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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.
Die vorliegende Dissertation mit dem Titel: Ecophysiological monitoring of Oaks in Central Europe, introduced in the framework of proactive climate change mitigation beschäftigt sich mit der Anwendung zerstörungsfreier, radiometrischer Methoden zur Bestimmung von Pigment- und Stickstoffkonzentrationen und der photosynthetischen Funktionalität in Blättern von heimischen und gebietsfremden Eichen und ihre Beeinflussung durch Trocken-, Hitze- und Kältestress.
Die Eichenarten Quercus robur L. (Stieleiche), Q. pubescens Willd. (Flaumeiche), Q. frainetto Ten. (Ungarische Eiche), Q. ilex L. (immergrüne Steineiche) und Q. rubra L. (amerikanische Roteiche) wurden im Frühjahr 2011 auf einer Versuchsfläche im Frankfurter Stadtwald gepflanzt, um ihre Nutzung als potentielle Waldbäume in einem sich ändernden Klima zu untersuchen. Über eine Dauer von zwei Jahren wurden diese Arten mit einem hohen Maß an blattspezifischer Merkmalsvariabilität beobachtet und beprobt. Ziel war es, die interspezifischen Unterschiede und die jahreszeitliche Dynamik von morphologischen und chemischen Blattmerkmalen sowie die Beeinflussung der radiometrischen Bestimmung des Chlorophyllgehaltes (und damit assoziierten Komponenten wie z.B. Blattstickstoffgehalt und Karotinoiden) und der photosynthetischen Funktionalität durch klimatische Umweltbelastungen in Eichen zu untersuchen. Die Analyse der Blattproben zielte neben der Bestimmung der Beziehung zwischen absoluten und optisch ermittelten Pigmentgehalten auf die Ermittlung des Einflusses der Blattstruktur auf die Lichttransmission im roten und infrarotem Bereich des Elektromagnetischen Spektrums ab, sowie auf die artspezifische Korrelation von Blattstickstoff zu Blattchlorophyll zu dessen indirekte Quantifizierung. Des Weiteren wurden Versuche zur Trocken- und Hitzestressanpassung durchgeführt, um eine potentiell artspezifische Stressantwort, sowie eine mögliche Beeinflussung der aufgenommenen radiometrischen Messwerte zu ermitteln. Ein zusätzliches Monitoringprogramm im Winter 2012/2013 mit einer Dauer von sechs Monaten ermöglichte die Überprüfung der Anpassungsfähigkeit der immergrünen Steineiche (Q. ilex) an mitteleuropäische Winterbedingungen und die Veränderung der photosynthetischen Funktionalität unter Kältestress. Messungen im Zusammenhang mit der praktischen Anwendbarkeit der zerstörungsfreien, optischen Methode und zur Bereitstellung von Referenzdaten für zukünftige Evaluierungen komplementieren die Untersuchungen.
Signifikante, artspezifische Unterschiede wurden in den blattmorphologischen Schlüsselmerkmalen in den Quercus-Arten ermittelt. Die artspezifischen Unterschiede in den morphologischen Blattmerkmalen beeinflussten auf signifikante Weise die Beziehung zwischen absoluten, massebasierten Pigment- und Stickstoffgehalten und deren radiometrischen Bestimmung. Wurden die Pigmentgehalte hingegen auf die Blattfläche bezogen und die Stickstoffgehalte mittels des Verhältnisses von Blattfläche zu Trockenmasse korrigiert, zeichnete sich eine Beziehung zwischen absoluten und optisch ermittelten Werten ab, der jegliche jahreszeitliche oder artspezifisch morphologische Variabilität fehlte und die somit für alle Quercus-taxa anwendbar ist. Koeffizienten für die Berechnung von flächenbezogenen Gehalten von Gesamtchlorophyll, Chl a, Chl b und Carotinoiden für die jeweiligen Quercus-taxa, wie auch für ein artübergreifendes Modell wurden ermittelt, um die Bestimmung dieser Gehalte während aller Entwicklungsstufen zu ermöglich. Aus der jahreszeitlichen Entwicklung der Pigmentgehalte konnten drei deutliche Phasen abgeleitet werden: Die Phase der Blattentwicklung im Frühling, einer Plateauphase mit geringen Veränderungen (“core vegetation time”) und die Phase des Pigmentabbaus während der Herbstlaubfärbung. Die Übergänge zwischen diesen Phasen variierten zum Teil erheblich zwischen einzelnen Individuen einer Art sowie zwischen den Arten, was Unterschiede in der potentiellen, jährlichen Kohlenstoffaufnahme nach sich zieht. Stressbedingungen, wie Hitze- Kälte- oder Trockenstress, können zu Veränderung von Fluoreszenzparametern ohne gleichzeitige Änderung des Pigmentgehaltes führen, wie auch die indirekte Bestimmung von mit Chl assoziierten Komponenten (Carotinoide, Chl a, Chl b) mittels optischer Bestimmung (durch die Veränderungen von Pigmentverhältnissen) beeinflussen.
