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- Institut für Biochemie und Biologie (215) (entfernen)
Wild bee species are important pollinators in agricultural landscapes. However, population decline was reported over the last decades and is still ongoing. While agricultural intensification is a major driver of the rapid loss of pollinating species, transition zones between arable fields and forest or grassland patches, i.e., agricultural buffer zones, are frequently mentioned as suitable mitigation measures to support wild bee populations and other pollinator species. Despite the reported general positive effect, it remains unclear which amount of buffer zones is needed to ensure a sustainable and permanent impact for enhancing bee diversity and abundance. To address this question at a pollinator community level, we implemented a process-based, spatially explicit simulation model of functional bee diversity dynamics in an agricultural landscape. More specifically, we introduced a variable amount of agricultural buffer zones (ABZs) at the transition of arable to grassland, or arable to forest patches to analyze the impact on bee functional diversity and functional richness. We focused our study on solitary bees in a typical agricultural area in the Northeast of Germany. Our results showed positive effects with at least 25% of virtually implemented agricultural buffer zones. However, higher amounts of ABZs of at least 75% should be considered to ensure a sufficient increase in Shannon diversity and decrease in quasi-extinction risks. These high amounts of ABZs represent effective conservation measures to safeguard the stability of pollination services provided by solitary bee species. As the model structure can be easily adapted to other mobile species in agricultural landscapes, our community approach offers the chance to compare the effectiveness of conservation measures also for other pollinator communities in future.
Artificial light at night (ALAN) is altering the behaviour of nocturnal animals in a manifold of ways. Nocturnal invertebrates are particularly affected, due to their fatal attraction to ALAN. This selective pressure has the potential to reduce the strength of the flight-to-light response in insects, as shown recently in a moth species. Here we investigated light attraction of ground beetles (Coleoptera: Carabidae).We compared among animals (three genera) from a highly light polluted (HLP) grassland in the centre of Berlin and animals collected at a low-polluted area in a Dark Sky Reserve (DSR), captured using odour bait. In an arena setting tested at night time, HLP beetles (n = 75 across all genera) showed a reduced attraction towards ALAN. Tested during daytime, HLP beetles were less active in an open field test (measured as latency to start moving), compared to DSR (n = 143). However, we did not observe a reduced attraction towards ALAN within the species most common at both sides, Calathus fuscipes (HLP = 37, DSR = 118 individuals) indicating that not all species may be equally affected by ALAN. Reduced attraction to ALAN in urban beetles may either be a result of phenotypic selection in each generation removing HLP individuals that are attracted to light, or an indication for ongoing evolutionary differentiation among city and rural populations in their light response. Reduced attraction to light sources may directly enhance survival and reproductive success of urban individuals. However, decrease in mobility may negatively influence dispersal, reproduction and foraging success, highlighting the selective pressure that light pollution may have on fitness, by shaping and modifying the behaviour of insects.
Novel algorithms for prediction of protein complexes from protein-protein interacton networks
(2022)
The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.
Extreme habitats often harbor specific communities that differ substantially from non-extreme habitats. In many cases, these communities are characterized by archaea, bacteria and protists, whereas the number of species of metazoa and higher plants is relatively low. In extremely acidic habitats, mostly prokaryotes and protists thrive, and only very few metazoa thrive, for example, rotifers. Since many studies have investigated the physiology and ecology of individual species, there is still a gap in research on direct, trophic interactions among extremophiles. To fill this gap, we experimentally studied the trophic interactions between a predatory protist (Actinophrys sol, Heliozoa) and its prey, the rotifers Elosa woralli and Cephalodella sp., the ciliate Urosomoida sp. and the mixotrophic protist Chlamydomonas acidophila (a green phytoflagellate, Chlorophyta). We found substantial predation pressure on all animal prey. High densities of Chlamydomonas acidophila reduced the predation impact on the rotifers by interfering with the feeding behaviour of A. sol. These trophic relations represent a natural case of intraguild predation, with Chlamydomonas acidophila being the common prey and the rotifers/ciliate and A. sol being the intraguild prey and predator, respectively. We further studied this intraguild predation along a resource gradient using Cephalodella sp. as the intraguild prey. The interactions among the three species led to an increase in relative rotifer abundance with increasing resource (Chlamydomonas) densities. By applying a series of laboratory experiments, we revealed the complexity of trophic interactions within a natural extremophilic community.
Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
Malagasy shrew tenrecs (Microgale) have increasingly been used to study speciation genetics over the last years. A previous study recently uncovered gene flow between the Shrew-toothed shrew tenrec (M. soricoides) and sympatric southern population of the Pale shrew tenrec (M. fotsifotsy). This gene flow has been suggested to be accompanied by complete mitochondrial replacement in M. fotsifotsy. To explore the temporal framework of this replacement, we assembled mitogenomes from publicly available sequencing data of ultra-conserved elements. We were able to assemble complete and partial mitogenomes for 19 specimens from five species of shrew tenrecs, which represents a multifold increase in mitogenomic resources available for all tenrecs. Phylogenetic inferences and sequence simulations support the close relationship between the mitochondrial lineages of M. soricoides and the southern population of M. fotsifotsy. Based on the nuclear divergence of northern and southern populations of M. fotsifotsy and the mitochondrial divergence between the latter and M. soricoides, there was a mean time window for replacement of similar to 350,000 years. This timeframe implies that the effective size of the ancestral M. fotsifotsy southern population was less 70,000.
Large quantities of the antibiotic florfenicol are used in animal farming and aquaculture, contaminating the ecosystem with antibiotic residues and promoting antimicrobial resistance, ultimately leading to untreatable multidrug-resistant pathogens. Florfenicol-resistant bacteria often activate export mechanisms that result in resistance to various structurally unrelated antibiotics. We devised novel strategies for the enzymatic inactivation of florfenicol in different media, such as saltwater or milk. Using a combinatorial approach and selection, we optimized a hydrolase (EstDL136) for florfenicol cleavage. Reaction kinetics were followed by time-resolved NMR spectroscopy. Importantly, the hydrolase remained active in different media, such as saltwater or cow milk. Various environmentally-friendly application strategies for florfenicol inactivation were developed using the optimized hydrolase. As a potential filter device for cost-effective treatment of waste milk or aquacultural wastewater, the hydrolase was immobilized on Ni-NTA agarose or silica as carrier materials. In two further application examples, the hydrolase was used as cell extract or encapsulated with a semi-permeable membrane. This facilitated, for example, florfenicol inactivation in whole milk, which can help to treat waste milk from medicated cows, to be fed to calves without the risk of inducing antibiotic resistance. Enzymatic inactivation of antibiotics, in general, enables therapeutic intervention without promoting antibiotic resistance.
