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Understanding global biodiversity patterns is one of the main objectives of ecology. Spatial variation in species richness can be explained by several environmental factors. The relationships between species richness and environmental factors have been associated with latitudinal, longitudinal and elevational gradients. The number of species is determined by birth, death and migration rates of species in a given area. These rates are affected by abiotic and biotic factors acting at local and regional scales. Climatic seasonal variation may also influence biodiversity, directly through physiological limitations and indirectly through biotic interactions, vegetation structure and food availability. Climate and land use change are the main factors for landscape simplification and biotic homogenization. Thus, the study of community patterns across environmental gradients may help to predict the effect of projected environmental change.
I investigated how abiotic and biotic factors influence different facets of bird diversity across an elevational gradient. My study was conducted along an elevational gradient spanning 2000 m within and around Podocarpus National Park and San Francisco reserve on the southeastern slope of the Andes in Ecuador. The climate is humid tropical montane with a bimodal rain regime. The region is characterized by evergreen premontane forest at low elevations, evergreen lower montane forest at mid elevations and upper montane forest at high elevations. The elevational gradient has natural continuous forests within the protected reserves and fragmented forests surrounding the reserves in a matrix of cattle pastures. To monitor bird diversity, I placed nine 20-m radius point counts within 18 one-hectare plots, in continuous and fragmented forest at 1000, 2000 and 3000 m a.s.l. I recorded and identified all birds for 10 minutes within each point count. Bird communities were sampled eight times per plot, in the most humid season and in the least humid season of 2014 and 2015. To estimate flower and fruit availability, I recorded all plants with open flowers and ripe fruits within each point count. To obtain the relative invertebrate availability, I assessed understory invertebrate fresh biomass using a standardized sweep-netting design along 100-metre borders of each plot. Vertical vegetation heterogeneity was estimated at eight layers above the ground within each point count. Temperature for each plot was obtained using an air temperature regionalization tool and precipitation through remote sensing techniques and meteorological data.
In the first chapter of this thesis, I explored the effects of elevation, climate and vegetation structure on overall bird communities as well as on frugivorous and insectivorous birds. I found that elevation was mostly indirectly associated with bird diversity, jointly mediated via temperature, precipitation and vegetation structure. Additionally, elevation was directly and positively associated with both the overall bird community and with insectivores, but not with frugivores. My findings indicate a reduction of bird diversity due to climatic factors and vegetation structure with increasing elevation. However, the direct, positive effect of elevation suggests that bird diversity was higher than expected towards high elevations, probably due to spatial, biotic and evolutionary settings.
In the second chapter, I analysed the influence of climate and resource availability on temporal variation of bird communities. I found a higher bird diversity in the least humid season than in the most humid season. The seasonality of the bird communities was mainly driven by temperature and precipitation. While temperature had a significant positive effect at high elevations, precipitation had a significant negative effect at low elevations. Resource availability had no significant effect. My findings suggest that the temporal fluctuations in bird communities likely occur due to climate
constraints rather than due to resource limitations.
In the third chapter, I studied the effect of forest fragmentation on taxonomic and functional bird diversity. I found that taxonomic diversity was higher in fragmented compared to continuous forests, while functional diversity was negatively affected by fragmentation, but only at low elevations. The increase of taxonomic diversity in disturbed habitats suggests an increase of habitat generalists, which may compensate the loss of forest specialists. My findings suggest that taxonomic diversity can be uncoupled from functional diversity in diverse communities at low elevations.
My results show the effects of environmental factors on the spatio-temporal patterns of bird communities and the potentially uncoupled responses of taxonomic and functional diversity to forest fragmentation. My findings highlight that bird communities respond differently to abiotic and biotic factors across elevational gradients. Overall, my study helps to better understand the mechanisms that drive species communities in response to complex environmental conditions, which could be an essential contribution for the conservation of bird communities in the tropical Andes.
The prefrontal cortex (PFC) is considered the cognitive center of the mammalian brain. It is involved in a variety of cognitive functions such as decision making, working memory, goal-directed behavior, processing of emotions, flexible action selection, attention, and others (Fuster, 2015). In rodents, these functions are associated with the medial prefrontal cortex (mPFC). Experiments in mice and rats have shown that neurons in the mPFC are necessary for successful performance of many cognitive tasks. Moreover, measurements of neural activity in the mPFC show excitation or inhibition in different cells in relation to specific aspects of the tasks to be solved. To date, however, it is largely unknown whether prefrontal neurons are stably activated during the same behaviors within a task and whether similar aspects are represented by the same neurons in different tasks. In addition, it is unclear how specifically neurons are activated, for example, whether cells that are activated in response to reward are activated in a different task without reward in a different situation or remain inactive. To address these questions, we recorded the same neurons in the mPFC of mice over the course of several weeks while the animals performed various behaviors.
To do this, we expressed GCaMP6 in pyramidal neurons in the mPFC of mice. A small lens was implanted in the same location and a miniature microscope ("miniscope") was used to record neural activity. Later the extracted neurons got aligned based on their shape and position across multiple days and sessions. The mice performed five different behavioral tests while neural activity was measured: A spatial working memory test in a T-maze, exploration of the elevated plus maze (EPM), a novel object recognition (NO) test including free open field (OF) exploration, a social interaction (SI) test and discriminatory auditory fear conditioning (FC). Each task was repeated at least twice to check for stable task encoding across sessions. Behavioral performance and neural correlates to specific task events were similar to earlier studies across all tasks. We utilized generalized linear models (GLM) to determine which behavioral variables most strongly influence neural activity in the mPFC. The position of the mouse in the environment was found to explain most of the variance in neural activity, together with movement speed they were the strongest predictors of neural activity across all tasks. Reward time points in the working memory test, the conditioned stimulus after fear conditioning, or head direction in general were also strongly encoded in the mPFC.
Many of the recorded neurons showed a stable spatial activity profile across multiple sessions of the same task. Similarly, cells that coded for position in one task tended to code for position in other tasks. Not only did the same cells code for position across multiple tasks, but cells also coded for movement speed and head direction. This indicates that at least these general behavioral variables are each represented by the same neurons in the mPFC. Interestingly, the stability of position or speed coding did not depend on the time between two sessions, but only on whether it was within the same or across different tasks. Within the same task, stability was slightly higher than across different tasks.
To find out whether task-specific behavioral aspects were also stably encoded in the mPFC, difference scores as the difference in neural activity between two task aspects like left- and right-choice trials or exposed and enclosed locations were calculated. Many cells encoded these aspects stably across different sessions of each task. Both the left-right differences in the different phases of the working memory test, the open-closed-arm differences in the elevated plus maze, the different activity between center and corners in the open field, the social target-object differences in the social interaction test, and the differences between the two tones during fear conditioning were all stably encoded across the population of mPFC cells. Only the distinction between the novel and the familiar object during object recognition was not stably encoded, but also the preference for the novel object was not present in the second session of novel object exploration.
There was also an overlap in coding for different aspects within a task across multiple sessions. For example, cells stably encoded left-right differences in the T-maze between different sessions as a function of walking direction across different phases of working memory, an aspect that we could already show within one session (Vogel, Hahn et al., 2022). During fear conditioning, the same cells showed a discrimination between CS+ and CS- that also responded to the start of CS+.
Consistency in the neurons activity across different tasks was also found, but only between tasks with similar demands, the elevated plus-maze and free exploration of the open field. Cells that were more active in the open arms also showed more activity in the center of the open field and vice versa. This could be an indicator that the cells were coding for anxiety or exposure across those tasks, indicating that neurons in the mPFC also stably encode general task aspects independent of the specific environment. However, it remains unclear what exactly these neurons encode; in the case of a general fear signal, one would also expect activation during fear conditioning which could not be found.
Overall, we found that neurons in the mPFC of mice encoded multiple general behavioral variables across multiple tasks and task-specific variables were encoded stably within each of the tested tasks. However, we found little task-specific variables that were systematically encoded by the same neurons with the exception being the elevated plus-maze and open field exploration, two tasks with similar features.
Evidence is increasingly pointing towards a significant global decline in biodiversity. The drivers of this decline are numerous, including habitat change and overexploitation, rapid deforestation, pollution, exotic species and disease, and finally climate change as an emerging driver of biodiversity change (Nakamura, et al., 2013; Hancocks, 2001; Pereira, Navarro & Martins, 2012). Raising public awareness of the need to conserve biological diversity is essential to safeguard the richness of life forms all over the world (Lindemann-Matthies, 2002). In this regard, institutions such as science museums, zoos and aquariums have the potential to play an important role (Rennie & Stocklmayer, 2003). Especially, zoos can provide a productive learning environment (Miles & Tout, 1992), facilitating the promotion of public conservation awareness and the adoption of pro-environmental behaviours that would reduce negative human impacts on biodiversity (Barongi, et al., 2015).
Based on these concepts, my study contributes to the developing field of visitor studies. Taking as reference non-zoo visitors and zoo visitors, I have focused on reviewing some aspects of conservation education, such as people's awareness of conservation, people's interest in animals and people's feelings towards animals and attitudes towards zoos. The study identified differences between non-regular and regular zoo visitors in interests in animals, as well as visitor attitudes towards conservation issues and zoos. Therefore, the present study indicated that positive emotional reactions and, in particular, a perceived sense of connection to the animal were linked and depended on the frequency of zoo visits. It was as well remarkable, that conservation awareness was influenced by the interest in animals, the interest in visiting zoos, the attitudes towards these institutions, and the age and the country of origin. All these variables had a greater effect in the conservation consciousness of the participants. Additionally interestingly, the main reason for visiting zoos in every country was to learn something about animals. This highlights the educational role of zoos and broadly supports the idea that people want to visit zoos to learn something about animals, in turn facilitating pro-conservation learning and changes in attitude. They are uniquely positioned to interact with visitors, communities, and society and to contribute by providing an informative and entertaining environment. Visiting zoos could led to contribute to promoting animal connectedness and interest in species.
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.