Im Rahmen des Forschungsprojektes konnten, Modelle zur Berechnung von Blattpigmenten und Blattstickstoff aus optischem Messdaten, Veränderungen der photosynthetischen Funktionalität, sowie Referenzdaten für die zukünftig nutzbaren Eichenarten hinsichtlich artspezifischer und jahreszeitlicher Variabilität unter mitteleuropäischen Umweltbedingungen ermittelt werden, die eine Nutzung und Einordnung von zerstörungsfreien, optischen Messwerten zur Ermittlung von Vitalitätsunterschieden in Eichen ermöglichen.
Photosynthesis is one of the most vital processes that takes place on Earth. Due to its global significance related to food, energy and material production, photosynthesis research is one of the leading scientific fields in the contemporary world. Particular interest in photosynthesis research is focused on diatoms and as one of the major players of marine phytoplankton, diatoms have a huge impact on global photosynthesis.
Diatoms originated from a secondary endosymbiosis that took place between a putative photosynthetic red algal ancestor and a heterotrophic eukaryote. Secondary endosymbiosis resulted in the formation of chloroplasts with four membranes. Centric diatoms (e.g. Thalassiosira pseudonana or Cyclotella meneghiniana) usually possess many small chloroplasts, while pennates (e.g. Phaeodactylum tricornutum) have several larger ones, or even only one which can occupy half of the cell volume...
In honeybees, reproductive females usually mate early in their life with more than 10 males in free flight, often within 10 minutes, and then store male gametes for up to five years. Because of the extreme polyandry and mating in free flight special adaptations in males are most likely. We present here the results of an investigation of the protein content of four types of male reproductive glands from the Western honeybee (Apis mellifera) drone, namely seminal vesicles (secretion in ejaculate), as well as bulbus, cornua and mucus glands (secretions for the mating plug). Using high resolution and accuracy mass spectrometry and a combination of database searching and de novo sequencing techniques it was possible to identify 50 different proteins in total, inside all mentioned glands, except in the mucus gland. Most of the proteins are unique for a specific gland type, only one of them (H9KEY1/ATP synthase subunit O) was found in three glands, and 7 proteins were found in two types of glands. The identified proteins represent a wide variety of biological functions and can be assigned to several physiological classes, such as protection, energy generation, maintaining optimal conditions, associated mainly with vesicula seminalis; signaling, cuticle proteins, icarpin and apolipoproteins located mainly in the bulbus and cornua glands; and some other classes. Most of the discovered proteins were not found earlier during investigation of semen, seminal fluid and tissue of reproductive glands of the bee drone. Moreover, we provide here the origin of each protein. Thus, the presented data might shed light on the role of each reproductive gland.
The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.
Australia has experienced dramatic declines and extinctions of its native rodent species over the last 200 years, particularly in southern Australia. In the tropical savanna of northern Australia significant declines have occurred only in recent decades. The later onset of these declines suggests that the causes may differ from earlier declines in the south. We examine potential regional effects (northern versus southern Australia) on biological and ecological correlates of range decline in Australian rodents. We demonstrate that rodent declines have been greater in the south than in the tropical north, are strongly influenced by phylogeny, and are consistently greater for species inhabiting relatively open or sparsely vegetated habitat. Unlike in marsupials, where some species have much larger body size than rodents, body mass was not an important predictor of decline in rodents. All Australian rodent species are within the prey-size range of cats (throughout the continent) and red foxes (in the south). Contrary to the hypothesis that mammal declines are related directly to ecosystem productivity (annual rainfall), our results are consistent with the hypothesis that disturbances such as fire and grazing, which occur in non-rainforest habitats and remove cover used by rodents for shelter, nesting and foraging, increase predation risk. We agree with calls to introduce conservation management that limits the size and intensity of fires, increases fire patchiness and reduces grazing impacts at ecological scales appropriate for rodents. Controlling feral predators, even creating predator-free reserves in relatively sparsely-vegetated habitats, is urgently required to ensure the survival of rodent species, particularly in northern Australia where declines are not yet as severe as those in the south.
Genetic signatures of adaptation revealed from transcriptome sequencing of Arctic and red foxes
(2015)
Background: The genus Vulpes (true foxes) comprises numerous species that inhabit a wide range of habitats and climatic conditions, including one species, the Arctic fox (Vulpes lagopus) which is adapted to the arctic region. A close relative to the Arctic fox, the red fox (Vulpes vulpes), occurs in subarctic to subtropical habitats. To study the genetic basis of their adaptations to different environments, transcriptome sequences from two Arctic foxes and one red fox individual were generated and analyzed for signatures of positive selection. In addition, the data allowed for a phylogenetic analysis and divergence time estimate between the two fox species.