A temperature-inducible epigenome editing system to knock down histone methylation can be used to study the role of histone H3K4 methylation during heat stress memory in Arabidopsis. <br /> Histone modifications play a crucial role in the integration of environmental signals to mediate gene expression outcomes. However, genetic and pharmacological interference often causes pleiotropic effects, creating the urgent need for methods that allow locus-specific manipulation of histone modifications, preferably in an inducible manner. Here, we report an inducible system for epigenome editing in Arabidopsis (Arabidopsis thaliana) using a heat-inducible dCas9 to target a JUMONJI (JMJ) histone H3 lysine 4 (H3K4) demethylase domain to a locus of interest. As a model locus, we target the ASCORBATE PEROXIDASE2 (APX2) gene that shows transcriptional memory after heat stress (HS), correlating with H3K4 hyper-methylation. We show that dCas9-JMJ is targeted in a HS-dependent manner to APX2 and that the HS-induced overaccumulation of H3K4 trimethylation (H3K4me3) decreases when dCas9-JMJ binds to the locus. This results in reduced HS-mediated transcriptional memory at the APX2 locus. Targeting an enzymatically inactive JMJ protein in an analogous manner affected transcriptional memory less than the active JMJ protein; however, we still observed a decrease in H3K4 methylation levels. Thus, the inducible targeting of dCas9-JMJ to APX2 was effective in reducing H3K4 methylation levels. As the effect was not fully dependent on enzyme activity of the eraser domain, the dCas9-JMJ fusion protein may act in part independently of its demethylase activity. This underlines the need for caution in the design and interpretation of epigenome editing studies. We expect our versatile inducible epigenome editing system to be especially useful for studying temporal dynamics of chromatin modifications.
High-throughput proteomics approaches have resulted in large-scale protein–protein interaction (PPI) networks that have been employed for the prediction of protein complexes. However, PPI networks contain false-positive as well as false-negative PPIs that affect the protein complex prediction algorithms. To address this issue, here we propose an algorithm called CUBCO+ that: (1) employs GO semantic similarity to retain only biologically relevant interactions with a high similarity score, (2) based on link prediction approaches, scores the false-negative edges, and (3) incorporates the resulting scores to predict protein complexes. Through comprehensive analyses with PPIs from Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens, we show that CUBCO+ performs as well as the approaches that predict protein complexes based on recently introduced graph partitions into biclique spanned subgraphs and outperforms the other state-of-the-art approaches. Moreover, we illustrate that in combination with GO semantic similarity, CUBCO+ enables us to predict more accurate protein complexes in 36% of the cases in comparison to CUBCO as its predecessor.
The Annamites mountain range of Southeast Asia which runs along the border of Viet Nam and Laos is an important biodiversity hotspot with high levels of endemism. However, that biodiversity is threatened by unsustainable hunting, and many protected areas across the region have been emptied of their wildlife. To better protect the unique species in the Annamites, it is crucial to have a better understanding of their ecology and distribution. Additionally, basic genetic information is needed to provide conservation stakeholders with essential information to facilitate conservation breeding and counteract the illegal wildlife trade. To date, this baseline information is lacking for many Annamites species.
This thesis aims to assess the effectiveness of using non-invasive collection methods, i.e. camera-trap surveys and leech-derived wildlife host DNA, in order to improve and enhance our understanding of ecology, distribution, and genetic diversity of the Annamites terrestrial mammals.
In chapter 1, we analysed data from a systematic landscape camera-trap survey using single-species occupancy models to assess the ecology and distribution of two little-known Annamite endemics, the Annamite dark muntjac (Muntiacus rooseveltorum / truongsonensis) and Annamite striped rabbit (Nesolagus timminsi), in multiple protected areas across the Annamites. This chapter provided the first in-depth information on their ecology, as well as distribution patterns at large spatial scales. Most notably, we found that the Annamite dark muntjac was predominantly found at higher elevations, while responses to elevation varied among study areas for the Annamite striped rabbit. We estimated occupancy probabilities for both endemics by using their responses to environmental and anthropogenic influences and used this information to make recommendations for targeted conservation actions. We discuss how the approach we used for these two Annamites endemics can be expanded for other little-known and threatened species in other tropical regions.
As is the case with ecology and distribution, very little is known about the genetic diversity of the Annamite striped rabbit and other mammals of the Annamites. This poor understanding is mainly attributed to the lack of a comprehensive DNA sample collection that covers the species’ entire distribution range, which is believed to be a consequence of the low density of mammals or the remoteness of species’ habitat. In order to overcome the difficulties when trying to collect DNA samples from elusive mammals, we applied invertebrate-derived DNA (iDNA) sampling via hematophagous leeches to indirectly obtain genetic materials of their terrestrial host mammals.
In chapter 2, leech-derived DNA was used to study the genetic diversity of the Annamite striped rabbit population. By analysing the DNA extracted from leech samples collected at multiple study areas of the central Annamites, we found a genetic variation with five haplotypes among nine obtained sequences. Despite this diversity, we found no clear phylogeographic pattern among the lagomorph’s populations in central Annamites. The findings have direct conservation implications for the species, as local stakeholders are currently establishing a conservation rescue and breeding facility for Annamite endemic species. Thus our results suggested that Annamite striped rabbits from multiple protected areas in central Annamites can be used as founders for the breeding program.
In chapter 3, the genetic material of six mammals, which are frequently found in Indochina's illegal wildlife trade, was extracted from leeches collected at six study sites across the Anamites. Species-specific genetic markers were used to obtain DNA fragments that were analysed together with Genbank reference sequences from other parts of the species’ distribution range. Our results showed that invertebrate-derived DNA can be used to fill the sampling gaps and provide genetic reference data that is needed for conservation breeding programmes or to counteract the illegal wildlife trade.
Overal, this dissertation provides the first insights in the ecology, distribution, and genetics of rare and threatened species of the Annamites by utilising camera traps and leech-derived DNA as two non-invasive collection methods. This information is essential for improving conservation efforts of local stakeholders and managers, especially for the Annamite endemics. Results in this dissertation also show the effectiveness of both non-invasive methods for studying terrestrial mammals at a landscape level. By expanding the application of these methods to other protected areas across the Annamites, we will further our understanding of ecology, distribution, and genetics of Annamite endemics. With such landscape-scale surveys, we are able to provide stakeholders with an overview of the current status of wildlife in the Annamites which supports efforts to protect these secretive species from illegal hunting and thus their extinction.