The main aim of this thesis work was to elucidate the catalytic mechanism of several enzyme complexes on the basis of their three-dimensional structure. All investigated enzyme complexes occur in the anaerobic energy metabolism and have an essential function by the challenging degradation of aromatic compounds and the flavin-based electron bifurcation (FBEB)/confurcation, an energy-coupling mechanism. More specifically, I studied the phthaloyl-CoA decarboxylase of Thauera chlorobenzoica (Pcd) involved in phthalate ester decomposition, the FBEB protein complexes lactate dehydrogenase/electron-transfer flavoprotein (Ldh/EtfAB) of Acetobacterium woodii, the heterodisulfide-related subunit HdrA of the sulfur- oxidizing bacteria Hyphomicrobium denitrificans (sHdrA). In addition, I contributed to the structure determination of the caffeyl-CoA reductase- EtfAB complex of A. woodii and the naphthoyl-CoA reductase of the sulfate-respiring enrichment culture N47 (mentioned in the Appendix E and F).
Structured illumination microscopy (SIM) is part of the super-resolution methods developed at the beginning of this century. To produce a super-resolution image SIM requires three things: 1) illumination of the sample with a periodic pattern, 2) acquisition of multiple images per plane under different pattern’s phases and orientations and 3) the processing of these images has to be carried with a reconstruction algorithm. The result of the reconstruction is an image with a resolution gain that is proportional to the frequency of the pattern (po). The typical SIM set-up uses an epi-fluorescence configuration, thus the interference angle of the beams that create the pattern is restricted by the angular aperture of the objective. Under this restriction the maximum value of po is given by the cut-off frequency of the objective lens and sets at 2 the maximum resolution gain of SIM under linear illumination.
In the first part of this thesis we present the implementation and characterization of the 2D-SIM set-up designed by Dr. Bo-Jui Chang (B-J. Chang et al., PNAS 2017), this design exploits the concept introduced by light-sheet microscopy, i.e. separation of illumination and detection paths to obtain resolution gains larger than the usual two-fold (Chapter 3). The set-up is named coherent structured illumination light-sheet based fluorescence microscopy (csiLSFM) and it consists of a triangular array of three objectives, such that two are used for illumination and one for detection. With the independent illumination arms is possible to interfere two coherent light-sheets at angles beyond the angular aperture of the detection lens, attaining the maximum interference angle of 180° when the light-sheets counter-propagate. This condition delivers a pattern with a po 1.4 times larger than the cut-off frequency (ωo), hence our set-up provides generic resolution gains of 2.4.
The extraction of the high spatial frequencies that produce the resolution gain in the csiLSFM is a challenge due to a low pattern modulation. The low modulation inherently arises because the frequency associated to the pattern period lies beyond the cut-off frequency of the detection lens. To overcome this challenge we developed a filtering strategy that facilitates the withdrawal of information from a SIM data set, simultaneously the proposed filtering process optimizes the reconstruction algorithm by reducing the periodic artifacts that are recurrent in SIM images. In this same chapter we also performed an spectral analysis of the artifacts and determined that they originate from irregularities in the power spectrum that occur due to the partial or total lack of certain spatial frequencies (fig.4.2 and 4.3), our reconstruction reduces this information drops and diminishes the artifact occurrence. The relevance of our reconstruction pipeline is that it delivers a standardized process to enhance the SIM image in a current context in which the commonly used reconstruction algorithms employ empirical tuning to improve it (fig.4.13). Moreover, the pipeline is applicable to the csiLSFM data and also to images acquired with any other 2D-/3D-SIM set-up (fig.4.10 and 4.11).
The processing of various image data sets acquired with the csiLSFM exposed us to the question of how low the modulation of the illumination pattern can be before no super-resolution frequencies can be extracted. Answering this question is important to guarantee that the SIM data contains enough spatial frequencies to provide significant resolution gains. Thus in chapter 5 we developed a quantitative metric to indirectly determine the pattern modulation from the SIM data and find its critical value to use it as evaluation criterion. We called this metric the quality factor (Q-factor) and it represents the normalized strength (amplitude) of the extracted frequencies respect to the Gaussian noise contained in the images. Through simulations we estimated that Q=0.11 is a critical value and a SIM data set requires this as minimum value is to deliver a significant resolution gain. Q works then as an assessment tool for classifying SIM data as optimal or sub-optimal, i.e. Q≥0.11 or Q<0.11. We demonstrated such application with data acquired in various SIM commercial set-ups to prove its feasibility in the field (fig.5.6-5.11)
As mentioned at the beginning of this abstract SIM requires a specialized set-up and a processing algorithm to produce super-resolution images. This thesis contributes to these two areas in the following aspects: first, in its linear version a structured illumination microscope is highly associated to a 2-fold resolution gain. Here we demonstrated the possibility of extending this gain to 2.4 using our custom set-up the csiLSFM. Second, a reconstructed SIM image is prone to artifacts due to the mathematical process it undergoes, here we analyzed the artifact sources and identified them with drops of spatial information in the reconstructed spectrum, based on these conclusions we designed a processing pipeline to facilitate the extraction of spatial frequencies and directly reduce artifacts. A third and final outcome of this thesis is the development and practical implementation of a quantitative index to evaluate the quality of SIM data in terms of its relevant information content (Q-factor). Accordingly, the overall contributions of this work were done in the areas of SIM set-up, SIM reconstruction procedure and SIM data evaluation.
Es gibt viele Theorien, die sich mit der Auswirkung einer zunehmenden carnivoren Ernährung von Homininen auf Carnivorengilden beschäftigen. Aussterbeereignisse in der Carnivorengilde werden oft mit carnivoren Homininen in Verbindung gebracht. Um zu prüfen, ob solche Theorien überhaupt zutreffen, benötigt man zunächst ein Modell, das Effekte von Konkurrenzbeziehungen innerhalb von Carnivorengilden quantifiziert darstellt.
In dieser Arbeit ist daher ein Modell entwickelt worden, das die Konkurrenz um Beute innerhalb einer Carnivorengilde darstellt und ermöglicht Veränderungen durch das Eintreten neuer Mitglieder in die Gilde zu modellieren. Dieses Modell wurde zur Analyse der rezenten Großcarnivorengilden der Serengeti, des Krüger-National-Parks und des Bandipur-Biosphärenreservat verwendet. Ebenso ist es zur Analyse pleistozäner Großcarnivorengilden Javas eingesetzt worden.
In dem Modell wird die verfügbare Beutemasse als limitierende Ressource für die Carnivorengilde betrachtet. Im ersten Schritt wird die Beute kategorisiert – in dieser Arbeit nach ihrer Körpermasse – und geprüft, welche Mitglieder dieselben Beutekategorien nutzen und welche für sie essentiell sind. Im zweiten Schritt wird die konkurrenzfreie Kapazität der Gildenmitglieder berechnet. Hierzu wird die für die gesamte Gilde verfügbare Beutemasse unter der Annahme verwendet, sie stehe einem Gildenmitglied allein zur Verfügung. Die konkurrenzfreie Kapazität ist daher die Populationsgröße, die ein Gildenmitglied mit dieser Beutemasse erreichen kann und stellt einen Referenzwert dar. Basierend auf diesem Referenzwert und der tatsächlichen Populationsgröße kann nun berechnet werden, zu welchem Anteil ein Mitglied diese Kapazität ausschöpft. Ist der Konsum an Beutemasse der übrigen Mitglieder in den essentiellen Beutekategorien bekannt, kann berechnet werden, zu welchem Anteil ein Mitglied durch ein anderes Mitglied von dieser Kapazität verliert. Dieser Verlust an Kapazität wird als Konkurrenzeffekt bezeichnet.
Dieses Modell ist sowohl auf rezente als auch fossile Gilden anwendbar. Um mit dem Modell die Konkurrenzeffekte zu berechnen, werden die Häufigkeit bzw. Populationsgröße, das Beutemassenspektrum sowie der tägliche Bedarf an Beutemasse benötigt.
Diese Größen können bei der Strukturanalyse von rezenten Gilden aus Freilandstudien entnommen werden. Im Falle fossiler Gilden müssen diese Größen erst rekonstruiert werden. Dafür sind in dieser Arbeit vorhandene Rekonstruktionsmethoden ergänzt, aber auch entwickelt worden, mit denen man basierend auf der Körpermasse fossiler Carnivora die benötigten Parameter rekonstruieren kann. Hierzu sind verschiedene Regressionen berechnet worden, die einen Zusammenhang zwischen verschiedenen Zahnparametern und der Körpermasse darstellen. Weiterhin sind Muster der Beutemassenspektren rezenter Carnivora untersucht worden und Regressionen berechnet worden, die zur Rekonstruktion der mittleren Beutemasse eines Carnivoren verwendet werden.
Die benötigten Daten der javanischen Gilden werden mit den eben genannten Regressionen rekonstruiert. Anschließend wird eine Strukturanalyse der genannten rezenten und fossilen Großcarnivorengilden durchgeführt.
Bei den drei rezenten Gilden ist eine generelle sich wiederholende Struktur erkennbar. Die erfolgreichsten Mitglieder schöpfen ihre Kapazität zu ca. 60 % aus und verfolgen eine soziale Lebensweise.
Dennoch werden die erfolgreichsten Mitglieder der Gilden von unterschiedlichen Arten repräsentiert. So sind dies der Löwe im Krüger-Nationalpark, die Tüpfelhyäne in der Serengeti oder der Rothund in Bandipur.
Bei den fossilen Gilden war diese Struktur allerdings nicht erkennbar. Hier schöpft der Tiger seine Kapazität in allen Gilden am stärksten aus und hat extrem hohe Konkurrenzeffekte (bis zu ca. 98 %) auf die übrigen Gildenmitglieder.
Diese Unterschiede können mit Isolationsbedingungen Javas als Insel zusammenhängen, die sich grundsätzlich auf Strukturen der Säugergemeinschaften auswirken.
Vermutlich konnte der Tiger durch Veränderungen der Körpermasse seine konkurrenzstarke Position in der Großcarnivorengilde Javas halten.