Results: The de novo assembly of reads resulted in more than 160,000 contigs/transcripts per individual. Approximately 17,000 homologous genes were identified using human and the non-redundant databases. Positive selection analyses revealed several genes involved in various metabolic and molecular processes such as energy metabolism, cardiac gene regulation, apoptosis and blood coagulation to be under positive selection in foxes. Branch site tests identified four genes to be under positive selection in the Arctic fox transcriptome, two of which are fat metabolism genes. In the red fox transcriptome eight genes are under positive selection, including molecular process genes, notably genes involved in ATP metabolism. Analysis of the three transcriptomes and five Sanger re-sequenced genes in additional individuals identified a lower genetic variability within Arctic foxes compared to red foxes, which is consistent with distribution range differences and demographic responses to past climatic fluctuations. A phylogenomic analysis estimated that the Arctic and red fox lineages diverged about three million years ago.
Conclusions: Transcriptome data are an economic way to generate genomic resources for evolutionary studies. Despite not representing an entire genome, this transcriptome analysis identified numerous genes that are relevant to arctic adaptation in foxes. Similar to polar bears, fat metabolism seems to play a central role in adaptation of Arctic foxes to the cold climate, as has been identified in the polar bear, another arctic specialist.
Species recognition in lichen-forming fungi has been a challenge because of unsettled species concepts, few taxonomically relevant traits, and limitations of traditionally used morphological and chemical characters for identifying closely related species. Here we analyze species diversity in the cosmopolitan genus Protoparmelia s.l. The ~25 described species in this group occur across diverse habitats from the boreal -arctic/alpine to the tropics, but their relationship to each other remains unexplored. In this study, we inferred the phylogeny of 18 species currently assigned to this genus based on 160 specimens and six markers: mtSSU, nuLSU, ITS, RPB1, MCM7, and TSR1. We assessed the circumscription of species-level lineages in Protoparmelia s. str. using two coalescent-based species delimitation methods – BP&P and spedeSTEM. Our results suggest the presence of a tropical and an extra-tropical lineage, and eleven previously unrecognized distinct species-level lineages in Protoparmelia s. str. Several cryptic lineages were discovered as compared to phenotype-based species delimitation. Many of the putative species are supported by geographic evidence.
Little work has been done on large-scale patterns of stream insect richness in China. We explored the influence of climatic and catchment-scale factors on stream insect (Ephemeroptera, Plecoptera, Trichoptera; EPT) richness across mid-latitude China. We assessed the predictive ability of climatic, catchment land cover and physical structure variables on genus richness of EPT, both individually and combined, in 80 mid-latitude Chinese streams, spanning a 3899-m altitudinal gradient. We performed analyses using boosted regression trees and explored the nature of their influence on richness patterns. The relative importance of climate, land cover, and physical factors on stream insect richness varied considerably between the three orders, and while important for Ephemeroptera and Plecoptera, latitude did not improve model fit for any of the groups. EPT richness was linked with areas comprising high forest cover, elevation and slope, large catchments and low temperatures. Ephemeroptera favoured areas with high forest cover, medium-to-large catchment sizes, high temperature seasonality, and low potential evapotranspiration. Plecoptera richness was linked with low temperature seasonality and annual mean, and high slope, elevation and warm-season rainfall. Finally, Trichoptera favoured high elevation areas, with high forest cover, and low mean annual temperature, seasonality and aridity. Our findings highlight the variable role that catchment land cover, physical properties and climatic influences have on stream insect richness. This is one of the first studies of its kind in Chinese streams, thus we set the scene for more in-depth assessments of stream insect richness across broader spatial scales in China, but stress the importance of improving data availability and consistency through time.
Membranes are central for cells as borders to the environment or intracellular organelle definition. They are composed of and harbor different molecules like various lipid species and sterols, and they are generally crowded with proteins. The membrane system is very dynamic and components show lateral, rotational and translational diffusion. The consequence of the latter is that phase separation can occur in membranes in vivo and in vitro. It was documented that molecular dynamics simulations of an idealized plasma membrane model result in formation of membrane areas where either saturated lipids and cholesterol (liquid-ordered character, Lo) or unsaturated lipids (liquid-disordered character, Ld) were enriched. Furthermore, current discussions favor the idea that proteins are sorted into the liquid-disordered phase of model membranes, but experimental support for the behavior of isolated proteins in native membranes is sparse. To gain insight into the protein behavior we built a model of the red blood cell membrane with integrated glycophorin A dimer. The sorting and the dynamics of the dimer were subsequently explored by coarse-grained molecular dynamics simulations. In addition, we inspected the impact of lipid head groups and the presence of cholesterol within the membrane on the dynamics of the dimer within the membrane. We observed that cholesterol is important for the formation of membrane areas with Lo and Ld character. Moreover, it is an important factor for the reproduction of the dynamic behavior of the protein found in its native environment. The protein dimer was exclusively sorted into the domain of Ld character in the model red blood cell plasma membrane. Therefore, we present structural information on the glycophorin A dimer distribution in the plasma membrane in the absence of other factors like e.g. lipid anchors in a coarse grain resolution.
