Refine
Year of publication
Document Type
- Article (1133)
- Doctoral Thesis (831)
- Preprint (69)
- Book (59)
- Contribution to a Periodical (44)
- Conference Proceeding (10)
- Diploma Thesis (10)
- Review (8)
- diplomthesis (4)
- Report (3)
Has Fulltext
- yes (2172)
Is part of the Bibliography
- no (2172)
Keywords
- Podospora anserina (17)
- aging (17)
- mitochondria (12)
- autophagy (10)
- Archaea (9)
- Haloferax volcanii (9)
- Saccharomyces cerevisiae (9)
- Phylogeny (8)
- heat stress (8)
- Mitochondria (7)
Institute
- Biowissenschaften (2172) (remove)
Photorhabdus and Xenorhabdus bacteria live in a highly specific symbiosis with nematodes that belong to the genus of Heterorhabditis and Steinernema, respectively. These cruiser type nematodes actively search for soil-dwelling insects and infect them via natural openings. Inside of the insect, the bacteria are released into the hemocoel where they start producing an array of secondary metabolites to bypass the insect immune system and kill the prey within 48 hours. Many of those natural products possess bioactivities against other bacteria, fungi, protozoa or insects, which makes them interesting candidates for pharmaceutical applications. Even though advanced molecular biological methods in combination with bioinformatics tools can now be used to predict biosynthetic gene clusters (BGCs) and their products, there are still many BGCs with unknown products. Even for the plethora of natural products that were successfully identified in the last couple of years, the exact ecological function often remains elusive, as laboratory conditions can vary considerably from the natural environment of the bacteria. Knowledge about the natural conditions that stimulate, or repress production of certain natural products and their underlying regulatory mechanisms yield new approaches for natural product research and enables possibilities for selective manipulations of the regulatory cascades.
The overarching goal of this work was to examine the regulatory networks in Photorhabdus and Xenorhabdus strains. The first part of this work focused on the Hfq-dependent regulation of specialized metabolite production. In those genera, the RNA chaperone, Hfq, represses expression of hexA, which encodes for a global transcriptional regulator that acts as the master repressor for SM production. Multiple global approaches were used to identify the sRNA ArcZ, which targets a specific region in the 5’-untranslated region of the hexA mRNA and ultimately guides Hfq in order to repress its expression. It was shown that a deletion of arcZ led to a drastic reduction of SM production in Photorhabdus and Xenorhabdus, consistent with the phenotype of their respective hfq deletion mutants. Transcriptomic profiling revealed far-reaching effects on the transcriptome, with up to 735 coding sequences significantly affected in the arcZ deletion strain. Finally, it was shown that the resulting chemical background, devoid of SMs, in combination with targeted promotor exchange can be used to exclusively overproduce a desired natural product, representing an alternative route of genetic manipulation.
The second part of this work focused on the influence and identification of insect related compounds that affect SM production in P. laumondii, X. szentirmaii and X. nematophila. Insect homogenate was generated from G. mellonella larvae, a model host for these bacteria. Supplementation of the cultivation medium with homogenate induced considerable shifts in the SM profiles of those bacteria. A global effect on the transcriptional output was determined by transcriptomic profiling. The core response to the simulation of an insect environment consisted of ten CDS, eight of which are involved in the degradation of fatty acids or the import of maltose and maltodextrin into the cells. Two abundant components in the insect homogenate, trehalose and putrescin, were added to the cultivation medium of those strains and subsequent HPLC-MS analysis revealed a direct correlation of their concentration in the medium and the production titres of certain SMs. These results indicated that the bacteria sense the insect environment via different insect specific components in order to initiate a metabolic adjustment, which is probably required for adaptation to the insect host.
The last part of this work examined the influence of other, so far not directly related genes on SM production, based on the isolation of P. laumondii transposon-insertion mutants with clear phenotypic alterations. Re-sequencing and SM profiling of the mutant strains revealed that a transposon-insertion in the gene encoding for a putative DNA-adenine methyltransferase affected SM production. The phenotype was confirmed by deleting this gene. Based on Single-Molecule Real-Time sequencing, the complete methylome of the WT, deletion- and complementation mutant were analysed (experimental work performed by Sacha J. Pidot, Melbourne, Australia). No obvious alterations were detected in the methylation patterns of the strains, indicating that the dam gene product does not methylate the adenine in GATC-motifs, as it was described in literature for E. coli. This data raises the question what the function of the putative DNA-adenine methyltransferase is in P. laumondii and how it can influence the secondary metabolism. Even though there is currently no clear evidence, the potential role of epigenetic gene regulation mechanisms should be considered in further work.
Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete Saccharomyces cerevisiae and the basidiomycete Ustilago maydis. One extract that inhibited the growth of S. cerevisiae much stronger than that of U. maydis was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against S. cerevisiae but reduced activity against U. maydis were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether U. maydis could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in U. maydis.
Characterization of a dual BET/HDAC inhibitor for treatment of pancreatic ductal adenocarcinoma
(2020)
Pancreatic ductal adenocarcinoma (PDAC) is resistant to virtually all chemo‐ and targeted therapeutic approaches. Epigenetic regulators represent a novel class of drug targets. Among them, BET and HDAC proteins are central regulators of chromatin structure and transcription, and preclinical evidence suggests effectiveness of combined BET and HDAC inhibition in PDAC. Here, we describe that TW9, a newly generated adduct of the BET inhibitor (+)‐JQ1 and class I HDAC inhibitor CI994, is a potent dual inhibitor simultaneously targeting BET and HDAC proteins. TW9 has a similar affinity to BRD4 bromodomains as (+)‐JQ1 and shares a conserved binding mode, but is significantly more active in inhibiting HDAC1 compared to the parental HDAC inhibitor CI994. TW9 was more potent in inhibiting tumor cell proliferation compared to (+)‐JQ1, CI994 alone or combined treatment of both inhibitors. Sequential administration of gemcitabine and TW9 showed additional synergistic antitumor effects. Microarray analysis revealed that dysregulation of a FOSL1‐directed transcriptional program contributed to the antitumor effects of TW9. Our results demonstrate the potential of a dual chromatin‐targeting strategy in the treatment of PDAC and provide a rationale for further development of multitarget inhibitors.
The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD‐glucose and glucose‐6‐phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose‐6‐phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose‐6‐phosphate phosphatase activity accumulated trehalose‐6‐phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose‐6‐phosphate. Our results demonstrate that trehalose‐6‐phosphate affects multiple physiological activities in A. baumannii ATCC 19606.
Nonribosomal peptides produced by minimal and engineered synthetases with terminal reductase domains
(2020)
Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2‐electron reduction of the enzyme‐bound thioester releasing the generated peptides as C‐terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde‐generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.
Climate change forces many species to move their ranges to higher latitudes or elevations. Resulting immigration or emigration of species might lead to functional changes, e.g., in the trait distribution and composition of ecological assemblages. Here, we combined approaches from biogeography (species distribution models; SDMs) and community ecology (functional diversity) to investigate potential effects of climate-driven range changes on frugivorous bird assemblages along a 3000 m elevational gradient in the tropical Andes. We used SDMs to model current and projected future occurrence probabilities of frugivorous bird species from the lowlands to the tree line. SDM-derived probabilities of occurrence were combined with traits relevant for seed dispersal of fleshy-fruited plants to calculate functional dispersion (FDis; a measure of functional diversity) for current and future bird assemblages. Comparisons of FDis between current and projected future assemblages showed consistent results across four dispersal scenarios, five climate models and two representative concentration pathways. Projections indicated a decrease of FDis in the lowlands, an increase of FDis at lower mid-elevations and little changes at high elevations. This suggests that functional dispersion responds differently to global warming at different elevational levels, likely modifying avian seed dispersal functions and plant regeneration in forest ecosystems along tropical mountains.
The ongoing biodiversity crisis becomes evident in the widely observed decline in abundance and diversity of species, profound changes in community structure, and shifts in species’ phenology. Insects are among the most affected groups, with documented decreases in abundance up to 76% in the last 25–30 years in some terrestrial ecosystems. Identifying the underlying drivers is a major obstacle as most ecosystems are affected by multiple stressors simultaneously and in situ measurements of environmental variables are often missing. In our study, we investigated a headwater stream belonging to the most common stream type in Germany located in a nature reserve with no major anthropogenic impacts except climate change. We used the most comprehensive quantitative long‐term data set on aquatic insects available, which includes weekly measurements of species‐level insect abundance, daily water temperature and stream discharge as well as measurements of additional physicochemical variables for a 42‐year period (1969–2010). Overall, water temperature increased by 1.88 °C and discharge patterns changed significantly. These changes were accompanied by an 81.6% decline in insect abundance, but an increase in richness (+8.5%), Shannon diversity (+22.7%), evenness (+22.4%), and interannual turnover (+34%). Moreover, the community's trophic structure and phenology changed: the duration of emergence increased by 15.2 days, whereas the peak of emergence moved 13.4 days earlier. Additionally, we observed short‐term fluctuations (<5 years) in almost all metrics as well as complex and nonlinear responses of the community toward climate change that would have been missed by simply using snapshot data or shorter time series. Our results indicate that climate change has already altered biotic communities severely even in protected areas, where no other interacting stressors (pollution, habitat fragmentation, etc.) are present. This is a striking example of the scientific value of comprehensive long‐term data in capturing the complex responses of communities toward climate change.
DnaK3, a highly conserved cyanobacterial chaperone of the Hsp70 family, binds to cyanobacterial thylakoid membranes, and an involvement of DnaK3 in the biogenesis of thylakoid membranes has been suggested. As shown here, light triggers synthesis of DnaK3 in the cyanobacterium Synechocystis sp. PCC 6803, which links DnaK3 to the biogenesis of thylakoid membranes and to photosynthetic processes. In a DnaK3 depleted strain, the photosystem content is reduced and the photosystem II activity is impaired, whereas photosystem I is regular active. An impact of DnaK3 on the activity of other thylakoid membrane complexes involved in electron transfer is indicated. In conclusion, DnaK3 is a versatile chaperone required for biogenesis and/or maintenance of thylakoid membrane-localized protein complexes involved in electron transfer reactions. As mentioned above, Hsp70 proteins are involved in photoprotection and repair of PS II in chloroplasts.
Peronospora salviae‐officinalis, the causal agent of downy mildew on common sage, is an obligate biotrophic pathogen. It grows in the intercellular spaces of the leaf tissue of sage and forms intracellular haustoria to interface with host cells. Although P. salviae‐officinalis was described as a species of its own 10 years ago, the infection process remains obscure. To address this, a histological study of various infection events, from the adhesion of conidia on the leaf surface to de novo sporulation is presented here. As histological studies of oomycetes are challenging due to the lack of chitin in their cell wall, we also present an improved method for staining downy mildews for confocal laser scanning microscopy as well as evaluating the potential of autofluorescence of fixed nonstained samples. For staining, a 1:1 mixture of aniline blue and trypan blue was found most suitable and was used for staining of oomycete and plant structures, allowing discrimination between them as well as the visualization of plant immune responses. The method was also used to examine samples of Peronospora lamii on Lamium purpureum and Peronospora belbahrii on Ocimum basilicum, demonstrating the potential of the presented histological method for studying the infection processes of downy mildews in general.
Active species reintroduction is an important conservation tool when aiming for the restoration of biological communities and ecosystems. The effective monitoring of reintroduction success is a crucial factor in this process. Here, we used a combination of environmental DNA (eDNA) techniques and species distribution models (SDMs) to evaluate the success of recent reintroductions of the freshwater fish Alburnoides bipunctatus in central Germany. We built SDMs without and with eDNA presence data to locate further suitable reintroduction sites and potentially overlooked populations of the species. We successfully detected eDNA of A. bipunctatus at all reintroduction sites, as well as several adjacent sites mostly in downstream direction, which supports the success of reintroduction efforts. eDNA‐based species detection considerably improved SDMs for A. bipunctatus, which allowed to identify species presence in previously unknown localities. Our results confirm the usefulness of eDNA techniques as standard tool to monitor reintroduced fish populations. We propose that combining eDNA with SDMs is a highly effective approach for long‐term monitoring of reintroduction success in aquatic species.