Das entwickelte Modell ermöglicht auch eine Modellierung von Szenarien, die verschiedene Möglichkeiten berücksichtigt. Diese sind vor allem Veränderungen der Populationsgrößen, aber auch Veränderungen der Körpermasse und daraus resultierende Verschiebungen der Beutemassenspektren.
In Beispielen der Trinil-Gilde wird gezeigt, dass die Rolle eines hyper- bzw. hypocarnivoren Homo erectus in der Gilde mit dem entwickelten Modell dargestellt werden kann. Auch lassen sich Szenarien modellieren, in denen ein hyper- bzw. hypocarnivorer Homo erectus in die Gilde eindringt und so die übrigen Mitglieder von bei ihrer Kapazitätsausschöpfung Einbuße hinnehmen müssen.
In dem Szenarium von Trinil wird erkennbar, dass nur ein hypercarnivorer Homo erectus einen starken Effekt auf die Gildenmitglieder hatte. Geht man von einem omnivoren Homo erectus aus, ist der Konkurrenzeffekt geringer und es sind keine Aussterbeereignisse zu erwarten.
Das Modell kann in weiteren Studien zur Testung von Hypothesen zu Aussterbeereignissen Aufklärung bieten. Durch Einbeziehung weiterer Faktoren wie Kleptoparasitismus und interspezifische Tötungen kann es noch erweitert werden. Auch eine Dynamisierung des Modells, die eine kontinuierlich zeitliche Veränderung der Gilden modellieren kann, ist in zukünftigen Studien möglich.
Es ist wohl unumstritten, dass das Leben, wie wir es kennen, ohne die sauerstoffproduzierenden Organismen unserer Erde nicht möglich wäre. Zu ihnen gehören nicht nur die Landpflanzen, deren mannigfaltige Nutzung wichtiger Bestandteil unseres Alltags ist. Auch mikroskopisch kleine Algenarten leisten einen entscheidenden Beitrag zu den Stoffwechselkreisläufen dieser Welt. Unter ihnen befinden sich die Kieselalgen (Diatomeen), die mit einer Varietät von bis zu 10000 Spezies etwa 40 % der marinen Primärproduktion verantworten. Der Ursprung der heutigen zur oxygenen Photosynthese befähigten Eukaryoten geht auf Endosymbioseereignisse zurück, von denen aus sich diese Organismen ausgesprochen vielfältig entwickelt haben. Diese Vielfalt wird dabei nicht nur anhand ihrer äußeren Morphologie, sondern auch auf subzellulärer Ebene, deutlich. So zum Beispiel durch die unterschiedlichen Strukturen der Thyakoidmembranen, die sich in Kieselalgen wie Cyclotella meneghiniana in dreilagigen Bändern arrangieren. In Pflanzen wie Nicotiana tabacum (Tabak) hingegen bilden sie große, stapelartige Bereich aus, die zur räumlichen Separation der in den Thylakoiden eingebetteten Photosystemen beitragen. Auch die an die Photosysteme (PS) gebundenen Lichtsammelproteine (Lhcs) haben sich in Tabak und Cyclotella unterschiedlich entwickelt. Gemäß ihrem Namen zeichnen sie sich zwar allesamt durch die Sammlung und Weiterleitung der Lichtenergie an die Photosysteme aus, grenzen sich aber in Hinblick auf Proteingröße und Pigmentierung voneinander ab.
Die Lhcs der höheren Pflanzen werden entsprechend ihrer Zuordnung zu den Photosystemen in den aus zwei Heterodimeren bestehenden LHCI des PSI und die Lhcb-Antennenproteine des PSII unterschieden. Zu letzteren gehören der trimere Hauptantennenkomplex LHCII und die monomeren, minoren Antennenproteine. Die Lhcs binden die zur Lichtsammlung benötigten Pigmente, vor allem Chlorophyll a und Chlorophyll b, aber auch primäre Carotinoide wie Violaxanthin, Lutein und Neoxanthin, in unterschiedlichen Stöchiometrien. Es ist bereits bekannt, dass die Pigmentierung entscheidend zur Stabilität der Lichtsammelproteine beiträgt, wenngleich zum Teil auch eine gewisse Flexibilität in Bezug auf die Art der gebundenen Pigmente an den entsprechenden Bindestellen der Proteine besteht.
Im Rahmen dieser Arbeit liegt der Fokus auf der Fragestellung inwieweit die in der Regel nicht in Pflanzen vorkommenden Ketocarotinoide die Struktur und Funktion des LHCII aus einer Ketocarotinoide produzierenden N. tabacum - Transformante (bkt-Linie) beeinflussen und welche Auswirkungen sie auf dessen Photosyntheseapparat im Allgemeinen haben. Die bkt-Linie bildet dabei zum Teil auf Kosten ihrer primären Carotinoide sowohl das als antioxidativ und als anti-kanzerogen beschriebene Astaxanthin, als auch dessen Vorstufe Canthaxanthin und einige Derivate dieser Pigmente, die, nach vergleichenden HPLC-Analysen von Blättern und Thylakoidfraktionen, zu einem großen Teil mit der Thylakoidmembran assoziiert sind. Durch spektroskopische Untersuchungen konnte gezeigt werden, dass diese Ketocarotinoide in Hinblick auf die Energieweiterleitung zum Chlorophyll a nicht funktionell an den LHCII binden, ihre Produktion aber die Trimerisierung dieses Lichtsammelkomplexes in N. tabacum nachhaltig beeinträchtigt. Auch die Assemblierung der PSII-LHCII-Superkomplexe wird dadurch maßgeblich gestört. Elektronenmikroskopische Aufnahmen von Chloroplasten der bkt-Linie verdeutlichten zudem die Beeinträchtigung der Granathylakoid-Stapelung: Sie fällt ungeordneter aus als im Wildtyp, was durch den Mangel an intakten LHCII-Trimeren begründet sein kann.
In funktioneller Hinsicht stören die Ketocarotinoide die Energieweiterleitung innerhalb des PSII und bewirken die Reduktion der photoprotektorischen, nicht-photochemischen Fluoreszenzlöschung des Wirtsorganismus nachhaltig. Zeitgleich reduzieren sie durch einen abschirmenden Effekt auf Grund ihrer Assoziation mit der Thylakoidmembran und/oder durch einen eventuellen S1-S1-Energietransfer von Chl a auf die Ketocarotinoide aber auch die Menge der Lichtenergie, die über die Lhcs an die Photosysteme weitergeleitet wird. Dadurch kommt ihnen neben dem nachhaltig störenden Einfluss auf die Intaktheit des Photosyntheseapparats zugleich auch eine schützende Wirkung vor einem Übermaß an Lichtenergie zu.
Aus Cyclotella meneghiniana sind zwei Hauptantennenkomplexe bekannt: FCPa und FCPb. Im Gegensatz zu den Lhcs der Chl a/b-haltigen Organismen binden die Lichtsammelproteine der Diatomeen das Xanthophyll Fucoxanthin anstelle des Luteins, und Chlorophyll c anstelle des Chlorophyll b. Im Gegensatz zu der bereits sehr detailliert aufgeklärten Struktur des trimeren LHCII in höheren Pflanzen, existieren für den Aufbau des FCPb in C. meneghiniana bisher nur fundierte Modellvorschläge. Diese postulieren eine homotrimere Grundstruktur für den FCPb, die zu höheren Oligomeren assembliert.
In der vorliegenden Arbeit konnte anhand elektronenmikroskopischer Aufnahmen und der anschließenden Einzelpartikelanalyse nun erstmalig die Struktur des etwa 6-7 nm großen, trimeren FCPb gezeigt und die Richtigkeit der bisher postulierten Modellvorschläge in Hinblick auf die Struktur des Trimers bewiesen werden. Nach den hier dargelegten Erkenntnissen gleicht die Anordnung der Untereinheiten des FCPb-Trimers der des LHCII. Zudem ergibt sich aus dem Zusammenhang der hier erhobenen Daten und den in der Fachliteratur veröffentlichten Ergebnissen zum Thema FCPb ein klares Bild über die Anordnung der höheren Oligomere in Form von Nonameren. Auch diese Erkenntnisse unterstützen das ursprünglich von C. Büchel vorgeschlagene Modell für die oligomere Struktur des FCPb in C. meneghiniana.
Sympathetic influences on articular cartilage regeneration capacity and osteoarthritis manifestation
(2021)
The pathogenesis of osteoarthritis (OA) involves articular cartilage, synovial tissue and subchondral bone and is therefore a disease of the whole joint. OA is characterized by progressive degradation of cartilage, synovial inflammation, osteophyte formation and subchondral bone sclerosis. Cartilage-surrounding tissues are innervated by tyrosine hydroxylase (TH)-positive sympathetic nerve fibers with the most important sympathetic neurotransmitter norepinephrine (NE) detected in the synovial fluid of OA patients. Furthermore, adrenergic receptors are expressed in different knee joint tissues. Most in vitro studies indicate a potential role of the β2-adrenergic receptor, which has been not investigated during OA pathogenesis in vivo. The role of the sympathetic nervous system (SNS) in OA progression has not yet been studied. Therefore, the objective of this study was to analyze how the SNS and NE influence the MSC dependent cartilage regeneration in vitro and the OA pathogenesis and manifestation in vivo.
In the first part of this study, the effect of NE on the chondrogenesis of sASC, which are known to play an important role in cartilage regeneration was analyzed in vitro. In the second part of this study, the role of the SNS was studied in vivo in mice that were sympathectomized chemically followed by surgically induced OA. The specific focus was on the β2-adrenergic receptor effects on OA pathogenesis, which were analyzed in β2-adrenergic receptor-deficient mice.
The in vitro experiments have shown that NE reduced the chondrogenic potential of sASCs by decreasing the expression of type II collagen and sGAG. NE mediated these effects mainly by the α2-AR signalling. Furthermore, NE treatment led to activation of the ERK1/2 signal pathway. These findings suggested that the sympathetic neurotransmitter NE might suppress the chondrogenic capacity of MSC and their dependent cartilage regeneration and may also play a role in OA progression and manifestation.