Uncovering the interplay between nutrient availability and cellulose biosynthesis inhibitor activity
(2022)
All plant cells are surrounded by a dynamic, carbohydrate-rich extracellular matrix known as the cell wall. Nutrient availability affects cell wall composition via uncharacterized regulatory mechanisms, and cellulose deficient mutants develop a hypersensitive root response to growth on high concentrations of nitrate. Since cell walls account for the bulk of plant biomass, it is important to understand how nutrients regulate cell walls. This could provide important knowledge for directing fertilizer treatments and engineering plants with higher nutrient use efficiency. The direct effect of nitrate on cell wall synthesis was investigated through growth assays on varying concentrations of nitrate, measuring cellulose content of roots and shoots, and assessing cellulose synthase activity (CESA) using live cell imaging with spinning disk confocal microscopy. A forward genetic screen was developed to isolate mutants impaired in nutrient-mediated cell wall regulation, revealing that cellulose biosynthesis inhibitor (CBI) activity is modulated by nutrient availability. Various non-CESA mutants were isolated that displayed CBI resistance, with the majority of mutations causing perturbation of mitochondria-localized proteins. To investigate mitochondrial involvement, the CBI mechanism of action was investigated using a reverse genetic screen, a targeted pharmacological screen, and -omics approaches. The results generated suggest that CBI-induced cellulose inhibition is due to off-target effects. This provides the groundwork to investigate uncharacterized processes of CESA regulation and adds valuable knowledge to the understanding of CBI activity, which could be harnessed to develop new and improved herbicides.
Biological invasions may result from multiple introductions, which might compensate for reduced gene pools caused by bottleneck events, but could also dilute adaptive processes. A previous common-garden experiment showed heritable latitudinal clines in fitness-related traits in the invasive goldenrod Solidago canadensis in Central Europe. These latitudinal clines remained stable even in plants chemically treated with zebularine to reduce epigenetic variation. However, despite the heritability of traits investigated, genetic isolation-by-distance was non-significant. Utilizing the same specimens, we applied a molecular analysis of (epi)genetic differentiation with standard and methylation-sensitive (MSAP) AFLPs. We tested whether this variation was spatially structured among populations and whether zebularine had altered epigenetic variation. Additionally, we used genome scans to mine for putative outlier loci susceptible to selection processes in the invaded range. Despite the absence of isolation-by-distance, we found spatial genetic neighborhoods among populations and two AFLP clusters differentiating northern and southern Solidago populations. Genetic and epigenetic diversity were significantly correlated, but not linked to phenotypic variation. Hence, no spatial epigenetic patterns were detected along the latitudinal gradient sampled. Applying genome-scan approaches (BAYESCAN, BAYESCENV, RDA, and LFMM), we found 51 genetic and epigenetic loci putatively responding to selection. One of these genetic loci was significantly more frequent in populations at the northern range. Also, one epigenetic locus was more frequent in populations in the southern range, but this pattern was lost under zebularine treatment. Our results point to some genetic, but not epigenetic adaptation processes along a large-scale latitudinal gradient of S. canadensis in its invasive range.
Phenotypic plasticity can increase individual fitness when environmental conditions change over time. Inducible defences are a striking example, allowing species to react to fluctuating predation pressure by only expressing their costly defended phenotype under high predation risk. Previous theoretical investigations have focused on how this affects predator–prey dynamics, but the impact on competitive outcomes and broader community dynamics has received less attention. Here we use a small food web model, consisting of two competing plastic autotrophic species exploited by a shared consumer, to study how the speed of inducible defences across three trade-off constellations affects autotroph coexistence, biomasses across trophic levels, and temporal variability. Contrary to the intuitive idea that faster adaptation increases autotroph fitness, we found that higher switching rates reduced individual fitness as it consistently provoked more maladaptive switching towards undefended phenotypes under high predation pressure. This had an unexpected positive impact on the consumer, increasing consumer biomass and lowering total autotroph biomass. Additionally, maladaptive switching strongly reduced autotroph coexistence through an emerging source-sink dynamic between defended and undefended phenotypes. The striking impact of maladaptive switching on species and food web dynamics indicates that this mechanism may be of more critical importance than previously recognized.
For starch metabolism to take place correctly, various enzymes and proteins acting on the starch granule surface are crucial. Recently, two non-catalytic starch-binding proteins, pivotal for normal starch turnover in Arabidopsis leaves, namely, EARLY STARVATION 1 (ESV1) and its homolog LIKE EARLY STARVATION 1 (LESV), have been identified. Both share nearly 38% sequence homology. As ESV1 has been found to influence glucan phosphorylation via two starch-related dikinases, alpha-glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD), through modulating the surface glucan structures of the starch granules and thus affecting starch degradation, we assess the impact of its homolog LESV on starch metabolism. Thus, the 65-kDa recombinant protein LESV and the 50-kDa ESV1 were analyzed regarding their influence on the action of GWD and PWD on the surface of the starch granules. We included starches from various sources and additionally assessed the effect of these non-enzymatic proteins on other starch-related enzymes, such as starch synthases (SSI and SSIII), starch phosphorylases (PHS1), isoamylase and beta-amylase. The data obtained indicate that starch phosphorylation, hydrolyses and synthesis were affected by LESV and ESV1. Furthermore, incubation with LESV and ESV1 together exerted an additive effect on starch phosphorylation. In addition, a stable alteration of the glucan structures at the starch granule surface following treatment with LESV and ESV1 was observed. Here, we discuss all the observed changes that point to modifications in the glucan structures at the surface of the native starch granules and present a model to explain the existing processes.
Patterns of space use are often subject to large temporal and individual-level variation, due to seasonality in behaviour and environmental conditions as well as age- or sex-specific needs. Especially in temperate regions, seasonality likely influences space use even in non-migratory birds. In waterfowl of the family Anatidae, however, few studies have analyzed space use of the same individuals across the full annual cycle. We used a resident population of Mandarin Ducks (Aix galericulata) in northeast Germany to study their year-round space use in relation to season, sex, and age. We marked 172 birds with colour rings and surveyed relevant water bodies for re-encounters for several years. As space-use patterns we derived home ranges from minimum convex polygons and the number of water bodies used by individual birds. Our analysis revealed that individuals shifted their space use between seasons, in particular extending their home ranges during the non-breeding season. Between years, in contrast, birds tended to show season-specific site fidelity. Sex differences were apparent during both breeding and non-breeding season, males consistently having larger home ranges and using slightly more water bodies. No difference was found between first-year and adult birds. Our study demonstrates that mark-resighting can provide valuable information about space use in species with suitable behaviour and readily accessible habitat. In such cases, it may be a valid alternative to more expensive GPS-tracking or short-term manual radio telemetry, particularly within citizen-science projects.
Understanding the complexity of metabolic networks has implications for manipulation of their functions. The complexity of metabolic networks can be characterized by identifying multireaction dependencies that are challenging to determine due to the sheer number of combinations to consider. Here, we propose the concept of concordant complexes that captures multireaction dependencies and can be efficiently determined from the algebraic structure and operational constraints of metabolic networks. The concordant complexes imply the existence of concordance modules based on which the apparent complexity of 12 metabolic networks of organisms from all kingdoms of life can be reduced by at least 78%. A comparative analysis against an ensemble of randomized metabolic networks shows that the metabolic network of Escherichia coli contains fewer concordance modules and is, therefore, more tightly coordinated than expected by chance. Together, our findings demonstrate that metabolic networks are considerably simpler than what can be perceived from their structure alone.
