chain constructs. The studies showed that when Get1 is expressed alone, Get1 does not seems to be located in the ER membrane but rather in microbodies like shape organelles (or peroxisome). Interestingly, Get1 seems to be located in the ER membrane when it is linked to Get2 as single chain construct.
The localization study of Get2/Get1 fused to GFP shows from the fluorescence intensity that Get2/Get1.GFP has a tube-like morphology or membrane-enclosed sacs (cisterna), implying that Get2/Get1 is actually targeted to the ER membrane and is likely functional. In other words, Get1 and Get2 stabilize each other in the ER membrane.
The expression of Get2/Get1 was found to be already optimum when expressed as single chain construct because the fluorescence counts did not improve when additives such as DMSO or histidine were added. However, when Get1 and Get2 are expressed separately, additives improve their protein production yield. In 1 liter culture, Get1 yield is increased by about 3 mg and Get2 by 1.8 mg. This can be explained by the space that Get1 and Get2 should occupy within the ER membrane as they must coexist with other membrane components to maintain the homeostasis of the cell. Hence, if there were no gain for single chain construct expression, it meant that Get2/Get1 was already well expressed on its own in ER membrane and has reached its optimum expression without the help of additives. The Get2/Get1 overexpression is more stable, tolerated and less toxic for the cells to express it at a high level.
DDM has proved to be the best detergent from the detergents tested to solubilize Get1, Get2, and Get2/Get1.
Thereafter, Get1, Get2 (data not shown), and Get2/Get1 were successfully purified in DDM micelles.
Furthermore, for the first time using LILBID, the actual study has shown that Get1 and Get2 are predominantly a heterotetramer (2xGet1 and 2xGet2) but higher oligomerization may exist as well.
Get3 binds to Get1 in a biphasic way with a specific strong binding of an affinity of 57 nM and the second of 740 nM nonspecific indicative of heterogeneity within the interaction between Get1 and Get3. This heterogeneity is caused by the presence of different conformation of either protein. However, in order to characterize a high-resolution structure model of a specific target one needs highly homogenous and identical molecules of the target protein or complex in solution. The homogeneity increases the chances of growing crystals during crystallography as the good homogeneity will likely generate a perfect packing of unit cells stack (also known as crystal lattice) in the three-dimensional spaces. The same truth goes for the single particles analysis Cryo-EM, especially for smaller complexes where having less or no conformation alterations of specific targets will enable the researcher to classify the particles in 2D and 3D, therefore improving the signal-to-noise-ratio that will ultimately lead to high-resolution structure determination.
Get1, Get2/Get1 and chimeric variants (tGet2/Get1, T4l.Get2/Get1, T4l.Get2.apocyte.Get1) were crystallized but none of the crystals could diffract due to heterogeneity.
This heterogeneity was not only occurring upon the binding of Get3 to its membrane receptors, but seems to be already present within the receptors themselves through possibly different conformation.
In this Ph.D. thesis, the heterogeneity of purified Get2 and Get1 as complex or individually in detergent is then, so far, the limiting factor for obtaining a high-resolution structure model of Get1 and Get2. As mentioned above, the heterogeneity observed was not due to the quality of the sample preparation but rather to the effect of different conformations that could have been native, or just because of the micelle used, as it was proven by the 3-D heterogeneity classification by Cryo-EM.
In general, crosslinking is one way to keep the integrity of protein complexes, however it appeared not to improve the sample quality when it was analyzed in micelles. Often the integrity of some membrane proteins is affected when they are solubilized and purified in detergents.
Finally, in this study, the structural map of Get2 and Get1 complex linked with chimeric protein T4 lysozyme and apocytochrome C b562RIL gene was obtained at 10 Å. However, this single chain construct has a density map corresponding to heterodimer species (one Get1 and Get2). Therefore, based on those data the tertiary structure of Get2/Get1 in micelle is poorly defined. It could be that the membrane extraction in DDM and the purification destabilizes the structure of the complex.
Multicellular organisms require that cells adhere to each other. This cell-cell adhesion is indispensable for the formation and the integrity of epithelial structures, tissues and organs. Mammals have developed four different cell-cell adhesion structures, the adhering junctions, which ensure the tight contact between cells but are also important platforms for communication and exchange in tissues. Two of these adhering junctions are cadherin based, the belt-like adherens junctions and the spot-like desmosomes. Both structures have in common that they are composed of single membrane spanning proteins, the cadherins, which accomplish adhesion in a calcium-dependent manner. The intracellular parts of classical as well as desmosomal cadherins bind to different adaptor proteins of the armadillo-protein family and others which build a protein plaque underneath the membrane and link the cadherins to the actin or intermediate filament cytoskeleton.