The in vivo study has shown that sympathectomy reduced synovial TH-positive nerve fibers in the synovium and the NE concentration in the spleen significantly. In WT mice, DMM leads to increased NE concentrations in the spleen compared to sham mice indicating an increased SNS activity after mechanical stress or inflammation due to DMM. Sympathectomy leads to less pronounced cartilage degeneration (OARSI score) after DMM compared to DMM in WT mice. Furthermore, the release of the type II collagen degradation fragment CTX-II was abolished in Syx DMM mice compared to WT DMM mice, suggesting that less SNS activity due to sympathectomy reduced the cartilage degeneration during OA pathogenesis. Similarly, sympathectomy decreased the synovitis score significantly after DMM compared to DMM in
WT mice. Synovitis in WT mice was accompanied by increased MMP-13 expression in the synovium after DMM, compared to Syx mice. Cartilage degeneration seemed to be driven mainly by the increased synovial inflammation accompanied by an increased MMP13 expression in synoviocytes and not in chondrocytes. The pathological changes in synovium and cartilage might also be linked to each other, as indicated by the moderate correlation between the synovial inflammation (synovitis score) and cartilage degeneration (OARSI score). Subchondral bone volume as well the thickness of the subchondral bone plate (SCBP) and calcified cartilage (CC) were increased in Syx mice compared to WT after DMM. The data on DMM induction in β2-AR deficient mice revealed that the β2-AR signaling is involved in cartilage degeneration and the aggravated subchondral bone changes as these mice had less pronounced cartilage degeneration compared to WT mice. While the cartilage degeneration was similar, the subchondral bone changes were more pronounced in β2-AR deficient mice compared to the Syx mice.
Overall, the SNS had differential effects in cartilage, synovium and subchondral bone. A reduced SNS activity by sympathectomy attenuated cartilage degeneration and synovitis but aggravated the OA specific subchondral bone changes. These findings provide new insights into the development of novel therapeutic strategies for OA by targeting the SNS in a tissue- specific manner.
Xenorhabdus and Photorhabdus are bacterial genera that live in symbiosis with entomopathogenic nematodes of the genera Steinernema and Heterorhabditis, respectively. These nematodes infect insect larvae through the trachea and then enter the hemocoel. Once inside the hemocoel, the nematodes release the bacteria through their intestine. Thereafter, the bacteria become active and kill the larvae within 48 h. During this process, the immune system of the insect host is compromised by molecules produced and secreted by the bacteria. This illustrates that the bacteria possess not only a large arsenal of biological weaponry such as antibiotics and fungicides but also lipases, proteases, etc. Therefore, they are not only able to kill the insect but also protect the cadaver from other food competitors.
During the past decades, a large number of natural products have been identified from Xenorhabdus and Photorhabdus. However, the targets and functions for many of these biological molecules are still unknown. Therefore, the goal of the doctoral thesis is to elucidate the modes of action of these natural products from Xenorhabdus and Photorhabdus with the main focus on non-ribosomal peptides (NRPs). The work can be divided into two parts. Initially, it starts with the synthesis of natural compounds and various chemically modified derivatives. Besides that, a number of peptides were synthesized for other projects to either verify their structures or quantify the amount produced by the bacteria. Then, secondary analysis methods are applied and provide additional insight into the modes of action of these compounds.
During the thesis, I carried out peptide synthesis either manually or with an automatic synthesizer system from Biotage. Here, the Fmoc-protecting group strategy was preferred in most cases. Natural products, such as silathride, xenoautoxin, phenylethylamide, tryptamide, rhabdopeptide, 3-hydroxyoctanoic acid, and PAX, were produced during this process. Furthermore, new peptide derivatives derived from synthetic NRPS approaches using the XU concept or SYNZIP were generated as standards.
Most of these natural compounds were experimentally verified by MIC tests (broth microdilution, plate diffusion) to be biologically active. For example, silathride, phenylethylamide, and tryptamide showed quorum quenching effects when tested against Chromobacterium violaceum. Initial results from collaborators (PD Dr. Nadja Hellmann/Mainz) showed that tryptamide and phenylethylamide interact with membrane or membrane proteins.
(R)-3-hydroxyoctanoic acid was synthesized to verify the molecule structure of phototemtide A, a cyclic lipopeptide with antiprotozoal activity. The rhabdopeptides are another class, which showed remarkable antiprotozoal effects. However, their mode of action was unknown. These compounds are relatively short peptide sequences, which contain hydrophobic residues, such as valine, leucine, or phenylalanine. Moreover, they possess N methylation, resulting in a rod-shaped highly hydrophobic structure. In this work, I synthesized eight new derivatives of rhabdopeptides for photo-affinity labeling (PAL). These molecules should react covalently under UV-light irradiation with the biological target of the peptides. In addition, these derivatives can be enriched in a pull-down assay using click chemistry. Afterward, analytic methods such as mass detection (proteome analysis) can be applied to elucidate the protein targets.
The PAX peptides derivatives are well-known to have anti-microbial activities and believed to be secreted into the environment by the producing bacteria. However, I found that the majority of these peptides are located in the cell pellet fraction and not in the supernatant. This has been shown through quantification using HPLC MS. New PAX derivatives were synthesized, which carry a moiety suitable for covalent modification using click-chemistry, therefore being functionalizable with a fluorescence dye. In collaboration with Dr. Christoph Spahn (Prof. Dr. Mike Heilemann group), we used confocal, as well as super-resolution microscopy, in particular, single-molecule localization microscopy (SMLM) to investigate the spatial distribution of clickable PAX molecules and revealed that they localize at the bacterial membrane. Furthermore, bioactivity assays revealed that the promotor exchanged X. doucetiae PAX mutants, which do not produce PAX molecules without chemical induction (hereby termed as pax-), were more susceptible to several insect AMPs tested. Based on these findings, a new dual mechanism of action for PAX was proposed. Besides the previously shown antimicrobial activity, these molecules with a positive net charge of +5 (pH = 7) would bind to the negatively charged bacterial surface. Hereby, the surface charge (typically negative) would be inversed resulting in a protective effect for Xenorhabdus against other positively charged AMPs. Furthermore, PAX was investigated as AMP against E. coli to study its antimicrobial mechanism of action. Here, the results show that PAX can disrupt the E. coli membrane at higher concentrations (> 30 µg/ml), enter the cytosol, and lead to reorganization of subcellular structures, such as the nucleoid during this process.
Another aspect of secondary analysis is the application of proteomic analysis. Therefore, I induced X. nematophila, X. szentirmaii, and P. luminescens with insect lysate. These samples were analyzed using HPLC-MS/MS (Q Exactive) together with a database approach (Maxquant/Andromeda). The results showed that in all strains the lipid degradation and the glyoxylate pathway were induced. This is in line with the given insect lysate diet, which mostly contained lipids. Moreover, several interesting unknown peptides and proteins were also upregulated and might get into the focus of future research.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
The division Ascomycota(Fungi) contains a large number of taxa known to reproduce only asexually by the formation of conidia or other non-motile propagules produced by mitotic cellular devisions. They are called anamorphic, mitosporic, asexual or conidial fungi and ecologically, they are often found associated with plant debris in different stages of decay. In general, saprobic anamorphs of ascomycetous affinities are poorly studied and their outstanding diversity is currently underexplored. Phylogenetic relationships are unknown for many of them and they are still largely underrepresented in the current phylogenetic classification system of Fungi, with many morphologically defined anamorphic taxa still awaiting taxonomic reassessment in the light of molecular approaches. The increasing usage of molecular markers combined with robust statistical methods has allowed their phylogenetic affinities to be revealed and to gradually incorporate many of them into the different taxonomic groups of the division Ascomycota. However, the phylogenetic placement and taxonomic status of a large number of saprobic taxa remain unresolved due to the lack of DNA sequence data.
The present dissertation aims to explore the rich but understudied diversity of those anamorphic fungi traditionally known as hyphomycetes that inhabit dead plant debris. It consists of five publications in which a polyphasic approach integrating morphological, developmental, cultural and molecular data was used to incorporate novel or incertae sedis taxa within Ascomycota and to make more sound decisions regarding their taxonomic status. Specific objectives include: 1. the collection, isolation and morphological characterization of selected anamorphic fungi representing putative new or interesting taxa of uncertain phylogenetic placement; 2. the generation of novel DNA sequence data to infer their phylogenetic relationships and to resolve their taxonomic affinities within Ascomycota; 3. the testing of any previously available morphologically based hypotheses on their putative position, generic placement or relationships with teleomorphic, pleomorphic or other anamorphic taxa; and 4. the determination of their generic validity, monophyly and taxonomic boundaries using molecular data and phylogenetic analyses methods.
Materials studied in these five projects consisted of specimens collected during field work carried out by the author or collaborators in different countries including USA, the Czech Republic and Panama between the years 2014 and 2017. The target substrates were dead leaves of different palm trees, dead wood and bark of pines and twigs or stems of unknown shrubs and woody vines that are all known to harbor a rich saprobic mycobiota. Putative novelties or anamorphic taxa with unknown or poorly studied phylogenetic affinities were selected for further morphological and molecular investigation. Micromorphological studies were based on fungal structures observed on natural substrate, herbarium specimens and in culture. DNA was extracted from cultures and PCR amplification followed by Sanger sequencing was carried out using relevant molecular markers employed in fungal phylogenetic studies. Newly obtained DNA sequence data were analyzed following a standard phylogenetic analysis pipeline and phylogenetic relationships were reconstructed using character-based methods such as Maximum Likelihood and Bayesian inference.