Even though the microevolution of plant hosts and pathogens has been intensely studied, knowledge regarding macro-evolutionary patterns is limited. Having the highest species diversity and host-specificity among Oomycetes, downy mildews are a useful a model for investigating long-term host-pathogen coevolution. We show that phylogenies of Bremia and Asteraceae are significantly congruent. The accepted hypothesis is that pathogens have diverged contemporarily with their hosts. But maximum clade age estimation and sequence divergence comparison reveal that congruence is not due to long-term coevolution but rather due to host-shift driven speciation (pseudo-cospeciation). This pattern results from parasite radiation in related hosts, long after radiation and speciation of the hosts. As large host shifts free pathogens from hosts with effector triggered immunity subsequent radiation and diversification in related hosts with similar innate immunity may follow, resulting in a pattern mimicking true co-divergence, which is probably limited to the terminal nodes in many pathogen groups.
The canonical Wnt pathway, also known as Wnt/β-‐catenin pathway, comprises a network of proteins which control diverse developmental and adult processes in all metazoan organisms. The binding of canonical Wnt ligands to a cell surface receptor complex, consisting of frizzled family members and low density lipoprotein receptor-‐ related protein 5 or 6 co‐receptors, triggers a signaling cascade which results in a β-catenin-‐mediated transcriptional activation of different target genes, implicated in cellular proliferation, apoptosis, migration and differentiation. A couple of years ago, several groups including us, iden2fied transient activation of the canonical Wnt-pathway in endothelial cells (ECs) of the developing central nervous system (CNS). In this context, Wnt/β-‐catenin signaling could be demonstrated to be crucial for brain angio genesis as well as for the establishment of the blood-brain barrier (BBB) phenotype in the newly formed vessels.
Gliomas, in particular the glioblastoma (GBM), belong to the group of highly vascularized solid tumors which gain their vascularization due to an angiogenic switch occurring during tumor progression. Interestingly, nuclear localized β-‐catenin could be exclusively detected in the activated endothelium of induced rat gliomas and of human GBM, suggesting a so far unknown and not further characterized involvement of the canonical Wnt pathway in pathological angiogenesis. In order to systematically decipher the precise role of endothelial Wnt/β-‐catenin signaling in tumor angiogenesis, I established
murine GL261 glioma cell lines overexpressing either Wnt1 or Dickkopf (Dkk) 1 in a doxycycline-‐dependent manner, an activator and potent inhibitor of Wnt/β-‐catenin signaling, respectively. In subcutaneous and intracranial transplantations, tumor-derived Wnt1 reduced, while Dkk1 increased GL261 tumor growth without affecting in vitro proliferation, cell cycle or cell death of the established cell lines. Nowadays, it is well accepted that solid tumors are dependent on vascular support allowing them to grow beyond a certain size. In my work I could show that tumor-‐derived Wnt1 targets the tumor vasculature by increasing endothelial Wnt/β-‐catenin signaling, which reduced tumor vessel density and resulted in a more quiescent tumor vasculature. Furthermore, Wnt1-‐expression mediated tight association of smooth muscle cells (SMCs) and pericytes to the tumor endothelium, a phenotype which is unusual for tumor vessels and a described hallmark of tumor vessel normalization. In contrast, inhibition of endothelial Wnt/β-‐catenin signaling by Dkk1 mediated an opposing effect, characterized by endothelial hyper-proliferation and a tumor vasculature with a rough basal lamina distribution and loosely anached mural cells, indicative of a strong angiogenic activity. The described vascular effects in Wnt1-expressing GL261 tumors could be verified by subcutaneous transplantations of a rat glioma cell line constitutively expressing Wnt1. Furthermore, an applied in vivo MatrigelTM plug assay uncovered the reduction in vessel density upon Wnt1 simulation to be tumor cell independent, suggesting an EC-‐autonomous effect. This hypothesis was confirmed by subcutaneous transplantations of parental GL261 cells into mice with genetically generated endothelial β-‐catenin gain-of-function (GOF). The derived GOF tumor from this experiment comprised a quiescent and normalized tumor vasculature and phenocopied the vascular effects observed in Wnt1-expressing tumors.
Our previous work provided evidence that Wnt/β-‐catenin signaling contributes to the BBB phenotype of the developing CNS through the transcriptional regulation of the tight junction protein claudin-‐3. Furthermore, the coverage of pericytes to brain vessels has been described to correlate with BBB integrity. In agreement with these publications, vessels of intracranial Wnt1-‐expressing GL261 tumors retained or regained barrier properties, indicated by a reduced leakage of the tracer Evans blue and endogenous mouse immunoglobulin G and increased junctional localiza2on of the tight junction proteins claudin-‐3, -‐5 and zonula occludens-‐1.