Morphological malformations induced by tributyltin (TBT) exposure during embryonic development have already been characterized in various taxonomic groups, but, nonetheless, the molecular processes underlying these changes remain obscure. The present study provides the first genome-wide screening for differentially expressed genes that are linked to morphological alterations of gonadal tissue from chicken embryos after exposure to TBT. We applied a single injection of TBT (between 0.5 and 30 pg as Sn/g egg) into incubated fertile eggs to simulate maternal transfer of the endocrine disruptive compound. Methyltestosterone (MT) served as a positive control (30 pg/g egg). After 19 days of incubation, structural features of the gonads as well as genome-wide gene expression profiles were assessed simultaneously. TBT induced significant morphological and histological malformations of gonadal tissue from female embryos that show a virilization of the ovaries. This phenotypical virilization was mirrored by altered expression profiles of sex-dependent genes. Among these are several transcription and growth factors (e.g. FGF12, CTCF, NFIB), whose altered expression might serve as a set of markers for early identification of endocrine active chemicals that affect embryonic development by transcriptome profiling without the need of elaborate histological analyses.
Ryanodine receptor 1 (RyR1) mediates excitation–contraction coupling by releasing Ca2+ from sarcoplasmic reticulum (SR) to the cytoplasm of skeletal muscle cells. RyR1 activation is regulated by several proteins from both the cytoplasm and lumen of the SR. Here, we report the structure of RyR1 from native SR membranes in closed and open states. Compared to the previously reported structures of purified RyR1, our structure reveals helix‐like densities traversing the bilayer approximately 5 nm from the RyR1 transmembrane domain and sarcoplasmic extensions linking RyR1 to a putative calsequestrin network. We document the primary conformation of RyR1 in situ and its structural variations. The activation of RyR1 is associated with changes in membrane curvature and movement in the sarcoplasmic extensions. Our results provide structural insight into the mechanism of RyR1 in its native environment.
The present study assessed the diurnal variation in salivary cortisol in captive African elephants during routine management (baseline) and in relation to a potential stressor (translocation) to evaluate to what extent acute stress may affect diurnal cortisol patterns. Under baseline conditions, we collected morning and afternoon saliva samples of 10 animals (three zoos) on different days in two study periods (n = 3–10 per animal, daytime and period). Under stress conditions, we sampled the transported cow (newcomer) and the two cows of the destination zoo before and after the transport in the morning and afternoon (n = 3–9 per animal, daytime and transport phase), as well as after the first introduction of the newcomer to the bull (n = 1 per animal). Cortisol was measured in unextracted samples by enzyme immunoassay. Under baseline conditions, we observed the expected diurnal variation with higher cortisol levels in the morning than in the afternoon. Under stress conditions, neither a significant difference between pre‐ and posttransport, nor between morning and afternoon levels was found. The percentage difference between morning and afternoon cortisol after the transport, however, was remarkably lower than before the transport in the newcomer potentially indicating a stress response to familiarization. Saliva samples taken immediately after the introduction of the newcomer to the bull revealed a marked cortisol increase. Our findings indicate that stressors may disturb the diurnal cortisol rhythm. Furthermore, provided that samples can be collected promptly, salivary cortisol is a useful minimally invasive measure of physiological stress in the African elephant.
The opportunistic human pathogen Acinetobacter baumannii is one of the leading causes of nosocomial infections. The high prevalence of multidrug‐resistant strains, a high adaptability to changing environments and an overall pronounced stress resistance contribute to persistence and spread of the bacteria in hospitals and thereby promote repeated outbreaks. Altogether, the success of A. baumannii is mainly built on adaptation and stress resistance mechanisms, rather than relying on ‘true’ virulence factors. One of the stress factors that pathogens must cope with is osmolarity, which can differ between the external environment and different body parts of the human host. A. baumannii ATCC 19606T accumulates the compatible solutes glutamate, mannitol and trehalose in response to high salinities. In this work, it was found that most of the solutes vanish immediately after reaching stationary phase, a very unusual phenomenon. While glutamate can be metabolized, mannitol produced by MtlD is excreted to the medium in high amounts. First results indicate that A. baumannii ATCC 19606T undergoes a rapid switch to a dormant state (viable but non‐culturable) after disappearance of the compatible solutes. Resuscitation from this state could easily be achieved in PBS or fresh medium.
Aim: To provide distribution information and preliminary conservation assessments for all species of the pineapple family (Bromeliaceae), one of the most diverse and ecologically important plant groups of the American tropics—a global biodiversity hotspot. Furthermore, we aim to analyse patterns of diversity, endemism and the conservation status of the Bromeliaceae on the continental level in the light of their evolutionary history.
Location: The Americas.
Methods: We compiled a dataset of occurrence records for 3,272 bromeliad species (93.4% of the family) and modelled their geographic distribution using either climate‐based species distribution models, convex hulls or geographic buffers dependent on the number of occurrences available. We then combined this data with information on taxonomy and used the ConR software for a preliminary assessment of the conservation status of all species following Criterion B of the International Union for the Conservation of Nature (IUCN).
Results: Our results stress the Atlantic Forest in eastern Brazil, the Andean slopes, Central America and the Guiana Highlands as centres of bromeliad diversity and endemism. Phylogenetically ancient subfamilies of bromeliads are centred in the Guiana highlands whereas the large radiations of the group spread across different habitats and large geographic area. A total of 81% of the evaluated bromeliad species are Possibly Threatened with extinction. We provide range polygons for 3,272 species, as well as newly georeferenced point localities for 911 species in the novel “bromeliad” r package, together with functions to generate diversity maps for individual taxonomic or functional groups.
Main conclusions: Diversity centres of the Bromeliaceae agreed with macroecological patterns of other plant and animal groups, but show some particular patterns related to the evolutionary origin of the family, especially ancient dispersal corridors. A staggering 2/3rds of Bromeliaceae species might be threatened with extinction, especially so in tropical rain forests, raising concerns about the conservation of the family and bromeliad‐dependent animal species.
Summary
Wild relatives of crops thrive in habitats where environmental conditions can be restrictive for productivity and survival of cultivated species. The genetic basis of this variability, particularly for tolerance to high temperatures, is not well understood. We examined the capacity of wild and cultivated accessions to acclimate to rapid temperature elevations that cause heat stress (HS).
We investigated genotypic variation in thermotolerance of seedlings of wild and cultivated accessions. The contribution of polymorphisms associated with thermotolerance variation was examined regarding alterations in function of the identified gene.
We show that tomato germplasm underwent a progressive loss of acclimation to strong temperature elevations. Sensitivity is associated with intronic polymorphisms in the HS transcription factor HsfA2 which affect the splicing efficiency of its pre‐mRNA. Intron splicing in wild species results in increased synthesis of isoform HsfA2‐II, implicated in the early stress response, at the expense of HsfA2‐I which is involved in establishing short‐term acclimation and thermotolerance.
We propose that the selection for modern HsfA2 haplotypes reduced the ability of cultivated tomatoes to rapidly acclimate to temperature elevations, but enhanced their short‐term acclimation capacity. Hence, we provide evidence that alternative splicing has a central role in the definition of plant fitness plasticity to stressful conditions.
Aim: Predicting future changes in species richness in response to climate change is one of the key challenges in biogeography and conservation ecology. Stacked species distribution models (S‐SDMs) are a commonly used tool to predict current and future species richness. Macroecological models (MEMs), regression models with species richness as response variable, are a less computationally intensive alternative to S‐SDMs. Here, we aim to compare the results of two model types (S‐SDMS and MEMs), for the first time for more than 14,000 species across multiple taxa globally, and to trace the uncertainty in future predictions back to the input data and modelling approach used.
Location: Global land, excluding Antarctica.
Taxon: Amphibians, birds and mammals.
Methods: We fitted S‐SDMs and MEMs using a consistent set of bioclimatic variables and model algorithms and conducted species richness predictions under current and future conditions. For the latter, we used four general circulation models (GCMs) under two representative concentration pathways (RCP2.6 and RCP6.0). Predicted species richness was compared between S‐SDMs and MEMs and for current conditions also to extent‐of‐occurrence (EOO) species richness patterns. For future predictions, we quantified the variance in predicted species richness patterns explained by the choice of model type, model algorithm and GCM using hierarchical cluster analysis and variance partitioning.
Results: Under current conditions, species richness predictions from MEMs and S‐SDMs were strongly correlated with EOO‐based species richness. However, both model types over‐predicted areas with low and under‐predicted areas with high species richness. Outputs from MEMs and S‐SDMs were also highly correlated among each other under current and future conditions. The variance between future predictions was mostly explained by model type.
Main conclusions: Both model types were able to reproduce EOO‐based patterns in global terrestrial vertebrate richness, but produce less collinear predictions of future species richness. Model type by far contributes to most of the variation in the different future species richness predictions, indicating that the two model types should not be used interchangeably. Nevertheless, both model types have their justification, as MEMs can also include species with a restricted range, whereas S‐SDMs are useful for looking at potential species‐specific responses.
In 2010, the Conference of the Parties of the Convention on Biological Diversity agreedon the Strategic Plan for Biodiversity 2011–2020 in Aichi Prefecture, Japan. As this planapproaches its end, we discussed whether marine biodiversity and prediction studieswere nearing the Aichi Targets during the 4th World Conference on Marine Biodiversityheld in Montreal, Canada in June 2018. This article summarises the outcome of a five-day group discussion on how global marine biodiversity studies should be focusedfurther to better understand the patterns of biodiversity. We discussed and reviewedseven fundamental biodiversity priorities related to nine Aichi Targets focusing onglobal biodiversity discovery and predictions to improve and enhance biodiversitydata standards (quantity and quality), tools and techniques, spatial and temporal scaleframing, and stewardship and dissemination. We discuss how identifying biodiversityknowledge gaps and promoting efforts have and will reduce such gaps, including via theuse of new databases, tools and technology, and how these resources could be improvedin the future. The group recognised significant progress toward Target 19 in relationto scientific knowledge, but negligible progress with regard to Targets 6 to 13 whichaimed to safeguard and reduce human impacts on biodiversity.
Signal transduction and the regulation of gene expression are fundamental processes in every cell. RNA-binding proteins (RBPs) play a key role in the post-transcriptional modulation of gene expression in response to both internal and external stimuli. However, how signaling pathways regulate the assembly of RBPs with mRNAs remains largely unknown. Here, we summarize observations showing that the formation and composition of messenger ribonucleoprotein particles (mRNPs) is dynamically remodeled in space and time by specific signaling cascades and the resulting post-translational modifications. The integration of signaling events with gene expression is key to the rapid adaptation of cells to environmental changes and stress. Only a combined approach analyzing the signal transduction pathways and the changes in post-transcriptional gene expression they cause will unravel the mechanisms coordinating these important cellular processes.
The strictly anaerobic acetogenic bacterium Acetobacterium woodii is metabolically diverse and grows on variety of substrates which includes H2 + CO2, sugars, alcohols and diols. It is unique in producing bacterial microcompartments (BMC) during growth on different substrates such as 1,2-propanediol, 2,3-butanediol, ethanol or fructose. In this study, we analyzed the genetic organization and expression of the BMC genes within the A. woodii genome, the previously described 18 gene pdu cluster as well as four other cluster potentially encoding one or two shell proteins. Expression analysis of respective gene clusters revealed that the pdu gene cluster is highly expressed during growth on 1,2-PD, 2,3-BD, ethanol and ethylene glycol. The promoter region upstream of the pduA gene was identified and used to establish a reporter gene assay based on chloramphenicol acetyl transferase as a reporter protein. The reporter gene assay confirmed the qPCR data and demonstrated that 1,2-PD is superior over ethanol and ethylene glycol as inducer. BMCs were enriched from cells grown on 2,3- BD and 1,2-PD and shown to have typical structure in electron micrographs. Biochemical analyses revealed several of the protein encoded by the pdu cluster to be part of the isolated BMCs. These data demonstrate a very unique situation in A. woodii in which apparently one BMC gene cluster in expressed during growth on different substrates.