Conclusion is that anamorphic Ascomycota inhabiting dead plant debris represents a largely untapped source of biodiversity and information still in need of further exploration. A new capnodiaceous genus Castanedospora, seven new species named Taeniolella sabalicola, Hermatomyces bifurcatus, H. constrictus, H. megasporus, H. sphaericoides, H. verrucosus and Septonema lohmanii, and two new combinations, Castanedospora pachyanthicola and H. reticulatus, are proposed based on morphological and DNA sequence data. Molecular phylogenetics was confirmed as the tool of choice for the inference of relationships in novel or incertae sedis anamorphic fungi that are otherwise difficult to assess in the absence of a teleomorphic state. They were first resolved or revisited for several saprobic species such as Ernakulamia cochinensis, H. sphaericus, H. tucumanensis or Septonema fasciculare in a suitable framework for phylogenetic hypothesis testing. Molecular data allowed to fully incorporate all these taxa in Ascomycota, particularly within the classes Dothideomycetes and Sordariomycetes, and to provide a foundation for better taxonomic decisions on their classification. Large and polyphyletic genera such as Taeniolella, Sporidesmium and Septonema, partially treated in this work and containing mostly saprobic species of obscure affinities, remained in need of further investigation.
The spider genus Eusparassus Simon, 1903 (Araneae: Sparassidae: Eusparassinae; stone huntsman spider) is revised worldwide to include 30 valid species distributed exclusively in Africa and Eurasia. The type species E. dufouri Simon, 1932 is redescribed and a neotype is designated from Portugal. An extended diagnosis for the genus is presented. Eight new species are described: Eusparassus arabicus Moradmand, 2013 (male, female) from Arabian Peninsula, E. educatus Moradmand, 2013 (male, female) from Namibia, E. reverentia Moradmand, 2013 (male, female) from Burkina Faso and Nigeria, E. jaegeri Moradmand, 2013 (male, female) from South Africa and Botswana, E. jocquei Moradmand, 2013 (male, female) from Zimbabwe, E. borakalalo Moradmand, 2013 (female) from South Africa, E. schoemanae Moradmand, 2013 (male, female) from South Africa and Namibia and E. mesopotamicus Moradmand and Jäger, 2012 (male and female) from Iraq, Iran and Turkey. 22 species are re-described six of them are transferred from the genus Olios Walckenaer, 1837. Six species-groups are proposed: the dufouri-group [8 species: E. dufouri, E. levantinus Urones, 2006, E. barbarus (Lucas, 1846), E. atlanticus Simon, 1909, E. syrticus Simon, 1909, E. oraniensis (Lucas, 1846), E. letourneuxi (Simon, 1874), E. fritschi (Koch, 1873); Iberian Peninsula to parts of north-western Africa], walckenaeri-group [3 species: E. walckenaeri (Audouin, 1826), E. laevatus (Simon, 1897), E. arabicus; eastern Mediterranean to Arabia and parts of north-eastern Africa], doriae-group [7 species: E. doriae (Simon, 1874), E. kronebergi Denis, 1958, E. maynardi (Pocock, 1901), E. potanini (Simon, 1895), E. fuscimanus Denis, 1958, E. oculatus (Kroneberg, 1846) and E. mesopotamicus; Middle East to Central and South Asia], vestigator-group (3 species: E. vestigator (Simon, 1897), E. reverentia, E. pearsoni (Pocock, 1901); central to eastern Africa and an isolated area in NW India], jaegeri-group [4 species: E. jaegeri, E. jocquei, E. borakalalo, E. schoemanae; southern and south-eastern Africa], tuckeri-group [2 species: E. tuckeri (Lawrence, 1927), E. educatus; south-western Africa). Two species, E. pontii Caporiacco, 1935 and E. xerxes (Pocock, 1901) cannot be placed in any of the above groups. Two species are transferred from Eusparassus to Olios: O. flavovittatus (Caporiacco, 1935) and O. quesitio Moradmand, 2013. 14 species are recognized as misplaced in Eusparassus, thus nearly half of the described species prior to this revision were placed mistakenly in this genus. Neotypes are designated for E. walckenaeri from Egypt, E. barbarus, E. oraniensis and E. letourneuxi (all three from Algeria) to establish their identity. The male and female of Cercetius perezi Simon, 1902, which was known only from the immature holotype, are described for the first time. It is recognized that the monotypic and little used generic name Cercetius Simon, 1902 — a species, which had been known only from the immature holotype — as a synonym of the widely used name Eusparassus. The case proposal 3596 (conservation of name Eusparassus) is under consideration by ICZN.
The first comprehensive molecular phylogeny of the family Sparassidae with focus on the genus Eusparassus is investigated using four molecular markers (mitochondrial COI and 16S; nuclear H3 and 28S). The monophyly of Eusparassus and the dufouri, walckenaeri and doriae species-groups are recovered with the latter two groups more closely related. The monophyly of the tuckeri-group is not supported and the position of E. jaegeri as the only available member of the jaegeri-group is not resolved within the Eusparassus clade. DNA samples of the vestigator-group were not accessible for this study. The origination of the genus Eusparassus around 70 million years ago (MA) is estimated according to molecular clock analyses. Using this recent result in combination with some biogeographic and geological data, the Namib Desert is proposed as the place of ancestral origin for Eusparassus and putative Eusparassinae genera.
Further analyses are done on the phylogenetic relationships of Sparassidae and its subfamilies. The Eusparassinae are not confirmed as monophyletic, with the two original genera Eusparassus and Pseudomicrommata in separate clades and only the latter clusters with most other assumed Eusparassinae, here termed the "African clade". Monophyly of the subfamilies Sparianthinae, Heteropodinae sensu stricto, Palystinae and Deleninae is recovered. The Sparianthinae are supported as the most basal clade, diverging considerably early (143 MA) from all other Sparassidae. The Sparassinae and genus Olios are found to be polyphyletic. The Sparassidae are confirmed as monophyletic and as most basal group within the RTA-clade. The divergence time of Sparassidae from the RTA-clade is estimated with 186 MA in the Jurassic. No affiliation of Sparassidae to other members of the "Laterigradae" (Philodromidae, Selenopidae and Thomisidae) is observed, thus the crab-like posture of this group was proposed a result of convergent evolution. Only the families Philodromidae and Selenopidae are found members of a supported clade. Including a considerable amount of RTA-clade representatives, the higher-level clade Dionycha is not but monophyly of the RTA-clade itself is supported.
Termites are important ecosystem engineers of the savanna biome, with the large mounds of fungus-cultivating termites being sources of habitat heterogeneity and structural complexity in African savanna landscapes. Studies from different localities throughout Africa have shown that termite mounds have a strong influence of diversity and composition of plant communities. However, most research has been conducted only at the local scale, and integrating knowledge across Africa is hampered by different methodology of studies and differing environmental context. Little is known about the variation in vegetation composition on termite mounds compared to the surrounding savanna at the regional scale and at the landscape scale, and the main determinants of plant communities on mounds are yet to be ascertained.
This thesis aimes at better understanding the influence of termite mounds on vegetation compared to the surrounding savanna across spatial scales. Three research projects analyse vegetation data and soil data from paired mound and savanna plots in West Africa. The first project examines the influence of termite-induced heterogeneity on plant diversity and vegetation composition at a regional scale, following a bioclimatic gradient from the Sahel of Burkina Faso to the Sudanian vegetation zone in North Benin. The second Project analysed variation of vegetation on and off mounds at the landscape scale in Pendjari National Park, North Benin. The third is a monitoring study over the course of two years, exploring dynamics of juvenile woody plant communities on mounds and in the surrounding savanna at a local scale. The thesis thus provides the first comparative quantitative analysis across scales of mound and savanna vegetation and the drivers of the mound–savanna difference in vegetation.
Synthesizing across scales, its results confirm that termite mounds strongly contribute to savanna plant diversity, even though mounds are not generally more species rich than the surrounding savanna. Variation in mound vegetation is much higher along climatic and soil gradients than previously acknowledged. Mound vegetation differs from the surrounding savanna in the whole study area and in each sampled savanna type, with the strongest differences occurring at the most humid study sites. A large proportion of the differences between mound and savanna vegetation is explained by clay enrichment and related soil factors, such as cation concentrations. Plants on mounds thus benefit from favourable soil conditions, including higher fertility and higher water availability, which is also mirrored by the higher abundance and basal area of juvenile woody plants found on mounds. The variation in mound vegetation between study sites across scales results in part from local differences in soil composition and from climatic differences that influence the regional distribution of species. Different sets of characteristic mound species are identified in each project. Specific plant families and traits like succulency, lianescence, and adaptations to zoochory are found to be overrepresented in mound communities.
In addition to the findings in this thesis, remaining parts of the variation in mound vegetation between study sites could likely be explained by investigating further factors. Specifically, mound vegetation depends on habitat context, which includes available species pools, spatial distribution of mounds, biotic interactions with dispersers and herbivores, fire, and also anthropogenic influence. The high proportion of species with adaptations to zoochory found on mounds, for example, indicates that animal dispersers should be of particular importance for vegetation on termite mounds. Herbivory and fire regime, which are known to contribute to the diversity and community composition of the mound–savanna system, also show strong local variation, not least because of anthropogenic influence.
In conclusion, termite mounds play a crucial role in maintaining heterogeneity and plant diversity in the savanna across scales. Ecosystem services provided by termites, especially considering long-term effects on soil fertility and ecosystem resilience, are most likely undervalued. Mounds should be considered in management plans from local to regional, transnational scales as a matter of course, accompanied by further research on the role of termite mounds in savanna ecology on a longer temporal scale. The research presented here thus provides a basis for future studies on termite mound vegetation that should specifically consider the biotic and abiotic context of the mound–savanna system.
Baleen whales (Mysticeti) are a clade of highly adapted carnivorous marine mammals that can reach extremely large body sizes and feature characteristic keratinaceous baleen plates used for obligate filter feeding. From a conservation perspective, nearly all baleen whale species were hunted extensively over a roughly 100 years lasting time period that depleted many of the respective whale stocks with so far unknown consequences for e.g. their molecular viability. From an evolutionary perspective, the lack of fossil records together with conflicting molecular patterns resulted in a still unclear and debated phylogeny of modern baleen whales, particularly in rorquals (Balaenopteridae). In this dissertation, I will demonstrate the application of baleen whale genomes to tackle these open questions by using modern approaches of conservation and evolutionary genomics.