This dissertation aimed to determine differential expressed miRNAs in the context of chronic pain in polyneuropathy. For this purpose, patients with chronic painful polyneuropathy were compared with age matched healthy patients. Taken together, all miRNA pre library preparation quality controls were successful and none of the samples was identified as an outlier or excluded for library preparation. Pre sequencing quality control showed that library preparation worked for all samples as well as that all samples were free of adapter dimers after BluePippin size selection and reached the minimum molarity for further processing. Thus, all samples were subjected to sequencing. The sequencing control parameters were in their optimal range and resulted in valid sequencing results with strong sample to sample correlation for all samples. The resulting FASTQ file of each miRNA library was analyzed and used to perform a differential expression analysis. The differentially expressed and filtered miRNAs were subjected to miRDB to perform a target prediction. Three of those four miRNAs were downregulated: hsa-miR-3135b, hsa-miR-584-5p and hsa-miR-12136, while one was upregulated: hsa-miR-550a-3p. miRNA target prediction showed that chronic pain in polyneuropathy might be the result of a combination of miRNA mediated high blood flow/pressure and neural activity dysregulations/disbalances. Thus, leading to the promising conclusion that these four miRNAs could serve as potential biomarkers for the diagnosis of chronic pain in polyneuropathy.
Since TRPV1 seems to be one of the major contributors of nociception and is associated with neuropathic pain, the influence of PKA phosphorylated ARMS on the sensitivity of TRPV1 as well as the part of AKAP79 during PKA phosphorylation of ARMS was characterized. Therefore, possible PKA-sites in the sequence of ARMS were identified. This revealed five canonical PKA-sites: S882, T903, S1251/52, S1439/40 and S1526/27. The single PKA-site mutants of ARMS revealed that PKA-mediated ARMS phosphorylation seems not to influence the interaction rate of TRPV1/ARMS. While phosphorylation of ARMST903 does not increase the interaction rate with TRPV1, ARMSS1526/27 is probably not phosphorylated and leads to an increased interaction rate. The calcium flux measurements indicated that the higher the interaction rate of TRPV1/ARMS, the lower the EC50 for capsaicin of TRPV1, independent of the PKA phosphorylation status of ARMS. In addition, the western blot analysis confirmed the previously observed TRPV1/ARMS interaction. More importantly, AKAP79 seems to be involved in the TRPV1/ARMS/PKA signaling complex. To overcome the problem of ARMS-mediated TRPV1 sensitization by interaction, ARMS was silenced by shRNA. ARMS silencing resulted in a restored TRPV1 desensitization without affecting the TRPV1 expression and therefore could be used as new topical therapeutic analgesic alternative to stop ARMS mediated TRPV1 sensitization.
Throughout their lifetime plants need to adapt to temperature changes. Plants adapt to nonfreezing cold temperatures in a process called cold priming (cold acclimation) and lose the acquired freezing tolerance during warmer temperatures through deacclimation. The alternation of both processes is essential for plants to achieve optimal fitness in response to different temperature conditions. Cold acclimation has been extensively studied, however, little is known about the regulation of deacclimation. This thesis elucidates the process of deacclimation on a physiological and molecular level in Arabidopsis thaliana. Electrolyte leakage measurements during cold acclimation and up to four days of deacclimation enabled the identification of four knockout mutants (hra1, lbd41, mbf1c and jub1) with a slower rate of deacclimation compared to the wild type. A transcriptomic study using RNA-Sequencing in A. thaliana Col-0, jub1 and mbf1c identified the importance of the inhibition of stress responsive and Jasmonate-ZIM-domain genes as well as the regulation of cell wall modifications during deacclimation. Moreover, measurements of alcohol dehydrogenase activity and gene expression changes of hypoxia markers during the first four days of deacclimation evidently showed that a hypoxia response is activated during deacclimation. Epigenetic regulation was observed to be extensively involved during cold acclimation and 24 h of deacclimation in A. thaliana. Further, both deacclimation studies showed that the previous hypothesis that heat stress might play a role in early deacclimation, is not likely. A number of DNA- and histone demethylases as well as histone variants were upregulated during deacclimation suggesting a role in plant memory. Recently, multiple studies have shown that plants are able to retain memory of a previous cold stress even after a week of deacclimation. In this work, transcriptomic and metabolomic analyses of Arabidopsis during 24 h of priming (cold acclimation) and triggering (recurring cold stress after deacclimation) revealed a uniquely significant and transient induction of DREB1D, DREB1E and DREB1F transcription factors during triggering contributing to fine-tuning of the second cold stress response. Furthermore, genes encoding Late Embryogenesis Abundant (LEA) and antifreeze proteins and proteins detoxifying reactive oxygen species were higher induced during late triggering (24 h) compared to primed samples, while cell wall remodelers of the class xyloglucan endotransglucosylase/hydrolase were early responders of triggering. The high induction of cell wall remodelers during deacclimation as well as triggering proposes that these proteins play an essential role in the stabilization of the cells during growth as well as the response to recurring stresses. Collectively this work gives new insights on the regulation of deacclimation and cold stress memory in A. thaliana and opens the door to future targeted studies of essential genes in this process.
Heat stress (HS) is one of the most common abiotic stresses, frequently affecting plant growth and crop production. With its fluctuating nature, HS episodes are frequently interspersed by stress-free intervals. Plants can be primed by HS, allowing them to survive better a recurrent stress episode. A memory of this priming can be maintained during stress-free intervals and this memory is closely correlated with transcriptional memory at several HS-inducible loci. This transcriptional memory is evident from hyper-induction of a locus upon a recurrent HS. ASCORBATE PEROXIDASE 2 (APX2) shows such hyper-induction upon recurring HS, however, the molecular basis of this transcriptional memory is not understood. Previous research showed that the HSinduced transcriptional memory at APX2 can last for up to seven days, and that it is controlled by cis-regulatory elements within the APX2 promoter.
To identify regulators involved in HS transcriptional memory, an unbiased forward genetic screening using EMS mutated seeds of pAPX2::LUC was performed from this screen. Two EMS mutants with affected transcriptional memory of LUC were identified. I confirmed that both two EMS mutants resulted from the gene mutations of HISTONE ACETYLTRANSFERASE 1 (HAC1). Besides pAPX2::LUC, the HS-induced transcription of other HS memory genes were also affected in hac1 mutants. Moreover, HAC1 may promote HS transcriptional memory by acetylating promoters of HS memory genes.
On the other hand, to identify cis-regulatory elements that are required for transcriptional memory of APX2, I performed promoter analysis of the four conserved HSEs identified within a functional APX2 promoter. I found out that one of the HSEs (HSE1) is necessary for both HS-induced APX2 transcription and transcriptional memory, while another one of HSEs (HSE2) is important for HS-induced APX2 transcriptional memory. I also found out that the HSE1 itself (with 10 bp of flanking sequence) is sufficient to confer HS-induced APX2 transcriptional memory, and HSE1 is also necessary for HSFA2 to bind on APX2 promoter and activate APX2 transcription. The findings will provide important clues for the molecular mechanism of transcriptional memory and will enable engineering of enhanced stress tolerance in crops.