Die CXCR4/CXCL12-Achse ist von entscheidender Bedeutung für die Entstehung und Aufrechterhaltung einer gesunden, reifen Hämatopoese. Erstmals beschrieben wurde der später als CXCR4 bezeichnete Rezeptor 1996 allerdings als Co-Rezeptor für den Eintritt humaner HI-Viren in Lymphozyten. Ein großes Interesse bestand daraufhin darin, sowohl natürliche Inhibitoren des G-Protein gekoppelten Rezeptors zu identifizieren, als auch synthetische herzustellen, um einen Eintritt des Virus in den menschlichen Organismus zu verhindern bzw. seine Ausbreitung zu unterbinden. Ein natürlich vorkommender CXCR4-Ligand, der 2015 von Zirafi und Kollegen erstmals beschrieben wurde, fand sich im Hämofiltrat von Dialysepatienten. Der im weiteren Verlauf als EPI-X4 bezeichnete CXCR4-Antagonist wurde als Spaltprodukt von Albumin identifiziert, welches über viele Spezies hochkonserviert ist. Diese Eigenschaft interpretieren wir als Hinweis auf eine relevante physiologische Funktion des Peptids. Da die Halbwertszeit von natürlich vorkommendem EPI-X4 beim Menschen vermutlich sehr kurz ist, sind in vivo- und darauffolgende in vitro-Analysen schwierig durchzuführen. In-vitro-Spike-Analysen von synthetischem EPI-X4 in humanem Plasma ergaben eine Halbwertszeit von nur 17 Minuten. Die geringen auftretenden Konzentrationen erschweren die Problematik zusätzlich. In dieser Arbeit sollen deshalb im Mausmodell in vivo-Analysen durchgeführt werden, um die Effekte von potentiell entstehendem EPI-X4 in verschiedenen experimentellen Ansätzen aufzudecken. Ein probates, hier verwendetes Mittel, ist die Analyse einer Knock-out (KO)-Maus. Die für die Bindung an CXCR4 entscheidende Aminosäure von EPI-X4, das am N-Terminus gelegene Leucin, wurde durch Alanin ersetzt, welches die Entstehung von EPI-X4 unterbindet und zusätzlich dessen Bindung an CXCR4 verhindert. Mit Hilfe zweier Mausmodelle können nun Analysen im EPI-X4-defizienten Modell durchgeführt werden, die im Umkehrschluss Informationen über die organismische Wirkung von EPI-X4 beinhalten. Zunächst wurde in beiden Modellen die physiologisch normale reife und unreife Hämatopoese charakterisiert. Hierbei zeigte sich kein signifikanter systematischer Einfluss von EPI-X4 auf reife Leukozyten (WBC), lediglich eine leichte Lymphozytose in der HR-Ala-Variante. Im weiteren Verlauf der homöostatischen Analyse der Hämatopoese der Ala-EPI-X4-Mäuse zeigten sich keine signifikanten Unterschiede zu wildtypischen Mäusen. Sowohl reife als auch unreife Zellen zeigten, außer in der T- und B-Zelllinie, keine zahlenmäßigen oder funktionalen Auffälligkeiten, weder im Blut, noch in der Milz oder im Knochenmark. Analysen der Zellzyklusaktivität unterschiedlicher Unreifestufen wiesen ebenfalls keine Auffälligkeiten auf. Diese Daten einer normalen, von einer C57Bl/6-Maus zu erwartenden Ergebnisse dienten als Grundlage zur Bewertung und Analyse von durchgeführten hämatopoetischen Stressmodellen. Hierfür wurden
zunächst hämatopoetische Stamm- und Vorläuferzellen (HSPC) mobilisiert. In den angewandten Mobilisierungsmodellen fanden sich lediglich unter G-CSF-Behandlung im Knochenmark eine größere Anzahl Granulozyten, was auf einen Einfluss von EPI-X4 auf HSPC schließen lässt. Um potentielle Auswirkungen von EPI-X4 im Knochenmark weiter zu untersuchen, wurde ein weiteres Stressmodell gewählt, welches ebenfalls mutmaßlich die Bedingungen zur EPI-X4-Generierung schafft: Subletale Bestrahlung der Mäuse sorgt für Schäden an allen Zellarten im Knochenmark, es wird ein steriles entzündliches Milieu kreiert. Unter diesen Umständen wurde die Regeneration von Blutzellen analysiert. Es zeigten sich keine nennenswerten Unterschiede sowohl in der akuten Phase des Schadens als auch in regelmäßigen Blutentnahmen während der Regenerierung.
Die Beschreibung von natürlich vorkommendem EPI-X4 in Vaginal- und Rektalschleimhaut zeigt seine Entstehung an Schleimhautbarrieren auf. Ala-EPI-X4-Muse werden deshalb auf deren Durchlässigkeit untersucht: LPS-Konzentrationen als Marker für eindringende pathogene Bakterien wurden im Plasma untersucht. Hierbei zeigten sich keine Unterschiede zwischen den Gruppen, eine Störung scheint hier nicht vorzuliegen. Zusätzlich wurde die Zusammensetzung des Mikrobioms im Darm untersucht, da beschrieben wurde, dass sich Mikrobiom und die Integrität der Darmschleimhaut gegenseitig beeinflussen. Im Falle der EPI-X4-defizienten Mäuse liegt zwar keine offensichtliche pathologische Veränderung vor, dennoch konnte in männlichen HR-Ala-Mäusen die Abwesenheit des Proteobakteriums Parasutterella nachgewiesen werden. Um eine mögliche Defizienz der Barrierefunktion weiter zu testen, wurden zwei Stressmodelle gewählt: Zunächst wurde den Mäusen eine akute, sterile Peritonitis zugefügt, woraufhin die Anzahl und Zusammensetzung der ins Peritoneum einströmenden Leukozyten analysiert wird. Die Reaktion auf diesen Entzündungsprozess war nicht verändert. Ähnliche Ergebnisse zeigten sich auch in einem akuten Colitis-Stressmodell.
Insgesamt konnte in dieser Arbeit mithilfe zweier KO-Mausmodelle die Rolle von EPI-X4 in der Hämatopoese und der Immunologie von Mäusen beginnend charakterisiert werden. Die homöostatische Hämatopoese scheint kaum von EPI-X4 abhängig zu sein, lediglich die Zahl der B- und T-Zellen, insbesondere der regulatorischen T-Zellen, scheint beeinflusst. Damit einhergehend konnten Veränderungen in Zytokinlevels bei inflammatorischen Ereignissen gezeigt werden. Experimente zur beeinflussten, eventuell gestörten Barrierefunktion von Ala-EPI-X4-Mäusen zeigten vielversprechende Ansätze und sollten in Zukunft weiter analysiert werden.
Background: More than 170 species of tabanids are known in Europe, with many occurring only in limited areas or having become very rare in the last decades. They continue to spread various diseases in animals and are responsible for livestock losses in developing countries. The current monitoring and recording of horseflies is mainly conducted throughout central Europe, with varying degrees of frequency depending on the country. To the detriment of tabanid research, little cooperation exists between western European and Eurasian countries.
Methods: For these reasons, we have compiled available sources in order to generate as complete a dataset as possible of six horsefly species common in Europe. We chose Haematopota pluvialis, Chrysops relictus, C. caecutiens, Tabanus bromius, T. bovinus and T. sudeticus as ubiquitous and abundant species within Europe. The aim of this study is to estimate the distribution, land cover usage and niches of these species. We used a surface-range envelope (SRE) model in accordance with our hypothesis of an underestimated distribution based on Eurocentric monitoring regimes.
Results: Our results show that all six species have a wide range in Eurasia, have a broad climatic niche and can therefore be considered as widespread generalists. Areas with modelled habitat suitability cover the observed distribution and go far beyond these. This supports our assumption that the current state of tabanid monitoring and the recorded distribution significantly underestimates the actual distribution. Our results show that the species can withstand extreme weather and climatic conditions and can be found in areas with only a few frost-free months per year. Additionally, our results reveal that species prefer certain land-cover environments and avoid other land-cover types.
Conclusions: The SRE model is an effective tool to calculate the distribution of species that are well monitored in some areas but poorly in others. Our results support the hypothesis that the available distribution data underestimate the actual distribution of the surveyed species.
Spinocerebellar ataxia type 2 (SCA2) is caused by polyglutamine expansion in Ataxin-2 (ATXN2). This factor binds RNA/proteins to modify metabolism after stress, and to control calcium (Ca2+) homeostasis after stimuli. Cerebellar ataxias and corticospinal motor neuron degeneration are determined by gain/loss in ATXN2 function, so we aimed to identify key molecules in this atrophic process, as potential disease progression markers. Our Atxn2-CAG100-Knock-In mouse faithfully models features observed in patients at pre-onset, early and terminal stages. Here, its cerebellar global RNA profiling revealed downregulation of signaling cascades to precede motor deficits. Validation work at mRNA/protein level defined alterations that were independent of constant physiological ATXN2 functions, but specific for RNA/aggregation toxicity, and progressive across the short lifespan. The earliest changes were detected at three months among Ca2+ channels/transporters (Itpr1, Ryr3, Atp2a2, Atp2a3, Trpc3), IP3 metabolism (Plcg1, Inpp5a, Itpka), and Ca2+-Calmodulin dependent kinases (Camk2a, Camk4). CaMKIV–Sam68 control over alternative splicing of Nrxn1, an adhesion component of glutamatergic synapses between granule and Purkinje neurons, was found to be affected. Systematic screening of pre/post-synapse components, with dendrite morphology assessment, suggested early impairment of CamKIIα abundance together with the weakening of parallel fiber connectivity. These data reveal molecular changes due to ATXN2 pathology, primarily impacting excitability and communication.
Das Gehirn weist in mehreren Bereichen anatomische Asymmetrien zwischen beiden Hemisphären auf, so auch in Bereichen der Hörrinde. Zudem ist bereits langjährig bekannt, dass menschliche Sprache vorrangig in der linken Gehirnhälfte, d.h. linksseitig lateralisiert, verarbeitet wird. Daraus folgend stellt sich die Frage, ob dies eine besondere Spezialisierung ist, oder ob es noch weitere lateralisierte Hirnfunktionen gibt. Viele akustische Signale haben dabei frequenzmodulierte (FM) Komponenten, die im Hörsystem für die Erkennung nach Parametern wie Richtung und Dauer der Modulation analysiert werden müssen. Ob die Analyse von FM-Komponenten oder einzelner Reizparameter im Gehirn lateralisiert stattfindet, wurde in der Literatur meist mit bildgebenden Verfahren untersucht.
Für das Erkennen und Unterscheiden der Modulationsrichtung weist eine Vielzahl von Studien auf eine erhöhte Aktivität in der rechten Hörrinde hin. Für die Analyse von Stimulusdauern ist es bisher allerdings noch unklar bzw. umstritten, ob diese lateralisiert erfolgt. Für die Untersuchung der Lateralisierung einfacher Sprachkomponenten werden häufig Konsonant-Vokal-Silben (CV-Silben) verwendet. In einer Vielzahl von Studien konnte eine linkslastige Lateralisierung, wie bei der Spracherkennung, gezeigt werden.
In der vorliegenden Arbeit wurde nun untersucht, ob ein eindeutigeres Muster von Lateralisierung zu finden ist, wenn diese in Wahrnehmungsexperimenten, untersucht wird. Dabei wurde ein zu untersuchender Teststimulus (FM-/CV-Stimulus) auf einem Ohr mit einem kontralateralen breitbandigen Rauschen auf dem anderen Ohr gleichzeitig präsentiert. Durch die Struktur der Hörbahn kann dabei davon ausgegangen werden, dass in einer Hemisphäre des Vorderhirns vorrangig Informationen aus dem kontralateralen Ohr verarbeitet und Informationen aus dem ipsilateralen Ohr unterdrückt werden und sich somit Rückschlüsse auf die Funktion/Beteiligung einer Hemishpäre ziehen lassen. Das Rauschen diente dabei zur unspezifischen Aktivierung der gegenüberliegenden Hemisphäre.
Die Lateralisierung wurde systematisch für unterschiedlich komplexe Reize untersucht. Dazu wurden in zwei Versuchsreihen Unterscheidungsexperimente durchgeführt, die sich in mehrere Messungen (mit mehreren Durchläufen) mit unterschiedlichen Parametereinstellungen gliederten. Pro Durchlauf musste sich die Versuchsperson immer zwischen zwei Antwortmöglichkeiten entscheiden (2-AFC-Verfahren). Der Schalldruckpegel des Rauschens war dabei für alle Messungen konstant. Der Schalldruckpegel der Teststimuli blieb zwar während einer Messung konstant, wurde jedoch innerhalb eines Experimentes von Messung zu Messung reduziert.
In einer gemeinsamen Analyse wurden jeweils die Fehlerraten und Reaktionszeiten beider Ohren, getrennt nach Seite und FM-/ CV-Stimulus, miteinander verglichen, um so auf eine mögliche Lateralisierung schließen zu können. Damit die Daten der Versuchspersonen bei vergleichbarer Schwierigkeit analysiert werden konnten, wurde als Vergleichswert zwischen allen Versuchspersonen der Schalldruckpegel der ersten Messung mit einer Fehlerrate von mindestens 15,0 % gewählt (15 %-Kriterium). Um auszuschließen, dass das Hörvermögen der Versuchspersonen Unterschiede zwischen beiden Ohren aufweist, wurde vor jeder Messung der „Punkt subjektiver Gleichheit“ für die Lautstärke-wahrnehmung zwischen linkem und rechten Ohr bestimmt.