Conservation genomic aspects of baleen whales were addressed in two projects, both using whole genome data of either an Icelandic fin whale (Balaenoptera physalus) population or multiple blue whale (Balaenoptera musculus) populations to evaluate the impact of the industrial whaling era on their molecular viability. The results suggest a substantial drop in effective population size of both species but also a lack of manifestation in genotypes of the fin whale population when compared to the blue whale populations. Especially the rare and short runs of homozygosity (ROH), usually indicative for inbreeding, suggest frequent outcrossing in fin whales while all analyzed blue whale populations featured long and frequent ROH. In addition to these analyses, genome data of blue whale populations was further used to evaluate if northern hemisphere blue whales diverged into different subspecies. Population genetic and gene flow analyses showed clearly separated and well isolated populations in accordance with their assumed geographical distance. In contrast, the genome-wide divergence between all blue whale populations was low compared to other cetacean populations and to the next closely related sei whale species. Because this includes the morphologically different and well recognized pygmy blue whale subspecies, a proposal was made to equally categorize the two northern-hemisphere blue whale populations as subspecies.
Evolutionary aspects were addressed in a third project, by constructing the genome of the pygmy right whale (Caperea marginata) and testing its potential in phylogenetics and cancer research. Phylogenomic analyses using fragments of a whole-genome alignment featuring nearly all extant baleen whales, allowed the revision of the complex evolutionary relationships of rorquals by quantifying and characterizing the amounts of conflicts in early diverging branches. These relationships were further used to identify phylogenetically independent pairs of baleen whales with a maximum of diverging body size differences to compare rates of positive selection between their genomes. The results suggest nearly evenly distributed frequencies of alternative topologies which supports the representation of the early divergence of rorquals as a hard polytomy with high amounts of introgression and incomplete lineage sorting. Within the set of available genomic data, three independent pairs of baleen whales with diverging body sizes were found and comparisons of positive selection rates resulted in many potentially body size and cancer related genes. The lack of conserved selection patterns, however, suggest a more convergent evolution of size and cancer resistance like previously discussed in paleontology.
In conclusion, the application of whole genome data using methods of conservation genetics allowed for a comprehensive estimation about the molecular viability of blue and fin whales as well as an assessment of the taxonomic status of northern-hemisphere blue whale populations. The rather different results between blue and fin whales underlines the importance of genomic monitoring of baleen whales because different species show rather different molecular consequences of their potentially varying depletions. Furthermore, as showcased for the northern-hemisphere blue whale, many important isolated populations of baleen whales may still be unknown to conservation management and genome-wide comparisons will most likely contribute to overcome this under-classification problem. The application of whole genome data in evolutionary research allowed the characterization of the complex patterns of molecular conflicts within baleen whales and especially rorquals that will contribute to the still rather unclear understanding of their evolution. The here found molecular support for the idea of convergent evolution of gigantism in whales will further guide the search for molecular patterns responsible for Peto’s paradox.
Bei Autismus-Spektrum-Störungen (ASS) handelt es sich um genetisch komplexe Störungen mit hoher Erblichkeit. Als zugrundeliegender Pathomechanismus von ASS werden unter anderem Veränderungen der neuronalen Entwicklung diskutiert. Der Phänotyp von ASS ist definiert durch Einschränkungen in der sozialen Interaktion und Kommunikation sowie repetitives und stereotypes Verhalten. Genkopiepolymorphismen (englisch „copy number variations“/CNVs), also Deletionen oder Duplikationen einer chromosomalen Region, wurden wiederholt in Probanden mit ASS identifiziert. Hierbei ist in ASS die Region 16p11.2 mit am häufigsten von CNVs betroffen. Einige Gene aus diesem chromosomalen Abschnitt wurden bereits funktionell charakterisiert. Dennoch können die Befunde der bisherigen Einzelgenstudien nicht alle Aspekte erklären, die durch 16p11.2 CNVs hervorgerufen werden. Ziel dieser Studie war es daher, ein weiteres neuronal assoziiertes Kandidatengen dieser Region zu identifizieren und im Anschluss funktionell im Kontext der neuronalen Differenzierung zu charakterisieren.
Das SH-SY5Y Neuroblastom-Zellmodell wurde auf Transkriptom- und morphologischer Ebene auf seine Eignung als Modell für neuronale Differenzierung untersucht und bestätigt. Eine Analyse der Expressionen aller Gene der 16p11.2-Region zeigte, dass das Gen Quinolinat-Phosphoribosyltransferase (QPRT) eine vergleichsweise hohe Expression mit der stärksten und robustesten Regulierung über die Zeit aufwies. Eine de novo Deletion der 16p11.2-Region wurde in einem Patienten im Vergleich zu seinen Eltern validiert. In Patienten-spezifischen lymphoblastoiden Zelllinien derselben Familie konnten wir eine Gendosis-abhängige Expression von QPRT auf RNA-Ebene bestätigen. In SH-SY5Y-Zellen korrelierte die Expression von QPRT signifikant mit der Entwicklung von Neuriten während der Differenzierung. Um QPRT funktionell zu charakterisieren, benutzten wir drei verschiedene Methoden zur Reduktion der QPRT-Gendosis: (i) knock down (KD) durch siRNA, (ii) chemische Inhibition durch Phthalsäure und (iii) knock out (KO) über CRISPR/Cas9-Geneditierung. Eine Reduktion von QPRT durch siRNA führte zu einer schwachen Veränderung der neuronalen Morphologie differenzierter SH-SY5Y-Zellen. Die chemische Inhibition sowie der genetische KO von QPRT waren letal für differenzierende aber nicht für proliferierende Zellen. Eine Metabolitenanalyse zeigte keine Veränderungen des QPRT-assoziierten Tryptophanstoffwechsels. Gene, welche auf Transkriptomebene im Vergleich zwischen KO- und Kontrollzellen differenziell reguliert vorlagen, waren häufig an Prozessen der neuronalen Entwicklung sowie an der Bildung, Stabilität und Funktion synaptischer Strukturen beteiligt. Die Liste differenziell regulierter Gene enthielt außerdem überdurchschnittlich viele ASS-Risikogene und ko-regulierte Gengruppen waren assoziiert mit der Entwicklung des dorsolateralen präfrontalen Cortex, des Hippocampus sowie der Amygdala.
In dieser Studie zeigten wir einen kausalen Zusammenhang zwischen QPRT und der neuronalen Differenzierung in vitro sowie einen Einfluss von QPRT auf die Regulation von ASS-assoziierten Genen und Gen-Netzwerken. Funktionell standen diese Gene im Kontext mit synaptischen Vorgängen, welche durch Veränderungen zu einem Exzitations-Inhibitions-Ungleichgewicht und letztendlich zum Zelltod von Neuronen führen können. Unsere Ergebnisse heben in Summe die wichtige Rolle von QPRT in der Krankheitsentstehung von ASS, insbesondere in Trägern einer 16p11.2 Deletion, hervor.
Carotinoide sind Pigmente, die in Pflanzen, Algen, einigen Pilzen und Bakterien vorkommen. Sie spielen eine wichtige Rolle bei der Photosynthese durch Absorption von Licht und beim Lichtschutz. Sie sind verantwortlich für die braunen, roten, orangen und gelben Farben von Obst, Gemüse, Herbstblättern und die Farbe einiger Blumen und Algen. Tiere können keine Carotinoide synthetisieren, daher ist ihre Anwesenheit auf die Nahrungsaufnahme zurückzuführen. Carotinoide sind Tetraterpenoide (40C), die aus Isoprenoidmolekülen (5C) synthetisiert werden. Der Methylerythritol-phosphatweg ist der Carotinoid-Vorläuferweg, der die Isoprenoideinheiten bildet. Carotinoide haben aufgrund ihrer gesundheitlichen Vorteile das Interesse der Nutrazeutika-Industrie geweckt.
Fucoxanthin ist ein Carotinoid, das nur in Kieselalgen, Braunalgen, Haptophyten und einigen Dinoflagellaten vorkommt. Aufgrund seiner Vorteile zur Vorbeugung von Krebs, kognitiven Erkrankungen und Fettleibigkeit sowie seiner antioxidativen Eigenschaften ist Fucoxanthin ein sehr interessantes Molekül fur die Nutrazeutikabranche.
Fucoxanthin hat eine komplexe chemische Struktur mit einer Allenbindung und einer Epoxyketogruppe. Daher wäre seine chemische Synthese kompliziert, da es auch eine stereokontrollierte Synthese erfordert86. Aus diesem Grund ist die Extraktion aus Makroalgen oder Mikroalgen die Methode der Wahl für die kommerzielle Herstellung von Fucoxanthin.
In dieser Arbeit bestand das Ziel darin, die Fucoxanthin-Produktivität in Kieselalgen mit gentechnischen Methoden zu steigern, damit die Zellen mehr Fucoxanthin produzieren. Zu diesem Zweck wurde der Effekt der Insertion zusätzlicher Kopien von Genen in das Genom untersucht, die für geschwindigkeitsbestimmende oder Schlüsselenzyme im Carotinoid- und MEP-Weg kodieren.
Zu Beginn wurden diese Effekte bei einzelnen Mutanten beobachtet. Letztendlich ist es jedoch das Ziel, eine Mutante zu erzeugen, die mehrere geschwindigkeitsbestimmende Enzyme überexprimiert, um auf diese Weise Engpässe zu vermeiden. In früheren Studien erreichten Eilers et al.54 durch die einmalige Überexpression der psy- und dxs-Gene in der Kieselalge P. tricornutum einen 2.4- und 1.8-fachen Anstieg der Fucoxanthin-Spiegel.