Overall, we detected sustained endothelial Wnt/β-‐catenin signaling to induce a quiescent and normalized tumor vascularization. Interestingly, the Notch signaling pathway has been shown to inhibit the angiogenic tip cell and to promote the quiescent stalk cell phenotype via its ligand Delta-like ligand 4 (Dll4) and the receptors Notch1 and 4. Mechanistically, my work demonstrated for the first time that overactivation of endothelial Wnt/β-‐catenin signaling reactivated expression of Dll4 in the tumor endothelium, which could be shown in vitro to increase Notch signaling and to favor a stalk cell-like gene signature. Furthermore, we uncovered the platelet-derived growth factor subunit B (pdgm) as a novel transcriptional target of Wnt/β-catenin signaling in ECs. Hence endothelial-‐derived PDGF-‐B is known to promote the recruitment of mural cells, the upregulation of this factor might explain the increased SMC/pericyte coverage observed in the tumor vasculature upon sustained endothelial Wnt/β-‐catenin signaling which additionally might promote a cycle of vascular normalization.
Taken together, my work reveals several vascular effects, being mediated by reinforced endothelial Wnt/β-‐catenin signaling during tumor angiogenesis. While a moderate level of canonical Wnt signaling, observed in vessels of human astrocytomas and murine control tumors, is considered to be associated with tumor angiogenesis, dominant activation of this pathway in ECs is shown to limit angiogenesis and to promote a quiescent and normalized tumor vasculature with increased barrier properties. Furthermore, my work discovers pdgm as a novel target of canonical Wnt signaling in ECs.
The work presented in this dissertation therefore not only uncovers the role of endothelial Wnt/β-‐catenin signaling in tumor angiogenesis but additionally reveals this pathway to be a novel modulator in pathological vessel development which might proof to be a valuable therapeutic target for anti-angiogenic and edema glioma therapy.
The biogenesis and function of photosynthetically active chloroplasts relies on the import of thousands of nuclear encoded proteins via the coordinated actions of two multiprotein translocon machineries in the outer and inner envelope membrane. Trafficking of preproteins across the soluble compartment of InterMembrane Space (IMS) is currently envisioned to be facilitated by an IMS complex composed of outer envelope proteins Toc64 and Toc12, a soluble IMS component, Tic22 and an IMS-localized Hsp70. Among them, currently Tic22 is the only component that stands undisputed in terms of its existence. Having two closely related homologs in A. thaliana, their biochemical and functional characterization was still lacking. A critical analysis of Tic22 knockout mutants displayed growth phenotype reminiscent of ppi1, the mutant of Toc33. However, both the genes have similar expression patterns with no clear preference for photosynthetic or nonphotosynthetic tissues, which explained the absence of a detectable phenotype in single mutants. In addition, transgenic complementation study with either of the homolog affirmed the identical localization of both proteins in the IMS which characterizes the two homologs as functionally redundant. Based on the pale-yellow phenotype exhibited by the double mutant plants, an attempt to analyze the import capacity of a stromal substrate in the double mutant revealed threefold reduction when compared to wild-type acknowledging the essential role of Tic22 in the import mechanism. Initially, Tic22 was identified together with another protein, Tic20, which has been heavily discussed as a protein conducting channel in the inner membrane. Despite being characterized, in A. thaliana, two out of four homologs of Tic20 are differentially localized with one being additionally localized in mitochondria and the other, exclusively residing in the thylakoids.
According to in silico analysis, for all the Tic20 proteins, a four-helix transmembrane topology was predicted. Accordingly, its topology was mapped by employing the recently established selfassembling GFP-based in vivo experiments. Astonishingly, the expression of one of the inner envelope localized Tic20 homolog enforces inner membrane proliferation affecting the shape and organization of the membrane. Therefore this study focuses on analyzing the effects of high envelope protein concentrations on membrane structures, which together with the existing results, an imbalance in the lipid to protein ratio and a possible role of signaling pathway regulating membrane biogenesis is discussed.
ß1-integrins are essential for angiogenesis but the mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. BRAG2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of BRAG2 in EC and angiogenesis and the underlying molecular mechanisms remains unclear. siRNA-mediated BRAG2-silencing reduced EC angiogenic sprouting and migration. BRAG2-siRNA-transfection differentially affected a5ß1- and aVß3-integrin function: specifically, BRAG2-silencing increased focal/fibrillar adhesions and EC adhesion on ß1-integrin-ligands (fibronectin and collagen), while reducing the adhesion on the aVß3-integrin-ligand, vitronectin. Consistent with these results, BRAG2-silencing enhanced surface expression of a5ß1-integrin, while reducing surface expression of aVß3-integrin. Mechanistically, BRAG2 mediated recycling of aVß3-integrins and endocytosis of ß1-integrins and specifically of the active/matrix bound a5ß1-integrin present in fibrillar/focal adhesions (FA), suggesting that BRAG2 contributes to the disassembly of FA via ß1-integrin-endocytosis. Arf5 and Arf6 are promoting downstream of BRAG2 angiogenic sprouting, ß1-integrin-endocytosis and the regulation of FA. In vivo silencing of the BRAG2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitral injection of plasmids containing BRAG2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveals that BRAG2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating ß1-integrin internalization and associates for the first time the process of ß1-integrin endocytosis with angiogenesis.