In der ersten Versuchsreihe wurde dabei die Verarbeitung der Modulationsrichtung und der Stimulusdauer von FM-Stimuli untersucht. Es zeigte sich für beide Experimente, dass ein sinkender Schalldruckpegel des FM-Stimulus zu einer steigenden Fehlerrate führte. Unter Anwendung des 15 %-Kriteriums waren die Fehlerraten für die Unterscheidung der Modulationsrichtung signifikant geringer, wenn der FM-Stimulus auf dem linken Ohr präsentiert wurde. Dies ist ein deutlicher Hinweis für eine rechtslastige Lateralisierung.
Für die Unterscheidung der Stimulusdauer gab es dagegen keinen signifikanten Unterschied zwischen den Fehlerraten beider Ohren. Somit muss davon ausgegangen werden, dass beide Hemisphären für diese Aufgabe benötigt werden und eine bilaterale Verarbeitung stattfindet. In den Reaktionszeiten konnten in beiden Experimente keine signifikanten Unterschiede gezeigt werden. Die Unterscheidung der Modulationsrichtung wurde dabei von allen Versuchspersonen als einfacher eingestuft als die Unterscheidung der Stimulusdauer, was sich auch in niedrigeren Antwortschnelligkeit und Fehlerraten bei vergleichbaren Schalldruckpegeln zeigte.
In der zweiten Versuchsreihe wurde als Referenzmessung nochmals die Unterscheidung der Modulationsrichtungen von FM-Stimuli durchgeführt. Anschließend wurde die Unterscheidung von „da“ und „ga“ untersucht. Diese CV-Silben differieren ausschließlich in der FM-Komponente. Die Untercheidung von CV-Silben ohne Unterschied in der FM-Komponente wurde mittels „ta“ und „ka“ getestet. Für alle drei Experimente zeigte sich, dass ein geringerer Schalldruckpegel des FM- oder CV-Stimulus zu einer steigenden Fehlerrate führte. Unter Anwendung des 15 %-Kriteriums zeigte sich für die Unterscheidung der Modulationsrichtung ein Trend zu niedrigeren Fehlerraten bei der Präsentation des FM-Stimulus auf dem linken im Vergleich mit dem rechten Ohr. In den Reaktionszeiten konnten keine signifikanten Unterschiede gezeigt werden.
Für die Unterscheidung von „da“ und „ga“ ließ sich unter Anwendung des 15 %-Kriteriums in den Fehlerraten und Reaktionszeiten kein Vorteil eines Ohres nachweisen. Dagegen zeigten sich klare Unterschiede bei einzelnen Versuchspersonen. So waren die Fehlerraten für Versuchspersonen, die vorwiegend „da“ erkannt bzw. gehört hatten signifikant höher, wenn der CV-Stimulus auf dem rechten Ohr präsentiert wurde, für „ga“-Hörer war das Gegenteil der Fall. In den Reaktionszeiten konnte kein signifikanter Zusammenhang nachgewiesen werden. Somit ließ sich zeigen, dass je nach Strategie der Versuchsperson bzw. deren individueller Wahrnehmung der CV-Silben, Unterschiede in der Lateralisierung erreicht werden können.
Für die Unterscheidung von „ta“ und „ka“ zeigten sich unter Anwendung des 15 %-Kriteriums signifikant niedrigere Fehlerraten und Reaktionszeiten, wenn der CV-Stimulus auf dem linken Ohr präsentiert wurde. Dies weist deutlich auf eine rechtslastige Lateralisierung hin. Vergleicht man alle drei Experimente ließ sich zudem zeigen, dass die Unterscheidung der Modulationsrichtung einfacher war als die Unterscheidung verschiedener CV-Stimuli. Dabei war die Unterscheidung von „da“ und „ga“ für die Versuchspersonen schwieriger als die Unterscheidung von „ta“ und „ka“. Allerdings konnte in den Lateralisierungsdaten kein direkter Zusammenhang zwischen den FM- und „da“-/„ga“-Stimuli gezeigt werden.
Zusammenfassend konnte in allen fünf Experimenten eine verschieden stark lateralisierte Verarbeitung von akustischen Stimuli bei gleichzeitigem kontralateralen Rauschen gezeigt werden. Der Vorteil eines Ohres (bzw. einer Hemisphäre) war sowohl von der Aufgabe als auch vom Stimulustyp abhängig. Dabei gab es zum Teil starke Unterschiede in der Effektstärke und dem Grad der Lateralisierung zwischen den einzelnen Versuchspersonen. Insgesamt konnte gezeigt werden, dass sich die hier angewendete psychophysische Methode gut eignet, um Ergebnisse zur Lateralisierung von akustischen Stimuli zu gewinnen und somit die Verhaltensrelevanz von Ergebnissen aus Studien mit bildgebenden Verfahren zu überprüfen.
The application of natural products (NPs) as drugs and lead compounds has greatly improved human health over the past few decades. Despite their success, we still need to find new NPs that can be used as drugs to combat increasing drug resistance via new modes of action and to develop safer treatments with less side effects.
Entomopathogenic bacteria of Xenorhabdus and Photorhabdus that live in mutualistic symbiosis with nematodes are considered as promising producers of NPs, since more than 6.5% of their genomes are assigned to biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites. The investigation on NPs from Xenorhabdus and Photorhabdus can not only provide new compounds for drug discovery but also help to understand the biochemical basis involved in mutualistic and pathogenic symbiosis of bacteria, nematode host and insect prey.
Nonribosomal peptides (NRPs) are a large class of NPs that are mainly found in bacteria and fungi. They are biosynthesized by nonribosomal peptide synthetases (NRPSs) and display diverse functions, representing more than 20 clinically used drugs. Although a large number of NRPs have been identified in Xenorhabdus and Photorhabdus, the advanced genome sequencing and bioinformatic analysis indicate that these bacteria still have many unknown NRPS-encoding gene clusters for NRP production that are worth to explore. Therefore, this thesis focuses on the discovery, biosynthesis, structure identification, and biological functions of new NRPs from Xenorhabdus and Photorhabdus.
The first publication describes the isolation and structure elucidation of seven new rhabdopeptide/xenortide-like peptides (RXPs) from X. innexi, incorporating putrescine or ammonia as the C-terminal amines. Bioactivity testing of these RXPs revealed potent antiprotozoal activity against the causative agents of sleeping sickness (Trypanosoma brucei rhodesiense) and malaria (Plasmodium falciparum), making them the most active RXP derivatives known to date. Biosynthetically, the initial NRPS module InxA might act iteratively with a flexible methyltransferase activity to catalyze the incorporation of the first five or six N-methylvaline/valine to these peptides.
The second publication focuses on the structure elucidation of seven unusual methionine-containing RXPs that were found as minor products in E. coli carrying the BGC kj12ABC from Xenorhabdus KJ12.1. To confirm the proposed structures from detailed HPLC-MS analysis, a solid-phase peptide synthesis (SPPS) method was developed for the synthesis of these partially methylated RXPs. These RXPs also exhibited good effects against T. brucei rhodesiense and P. falciparum, suggesting RXPs might play a role in protecting insect cadaver from soil-living protozoa to support the symbiosis with nematodes.
The third publication presents the identification of a new peptide library, named photohexapeptide library, which occurred after the biosynthetic gene phpS was activated in P. asymbiotica PB68.1 via promoter exchange. The chemical diversity of the photohexapeptides results from unusual promiscuous specificity of five out of six adenylation (A) domains being an excellent example of how to create compound libraries in nature. Furthermore, photohexapeptides enrich the family of the rare linear D-/L-peptide NPs.
The fourth publication concentrates on the structure elucidation of a new cyclohexapeptide, termed photoditritide, which was produced by P. temperata Meg1 after the biosynthetic gene pdtS was activated via promoter exchange. Photoditritide so far is the only example of a peptide from entomopathogenic bacteria that contains the uncommon amino acid homoarginine. The potent antimicrobial activity of photoditritide against Micrococcus luteus implies that photoditritide can protect the insect cadaver from food competitor bacteria in the complex life cycle of nematode and bacteria.
The last publication reports a new family of cyclic lipopeptides (CLPs), named phototemtides, which were obtained after the BGC pttABC from P. temperata Meg1 was heterologously expressed in E. coli. The gene pttA encodes an MbtH protein that was required for the biosynthesis of phototemtides in E. coli. To determine the absolute configurations of the hydroxy fatty acids, a total synthesis of the major compound phototemtide A was performed. Although the antimalarial activity of phototemtide A is only weak, it might be a starting point towards a selective P. falciparum compound, as it shows no activity against any other tested organisms.
In the fish embryo toxicity (FET) test with zebrafish (Danio rerio) embryos, 3,4-dichloroaniline (3,4-DCA) is often employed as a positive control substance. Previous studies have characterized bioconcentration and transformation of 3,4-DCA in this test under flow-through conditions. However, the dynamic changes of chemical concentrations in exposure media and embryos were not studied systematically under the commonly used semi-static exposure conditions in multiwell plates. To overcome these limitations, we conducted semi-static exposures experiments where embryolarval zebrafish were exposed to 0.5, 2.0, and 4.0 mg L−1 of 3,4-DCA for up to 120 hpf, with 24-h renewal intervals. During each renewal interval, concentrations of 3,4-DCA were quantified in water samples at 0, 6, 18, and 24 h using high-performance liquid chromatography with diode array detection. Levels of 3,4-DCA in larvae were measured after 120 h exposure. Concentrations of 3,4-DCA in the test vessels decreased rapidly during exposure. Taking these dynamics into account, bioconcentration factors in the present study ranged from 12.9 to 29.8 L kg−1, depending on exposure concentration. In summary, this study contributed to our knowledge of chemical dynamics in the FET test with embryolarval zebrafish, which will aid in defining suitable exposure conditions for future studies.
Our knowledge of early evolution of snakes is improving, but all that we can infer about the evolution of modern clades of snakes such as boas (Booidea) is still based on isolated bones. Here, we resolve the phylogenetic relationships of Eoconstrictor fischeri comb. nov. and other booids from the early-middle Eocene of Messel (Germany), the best-known fossil snake assemblage yet discovered. Our combined analyses demonstrate an affinity of Eoconstrictor with Neotropical boas, thus entailing a South America-to-Europe dispersal event. Other booid species from Messel are related to different New World clades, reinforcing the cosmopolitan nature of the Messel booid fauna. Our analyses indicate that Eoconstrictor was a terrestrial, medium- to large-bodied snake that bore labial pit organs in the upper jaw, the earliest evidence that the visual system in snakes incorporated the infrared spectrum. Evaluation of the known palaeobiology of Eoconstrictor provides no evidence that pit organs played a role in the predator–prey relations of this stem boid. At the same time, the morphological diversity of Messel booids reflects the occupation of several terrestrial macrohabitats, and even in the earliest booid community the relation between pit organs and body size is similar to that seen in booids today.
Fifty years ago, Zajonc, Heingartner, and Herman (1969) conducted a famous experiment on social enhancement and inhibition of performance in cockroaches. A moderating effect of task difficulty on the effect of the presence of an audience, as revealed by impaired performance in complex tasks and enhanced performance in simple tasks, was presented as the major conclusion of this research. However, the researchers did not test this interaction statistically. We conducted a preregistered direct replication using a 2 (audience: present vs. absent) × 2 (task difficulty: runway vs. maze) between-subjects design. Results revealed main effects for task difficulty, with faster running times in the runway than the maze, and for audience, with slower running times when the audience was present than when it was absent. There was no interaction between the presence of an audience and task difficulty. Although we replicated the social-inhibition effect, there was no evidence for a social-facilitation effect.
Das Ziel dieser Dissertation war es, die biologische Relevanz der F1Fo-ATP-Synthase für den Alterungsprozess des Ascomyceten P. anserina aufzuklären sowie die Funktion der Dimerisierungsuntereinheiten PaATPE und PaATPG genauer zu untersuchen. Folgende Ergebnisse wurden dabei erzielt:
1. Der Verlust einer Dimerisierungsuntereinheit führt in P. anserina zum Verlust der Dimerisierungsfähigkeit der F1Fo-ATP-Synthase. Dieses Ereignis resultiert in einer vorzeitigen Anhäufung von Seneszenzmerkmalen, einer starken Verkürzung der Lebensspanne und einem beschleunigten Alterungsprozess. Der Phänotyp der Stämme ∆PaAtpe und ∆PaAtpg lässt sich erfolgreich revertieren. Dadurch konnte bestätigt werden, dass der beobachtete Phänotyp der Deletionsstämme auf den Verlust der Dimerisierungsgene zurückzuführen ist.