Die aktuelle COVID-19-Pandemie zeigt deutlich, wie sich Infektionskrankheiten weltweit verbreiten können. Neben Viruserkrankungen breiten sich auch multiresistente bakterielle Erreger weltweit aus. Dementsprechend besteht ein hoher Bedarf, durch frühzeitige Erkennung Erkrankte zu finden und Infektionswege zu unterbrechen.
Herkömmliche kulturelle Verfahren benötigen minimalinvasive bzw. invasive Proben und dauern für Screeningmaßnahmen zu lange. Deshalb werden schnelle, nichtinvasive Verfahren benötigt.
Im klassischen Griechenland verließen sich die Ärzte unter anderem auf ihren Geruchssinn, um Infektionen und andere Krankheiten zu differenzieren. Diese charakteristischen Gerüche sind flüchtige organische Substanzen (VOC), die im Rahmen des Metabolismus eines Organismus entstehen. Tiere, die einen besseren Geruchssinn haben, werden trainiert, bestimmte Krankheitserreger am Geruch zu unterscheiden. Allerdings ist der Einsatz von Tieren im klinischen Alltag nicht praktikabel. Es bietet sich an, auf technischem Weg diese VOCs zu analysieren.
Ein technisches Verfahren, diese VOCs zu unterscheiden, ist die Ionenmobilitätsspektrometrie gekoppelt mit einer multikapillaren Gaschromatographiesäule (MCC-IMS). Hier zeigte sich, dass es sich bei dem Verfahren um eine schnelle, sensitive und verlässliche Methode handelt.
Es ist bekannt, dass verschiedene Bakterien aufgrund des Metabolismus unterschiedliche VOCs und damit eigene spezifische Gerüche produzieren. Im ersten Schritt dieser Arbeit konnte gezeigt werden, dass die verschiedenen Bakterien in-vitro nach einer kurzen Inkubationszeitzeit von 90 Minuten anhand der VOCs differenziert werden können. Hier konnte analog zur Diagnose in biochemischen Testreihen eine hierarchische Klassifikation der Bakterien erfolgen.
Im Gegensatz zu Bakterien haben Viren keinen eigenen Stoffwechsel. Ob virusinfizierte Zellen andere VOCs als nicht-infizierte Zellen freisetzen, wurde an Zellkulturen überprüft. Hier konnte gezeigt werden, dass sich die Fingerprints der VOCs in Zellkulturen infizierter Zellen mit Respiratorischen Synzytial-Viren (RSV) von nicht-infizierten Zellen unterscheiden.
Virusinfektionen im intakten Organismus unterscheiden sich von den Zellkulturen dadurch, dass hier neben Veränderungen im Zellstoffwechsel auch durch Abwehrmechanismen VOCs freigesetzt werden können.
Zur Überprüfung, inwiefern sich Infektionen im intakten Organismus ebenfalls anhand VOCs unterscheiden lassen, wurde bei Patienten mit und ohne Nachweis einer Influenza A Infektion als auch bei Patienten mit Verdacht auf SARS-CoV-2 (Schweres-akutes-Atemwegssyndrom-Coronavirus Typ 2) Infektion die Atemluft untersucht. Sowohl Influenza-infizierte als auch SARS-CoV-2 infizierte Patienten konnten untereinander und von nicht-infizierten Patienten mittels MCC-IMS Analyse der Atemluft unterschieden werden.
Zusammenfassend erbringt die MCC-IMS ermutigende Resultate in der schnellen nichtinvasiven Erkennung von Infektionen sowohl in vitro als auch in vivo.
The increasing introduction of non-native plant species may pose a threat to local biodiversity. However, the basis of successful plant invasion is not conclusively understood, especially since these plant species can adapt to the new range within a short period of time despite impoverished genetic diversity of the starting populations. In this context, DNA methylation is considered promising to explain successful adaptation mechanisms in the new habitat. DNA methylation is a heritable variation in gene expression without changing the underlying genetic information. Thus, DNA methylation is considered a so-called epigenetic mechanism, but has been studied in mainly clonally reproducing plant species or genetic model plants. An understanding of this epigenetic mechanism in the context of non-native, predominantly sexually reproducing plant species might help to expand knowledge in biodiversity research on the interaction between plants and their habitats and, based on this, may enable more precise measures in conservation biology.
For my studies, I combined chemical DNA demethylation of field-collected seed material from predominantly sexually reproducing species and rearing offsping under common climatic conditions to examine DNA methylation in an ecological-evolutionary context. The contrast of chemically treated (demethylated) plants, whose variation in DNA methylation was artificially reduced, and untreated control plants of the same species allowed me to study the impact of this mechanism on adaptive trait differentiation and local adaptation. With this experimental background, I conducted three studies examining the effect of DNA methylation in non-native species along a climatic gradient and also between climatically divergent regions.
The first study focused on adaptive trait differentiation in two invasive perennial goldenrod species, Solidago canadensis sensu latu and S. gigantea AITON, along a climate gradient of more than 1000 km in length in Central Europe. I found population differences in flowering timing, plant height, and biomass in the temporally longer-established S. canadensis, but only in the number of regrowing shoots for S. gigantea. While S. canadensis did not show any population structure, I was able to identify three genetic groups along this climatic gradient in S. gigantea. Surprisingly, demethylated plants of both species showed no change in the majority of traits studied. In the subsequent second study, I focused on the longer-established goldenrod species S. canadensis and used molecular analyses to infer spatial epigenetic and genetic population differences in the same specimens from the previous study. I found weak genetic but no epigenetic spatial variation between populations. Additionally, I was able to identify one genetic marker and one epigenetic marker putatively susceptible to selection. However, the results of this study reconfirmed that the epigenetic mechanism of DNA methylation appears to be hardly involved in adaptive processes within the new range in S. canadensis.
Finally, I conducted a third study in which I reciprocally transplanted short-lived plant species between two climatically divergent regions in Germany to investigate local adaptation at the plant family level. For this purpose, I used four plant families (Amaranthaceae, Asteraceae, Plantaginaceae, Solanaceae) and here I additionally compared between non-native and native plant species. Seeds were transplanted to regions with a distance of more than 600 kilometers and had either a temperate-oceanic or a temperate-continental climate. In this study, some species were found to be maladapted to their own local conditions, both in non-native and native plant species alike. In demethylated individuals of the plant species studied, DNA methylation had inconsistent but species-specific effects on survival and biomass production. The results of this study highlight that DNA methylation did not make a substantial contribution to local adaptation in the non-native as well as native species studied.