In dieser Arbeit führte die Insertion zusätzlicher Kopien der Gene idi und pds2 nicht dazu, dass die Zellen mehr Fucoxanthin produzieren. Im Gegensatz dazu erreichten die Mutanten mit zusätzlichen Kopien der Gen ggpps und mit zusätzlichen Kopien sowohl von psy als auch von dxs seine um 28% bzw. 10% höhere Fucoxanthin-Produktivität pro Million Zellen. Bei diesen Mutanten ist die Gesamtproduktivität jedoch geringer als beim Wildtyp, da ihr Wachstum langsamer als beim Wildtyp ist.
Unter Berücksichtigung der besten Zielgene wurden Mutanten erzeugt, die gleichzeitig zusätzliche Kopien von psy, dxs und ggpps enthielten. Die Mutanten hatten unter sehr niedriegen Lichtbedingungen eine um bis zu 61% höhere Produktivität pro Million Zellen als der Wildtyp. Ausnahmsweise wurden diese Mutanten bei sehr schwachem Licht (10 µE m-2 s-1) gezüchtet, da sie sehr gestresst waren und als Zellklumpen wuchsen. Obwohl die Gesamt-Fucoxanthin-Spiegel in diesen Mutanten unter diesen Bedingungen höher sind als im Wildtyp, sind sie daher niedriger als die Fucoxanthin-Spiegel bei den in anderen Experimenten verwendeten Lichtbedingungen (50 µE m-2 s-1). Als Ergebnis dieser Experimente kann gesagt werden, dass die Belastung der Zellen nach den genetischen Veränderungen untersucht werden muss, da dies zu einer Abnahme der Biomasse und folglich zu einer Abnahme der Fucoxanthinproduktion führt. Alternativ könnte auch eine 2-Stufen-Kultur etabliert werden, in der in einem ersten Schritt eine hohe Biomasse erreicht wird und im zweiten Schritt die Expression der interessierenden Gene induziert wird.
Aufgrund der antioxidativen Eigenschaften von Carotinoiden besteht eine übliche Strategie zur Akkumulation von Carotinoiden darin, die Zellen unter oxidative Stressbedingungen zu setzen. Diese Strategie ist jedoch nicht wirksam für die Anreicherung von Fucoxanthin unter hohen Salzkonzentrationen oder hohen Lichtbedingungen. Bessere Versuchspläne könnten jedoch eine 2-Stufen-Kultur oder adaptive Laborbedingungen gewesen sein.
Eine andere mögliche Strategie zur Erhöhung des Fucoxanthinspiegels wäre die Durchführung einer zufälligen Mutagenese der Zellen. Auf diese Weise sind keine Vorkenntnisse über den Carotinoidsyntheseweg und seine Regulation erforderlich und es kann zu Veränderungen in Genen führen, die keine offensichtlichen Ziele sind.
Experimente mit zufälliger Mutagenese erfordern ein Hochdurchsatz-Screeningsystem, da Hunderte oder sogar Tausende von Mutanten erhalten werden. Eine mögliche Strategie, um die Kultivierung der hohen Anzahl von Mutanten zu vereinfachen, ist die Einkapselung dieser Mutanten in Alginatkügelchen. Auf diese Weise können alle Mutanten in demselben Gefäß kultiviert werden. Die eingekapselten Zellen können dann beispielsweise mit einem Durchflusszytometer auf große Partikel durch Fluoreszenz- oder Absorptionsmessungen gescreent werden.
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Bei den meisten erwachsenen Säugetieren führt ein Herzinfarkt zu Fibrose und Verlust von funktionellem Herzgewebe. Einige Wirbeltiere, wie der Zebrabärbling, besitzen jedoch die bemerkenswerte Fähigkeit, nach einer Schädigung ihres Herzgewebes verlorenes Gewebe zu regenerieren und so schädliche Folgen zu verhindern. Die lokale Immunantwort auf eine Verletzung wird zunehmend als eine wichtige Determinante für das regenerative Potential eines Gewebes gesehen. Das Komplementsystem ist Teil des humoralen Immunsystems. Historisch ist es als eine Sammlung von Protein bekannt, den Komplementkomponenten, die in der Leber synthetisiert werden und im Blutkreislauf zirkulieren. Bei Exposition gegenüber einem Auslöser, wie z. B. einem Pathogen, wird eine Komplementkomponentproteinspaltungskaskade initiiert, die dazu führen kann, dass Immunzellen rekrutiert werden, und, dass die Phagozytose erleichtert, ggf. die Zielzelle lysiert wird. Studien legen nahe, dass das Komplementsystem an zellulären Prozessen beteiligt sei, die für Entwicklungs- und Krankheitsprozesse entscheidend sind, wie etwa Proliferation und Dedifferenzierung. Es gibt Hinweise, dass das Komplementsystem eine Rolle bei Krebserkrankungen und bei regenerativen Prozessen spielen könnte. In verschiedenen Arten wurde eine lokale verletzungsinduzierte Expression von komplementkomponentkodierenden Genen in regenerierendem Gewebe beobachtet.
Einzelne Studien legen nahe, dass Funktionsverlust einzelner Komplementkomponenten regenerative Prozesse beeinträchtigt.
Offene Fragen bleiben jedoch: Ist die lokale Expression von mehreren komplementkomponentkodierenden Genen ein Merkmal von regenerierendem Gewebe, das sie von Geweben unterscheidet, welchem die Fähigkeit zur Regeneration fehlt? Und welche Rolle könnte das Komplementsystem und seine Komponenten während des regenerativen Prozesses spielen? Um diesen Fragen nachzugehen, wurde eine Expressionsanalyse von Zebrabärblingsgewebe nach Verletzung mittels RT-qPCR und in situ Hybridisierung durchgeführt: kardiale Kryoverletzung, Larvenrumpfamputation und Schwanzflossenamputation. Ich beobachtete, dass mehrere komplementkomponentkodierende Gene in diesen Geweben nach Verletzung induziert wurden. Die Interpretation veröffentlichter single cell RNAseq Datensätze legt nahe, dass diese komplementkomponentenkodierenden Gene von verschiedenen Zelltypen exprimiert werden, darunter Immunzellen, Epikardzellen und Fibroblasten. Um transkriptionelle Unterschiede zwischen regenerierendem und nicht regenerierendem Gewebe zu identifizieren, verwendete ich ein nicht regeneratives Zebrabärblingmodell, die il11ra- Mutante. Dieser Mutante fehlt die Fähigkeit, verschiedene Organe zu regenerieren, das ist der Fall beim Herzen, dem larvalen Rumpf, und der Schwanzflosse. Ich stellte fest, dass die Mehrheit der verletzungsinduzierten komplementkomponentkodierenden Gene il11ra nachgeschaltet war. Darüber hinaus zeigten Experimente unter Verwendung chemischer Inhibitoren, dass speziell die Expression der komplementkomponentkodierenden Gene c3a.1,
c4b und c7a im Larvenrumpfamputationsmodell durch den Il11-Stat3-Signalweg moduliert wird.
Zur Klärung der Frage, ob das Komplementsystem und/ oder seine Komponenten eine Rolle während der Regeneration spielen, wurden verschiede Funktionsverlustmodelle generiert und im larvalen Rumpfamputationsmodell auf mögliche Aberrationen getestet. Zum einen generierte ich Überexpressionslinien von endogenen Inhibitoren der Komplementproteinspaltungskaskade. Überexpression eines etablierten Komplementsysteminhibitors rca2.1/ tecrem führte zu einer im Vergleich zu Wildtyp- Geschwistern verringerten Regeneration des larvalen Rumpfs. Zum anderen generierte ich Funktionsverlustmutanten von individuellen Komplementkomponenten durch CRISPR/Cas9 vermittelter Mutagenese, und zwar für masp1, masp2, cfd, c1s, c4b, c5 und c9. Die larvale Rumpfregeneration war in diesen Mutanten unauffällig. Allerdings zeigten c4b Mutanten eine verringerte Kardiomyozytenproliferation und eine differenzielle Expression von einigen Markergenen, einschließlich einer erhöhten Expression von inflammatorischen Zytokinen.
Meine Studien führten zu neuen Einblicken in das Komplementsystem im Kontext der Regeneration. Ich fand heraus, dass mehrere komplementkomponentenkodierenden Gene in regenerierendem Zebrabärblinggewebe exprimiert werden, und zwar im Herzgewebe, im larvalen Rumpf und in der adulten Flosse. Darüber hinaus zeige ich, dass die verletzungsinduzierte Expression von komplementkodierenden Genen in regenerierendem Gewebe dem Regenerationsmasterregulator il11ra nachgeschaltet ist. Speziell c3a.1, c4b und c7a wurden durch il11/ stat3 reguliert...
Die Parkinson Erkrankung ist die zweithäufigste neurodegenerative Erkrankung in industrialisierten Ländern. Die charakteristischen Symptome sind schwere Beeinträchtigungen des Bewegungsablaufes welche auf den Verlust dopaminerger Neurone der Substantia nigra und der damit einhergehenden Reduktion des striatalen Dopamin Gehaltes zurückzuführen sind. Alpha-Synuklein (SNCA) ist ein Protein welches zum einen mit sporadischen aber auch mit idiopathischen Erkrankungen assoziiert ist. Mutationen welche einen Funktionsgewinn von SNCA zur Folge haben konnten mit autosomal dominanten Varianten der Parkinson Erkrankung assoziiert werden und genetische Veränderungen an beiden Genenden agieren als Risikofaktor für sporadische Formen der Erkrankung. Des Weiteren wird SNCA als Hauptbestandteil der Lewy Körperchen gefunden, einem pathologischen Kennzeichen der parkinsonschen Erkrankung. Die charakteristischen Bewegungsstörungen können mittels L-DOPA, einer metabolischen Vorstufe von Dopamin, behandelt werden. Neben dem enorm positiven Effekt auf die Bewegungsstörungen, geht die Behandlung mit L-DOPA jedoch auch mit ernsten Nebenwirkungen einher, welche als Levodopa induzierte Dyskinesien (LID) beschrieben werden.