2. Die konstitutive Überexpression des PaAtpe-Gens führt zu einer Verlängerung der Lebensspanne, die allerdings nicht abhängig von einer Erhöhung der Dimer-Menge, sondern auf eine Verlängerung der Mitochondriennetzwerke und eine erhöhte Atmung zurückzuführen ist.
3. Obwohl die Lebensspannen der untersuchten PaAtpg_OEx-Stämme voneinander abweichen, ist der auf den Organismus ausgeübte Effekt der PaAtpg-Überexpression positiv. Dabei lässt sich eine Erhöhung der Dimer-Menge auch in diesen Stämmen nicht nachweisen und eine Veränderung der Mitochondrienmorphologie tritt nur in einem der fünf untersuchten Stämme auf, was hier allerdings mit dem größten Effekt auf die Lebensspanne korreliert.
4. Die gleichzeitige Überexpression der Gene PaAtpe und PaAtpg führt interessanterweise zu einer Aufhebung der durch die PaAtpe-Überexpression hervorgerufenen positiven Effekte. Die generierte Doppelmutante weist somit wildtypische Eigenschaften auf. Eine Erhöhung der Dimer-Menge kann trotz Überexpression beider Gene nicht festgestellt werden.
5. Trotz gleicher Funktion in der Dimerbildung der F1Fo-ATP-Synthase ist das Expressionsniveau der Dimerisierungsgene in P. anserina unterschiedlich. Bereits im Wildtyp wird das PaAtpe-Gen weniger transkribiert als das PaAtpg-Gen. Letzteres wird durch die PaAtpe-Überexpression sowohl in den PaAtpe_OEx-Mutanten als auch in der Doppelmutante herunterreguliert. Auch im Wildtyp kommt es zu einer altersabhängigen Herunterregulierung des PaAtpg-Gens.
6. Im Wildtyp nimmt die Dimer-Menge im Alter ab und es kommt zu einer altersbedingten Änderung der Superkomplexe von S1 zu S0. Dies sind Hinweise auf eine altersabhängige Remodellierung der Cristae-Membran, die möglicherweise zum Seneszenzphänotyp von P. anserina beiträgt. Somit ist die Aufrechterhaltung des Dimers von großer Bedeutung für die Gewährleistung mitochondrialer Funktionen des Organismus.
Die F1Fo-ATP-Synthase spielt in ihrer dimeren Form eine bedeutende Rolle in der Aufrechterhaltung der Mitochondrienfunktion und gewährleistet den Ablauf von lebenswichtigen mitochondrialen Prozessen, die für die Erhaltung der zellulären Homöostase von essentieller Bedeutung sind.
The neocortical microcircuit, a local network of excitatory and inhibitory neurons, is a highly complex information processing unit, which can flexibly be modulated to adapt to external context and internal state such as motivation or attention. The mechanisms underlying these adaptations for flexible processing are not sufficiently understood yet. The aim of this study is to further elucidate the role of inhibitory and excitatory components of the local neocortical microcircuit for the processing of sensory information in an awake, behaving animal.
Layer 1 of the neocortex is of particular importance because it contains afferents from the thalamus and more distant cortical regions, which relay top-down information that is important for processes such as learning and attention. The dendrites of the excitatory pyramidal neurons located in deeper layers extend into layer 1, and in addition to that layer 1 contains inhibitory neurons, as well as axons from inhibitory somatostatin expressing (SOM) neurons located in lower layers. These layer 1 inhibitory neurons and SOM axons are therefore well positioned to control top-down information transfer at the pyramidal dendrites, and thus to flexibly regulate information processing in the local circuit. To further investigate this, the stimulus responses in inhibitory (SOM axons) and excitatory (layer 2/3 pyramidal neurons) components of the neocortical microcircuit were measured in primary auditory cortex during learning, when auditory stimuli gain relevance.
For this purpose, I first established a suitable learning behaviour, an auditory GO-NOGO discrimination task, which can be performed by head-fixed mice under the microscope. The task also contains a visual start cue, which signals the start of every trial, as a multimodal element. Mice learn to distinguish two auditory stimuli by being rewarded with water after the GO stimulus and receiving no reward after the NOGO stimulus. They indicate that they have identified the stimuli accordingly by licking at a water dispenser during the GO stimulus and not during the NOGO stimulus. Licking during the NOGO stimulus is punished by an aversive air puff. As the mice learn this behaviour, the stimuli gain relevance. The activity in the same neuronal structures was observed over the course of all training sessions via 2-photon imaging in awake, behaving mice, and their stimulus responses were measured throughout the learning process, acquiring a comprehensive dataset. In these data, short-term and long-term plasticity of the stimulus responses can be detected and these changes in the stimulus responses differ for SOM axons and pyramidal neurons. Already from the first training day, stimulus responses change in the course of a single session, both in SOM axons and in pyramidal cells. With time over the course of task acquisition, the stimulus representation in a group of pyramidal neurons in layer 2/3 is enhanced and distal dendrites are less inhibited over training through reduced activation of the SOM axons, so that the integration of information along the somatodendritic axis shifts, increasing the relative impact of top-down information. This shift is even stronger for the NOGO stimulus in correct trials compared to the GO stimulus. This is the first study to show that this somato-dendritic shift by SOM-axon responses occurs at different strengths for the GO and NOGO stimulus, probably due to the different learned responses (action or refraining), which require different forms of circuit control. After learning, the neuronal responses to GO and NOGO stimuli also differ in pyramidal neurons, with the GO stimulus evoking stronger responses than the NOGO stimulus. This learned distinction is reversed in passive trials during which the mice have no possibility to respond to the stimuli, in both SOM axons and pyramidal neurons, resulting in similar response sizes for both stimuli. This indicates that not only learning over the long term, but also short-term changes regarding the state (active execution of the discrimination task or no active participation during the stimulus presentations) affect the processing of the stimuli in the local circuit. In addition, on an even shorter time scale pyramidal neurons show a modulation of responses from trial to trial, probably due to anticipation of reward, which is absent from SOM axon responses. Thus, there are various levels of plasticity that develop over the course of training: long-term changes in the response size of both the excitatory and inhibitory components that facilitate stimulus recognition when engaged, and short-term modulation (possibly in anticipation of reward) in excitatory neurons that could underlie sensorimotor transformation. Both pyramidal neurons and SOM axons in the primary auditory cortex respond to multimodal and reinforcement-related stimuli, likely contributing to the optimisation of circuit dynamics for goal-directed information processing. This shows that the circuit flexibly adjusts information processing under different circumstances, depending on the relevance the stimuli carry and whether the mouse is active or inactive and can use the presented information to achieve a goal.
Hypoxia is a condition in which cells are deprived of adequate oxygen supply and represents a main feature of solid tumours. Cells under hypoxic stress activate transcriptional responses driven by hypoxia-inducible factors (HIFs), which affect multiple cellular pathways, including angiogenesis, metabolic adaptation and cell proliferation. While the transcriptional changes induced in hypoxic tumours are well characterised, it is still poorly understood how hypoxia contributes to the aberrant post-transcriptional regulation observed in tumours. In this PhD thesis, I studied the RNA response to hypoxia in cancer, to provide novel insights into its regulation.
Using deep RNA-Sequencing (RNA-Seq), I investigated transcriptome changes of three human cell lines from lung, cervical and breast cancer under hypoxia, advancing our knowledge of post-transcriptional gene regulation in hypoxic cancer. I show that hypoxia induced consistent changes in transcript abundance in the three cancer types. This was coupled to divergent splicing responses, highlighting the cell type specificity of alternative splicing programs. While the mRNA levels of RNA-binding proteins were mainly reduced, hypoxia upregulated muscleblind-like protein 2 (MBNL2) in all three cell lines. Hypoxia control was specific for MBNL2, since it did not affect its paralogs MBNL1 and MBNL3. Via knockdown experiments of MBNL2 in hypoxic cells, I could show that MBNL2 induction promotes adaptation of cancer cells to low oxygen by regulating both transcript abundance and alternative splicing of hypoxia response genes. In addition, depletion of MBNL2 reduced the proliferation and migration of cancer cells, corroborating a function of MBNL2 as cancer driver.
In the last few years, a novel class of RNAs has gained attention, namely circular RNAs (circRNAs), which are produced by a particular splicing mechanism, known as back-splicing. CircRNAs have been reported to change their abundance in cancer and their high stability makes them promising candidates as diagnostic biomarkers. In this study, I took advantage of deep rRNA-depleted RNA-Seq data to comprehensively investigate the expression of circRNAs in human cancer cells and their changes in response to hypoxia. To reliably identify circRNAs, I established a pipeline that integrates two available tools. for circRNA detection with custom approaches for quantification and statistical analysis. Using this pipeline, I identified 12006 circRNAs in the three cancer cell lines. Their molecular features suggest an involvement of complementary RNA sequences as well as trans-acting factors in circRNA biogenesis, including the splicing factor HNRNPC. Remarkably, I detected 210 circRNAs that are more abundant than their linear counterparts. Upon hypoxic stress, 64 circRNAs were differentially expressed in cancer cells, in most cases in a cell type-specific manner. In summary, in this PhD thesis, I present a comparative transcriptome profiling in human cancer cell lines. It reveals MBNL2 as an important player in hypoxic cancer progression and provides novel insights into the biogenesis and regulation of circRNAs under hypoxic stress.
Cardiovascular diseases are still regarded as the main cause of death in the modern world. However, the generic term "cardiovascular diseases" is not uniformly defined. It essentially describes diseases of the cardiovascular system and includes diseases such as hypertension, arteriosclerosis, myocardial infarctions, heart failure, coronary heart diseases, rheumatic heart diseases and heart valve defects. In addition to the well-known risk factors such as obesity, smoking, hypercholesterolemia and lack of exercise, age is a further risk factor that plays an important role in the development of cardiovascular diseases. As the modern societies age; this becomes an increasing problem.
But why does the prevalence of cardiovascular diseases increase with age? In gen-eral, age-dependent changes at the cellular level are assumed to be responsible for the pathological changes in the cardiac and vascular tissues. Important mechanisms such as autophagy, oxidative stress, mitochondrial dysfunctions, genomic instability, cellular senescence and disturbances in signaling pathways of growth factors play a decisive role. In old age, myocardial hypertrophy occurs, which results in cardiac wall thickening and an altered geometry of the ventricle. Chronic inflammations, paracrine and age-dependent cell-intrinsic factors further lead to activation of cardiac fibro-blasts with increase cell proliferation, collagen secretion and matrix cross-linking. The consequences are interstitial and perivascular fibrosis, which stiffen the heart and blood vessels. Oxidative stress and inflammations additionally attack the blood ves-sels and impair endothelial function, which is further aggravated by possible pre-existing conditions such as diabetes mellitus and hypertension.
In the past decades, the main focus has therefore been on researching these age-dependent changes in the hope of better understanding cardiovascular ageing and developing possible regenerative interventions. By studying the repair mechanisms of other organs such as the lungs and the bone marrow, the endothelium in particular showed a high regenerative capacity, which influences the proliferation and cell func-tion of the surrounding cells.
For a long time, the general opinion was that the endothelium is only the internal lin-ing of blood and lymphatic vessels, as well as the heart chambers, which as a single-layer barrier guarantees the integrity of the blood vessels. However, endothelial cells are very heterogeneous, depending on the type of blood vessel and the type of tis-sue they serve. In addition to their barrier function, endothelial cells also regulate the exchange of substances between blood and tissue, stimulate the formation of new blood vessels and re-model existing vascular networks. They are also able to re-structure the extracellular matrix that surrounds them. They release not only matrix proteins, but also cytokines and growth factors into the extracellular space. On de-mand, these factors are then released and stimulate angiogenesis or cell prolifera-tion. In addition, the secretion of various matrix proteins not only stabilizes the cellu-lar neighborhood, but also regulates various cell functions.
By modelling the endothelial environment - the so-called vascular niche - endothelial cells are able to communicate with the surrounding cells. As a result, a regenerative effect of the vascular niche has already been described in various organs. In the liv-er, for example, it has been shown that increased concentrations of endothelial Ang2 and decreased endothelial activin A after partial hepatectomy stimulate the prolifera-tion of hepatocytes and thus liver regeneration. In the bone marrow, endothelial cells mobilize stem cells via nitric oxide and in the lungs, endothelial MMP14 releases growth factors from the extracellular matrix, which stimulate epithelial cell prolifera-tion after partial pneumectomy. Whether such a regenerative effect of the vascular niche also plays a role in the heart is largely unknown.
Since both the regenerative capacity of the heart and endothelial function decrease with age, the aim of this dissertation was to investigate the role of the vascular niche and endothelial cell communication in the aged heart. Human cell lines as well as mouse and artificial rat models were used for these investigations. Since this thesis is a cumulative dissertation with partially published papers, it is divided into three parts.