In summary, my work showed that DNA methylation plays a negligible role in both adaptive trait variation along climatic gradients and local adaptation in non-native plant species that either exhibit a high degree of genetic variation or rely mainly on sexual reproduction with low clonal propagation. I was able to show that the adaptive success of these non-native plant species can hardly be explained by DNA methylation, but could be a possible consequence of multiple introductions, dispersal corridors and meta-population dynamics. Similarly, my results illustrate that the use of plant species that do not predominantly reproduce clonally and are not model plants is essential to characterize the effect size of epigenetic mechanisms in an ecological-evolutionary context.
Biological invasions may result from multiple introductions, which might compensate for reduced gene pools caused by bottleneck events, but could also dilute adaptive processes. A previous common-garden experiment showed heritable latitudinal clines in fitness-related traits in the invasive goldenrod Solidago canadensis in Central Europe. These latitudinal clines remained stable even in plants chemically treated with zebularine to reduce epigenetic variation. However, despite the heritability of traits investigated, genetic isolation-by-distance was non-significant. Utilizing the same specimens, we applied a molecular analysis of (epi)genetic differentiation with standard and methylation-sensitive (MSAP) AFLPs. We tested whether this variation was spatially structured among populations and whether zebularine had altered epigenetic variation. Additionally, we used genome scans to mine for putative outlier loci susceptible to selection processes in the invaded range. Despite the absence of isolation-by-distance, we found spatial genetic neighborhoods among populations and two AFLP clusters differentiating northern and southern Solidago populations. Genetic and epigenetic diversity were significantly correlated, but not linked to phenotypic variation. Hence, no spatial epigenetic patterns were detected along the latitudinal gradient sampled. Applying genome-scan approaches (BAYESCAN, BAYESCENV, RDA, and LFMM), we found 51 genetic and epigenetic loci putatively responding to selection. One of these genetic loci was significantly more frequent in populations at the northern range. Also, one epigenetic locus was more frequent in populations in the southern range, but this pattern was lost under zebularine treatment. Our results point to some genetic, but not epigenetic adaptation processes along a large-scale latitudinal gradient of S. canadensis in its invasive range.
Biofilms are complex living materials that form as bacteria get embedded in a matrix of self-produced protein and polysaccharide fibres. The formation of a network of extracellular biopolymer fibres contributes to the cohesion of the biofilm by promoting cell-cell attachment and by mediating biofilm-substrate interactions. This sessile mode of bacteria growth has been well studied by microbiologists to prevent the detrimental effects of biofilms in medical and industrial settings. Indeed, biofilms are associated with increased antibiotic resistance in bacterial infections, and they can also cause clogging of pipelines or promote bio-corrosion. However, biofilms also gained interest from biophysics due to their ability to form complex morphological patterns during growth. Recently, the emerging field of engineered living materials investigates biofilm mechanical properties at multiple length scales and leverages the tools of synthetic biology to tune the functions of their constitutive biopolymers.
This doctoral thesis aims at clarifying how the morphogenesis of Escherichia coli (E. coli) biofilms is influenced by their growth dynamics and mechanical properties. To address this question, I used methods from cell mechanics and materials science. I first studied how biological activity in biofilms gives rise to non-uniform growth patterns. In a second study, I investigated how E. coli biofilm morphogenesis and its mechanical properties adapt to an environmental stimulus, namely the water content of their substrate. Finally, I estimated how the mechanical properties of E. coli biofilms are altered when the bacteria express different extracellular biopolymers.
On nutritive hydrogels, micron-sized E. coli cells can build centimetre-large biofilms. During this process, bacterial proliferation and matrix production introduce mechanical stresses in the biofilm, which release through the formation of macroscopic wrinkles and delaminated buckles. To relate these biological and mechanical phenomena, I used time-lapse fluorescence imaging to track cell and matrix surface densities through the early and late stages of E. coli biofilm growth. Colocalization of high cell and matrix densities at the periphery precede the onset of mechanical instabilities at this annular region. Early growth is detected at this outer annulus, which was analysed by adding fluorescent microspheres to the bacterial inoculum. But only when high rates of matrix production are present in the biofilm centre, does overall biofilm spreading initiate along the solid-air interface. By tracking larger fluorescent particles for a long time, I could distinguish several kinematic stages of E. coli biofilm expansion and observed a transition from non-linear to linear velocity profiles, which precedes the emergence of wrinkles at the biofilm periphery. Decomposing particle velocities to their radial and circumferential components revealed a last kinematic stage, where biofilm movement is mostly directed towards the radial delaminated buckles, which verticalize. The resulting compressive strains computed in these regions were observed to substantially deform the underlying agar substrates. The co-localization of higher cell and matrix densities towards an annular region and the succession of several kinematic stages are thus expected to promote the emergence of mechanical instabilities at the biofilm periphery. These experimental findings are predicted to advance future modelling approaches of biofilm morphogenesis.
E. coli biofilm morphogenesis is further anticipated to depend on external stimuli from the environment. To clarify how the water could be used to tune biofilm material properties, we quantified E. coli biofilm growth, wrinkling dynamics and rigidity as a function of the water content of the nutritive substrates. Time-lapse microscopy and computational image analysis revealed that substrates with high water content promote biofilm spreading kinetics, while substrates with low water content promote biofilm wrinkling. The wrinkles observed on biofilm cross-sections appeared more bent on substrates with high water content, while they tended to be more vertical on substrates with low water content. Both wet and dry biomass, accumulated over 4 days of culture, were larger in biofilms cultured on substrates with high water content, despite extra porosity within the matrix layer. Finally, the micro-indentation analysis revealed that substrates with low water content supported the formation of stiffer biofilms. This study shows that E. coli biofilms respond to the water content of their substrate, which might be used for tuning their material properties in view of further applications.
Biofilm material properties further depend on the composition and structure of the matrix of extracellular proteins and polysaccharides. In particular, E. coli biofilms were suggested to present tissue-like elasticity due to a dense fibre network consisting of amyloid curli and phosphoethanolamine-modified cellulose. To understand the contribution of these components to the emergent mechanical properties of E. coli biofilms, we performed micro-indentation on biofilms grown from bacteria of several strains. Besides showing higher dry masses, larger spreading diameters and slightly reduced water contents, biofilms expressing both main matrix components also presented high rigidities in the range of several hundred kPa, similar to biofilms containing only curli fibres. In contrast, a lack of amyloid curli fibres provides much higher adhesive energies and more viscoelastic fluid-like material behaviour. Therefore, the combination of amyloid curli and phosphoethanolamine-modified cellulose fibres implies the formation of a composite material whereby the amyloid curli fibres provide rigidity to E. coli biofilms, whereas the phosphoethanolamine-modified cellulose rather acts as a glue. These findings motivate further studies involving purified versions of these protein and polysaccharide components to better understand how their interactions benefit biofilm functions.