Desmosomes are of special importance for tissues that have to withstand mechanical stress. Although they are essential to stabilize tissues they have to be highly flexible and dynamic structures, as processes like wound healing or tissue remodeling require that adhesive interactions can be modulated. The molecular dynamics within desmosomes are not jet understood in detail, but it is assumed that two different membrane associated pools of desmosomal cadherins exist in cells. Cadherins that are incorporated in mature desmosomes are part of the junctional pool, whereas cadherins that are not associated with firm desmosomes and the intermediate filament cytoskeleton belong to the non-junctional pool. Lateral movements between the two pools results in a dynamic equilibrium and allows for example the exchange of old cadherins. Little is known about the breakdown of desmosomal cadherins. Several studies found that desmosome assembly or endocytosis are cholesterol dependent processes and claimed that membrane microdomains play a role in the regulation of desmosome dynamics. Moreover, membrane rafts may be involved in the pathomechanism of the desmosome associated disease pemphigus, were autoantibodies bind to the cadherin desmoglein-3 and trigger its internalization which results in a loss of adhesion in skin cells.
Membrane rafts are cholesterol dependent nanoscale structures of cellular membranes that are able to regulate the distribution of proteins within the plasma membrane and thus form platforms for cell signaling and membrane trafficking. Flotillins are proteins that are associated with membrane rafts and are reported to be involved in processes like endocytosis, endosomal sorting and a multitude of different signaling events. We could recently show that the membrane raft associated proteins flotillin-1 and flotillin-2 bind directly to the armadillo protein y-catenin which can be part of both, the adherens junction and the desmosome. The aim of this study was to eluciadate a possible role of flotillins in the regulation of desmosomes.
HaCaT keratinocytes were chosen as the main cell system for this study and at first the association of desmosomal components with flotillins was analyzed in detail. It was found that flotillins are clearly associated with desmosomal proteins. They colocalize with desmoglein-3 at cell borders and precipitate the other desmogleins. Further binding assays revealed that both flotillins bind to all desmogleins and the long isoforms of the second class of desmosomal cadherins, the desmocollins. The interaction is a direct one and was mapped to the ICS sequence within the cadherins. This close association rendered the question whether flotillins are functionally implicated in desmosome regulation. To address this issue, stable flotillin knockdown HaCaT cells were analyzed in detail. The molecular morphology of desmoglein-3, desmoglein-1 and two plaque proteins was clearly altered in the absence of flotillins. The membrane staining of all tested desmosomal proteins was derailed and disordered. Furthermoore, the loss of flotillins had an impact on the adhesive capacity of HaCaT keratinocytes. The cell-cell adhesion was weakened in the absence of flotillins, which was monitored by an increased fragmentation of knockdown cells in a cell dissociation assay.
In order to find out the mechanism by which flotillins influence the membrane morphology and the adhesiveness in keratinocytes, the association of desmosomal proteins with membrane microdomains was examined, at first. A predominant part of desmoglein-3 is associated with membrane rafts in HaCaT keratinocytes, whereas only a minor part of desmoglein-1 is found there. However, the raft-association of none of the examined proteins was altered in the absence of flotillins. Furthermore, flotillin depletion did not change the distribution of desmogleins with the two different cadherin pools. Less desmoglein-3 is found in the junctional pool of the flotillin depleted cells compared to the control cells, but this is due to an overall diminished desmoglein-3 protein level in these cells.
Flotillins are involved in endocytic processes but their exact role there is under debate. The endocytic uptake of desmosomal cadherins requires intact membrane rafts, but the precise mechanism is still unknown. A possible involvement of flotillins on the endocytosis of desmoglein-3 was addressed next. It is known that the internalization of desmoglein-2 is dependent on the GTPase dynamin, arguing for an involvement of dynamin in the endocytosis of desmoglein-3 as well. When dynamin and thus desmoglein-3 endocytosis was inhibited using chemical compounds, the mislocalization of desmoglein-3 that was observed in flotillin knockdown cells was restored. This suggest that inhibition of desmoglein-3 endocytosis enhances the amount and/or availability of desmoglein-3 at the plasma membrane, which then normalizes the morphological alterations caused by a knockdown of flotillins. Furthermore the morphological alterations in the flotillin knockdown HaCaT cells were found to be similar to the localization of desmoglein-3 that was observed upon treatment of keratinocytes with PV IgG These structures have been described before as linear arrays and are assumed to be sites of endocytic uptake. This strengthens the idea that enhanced desmoglein-3 internalization takes place in the absence of flotillins, which then results in a weakened adhesion.
Altogether this study revealed flotillins as novel players in desmosome mediated cell-cell adhesion processes. By binding to desmosomal cadherins and desmosomal plaque proteins, flotillins stabilize desmosomes at the plasma membrane and are required for a proper cell-cell adhesion.