In the first part of this thesis, the transcriptional signature of secretory genes in the aged cardiac endothelium was studied. Perfused endothelial cells from hearts of young (12-week-old animals) and old mice (20-month-old animals) were isolated and used for bulk RNA sequencing. The two matrix proteins laminin β1 and β2 were among the top-regulated genes. While laminin β2 was particularly expressed in the young cardiac endothelium, laminin β1 was predominantly found in the old endotheli-um. This change in laminin expression was confirmed histologically at protein level and its autocrine function was investigated in vitro. To mimic the in vivo situation in vitro, cell culture dishes were coated with human recombinant laminin 421 or laminin 411 and sutured with human endothelial cells from the umbilical vein (HUVEC). Di-verse functional investigations showed that endothelial cells migrated and adhered poorly in the presence of laminin 411, while in Matrigel tube formation assays HU-VEC formed reduced endothelial networks when cultured on LM 411.
...
Die klimatische Nische beschreibt die klimatischen Bedingungen, unter denen eine Art eine stabile Population aufrechterhalten kann. Die Quantifizierung von Klimanischen ist ein wichtiges Werkzeug, um tiefergehende Einsichten in individuelle Art-Umwelt Beziehungen zu erlangen, um den Effekt des Klimawandels effektiv zu bewerten, und um Arten- und Naturschutz zu unterstützen. Ein makroökologischer Ansatz ist von Vorteil um Ökosysteme über ein breites taxonomisches, geographisches und zeitliches Spektrum zu untersuchen, und damit die klimatischen Nischen vieler Arten auf eine konsistente Art und Weise zu quantifizieren und vergleichen.
Im Kontext des aktuellen Klimawandels ist es wichtig zu verstehen, ob Arten in der Lage sind ihre Klima-nische anzupassen. Viele bisherige Vorhersagen über klimawandelbedingte Veränderungen von Artverbreitungen beruhen auf der Annahme, dass die klimatische Nische einer Art konstant ist. Allerdings ist bekannt, dass Arten ihre klimatischen Präferenzen auf unterschiedlichen Zeitskalen verändern - sowohl über kurze (ökologische) als auch evolutionäre Zeiträume. Dies ist ein wichtiger, aber oft missachteter Faktor für die Nischenquantifizierung. Ein gutes Beispiel für solche ökologische Dynamiken sind Zugvögel, die etwa 20% aller Vogelarten ausmachen. Sie stellen eine interessante, aber auch herausfordernde Artengruppe für die Untersuchung klimatischer Nischen dar. Des Weiteren ist es wichtig klimatische Nischen über evolutionäre Zeiträume zu untersuchen, um die Prozesse zu verstehen, die Evolution, Diversifikation und Extinktion unterliegen, da sich Klimanischen mit der Anpassung einzelner Arten an neue klimatische Gegebenheiten ebenfalls wandeln. Bislang hat ein Mangel an geographisch expliziten Daten über terrestrische Umwelt-bedingungen durch evolutionäre Zeiträume eine explizite Überprüfung dieser Zusammenhänge verhindert.
Das übergeordnete Ziel dieser Dissertation war es, die ökologische (d.h. saisonale) und evolutionäre Dynamik klimatischer Nischen von Vögeln zu untersuchen. Dazu wurde ein Ansatz gewählt der makroökologische, und evolutionsbiologische Methoden vereint, um ein breites taxonomisches und zeitliches Spektrum abzudecken. Das erste Kapitel bearbeitet die Frage wie klimatische Nischen am besten zu quantifizieren sind, wenn man die Dynamik des Vogelzuges in Betracht zieht. Dazu wurde eine Datenbank erstellt, die das Zugverhalten aller 10.443 lebenden Vogelarten katalogisiert. Des Weiteren wurde eine Übersicht über die Methoden zur Quantifizierung klimatischer Nischen in der makroökologischen Literatur erstellt. Das Ergebnis derselben ist, dass die überwiegende Mehrzahl der Veröffentlichungen saisonalen Zugbewegungen nicht ausreichend berücksichtigt. Zuletzt habe ich anhand der Avifauna Australiens die Vor- und Nachteile der Verwendung von Verbreitungskarten gegenüber Punktverbreitungsdaten zur Erfassung saisonaler geographischer Muster der Artenvielfalt bewertet. Damit bietet dieses Kapitel Rahmenempfehlungen für die Datenanforderungen und Methoden, die je nach Zugverhalten einer Art, und dem geographischen, bzw. zeitlichen Fokus einer Studie für eine optimale Nischenquantifizierung notwendig sind.
Im zweiten Kapitel untersuchte ich die saisonale Dynamik klimatischer Nischen von Zugvögeln. Dabei überprüfte ich die Hypothese, dass Zugvögel in ihrem Jahreszyklus durch die Zugbewegung eine gewisse Klimanische verfolgen. Zu diesem Zweck habe ich mit Brut- und Überwinterungsarealkarten saisonale Klima-nischen für 437 Zug- und Standvogelarten aus acht Kladen der Sperlingsvögel (Passeriformes) charakterisiert. Mit Ordinationsmethoden wurde dann der innerartliche saisonale Nischenüberlapp quantifiziert. Der Beweis für die Verfolgung einer klimatischen Nische in einer Art war von mehreren Faktoren, z.B. der geographischen Verortung des Brutareals und der Zugrichtung, abhängig. Dies lässt darauf schließen, dass sich die Ursachen für den Vogelzug sowohl geographisch als auch saisonal (d.h. abhängig von der Zugrichtung) unterscheiden.
Im dritten Kapitel untersuchte ich die evolutionäre Dynamik klimatischer Nischen in Steinschmätzern (Gattung Oenanthe), um explizit zu untersuchen ob es einen Zusammenhang zwischen den Raten klimatischer Nischen-evolution und den Veränderungen paläoklimatischer Bedingungen gibt. Methoden der Klimanischen-quantifizierung wurden mit datierten molekularen Phylogenien verknüpft, um die Raten klimatischer Nischen-evolution mit einem variablen Ratenmodell abzuschätzen. Paläoklimatische Umweltbedingungen wurden mit paläobiologischen Methoden aus dem Fossilbericht altweltlicher Säugetiere der vergangenen 20 Millionen Jahre erschlossen. Die Fallstudie konnte keinen Zusammenhang zwischen Nischenevolution und Umwelt-bedingungen feststellen. Dies legt nahe, dass Vögel als überaus mobile Organismen, auf Klimaveränderungen eher durch Arealverschiebungen reagieren, als durch eine Anpassung ihrer klimatischen Nische. Die Klimanischen der Steinschmätzer waren allerdings an sich nicht statisch, so dass andere Faktoren wie z.B. biologische Wechselbeziehungen für die Nischenevolution dieser Gattung verantwortlich sein müssen.
Meine Dissertation beleuchtet die zentrale Bedeutung zeitlicher Dynamiken für den Nischenraum, den Arten über ökologische (d.h. saisonale) und evolutionäre Zeiträume einnehmen. Aus ihr ergeben sich methodische Konsequenzen für zukünftige Studien klimatischer Nischen. Der Befund, dass die klimatischen Nischen von Zugvögeln nicht saisonal konstant sind, zeigt dass es für mobile Kladen wie Vögel notwendig ist die klimatischen Bedingungen über den gesamten Jahreszyklus und das gesamte Verbreitungsgebiet in Betracht zu nehmen, um die jeweiligen klimatischen Nischen voll charakterisieren zu können.
Über diese methodischen Innovationen hinaus, hat meine Arbeit auch wichtige theoretische und praktische Schlussfolgerungen produziert. Zum einen zeigt die Betrachtung saisonaler Klimanischen, dass Zugvögel entgegen gängiger Annahmen nicht denselben Umweltbedingungen in ihren Brut- und Überwinterungsarealen ausgesetzt sind. Zum anderen zeigt meine Betrachtung von Klimanischen über evolutionäre Zeiträume, dass die Nischenevolution nicht von klimatischen Bedingungen angetrieben wird. Zusammengenommen zeigen diese Ergebnisse auf unterschiedlichen Zeitskalen, dass das Klima nicht der alleinige Faktor ist, der die Artverbreitung von Vögeln bestimmt. Während dieser Befund Raum für Optimismus schafft, was die Auswirkungen des aktuellen Klimawandels auf Vögel angeht, zeigt er auch auf, dass Faktoren wie wechselseitige Artbeziehungen und das Mobilitätspotential von Arten einen wichtigen Einfluss auf Artverbreitungen ausüben. Diese Faktoren könnten jedoch an sich vom Klimawandel beeinflusst sein, und Untersuchungen dieses Zusammenspiels zwischen Klima und anderen Faktoren und die daraus resultierenden Einflüsse auf Artareale bieten ein vielversprechendes Arbeitsfeld für zukünftige Studien.
Soil degradation can have an impact on the soil microbiota, but its specific effects on soil fungal communities are poorly understood. In this work, we studied the impact of soil degradation on the richness and diversity of communities of soil fungi, including three different degrees of degradation in Germany and Panama. Soil fungi were isolated monthly using the soil-sprinkling method for 8 months in Germany and 3 months in Panama, and characterized by morphological and molecular data. Soil physico-chemical properties were measured and correlated with the observed values of fungal diversity. We isolated a total of 71 fungal species, 47 from Germany, and 32 from Panama. Soil properties were not associated with fungal richness, diversity, or composition in soils, with the exception of soil compaction in Germany. The geographic location was a strong determinant of the soil fungal species composition although in both countries there was dominance by members of the orders Eurotiales and Hypocreales. In conclusion, the results of this work do not show any evident influence of soil degradation on communities of soil fungi in Germany or Panama.
Light is one of the most important abiotic factors for plant physiological processes. In addition to light intensity, the spectral quality of light can also influence the plant morphology and the content of secondary metabolites. In the horticultural industry, artificial light is used in to enable year-round production of herbs, ornamental plants and vegetables in winter terms.
Until today, discharge lamps like high-pressure sodium (HPS) lamps, emitting predominantly orange and red light and high amounts of infrared radiation, are the most common lamp systems in greenhouses. In the last decades, light-emitting diodes (LEDs) emerged as an efficient alternative light source. LEDs have the advantage of distinct adjustments to the light spectrum. For a usage in horticultural industry LEDs are often too expensive. Furthermore, reduced plant growth can occur due to incorrectly adjusted light spectra and lower leaf temperatures caused by the lack of infrared radiation.
In a research project (LOEWE, funding no. 487/15-29) funded by the Hessen State Ministry of Higher Education, Research and Arts, Microwave plasma lamps (MPL) were tested as new light sources for horticultural industry and plant research. The electrodeless lamp systems emit light in similar properties like sun light. The aim of the study was to determine the influence of artificial sunlight of the MPL on the accumulation of secondary metabolites, plant architecture and plant physiology of three different species (coleus, basil and potted roses). The MPL was compared with other light systems such as commercial HPS lamps, LEDs or ceramic metal halide lamps (CDM). In addition to morphological parameters such as plant height, internode length or fresh and dry weight, the phenolic content of leaves grown under the respective light sources were examined.
Overall an increased far-red light content in the emission spectra of the MPL showed high influence on the plant architecture which was observed in all three plant species. Artificial sunlight from MPL induced stem elongation in coleus and basil plants, compared to the other tested light sources. In potted roses a reduced branching degree was observed under MPL light compared to HPS grown plants.
In addition to the impact of far-red light also the blue light content of the emission spectra was found to be a strong influencing factor for plant physiological processes. A positive correlation between blue light content and leaf thickness was determined in coleus cultivated under MPL, LED, HPS and CDM lamps. Low blue light content in HPS emission spectra resulted in shade-adapted leaves with low photosynthetic capacity and susceptibility to high irradiances. Blue light was assumed to increase phenolic metabolites in basil and rose leaves. Furthermore, the different light treatments resulted in an alteration of the composition of essential oils of basil.
Experiments with coleus plants demonstrated that besides light color also the infrared radiation, had an influence on secondary metabolites by causing different leaf temperatures. Coleus plants grown with MPL showed the lowest content of phenolic compounds such as rosmarinic acid per dry weight. Infrared radiation resulted in a faster plant development indicated by increased biomass production and higher leaf formation rate as observed in coleus and basil plants.
The results obtained in this study show that the influence of leaf temperature should always be considered when comparing different lamp systems. Especially when LEDs are compared to discharge lamps an overestimation of light color can be a consequence since also infrared radiation influences the content of phenolic compounds and plant growth.