All three studies depict different aspects of biofilm morphogenesis, which are interrelated. The first work reveals the correlation between non-uniform biological activities and the emergence of mechanical instabilities in the biofilm. The second work acknowledges the adaptive nature of E. coli biofilm morphogenesis and its mechanical properties to an environmental stimulus, namely water. Finally, the last study reveals the complementary role of the individual matrix components in the formation of a stable biofilm material, which not only forms complex morphologies but also functions as a protective shield for the bacteria it contains. Our experimental findings on E. coli biofilm morphogenesis and their mechanical properties can have further implications for fundamental and applied biofilm research fields.
Plants can be primed to survive the exposure to a severe heat stress (HS) by prior exposure to a mild HS. The information about the priming stimulus is maintained by the plant for several days. This maintenance of acquired thermotolerance, or HS memory, is genetically separable from the acquisition of thermotolerance itself and several specific regulatory factors have been identified in recent years.
On the molecular level, HS memory correlates with two types of transcriptional memory, type I and type II, that characterize a partially overlapping subset of HS-inducible genes. Type I transcriptional memory or sustained induction refers to the sustained transcriptional induction above non-stressed expression levels of a gene for a prolonged time period after the end of the stress exposure. Type II transcriptional memory refers to an altered transcriptional response of a gene after repeated exposure to a stress of similar duration and intensity. In particular, enhanced re-induction refers to a transcriptional pattern in which a gene is induced to a significantly higher degree after the second stress exposure than after the first.
This thesis describes the functional characterization of a novel positive transcriptional regulator of type I transcriptional memory, the heat shock transcription factor HSFA3, and compares it to HSFA2, a known positive regulator of type I and type II transcriptional memory. It investigates type I transcriptional memory and its dependence on HSFA2 and HSFA3 for the first time on a genome-wide level, and gives insight on the formation of heteromeric HSF complexes in response to HS. This thesis confirms the tight correlation between transcriptional memory and H3K4 hyper-methylation, reported here in a case study that aimed to reduce H3K4 hyper-methylation of the type II transcriptional memory gene APX2 by CRISPR/dCas9-mediated epigenome editing. Finally, this thesis gives insight into the requirements for a heat shock transcription factor to function as a positive regulator of transcriptional memory, both in terms of its expression profile and protein abundance after HS and the contribution of individual functional domains.
In summary, this thesis contributes to a more detailed understanding of the molecular processes underlying transcriptional memory and therefore HS memory, in Arabidopsis thaliana.
Large quantities of the antibiotic florfenicol are used in animal farming and aquaculture, contaminating the ecosystem with antibiotic residues and promoting antimicrobial resistance, ultimately leading to untreatable multidrug-resistant pathogens. Florfenicol-resistant bacteria often activate export mechanisms that result in resistance to various structurally unrelated antibiotics. We devised novel strategies for the enzymatic inactivation of florfenicol in different media, such as saltwater or milk. Using a combinatorial approach and selection, we optimized a hydrolase (EstDL136) for florfenicol cleavage. Reaction kinetics were followed by time-resolved NMR spectroscopy. Importantly, the hydrolase remained active in different media, such as saltwater or cow milk. Various environmentally-friendly application strategies for florfenicol inactivation were developed using the optimized hydrolase. As a potential filter device for cost-effective treatment of waste milk or aquacultural wastewater, the hydrolase was immobilized on Ni-NTA agarose or silica as carrier materials. In two further application examples, the hydrolase was used as cell extract or encapsulated with a semi-permeable membrane. This facilitated, for example, florfenicol inactivation in whole milk, which can help to treat waste milk from medicated cows, to be fed to calves without the risk of inducing antibiotic resistance. Enzymatic inactivation of antibiotics, in general, enables therapeutic intervention without promoting antibiotic resistance.
Synthetische Transkriptionsfaktoren bestehen wie natürliche Transkriptionsfaktoren aus einer DNA-Bindedomäne, die sich spezifisch an die Bindestellensequenz vor dem Ziel-Gen anlagert, und einer Aktivierungsdomäne, die die Transkriptionsmaschinerie rekrutiert, sodass das Zielgen exprimiert wird. Der Unterschied zu den natürlichen Transkriptionsfaktoren ist, sowohl dass die DNA-Bindedomäne als auch die Aktivierungsdomäne wirtsfremd sein können und dadurch künstliche Stoffwechselwege im Wirt, größtenteils chemisch, induziert werden können. Optogenetische synthetische Transkriptionsfaktoren, die hier entwickelt wurden, gehen einen Schritt weiter. Dabei ist die DNA-Bindedomäne nicht mehr an die Aktivierungsdomäne, sondern mit dem Blaulicht-Photorezeptor CRY2 gekoppelt. Die Aktivierungsdomäne wurde mit dem Interaktionspartner CIB1 fusioniert. Unter Blaulichtbestrahlung dimerisieren CRY2 und CIB1 und damit einhergehend die beiden Domänen, sodass ein funktionsfähiger Transkriptionsfaktor entsteht. Dieses System wurde in die Saccharomyces cerevisiae genomisch integriert. Verifiziert wurde das konstruierte System mit Hilfe des Reporters yEGFP, welcher durchflusszytometrisch detektiert werden konnte. Es konnte gezeigt werden, dass die yEGFP Expression variabel gestaltet werden kann, indem unterschiedlich lange Blaulichtimpulse ausgesendet wurden, die DNA-Bindedomäne, die Aktivierungsdomäne oder die Anzahl der Bindestellen, an dem sich die DNA-Bindedomäne anlagert, verändert wurden. Um das System für industrielle Anwendungen attraktiv zu gestalten, wurde das System vom Deepwell-Maßstab auf Photobioreaktor-Maßstab hochskaliert. Außerdem erwies sich das Blaulichtsystem sowohl im Laborstamm YPH500 als auch im industriell oft verwendeten Hefestamm CEN.PK als funktional. Des Weiteren konnte ein industrierelevante Protein ebenso mit Hilfe des verifizierten Systems exprimiert werden. Schlussendlich konnte in dieser Arbeit das etablierte Blaulicht-System erfolgreich mit einem Rotlichtsystem kombiniert werden, was zuvor noch nicht beschrieben wurde.
In this thesis, a collection of studies is presented that advance research on complex food webs in several directions. Food webs, as the networks of predator-prey interactions in ecosystems, are responsible for distributing the resources every organism needs to stay alive. They are thus central to our understanding of the mechanisms that support biodiversity, which in the face of increasing severity of anthropogenic global change and accelerated species loss is of highest importance, not least for our own well-being.