In vitro investigation of genes identified by genome-wide association studies of Parkinson's disease
(2014)
Die Spinozerebelläre Ataxie Typ 2 (SCA2) ist eine autosomal dominant vererbte neurodegenerative Krankheit, welche durch die Expansion des Trinukleotids Cytosin-Adenin-Guanin von ~22/23 auf >32 im Ataxin-2 Gen (ATXN2) verursacht wird. Dieses Trinukleotid codiert für die Aminosäure Glutamin weshalb SCA2 auch zu den Polyglutaminerkrankungen zählt. Zu dieser Gruppe zählen außerdem fünf weitere SCA-Subtypen sowie drei weitere neurodegenerative Erkrankungen, darunter die Huntington-Krankheit.
SCA2 wurde 1971 zum ersten Mal von Wadia und Swami beschrieben und unterscheidet sich von den anderen SCAs aufgrund der typischen Störung der sakkadischen Augenbewegungen. Weitere klinische Symptome von SCA2 sind Ataxie, Tremor, Dysmetrie, Dysarthrie, Hyporeflexie und Dysdiadochokinese. Die Symptome gehen auf einen neuronalen Verlust insbesondere im Cerebellum, aber auch in anderen Hirnregionen wie zum Beispiel dem Hirnstamm zurück.
Atxn2 wird in weiten Teilen des Zentralnervensystems aber auch in vielen nicht-neuronalen Geweben exprimiert. Es handelt sich um ein überwiegend cytoplasmatisch lokalisiertes Protein, welches im Gegensatz zu vielen anderen SCA-Proteinen cytoplasmatische und nur selten nukleäre Aggregate bildet. Die exakte Funktion von Atxn2 ist bisher unklar, es wurde allerdings mehrfach gezeigt, dass es in die mRNA Translation involviert ist aufgrund seiner Interaktion mit dem PolyA-bindenden Protein PABPC1.
Eine Expansion des Trinukleotids in Ataxin-2 kann nicht nur zu SCA2 führen, sondern stellt bei Wiederholungen zwischen 27 und 32 CAGs auch ein erhöhtes Risiko für eine Erkrankung an Amyotropher Lateralsklerose (ALS) und anderen neurodegenerativen Krankheiten dar. Eine Interaktion zwischen ATXN2 und dem ALS-verursachenden TDP43 (Tardbp) wurde bereits zahlreich beforscht, da Aggregate von ATXN2 in Motoneuronen des Rückenmarks von ALS-Patienten und aggregiertes TDP43 in SCA2-Neuronen beobachtet wurden.
Generell sind die Mechanismen, die zur Pathologie von SCA2 und ALS führen, noch weitgehend unklar. Ziel dieser Arbeit war es daher auf der einen Seite einen Einblick in den Pathomechanismus von SCA2 zu erhalten, indem mögliche oder bereits bekannte Interaktoren in etablierten Atxn2-Mausmodellen untersucht wurden. Auf der anderen Seite wurden zwei neue Mausmodelle charakterisiert, um ihre Eignung für die Erforschung von ALS und SCA2 zu prüfen.
Für den ersten Teil der Arbeit dienten Daten aus mehreren Transkriptomstudien von Atxn2-Knock-Out (KO) und Atxn2-CAG42-Knock-In (KIN) Mäusen als Grundlage. Konnten die Daten mit einer unabhängigen Methode bestätigt werden, folgten weitere Untersuchungen auf mRNA und Proteinebene sowie unter zusätzlicher Verwendung von Zellkultur und Patientenmaterial. Dadurch konnten neue Interaktionspartner von ATXN2 identifiziert und bereits bekannte in diesen Mausmodellen bestätigt werden.
So wurde zum Beispiel eine Interaktion von ATXN2 mit der E3-Ubiquitin-Protein-Ligasekomponente FBXW8 gezeigt und deren Beteiligung am Abbau von expandiertem ATXN2. Außerdem wurde eine Interaktion von FBXW8 mit dem bereits bekannten ATXN2-degradierenden Protein PARK2 gezeigt. Eine Hochregulierung des Fbxw8 Transkripts wurde sowohl im Atxn2-CAG42-KIN-Mausmodell als auch in SCA2-Patientenfibroblasten gefunden, während Park2 in keinem der Modelle signifikant veränderte Transkriptspiegel aufwies. Diese Daten belegen die Relevanz von Fbxw8 für den Abbau von moderat-expandiertem Atxn2 und begründen weitere Studien zur genauen Funktion dieses Proteins im Pathomechanismus von Atxn2.