The Culex pipiens complex encompasses five species and subspecies of the genus Culex. Over time, a multitude of morphologically indistinguishable species has been assigned to this complex with several species being classified as important vectors for different diseases. Some species of this complex hibernate in subterranean habitats, and it has been proven that viruses can survive this phase of hibernation. However, studies focusing on the environmental requirements, ecology and spatial and temporal distribution patterns of mosquitos in underground habitats are sparse. Here, we investigate the main environmental factors and dependencies of Culex, considering the number of individuals and survival probabilities in underground habitats during the winter months. Methods. Since the State of Hesse, Germany harbors about 3500 to 4000 subterranean shelters ample availability of subterranean habitats there provides a good opportunity to conduct detailed investigations of the Culex pipiens complex. In this study, we identified a sample of 727 specimens of overwintering females within the Culex pipiens complex from 52 different underground sites collected over a period of 23 years using qPCR. A complete data set of samplings of hibernating mosquitos from 698 subterranean habitats in Central Germany over the same period was available to study the spatial and temporal patterns and the effect of temperature and precipitation conditions on these hibernating populations using a generalized linear model (GLM). Results. Our qPCR-results show, similar to aboveground studies of mosquitos, that Culex pipiens pipiens and Culex torrentium occur sympatrically. On the other hand, Culex pipiens molestus occurred very rarely. The GLM revealed no shifts in species composition over time, but different preferences for subterranean hibernacula, chemical effects on overwintering populations as well as effects of annual and seasonal mean temperature and precipitation during the active phase from March to November. Cx. p. pipiens and Cx. torrentium are the most common species within Hessian caves and other underground habitats during winter. They co-occur with different frequency without any patterns in species composition. Weather conditions influence the number of overwintering mosquitos during the activity phase. Depending on cave parameters, the number of mosquitos decreases during the winter months.
Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification
Iron is part of many redox and other enzymes and, thus, it is essential for all living beings. Many oxic environments have extremely low concentrations of free iron. Therefore, many prokaryotic species evolved siderophores, i.e., small organic molecules that complex Fe3+ with very high affinity. Siderophores of bacteria are intensely studied, in contrast to those of archaea. The haloarchaeon Haloferax volcanii contains a gene cluster that putatively encodes siderophore biosynthesis genes, including four iron uptake chelate (iuc) genes. Underscoring this hypothesis, Northern blot analyses revealed that a hexacistronic transcript is generated that is highly induced under iron starvation. A quadruple iuc deletion mutant was generated, which had a growth defect solely at very low concentrations of Fe3+, not Fe2+. Two experimental approaches showed that the wild type produced and exported an Fe3+-specific siderophore under low iron concentrations, in contrast to the iuc deletion mutant. Bioinformatic analyses revealed that haloarchaea obtained the gene cluster by lateral transfer from bacteria and enabled the prediction of enzymatic functions of all six gene products. Notably, a biosynthetic pathway is proposed that starts with aspartic acid, uses several group donors and citrate, and leads to the hydroxamate siderophore Schizokinen.
Diatoms are thought to provide about 40% of total global photosynthesis and diatoms of the genus Coscinodiscus are an important, sometimes dominant, cosmopolitan component of the marine diatom community. The oomycete parasitoid Lagenisma coscinodisci is widespread in the northern hemisphere on its hosts in the genus Coscinodiscus. Because of its potential ecological importance, it would be a suitable pathogen model to investigate plankton/parasite interactions, but the species cannot be cultivated on media without its host, so far. Thus, it was the aim of this study to explore the potential of dual culture of host and pathogen in the laboratory and to optimise cultivation to ensure a long-term cultivation of the pathogen. Here, we report successful cultivation of a single spore strain of L. coscinodisci (Isla), on several Coscinodiscus species and strains, as well as the establishment of a cultivation routine with Coscinodiscus granii (CGS1 and CG36), which enabled us to maintain the single spore strain for more than 3 years in 6 cm Petri dishes and 10 ml tissue culture flasks. This opens up the opportunity to study the processes and mechanism in plankton/parasitoid interactions under controlled conditions.
The oomycete genus Ectrogella currently comprises a rather heterogeneous group of obligate endoparasitoids, mostly of diatoms and algae. Despite their widespread occurrence, little is known regarding the phylogenetic affinities of these bizarre organisms. Traditionally, the genus was included within the Saprolegniales, based on zoospore diplanetism and a saprolegnia/achlya-like zoospore discharge. The genus has undergone multiple re-definitions in the past, and has often been used largely indiscriminately for oomycetes forming sausage-like thalli in diatoms. While the phylogenetic affinity of the polyphyletic genus Olpidiopsis has recently been partially resolved, taxonomic placement of the genus Ectrogella remained unresolved, as no sequence data were available for species of this genus. In this study, we report the phylogenetic placement of Ectrogella bacillariacearum infecting the freshwater diatom Nitzschia sigmoidea. The phylogenetic reconstruction shows that Ectrogella bacillariacearum is grouped among the early diverging lineages of the Saprolegniomycetes with high support, and is unrelated to the monophyletic diatom-infecting olpidiopsis-like species. As these species are neither related to Ectrogella, nor to the early diverging lineages of Olpidiopsis s. str. and Miracula, they are placed in a new genus, Diatomophthora, in the present study.
Holocarpic oomycetes are poorly known but widespread parasites in freshwater and marine ecosystems. Most of the holocarpic species seem to belong to clades that diverge before the two crown lineages of the oomycetes, the Saprolegniomycetes and the Peronosporomycetes. Recently, the genus Miracula was described to accommodate Miracula helgolandica, a holocarpic parasitoid of Pseudo-nitzschia diatoms, which received varying support for its placement as the earliest-diverging oomycete lineage. In the same phylogenetic reconstruction, Miracula helgolandica was grouped with some somewhat divergent sequences derived from environmental sequencing, indicating that Miracula would not remain monotypic. Here, a second species of Miracula is reported, which was found as a parasitoid in the limnic centric diatom Pleurosira leavis. Its life-cycle stages are described and depicted in this study and its phylogenetic placement in the genus Miracula revealed. As a consequence, the newly discovered species is introduced as Miracula moenusica.
Olpidiopsis is a genus of obligate holocarpic endobiotic oomycetes. Most of the species classified in the genus are known only from their morphology and life cycle, and a few have been examined for their ultrastructure or molecular phylogeny. However, the taxonomic placement of all sequenced species is provisional, as no sequence data are available for the type species, O. saprolegniae, to consolidate the taxonomy of species currently placed in the genus. Thus, efforts were undertaken to isolate O. saprolegniae from its type host, Saprolegnia parasitica and to infer its phylogenetic placement based on 18S rDNA sequences. As most species of Olpidiopsis for which sequence data are available are from rhodophyte hosts, we have also isolated the type species of the rhodophyte-parasitic genus Pontisma, P. lagenidioides and obtained partial 18S rDNA sequences. Phylogenetic reconstructions in the current study revealed that O. saprolegniae from Saprolegnia parasitica forms a monophyletic group with a morphologically similar isolate from S. ferax, and a morphologically and phylogenetically more divergent species from S. terrestris. However, they were widely separated from a monophyletic, yet unsupported clade containing P. lagenidioides and red algal parasites previously classified in Olpidiopsis. Consequently, all holocarpic parasites in red algae should be considered to be members of the genus Pontisma as previously suggested by some researchers. In addition, a new species of Olpidiopsis, O. parthenogenetica is introduced to accommodate the pathogen of S. terrestris.
The early-diverging oomycetes contain a large number of holocarpic obligate parasites of diatoms, algae, aquatic phycomycetes, and invertebrate animals. These organisms are diverse and widespread. However, taxonomic placement most of the early-diverging oomycetes remains provisional and unresolved, since many have not been sequenced and studied for molecular phylogeny. Here, we report the taxonomy and phylogeny of several holocarpic oomycetes that we have rediscovered and newly classified, including several new species combinations. Phylogenetic reconstructions revealed that the type species of genus Ectrogella (E. bacillariacearum) is a member of the early-diverging Saprolegniales, while the type species of Olpidiopsis (O. saprolegniae) and Pontisma (P. lagenidioides) grouped within the early-diverging lineage of oomycetes forming distinct clades. Since the monophyletic red-algae parasitoids are unrelated to the Olpidiopsis, these were reclassified to the genus Pontisma, while genus Diatomophthora was introduced to accommodate all the diatom parasitoids that were previously assigned to Olpidiopsis. In addition, four new oomycete parasitoids, Miracula helgolandica, Miracula moenusica, Diatomophthora drebesii and Olpidiopsis parthenogenetica and a single rediscovered species, Diatomophthora gillii, are also classified here, including eight new species combinations of red-algae parasites (Pontisma bostrychiae, P. heterosiphoniae, P. muelleri, P. palmariae, P. porphyrae, P. pyropiae) and diatom parasitoids (Diatomophthora drebesii, D. gillii). The results obtained in this study have further improved the resolution and expanded the knowledge on the phylogeny of the earlydiverging oomycetes, leading to the establishment of three new orders (Miraculales, Diatomophthorales, Pontismatales) and one order (Anisolpidiales) being reintroduced.
Zoos attract millions of visitors every year, many of whom are schoolchildren. For this reason, zoos are important institutions for the environmental education of future generations. Empirical studies on the educational impact of environmental education programs in zoos are still rare. To address this issue, we conducted two studies: In study 1, we investigated students’ interests in different biological topics, including zoos (n = 1,587). Data analysis of individual topics revealed large differences of interest, with advanced students showing less interest in zoos. In study 2, we invited school classes of this age group to visit different guided tours at the zoo and tested connection to nature before and after each educational intervention (n = 608). The results showed that the guided tours are an effective tool to raise students’ connection to nature. Add-on components have the potential to further promote connection to nature. The education programs are most effective with students with a low initial nature connection.
Brain aging is one of the major risk factors for the development of several neurodegenerative diseases. Therefore, mitochondrial dysfunction plays an important role in processes of both, brain aging and neurodegeneration. Aged mice including NMRI mice are established model organisms to study physiological and molecular mechanisms of brain aging. However, longitudinal data evaluated in one cohort are rare but are important to understand the aging process of the brain throughout life, especially since pathological changes early in life might pave the way to neurodegeneration in advanced age. To assess the longitudinal course of brain aging, we used a cohort of female NMRI mice and measured brain mitochondrial function, cognitive performance, and molecular markers every 6 months until mice reached the age of 24 months. Furthermore, we measured citrate synthase activity and respiration of isolated brain mitochondria. Mice at the age of three months served as young controls. At six months of age, mitochondria-related genes (complex IV, creb-1, β-AMPK, and Tfam) were significantly elevated. Brain ATP levels were significantly reduced at an age of 18 months while mitochondria respiration was already reduced in middle-aged mice which is in accordance with the monitored impairments in cognitive tests. mRNA expression of genes involved in mitochondrial biogenesis (cAMP response element-binding protein 1 (creb-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), nuclear respiratory factor-1 (Nrf-1), mitochondrial transcription factor A (Tfam), growth-associated protein 43 (GAP43), and synaptophysin 1 (SYP1)) and the antioxidative defense system (catalase (Cat) and superoxide dismutase 2 (SOD2)) was measured and showed significantly decreased expression patterns in the brain starting at an age of 18 months. BDNF expression reached, a maximum after 6 months. On the basis of longitudinal data, our results demonstrate a close connection between the age-related decline of cognitive performance, energy metabolism, and mitochondrial biogenesis during the physiological brain aging process.
High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.
Alternative splicing (AS) is a co- or post-transcriptional process by which one gene gives rise to multiple isoforms. This ‘split and combine’ step multiplies eukaryotic proteome diversity several fold and is implicated in several diseases given its pervasive impact. Control of alternative splicing is brought about by cis-regulatory elements, such as RNA sequence and structure, which recruit trans-acting RNA-binding proteins (RBPs). Although several of these interactions are already described in detail, we lack a comprehensive understanding of the regulatory code that underlies a splicing decision.