Ziel der Arbeit war die Analyse von Effekten eines SNCA Funktionsgewinns sowie des Pink1 Funktionsverlustes auf molekulare Signalwege der synaptischen Plastizität unter Verwendung dreier PD Mausmodelle (A53T-SNCA überexprimierendes Modell (PrPmtA), Pink1KO Modell sowie A53T-SNCA + Pink1KO Doppelmutante (DM)). Es wurden Kandidatengene welche eine Rolle für synaptische Plastizität spielen in 6 Monate alten Mäusen aller drei PD Mauslinien untersucht. Die Analyse von PrPmtA zeigte erhöhte mRNA Spiegel von Glutamatrezeptor-Untereinheiten und von Kandidatengenen welche eine Rolle bei der synaptischen Signalweiterleitung spielen, sowie reduzierte mRNA Spiegel von IEGs und Transkriptionsfaktoren. Die Analyse der DM zeigte nur geringe Expressionsänderungen der Glutamatrezeptor-Untereinheiten und die Analyse von IEGs und Transkriptionsfaktoren zeigte erneut reduziert mRNA Spiege. In Pink1KO Tieren konnten nur minimale Expressionsveränderungen der Kandidatengene gefunden werden, was den Schluss zulässt, dass die zuvor beschriebenen Expressionsveränderungen in PrPmtA und DM Mäusen eindeutig auf den SNCA Funktionsgewinn zurückzuführen sind. Um frühe Effekte des SNCA Funktionsgewinns zu studieren wurde die Analyse auf 3 Monate alte PrPmtA Mäuse ausgeweitet. Diese ergab, Expressionsveränderungen für Homer1, cFos, NOR1, Nurr1 und Nur77.
In einem weiteren Versuchsansatz wurde die Auswirkung des SNCA Funktionsgewinns auf das Verhalten sowie auf molekulare Parameter nach Apomorphin Behandlung analysiert. Die Analyse ergab ein erhöhtes Niveau an unwillkürlichen Bewegungsmustern mit stereotypen und dystonischen Eigenschaften in PrPmtA im Vergleich zu Wildtypen (wt). Die molekulare Analyse von striatalem Gewebe wurde zu zwei Zeitpunkten durchgeführt, 30 min nach Apomorphin Injektion und 100 min nach Injektion. Die Analyse von striatalem Gewebe welches zum Zeitpunkt 30 min nach Injektion entnommen wurde ergab eine erhöhte Apomorphin abhängige Phosphorylierung von ERK1/2, sowie eine erhöhte Apomorphin abhängige Expression von Dusp1, Dusp6 und cFos in transgenen und wt Tieren. Genotyp abhängige Unterschiede ergaben sich für cFos, welches signifikant höher in PrPmtA induziert wurde. 100 min nach Apomorphin Injektion ergab die gleiche Analyse eine erhöhte Apomorphin abhängige Phosphorylierung von ERK1 und eine erhöhte Apomorphin abhängige Expression von Dusp1, Dusp6, cFos und Nur77 in PrPmtA im Vergleich zu wt. Die Daten unterstreichen die fundamentale Rolle von SNCA auf die Neurotransmission und synaptische Plastizität und zeigen auf, dass PrPmtA ein zuverlässiges Modell für die Analyse von präsynaptischer Dysfunktion in Frühstadien der Parkinson Erkrankung darstellt.
Der letzte Versuchsansatz stellt die Charakterisierung des DM-Mausmodells welches sich durch einen starken Phänotyp auszeichnet, sowie die Analyse des Pink1 Effektes auf die SNCA induzierte Neurotoxizität dar. DM-Tiere zeigen deutlich reduzierte Spontanmotorik im Alter von 3 Monaten sowie einer progressiven Lähmung der Hinterläufe, was Anlass zu einer immunhistologischen Charakterisierung mittels Schnitten des Gehirns und Rückenmarks gab. Die histologische Analyse zeigte pSer129-SNCA, p62/SQSTM1 und Ubiquitin positive Aggregate innerhalb der grauen Substanz des Rückenmarks sowie innerhalb einer neuronalen Zellpopulation welche dorsal der Substantia nigra angeordnet ist. Das histologische Erscheinungsbild wurde spezifisch in gelähmten DM-Tieren gefunden und nicht in Einzelmutanten oder DM-Tieren ohne Lähmung. Dieses Modell stellt ein wertvolles Instrument für die Identifizierung von pathologischen Mechanismen und Signalkaskaden welche beiden Parkinson relevanten Genen gemeinsam sind, dar.
The functional and molecular role of transglutaminase 2 in hematopoietic stem and progenitor cells
(2023)
Long-term repopulating hematopoietic stem cells (LT-HSCs) that reside in the bone marrow (BM) give rise to all blood cell types including erythrocytes, leukocytes and platelets. LT-HSCs are mainly quiescent during steady state hematopoiesis. LT-HSCs can process self-renewal to expand and maintain stemness, or commit to differentiation into short-term (ST) repopulating HSC and multipotent progenitors (MPPs). MPPs differentiate into oligopotent lineagerestricted progenitors which eventually produce all mature blood cell lineages, and thereby regenerate hematopoietic system.
Previous studies have shown in transcription profiles and quantitative PCR (qPCR) analysis that transglutaminase 2 (Tgm2) is one of the most upregulated genes in quiescent LT-HSCs in comparison to active HSCs, mobilized HSCs, ST-HSCs, MPPs, as well as leukemic stem cells (LSC). However, the reason why Tgm2 is strongly upregulated in dormant mouse LTHSCs and what the role of Tgm2 is in LT-HSCs has not been investigated yet.
Tgm2, encoded by the Tgm2 gene, is a multi-functional protein within the transglutaminase family. It has been found to be widely expressed inside and outside the cells. It consists of four domains and two functionally exclusive forms that are regulated by the Ca2+ and GTP concentration. Besides the most well-known transglutaminase enzymatic activity for transamidation, deamidation and crosslinking, Tgm2 acts also as a GTPase/ATPase, kinase, adhesion/scaffold protein, as well as disulfide isomerase. The role of Tgm2 in hematopoiesis remains elusive. Accordingly, the aim of this dissertation is to investigate the role of Tgm2 in murine hematopoiesis, especially in murine LT-HSCs.
Firstly, the expression of Tgm2 was analyzed in highly purified murine hematopoietic stem and progenitor cell (HSPC) populations. Low input label-free mass spectrometric proteomics and WES protein analysis confirmed the highly specific expression of Tgm2 in LT-HSCs at protein level. Already at the state of MPPs, Tgm2 protein was almost absent with further decline towards oligopotent progenitors. These results indicated Tgm2 as a specific protein marker for LT-HSCs, justifying the future generation of a fluorescent reporter mouse line based on endogenous Tgm2 tagging.
To delineate the functional and molecular role of Tgm2 in LT-HSCs, a conditional Tgm2 knockout mouse model was generated using the Mx1-Cre/loxP system, with the loxP sites flanking the coding exons of the catalytic domain of Tgm2. After PolyIC-mediated induction, a more than 95% knockout efficiency was observed in purified LT-HSCs and the protein expression of Tgm2 was confirmed to be vanished in the purified LT-HSCs from conditional Tgm2-KO mice. Conditional knockout mice are viable and show no aberrant organ functions.
In steady state condition, the distribution of mature blood cell lineages and immunophenotypically-defined HSPC populations within the BM, the mitochondrial potential of HSPCs reflected by the non-invasive cationic dye JC-1, as well as the cell cycle status of HSPCs mirrored by the intracellular Ki67 staining did not show any significant variations upon loss of Tgm2. However, the in vitro continuous observation of prospectivly isolated LT-HSCs by time-lapse microscopy-based cell tracking revealed a delayed entry into cell cycle with a two fold increased apoptosis rate after knocking out Tgm2, indicating Tgm2 expression might be essential for survival of LT-HSCs. Moreover, while the absence of Tgm2 in LT-HSCs did not influence differentiation and lineage choice in vitro, overexpression of Tgm2 in LT-HSCs resulted in an increase of the most immature subpopulation upon cultivation. All these features were not observed in Tgm2-deleted MPPs, suggesting Tgm2 playing a specific function at the level of LT-HSCs. Upon stress hematopoiesis, induced by the administration of 5-fluorouracil (5-FU), there was a trend towards delayed recovery of LT-HSCs lacking Tgm2. Although Tgm2 express specificly in LT-HSCs, two rounds of competitive BM serial transplantation displayed an equal overall engraftment and multi-lineage reconstitution of LT-HSCs from Tgm2-WT and Tgm2-KO mice in peripheral blood (PB), BM and spleens. Interestingly, LT-HSCs from Tgm2-KO mice reconstituted to more myeloid cells and fewer B cells in the first four weeks after primary transplantation, which disappeared at later time points.
Gene expression profiling and simultaneous single cell proteo-genomic profiling indicated that HSPCs and LT-HSCs from Tgm2-KO mice were transcriptionally more active. A heterogeneity of Tgm2 expression within Tgm2-WT LT-HSCs was revealed by single cell data. Commonly up-regulated genes in Tgm2-KO LT-HSCs and MPPs were significantly involved in regulation of transcription from RNA polymerase II promoter in response to stress, positive regulation of cell death as well as negative regulation of mitogen-activated protein kinase (MAPK) signaling pathways. In Tgm2-KO LT-HSCs, 136 up-regulated genes demonstrated an enrichment of genes involved in apoptosis, as well as negative regulation of MAPK signaling pathway.
Taken together, this dissertation shows that Tgm2 protein is highly specifically expressed in LT-HSCs, but not in subsequent progenitor populations. However, Tgm2 is not essential for differentiation and maturation of myeloid lineages, the proliferation and the long-term multilineage reconstitution potential of LT-HSCs after transplantation. Tgm2 might be involved in accurate stress response of LT-HSCs and the transition from LT-HSCs into MPPs, meaning that the absence of Tgm2 results in poor survival, myeloid bias upon transplantation, as well as slower recovery upon chemotherapeutic treatment.