Des Weiteren wurden diverse Kalziumhomöostasefaktoren untersucht, welche eine konsistente Herunterregulierung der Transkripte in beiden Mausmodellen aufwiesen. Auf Proteinebene zeigten sich jedoch Unterschiede zwischen den Modellen. Diese Daten belegen, dass zwar ähnliche Transkriptveränderungen im KIN- und KO-Modell auftreten, diesen aber vermutlich verschiedene Mechanismen zugrunde liegen. Welche Mechanismen dies genau sind bleibt zu klären, es ist jedoch wahrscheinlich, dass im KIN-Modell die Aggregatbildung sowie in beiden Modellen die Beteiligung von ATXN2 an der Translationregulation eine Rolle spielen. Die Ergebnisse dieser Studie unterstreichen die Relevanz des Ca2+ Signalwegs für die Entwicklung von SCA2.
Der zweite Teil der Arbeit beinhaltet die Charakterisierung einer ATXN2/TDP43 Doppelmutante auf Verhaltensebene sowie die gründliche Evaluierung des Phänotyps einer vollkommen neuen SCA2 Mausmutante. Während in der Doppelmutante trotz doppelter Genmutation nur ein sehr schwacher Phänotyp auf Verhaltensebene festgestellt werden konnte und bis zu einem Alter von 12 Monaten keine Potenzierung der Mutationen zu beobachten war, zeigte die Atxn2-CAG100-KIN Maus signifikante und früh auftretende Pathologie. Neben einer verminderten Überlebensrate, einem Gewichtsverlust und diversen motorischen Störungen, konnten auch Aggregate des mutierten Proteins in diversen Hirnregionen identifiziert werden. Der Atxn2-CAG100-KIN Phänotyp spiegelt die humanen Symptome daher recht gut wider, weshalb diese Mausmutante ein wertvolles Modell für die weitere SCA2-Forschung darstellt.
Zusammengefasst zeigt diese Arbeit die Bedeutung des ATXN2-Interaktors FBXW8 im SCA2-Mausmodell als auch im Patientenmaterial. Sie betont die Relevanz des Atxn2-KO-Modells in Bezug auf Störungen der Kalziumhomöostase und dokumentiert die Alters- und Gewebespezifität dieser Veränderungen. Außerdem beinhaltet sie die vorläufige Beschreibung eines kombinierten Atxn2/TDP43-Mausmodells und schließlich die ausführliche Charakterisierung eines vollkommen neuen und äußerst wertvollen SCA2-Mausmodells.
Evolutionary genetics of bears and red foxes over phylogenetic and phylogeographic time scales
(2014)
Climatic fluctuations during the Pleistocene (2.6-0.01 million years) have played an important role during evolution of many species. Cyclic range contractions and expansions had demographic consequences within species, provided environmental conditions for population divergence and speciation and enabled secondary contact and interspecific hybridization. These and other evolutionary processes have left genetic signatures in the genomes of affected organisms. Comprehensive and unbiased estimates of evolutionary processes can be obtained using genetic markers from different parts of the genome and by integrating population genetic and phylogenetic concepts.
Suitable for studies on evolutionary processes and patterns over different evolutionary time scales are bears (Ursidae) and foxes (Vulpes), which occupy a wide range of habitats and evolved during the past few millions of years. In my thesis, I therefore used bears and red foxes as study species to investigate the genetic variation within and between species and to obtain estimates of evolutionary relationships and divergence times of populations and species that I interpreted in a climatic context. Further, I investigated population genetic processes during the evolution of bears. My thesis includes three publications and one submitted manuscript, spanning different evolutionary time scales - from evolutionary relationships and processes among species (phylogenetic time scales, Publications I & II), among populations and closely related species in a geographical context (phylogeographic time scales, Publications II & III), to ongoing processes within species (population genetic time scales, Publication IV).
In Publication I (Kutschera et al. 2014, Mol Biol Evol 31(8):2004-2017), I studied bears at several nuclear markers from several individuals per species, complemented with markers from the Y chromosome. Using approaches based on a population genetic concept (coalescent theory) I obtained a species tree with divergence time estimates. Further, I studied two evolutionary processes in bears, interspecific gene flow and incomplete lineage sorting (ILS). This study contributed to the growing evidence that population genetic processes can be relevant on time scales up to several millions of years.