Here, we have established a high-throughput screen to comprehensively identify and characterise cis-regulatory elements that control a specific splicing decision. A cancer-relevant splicing event in proto-oncogene RON was picked as a minigene prototype for initialising the screening approach. Then, we transfected a library of thousands of randomly mutagenised minigene variants as a pool into human cells, and subsequently quantified the spliced isoforms by RNA sequencing. Importantly, we used a barcode sequence to tag the minigene variants and thereby linked mutations to their corresponding spliced products. By using a linear regression-based modelling approach, we were able to determine the effects of single mutations on RON AS. In total, more than 700 mutations were found to significantly affect the splicing regulation of the RON alternative exon. In addition, mutation effects quantified from the screening approach correlate with RON alternative splicing in cancer patients. We discovered numerous previously unknown cis-regulatory elements in both introns and exons, and found that the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH) extensively regulates RON AS at multiple levels in both cell lines and cancer. Furthermore, the large number of RBPs involved in the process, point to a complex splicing regulatory network involved in the control of RON splicing. iCLIP and synergy analysis between mutations and HNRNPH knockdown data pinpointed the most relevant HNRNPH binding sites across RON. Finally, cooperative HNRNPH binding was shown to mediate a splicing switch of RON alternative exon. In summary, our results provide an unprecedented view on the complexity of splicing regulation of an alternative exon. The novel screening approach introduces a tool to study the relationship of RNA sequence variants along with trans-acting regulators to their impact on the splicing outcome, offering insights on alternative splicing regulation and the relevance of mutations in human disease.
Die Rheumatoide Arthritis (RA) ist die häufigste chronisch-entzündliche Gelenkerkrankung, die inadäquat therapiert zu Gelenkzerstörung und resultierender Invalidität führen kann. Genetische Risikofaktoren sowie Lebensstileinflüsse führen in präklinischen Erkrankungsstadien zu posttranslationalen Modifikationen körpereigener Strukturen, die die immunologische Selbst-Toleranz brechen und zur immunologischen Fehlerkennung von Gelenkstrukturen durch B- und T-Lymphozyten führen.
Das Ziel der hier vorliegenden Arbeit war die Aufklärung von Wirkmechanismen eines für die immunmodulatorische Therapie der RA entwickelten innovativen Ansatzes zur Rekonstitution der immunologischen Autotoleranz mittels rekombinant hergestellter MHC-Klasse-II/Peptidkomplexe durch Induktion regulatorischer T-Zellen. Im Mittelpunkt der in vitro Studien steht hierbei eine über Speziesbarrieren hinweg evolutionär konservierte, von T-Lymphozyten auf dem Kollagen Typ-II (CII) erkannte, durch Glykosylierung posttranslational modifizierte, autoantigene Strukturdeterminante. Dieses T-Zellepitop (CII-Peptid) stellt sowohl in der humanen RA als auch in der murinen Experimentalerkrankung der CIA (Collagen induced arthritis) eine immunodominante Struktur der arthritogenen Autoimmunität dar. Für die modellhaften in vitro Studien zur Aufklärung der Wirkweise rekombinanter MHC-II/Peptidkomplexe auf humane T-Zellen, standen über eine Kooperation mit Prof. Rikard Holmdahl (Karolinska Institut, Stockholm) T-Zell-Hydridome mit transgener Expression des humanen MHC-II/Moleküls DR4 (DRA1/DRB1*04:01) mit unterschiedlicher Epitopspezifität (T-Zell-Hybridom 3H8, Spezifität: unmodifiziertes CII-Peptid und mDR1.1, Spezifität: galaktosyliertes CII-Peptid an Position K264) zur Verfügung. Das aus einer α- und β-Kette bestehende MHC-II/Molekül DR4 ist durch das DRA1-Gen und allelische Varianten des DRB1-Locus (stärkste RA-Assoziation: DRB1*04:01) kodiert und bildet die Form seiner Bindungstasche für die Präsentation antigener Peptide an den T-Zell-Rezeptor (TCR) auf der Oberfläche antigenpräsentierender Zellen (APC). In den Studien zur Stimulation der Hybridomzellen konnte gezeigt werden, dass die T-Zellstimulation und die daraus resultierende Zytokinausschüttung (IL-2 und IL-10) kontextabhängig ist. Je nach Stimulationsart, ob festphasengebunden- oder löslich, erfolgt die Stimulusperzeption über differente TCR-Anordnungen in Mikrodomänen der Zelloberfläche und resultiert in entsprechend modulierten Signalstärken. So führt die Zellaktivierung über die festphasengebundene Stimulation mittels MHC-II/Peptidkomplexen zur Ausbildung einer hohen TCR-Dichte, die über hohe Signalstärken zu einer spezifischen IL-2 Sekretion als Antwort führen. Die Stimulation mit monomeren DR4/CII-Peptidkomplexen in gelöster Form adressiert dagegen die auf der gesamten Zelloberfläche verteilten T-Zell-Rezeptoren, was in einer geringeren Aktivierungsdichte und einer attenuierten Gesamtsignalstärke sowie der Sekretion des immunsupressiv wirkenden IL-10 resultiert. Für den angestrebten pharmakologischen Einsatz der DR4/CII-Peptidkomplexe ist bedeutsam, dass die aktivierende TCR-Bindung der gelösten monomeren Komplexe nur partiell agonistisch wirkt und die Induktion immunregulatorischer IL-10 Zytokinantworten begünstigt. Neben der direkten T-Zellinteraktion konnte auch die Möglichkeit einer indirekten Aktivierung unter Vermittlung von APCs nach Endozytose der DR4/CII-Peptidkomplexe, ihrer lysosomalen Prozessierung und Präsentation auf endogenen neusynthetisierten DR4/Molekülen experimentell u.a. unter Verwendung der HLA-DR4- exprimierenden murinen Makrophagenlinie BL25 als APC-Modell belegt werden. Im Hinblick auf die intendierte Weiterentwicklung zu therapeutischen Anwendungen der MHC-II/CII-Peptidkomplexe unter Gesichtspunkten der Arzneimittelsicherheit ist wichtig, dass der aufgezeigte indirekte Weg der T-Zellaktivierung nach vorausgehender Prozessierung durch APCs ineffizient ist. Dieser Weg erfordert nämlich sehr hohe Konzentrationen an MHC-II/Peptidkomplexen, welche weit oberhalb der in tierexperimentellen Studien unter therapeutisch wirksamen Dosierungen erreichten Gewebespiegel liegen.
Darüber hinaus ist es uns gelungen, methodisch den Nachweis CII-spezifischer T-Zellen, die im Gesamtrepertoire der CD4+ T-Zellen im peripheren Blut von RA-Patienten (HLA-DRB1*04:01) nur in sehr niedriger Frequenz vorkommen, mittels T-Zellaktivierung und spezifischer Tetramerbindung als phänotypischen Marker zu verbessern. Für die Tetramerbindung wurden Monomere mit dem galaktosylierten CII-Peptid (CIIgal259-273) beladenen DR4/Moleküle über einen aminoterminal konjugierten Biotinrest mittels eines Fluorochromgekoppelten Streptavidins tetramerisiert. Unter Einsatz dieser Methoden ist es gelungen, aus den durchflusszytometrisch sortierten CII-spezifischen Zellen, mittels Nukleotidsequenzierung, ihr TCR-Repertoire zu analysieren und hinsichtlich präferentieller V-Genverwendung zu charakterisieren. Für zwei humane DR4-restringiert gal264CII-spezifische T-Zell-Rezeptoren aus RA-Patienten konnte die Funktionalität und Epitopspezifität durch rekombinante Expression demonstriert werden. Auf Basis der gemeinsamen Vorarbeiten mit Prof. Rikard Holmdahl im murinen CIA-Modell und den bekannten Daten zur Induktion regulatorischer T-Zellen (Tr1-Zellen) durch MHC-II/CII-Peptidkomplexe, wurden in vitro Differenzierungsexperimente an humanen PBMCs DR4-positiver RA-Patienten unter dem Einfluss von DR4/gal264CII-Peptidkomplexen durchgeführt. Die Studien belegen, dass die Komplexe mit den antigenspezifischen T-Zellen interagieren und zur Induktion von Markern eines Tr1-Phänotyps, darunter PD-1 und IL-10 führen. Zukünftige Kristallstrukturanalysen eines TCR/DR4/gal264CII-Komplexes sollen dem verbesserten molekularen Verständnis der TCR-Erkennung von CII als Autoantigen insbesondere bzgl. des flexibleren Galaktoserestes für Arthritogenität und Tolerogenität dienen. Fernziel ist die Entwicklung einer wirksamen und sicheren immunmodulatorischen Therapie der RA durch Induktion regulatorischer T-Zellen.
The existence of all living organisms depends on their multidimensional adjustment to the conditions of the environment in which they live. Organisms must constantly deal with not only abiotic stress factors (such as water availability or extreme temperatures), but also with various biotic interactions (the competition between different organisms, both intraspecific and interspecies). When there is a consensus between an organism and the environment it means that this organism is well adjusted and increases its probability of survival.
Symbiotic organisms possess the ability to establish an intimate interaction with another species (symbiont) that provides benefits for survival. Organisms that are involved in obligate symbiosis may adapt to a new environment by switching to another symbiotic partner that is locally better adapted; or by reshuffling symbiont communities present in the holobiont. This ability potentially gives them the opportunity to flexibly react to changing environmental conditions.
In this thesis I studied the genetic diversity and geographic distribution of symbiont lineages in a lichen symbiosis to better understand environmental adaptation in symbiotic systems. Lichens are symbiotic associations of photobionts (one or several green-algal species or cyanobacteria), filamentous mycobionts (lichen-forming fungi) and co-inhabiting symbiotic microorganisms (lichen-associated bacteria, endolichenic fungi, and basidiomycete yeast). The coccoid green algae of the genus Trebouxia are the most common and the most studied lichen photobionts. However, the lack of formal Trebouxia taxonomy impedes our understanding of this photobiont diversity.
Different species of mycobionts may share the same photobionts and a single species of mycobiont may associate with multiple, genetically different photobionts. Interactions among symbionts are not random and are constrained by evolutionary and environmental processes. The ability to associate with specific symbiotic partner is considered as a lichen strategy to facilitate adaptation to the constantly changing environments.
The objectives of this thesis were to 1. Elucidate the intraspecific diversity of fungal and algal symbionts in the lichen Umbilicaria pustulata, given a range-wide (Europe-wide) sampling; 2. Evaluate species delimitation in trebouxioid photobionts based on molecular data, and 3. Quantify the climatic niches of photobiont lineages within U. pustulata, to establish whether the association with particular photobionts may modify the range and ecological niche of this lichen.
The main findings of this thesis are:
1. The genetic diversity within trebouxoid photobiont of U. pustulata is higher than within the mycobiont. The most variable photobiont loci are nrITS rDNA, psbJ-L, and COX2. RbcL is the least variable photobiont locus. The most variable mycobiont loci are MCM7 and TSR1. This study shows a lack of genetic variability in the mycobiont loci EF1, nrITS rDNA, RPB1, and RPB2.
2. U. pustulata shows a low level of selectivity and is associated with numerous (most likely six) putative algal species. All photobiont haplotypes found in U. pustulata are shared between other lichen-forming fungi species, showing different patterns of species-to-species and species-to-community interactions.
3. The geographic distribution of U. pustulata symbionts associations is strongly connected to changes in the climatic niches. The mycobiont-photobiont interactions change along latitudinal temperature gradients (cold-adapted hotspot) and in Mediterranean climate zones (warm-adapted hotspot). U. pustulata broadens its distribution range by switching between photobionts that posses specific environmental preferences.
Overall, this thesis contributes to the understanding of the symbiont diversity, fungal-algal association patterns and local adaptation linked to symbiont-mediated niche expansion in lichens. While identifying intraspecific diversity of both lichen symbionts is a key predisposition to understand symbiont interactions, population dynamics or co-evolution, my comparative study of the sequence-based molecular markers is relevant to reveal cryptic diversity in other lichen-forming fungi and their photobionts.
The determination of species boundaries in lichen symbionts is essential for the study of selectivity and specificity, co-distribution, and co-evolution. Whereas the phylogenetic relationships of Trebouxiophyceae are poorly understood, the application of a novel multifaceted approach based on phylogenetic relationships, coalescence methods and morphological traits presented in this thesis is a promising tool to address species boundaries within this heterogeneous genus.
This thesis provides evidence for symbiont-mediated niche expansion in lichens and highlights the preferential photobiont association from a niche-modeling perspective. My results shed light on symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation in the lichen symbiosis. The spatial genetic pattern found in U. pustulata symbionts supports the concept of ecological fitting and is consistent with patterns found in other lichen studies. Results presented here relate also to findings in different symbiotic systems, like reef-building corals, where different latitudinal patterns and symbiont switching has been reported as an adaptive response to severe bleaching events. Furthermore, this study is timely in light of global warming, because the identification of interaction hotspots among symbionts helps to understand how lichens or other symbiotic organisms adjust to the ongoing climate change. This knowledge will, in turn, facilitate the proper conservation of the most vulnerable lichen populations. My doctoral thesis provides a conceptual framework for analyzing symbiont diversity, interaction patterns, and symbiont-mediated niche expansion that could be applied to other types of lichen species as well as other organisms involved in facultative or obligate symbiosis.