In Publication II (Hailer, Kutschera et al. 2012, Science 336(6079):344-347), we complemented previous mitochondrial (mt) DNA-based inference of the evolutionary history of polar and brown bears with nuclear DNA. Coalescence-based species tree analyses of multiple nuclear markers from several individuals per species placed polar bears as sister lineage to brown bears and their divergence time to about 600 thousand years ago (ka). This contrasted previous mtDNA-based inference. We explained this discrepancy between mtDNA and nuclear DNA with interspecific gene flow between polar and brown bears.
In Publication III (Kutschera et al. 2013, BMC Evol Biol 13:114), I studied range-wide phylogeographic events and their timing in red foxes. A synthesis of newly generated and published mtDNA sequences was analyzed using a coalescence-based approach with multiple fossil calibration points. Thereby, I validated the identity and geographic distribution of several red fox lineages and showed that red foxes colonized North America and Japan several times independently during the late Pleistocene (126-11 ka) and around the last glacial maximum (26.5-19 ka). In a comparison of my results from red foxes to brown bears and grey wolves, I identified similar phylogeographic patterns.
In Publication IV (Kutschera et al., submitted to Biol Conserv), I found similar levels of genetic variability in vagrant polar bears that had reached Iceland compared to established subpopulations from across the range. Based on climate projections reported by the Intergovernmental Panel on Climate Change in 2014, polar bear habitat will markedly decline and become increasingly fragmented within the next decades. Dispersal will play an important role by connecting isolated subpopulations, thereby maintaining genetic diversity levels. My results indicate that vagrants could stabilize genetic variability when immigrating into established subpopulations.
In conclusion, my thesis provided a deeper understanding of evolutionary genetic processes and patterns and their timing in bears and red foxes in a climatic context, which can have conservation implications. Further, I showed that processes like ILS and interspecific gene flow can be relevant over different time scales and are important aspects of evolutionary history. Thereby, my thesis contributed to the knowledge on the evolutionary history of several carnivore species and on evolutionary processes acting within and between closely related species.
Background: The complex cellular networks within tumors, the cytokine milieu, and tumor immune escape mechanisms affecting infiltration and anti-tumor activity of immune cells are of great interest to understand tumor formation and to decipher novel access points for cancer therapy. However, cellular in vitro assays, which rely on monolayer cultures of mammalian cell lines, neglect the three-dimensional architecture of a tumor, thus limiting their validity for the in vivo situation.
Methods: Three-dimensional in vivo-like tumor spheroid were established from human cervical carcinoma cell lines as proof of concept to investigate infiltration and cytotoxicity of NK cells in a 96-well plate format, which is applicable for high-throughput screening. Tumor spheroids were monitored for NK cell infiltration and cytotoxicity by flow cytometry. Infiltrated NK cells, could be recovered by magnetic cell separation.
Results: The tumor spheroids were stable over several days with minor alterations in phenotypic appearance. The tumor spheroids expressed high levels of cellular ligands for the natural killer (NK) group 2D receptor (NKG2D), mediating spheroid destruction by primary human NK cells. Interestingly, destruction of a three-dimensional tumor spheroid took much longer when compared to the parental monolayer cultures. Moreover, destruction of tumor spheroids was accompanied by infiltration of a fraction of NK cells, which could be recovered at high purity.
Conclusion: Tumor spheroids represent a versatile in vivo-like model system to study cytotoxicity and infiltration of immune cells in high-throughput screening. This system might proof useful for the investigation of the modulatory potential of soluble factors and cells of the tumor microenvironment on immune cell activity as well as profiling of patient-/donor-derived immune cells to personalize cellular immunotherapy.
Background: Plant hormones are well known regulators which balance plant responses to abiotic and biotic stresses. We investigated the role of abscisic acid (ABA) in resistance of barley (Hordeum vulgare L.) against the plant pathogenic fungus Magnaporthe oryzae.
Results: Exogenous application of ABA prior to inoculation with M. oryzae led to more disease symptoms on barley leaves. This result contrasted the finding that ABA application enhances resistance of barley against the powdery mildew fungus. Microscopic analysis identified diminished penetration resistance as cause for enhanced susceptibility. Consistently, the barley mutant Az34, impaired in ABA biosynthesis, was less susceptible to infection by M. oryzae and displayed elevated penetration resistance as compared to the isogenic wild type cultivar Steptoe. Chemical complementation of Az34 mutant plants by exogenous application of ABA re-established disease severity to the wild type level. The role of ABA in susceptibility of barley against M. oryzae was corroborated by showing that ABA application led to increased disease severity in all barley cultivars under investigation except for the most susceptible cultivar Pallas. Interestingly, endogenous ABA concentrations did not significantly change after infection of barley with M. oryzae.
Conclusion: Our results revealed that elevated ABA levels led to a higher disease severity on barley leaves to M. oryzae. This supports earlier reports on the role of ABA in enhancing susceptibility of rice to the same pathogen and thereby demonstrates a host plant-independent function of this phytohormone in pathogenicity of monocotyledonous plants against M. oryzae.