The studies in the first part of the thesis are concerned with general mechanisms that determine the structure and stability of food webs. It is shown how the allometric scaling of metabolic rates with the species' body masses supports their persistence in size-structured food webs (where predators are larger than their prey), and how this interacts with the adaptive adjustment of foraging efforts by consumer species to create stable food webs with a large number of coexisting species. The importance of the master trait body mass for structuring communities is further exemplified by demonstrating that the specific way the body masses of species engaging in empirically documented predator-prey interactions affect the predator's feeding rate dampens population oscillations, thereby helping both species to survive. In the first part of the thesis it is also shown that in order to understand certain phenomena of population dynamics, it may be necessary to not only take the interactions of a focal species with other species into account, but to also consider the internal structure of the population. This can refer for example to different abundances of age cohorts or developmental stages, or the way individuals of different age or stage interact with other species.
Building on these general insights, the second part of the thesis is devoted to exploring the consequences of anthropogenic global change on the persistence of species. It is first shown that warming decreases diversity in size-structured food webs. This is due to starvation of large predators on higher trophic levels, which suffer from a mismatch between their respiration and ingestion rates when temperature increases. In host-parasitoid networks, which are not size-structured, warming does not have these negative effects, but eutrophication destabilises the systems by inducing detrimental population oscillations. In further studies, the effect of habitat change is addressed. On the level of individual patches, increasing isolation of habitat patches has a similar effect as warming, as it leads to decreasing diversity due to the extinction of predators on higher trophic levels. In this case it is caused by dispersal mortality of smaller and therefore less mobile species on lower trophic levels, meaning that an increasing fraction of their biomass production is lost to the inhospitable matrix surrounding the habitat patches as they become more isolated. It is further shown that increasing habitat isolation desynchronises population oscillations between the patches, which in itself helps species to persist by dampening fluctuations on the landscape level. However, this is counteracted by an increasing strength of local population oscillations fuelled by an indirect effect of dispersal mortality on the feeding interactions. Last, a study is presented that introduces a novel mechanism for supporting diversity in metacommunities. It builds on the self-organised formation of spatial biomass patterns in the landscape, which leads to the emergence of spatio-temporally varying selection pressures that keep local communities permanently out of equilibrium and force them to continuously adapt. Because this mechanism relies on the spatial extension of the metacommunity, it is also sensitive to habitat change.
In the third part of the thesis, the consequences of biodiversity for the functioning of ecosystems are explored. The studies focus on standing stock biomass, biomass production, and trophic transfer efficiency as ecosystem functions. It is first shown that increasing the diversity of animal communities increases the total rate of intra-guild predation. However, the total biomass stock of the animal communities increases nevertheless, which also increases their exploitative pressure on the underlying plant communities. Despite this, the plant communities can maintain their standing stock biomass due to a shift of the body size spectra of both animal and plant communities towards larger species with a lower specific respiration rate. In another study it is further demonstrated that the generally positive relationship between diversity and the above mentioned ecosystem functions becomes steeper when not only the feeding interactions but also the numerous non-trophic interactions (like predator interference or competition for space) between the species of an ecosystem are taken into account. Finally, two studies are presented that demonstrate the power of functional diversity as explanatory variable. It is interpreted as the range spanned by functional traits of the species that determine their interactions. This approach allows to mechanistically understand how the ecosystem functioning of food webs with multiple trophic levels is affected by all parts of the food web and why a high functional diversity is required for efficient transportation of energy from primary producers to the top predators.
The general discussion draws some synthesising conclusions, e.g. on the predictive power of ecosystem functioning to explain diversity, and provides an outlook on future research directions.
The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.
The deciduous needle tree larch (Larix Mill.) covers more than 80% of the Asian boreal forests. Only a few Larix species constitute the vast forests and these species differ markedly in their ecological traits, most importantly in their ability to grow on and stabilize underlying permafrost. The pronounced dominance of the summergreen larches makes the Asian boreal forests unique, as the rest of the northern hemisphere boreal forests is almost exclusively dominated by evergreen needle-leaf forests. Global warming is impacting the whole world but is especially pronounced in the arctic and boreal regions. Although adapted to extreme climatic conditions, larch forests are sensitive to varying climatic conditions. By their sheer size, changes in Asian larch forests as range shifts or changes in species composition and the resulting vegetation-climate feedbacks are of global relevance. It is however still uncertain if larch forests will persist under the ongoing warming climate or if they will be replaced by evergreen forests. It is therefore of great importance to understand how these ecosystems will react to future climate warmings and if they will maintain their dominance. One step in the better understanding of larch dynamics is to study how the vast dominant forests developed and why they only established in northern Asia. A second step is to study how the species reacted to past changes in the climate.
The first objective of this thesis was to review and identify factors promoting Asian larch dominance. I achieved this by synthesizing and comparing reported larch occurrences and influencing components on the northern hemisphere continents in the present and in the past. The second objective was to find a possibility to directly study past Larix populations in Siberia and specifically their genetic variation, enabling the study of geographic movements. For this, I established chloroplast enrichment by hybridization capture from sedimentary ancient DNA (sedaDNA) isolated from lake sediment records. The third objective was to use the established method to track past larch populations, their glacial refugia during the Last Glacial Maximum (LGM) around 21,000 years before present (ka BP), and their post-glacial migration patterns.
To study larch promoting factors, I compared the present state of larch species ranges, areas of dominance, their bioclimatic niches, and the distribution on different extents and thaw depths of permafrost. The species comparison showed that the bioclimatic niches greatly overlap between the American and Asian species and that it is only in the extremely continental climates in which only the Asian larch species can persist. I revealed that the area of dominance is strongly connected to permafrost extent but less linked to permafrost seasonal thaw depths. Comparisons of the paleorecord of larch between the continents suggest differences in the recolonization history. Outside of northern Asia and Alaska, glacial refugial populations of larch were confined to the southern regions and thus recolonization could only occur as migration from south to north. Alaskan larch populations could not establish wide-range dominant forest which could be related to their own genetically depletion as separated refugial population. In Asia, it is still unclear whether or not the northern refugial populations contributed and enhanced the postglacial colonization or whether they were replaced by populations invading from the south in the course of climate warming. Asian larch dominance is thus promoted partly by adaptions to extremely continental climates and by adaptations to grow on continuous permafrost but could be also connected to differences in glacial survival and recolonization history of Larix species.
Except for extremely rare macrofossil findings of fossilized cones, traditional methods to study past vegetation are not able to distinguish between larch species or populations. Within the scope of this thesis, I therefore established a method to retrieve genetic information of past larch populations to distinguish between species. Using the Larix chloroplast genome as target, I successfully applied the method of DNA target enrichment by hybridization capture on sedaDNA samples from lake records and showed that it is able to distinguish between larch species. I then used the method on samples from lake records from across Siberia dating back up to 50 ka BP. The results allowed me to address the question of glacial survival and post-glacial recolonization mode in Siberian larch species. The analyzed pattern showed that LGM refugia were almost exclusively constituted by L. gmelinii, even in sites of current L. sibirica distribution. For included study sites, L. sibirica migrated into its extant northern distribution area only in the Holocene. Consequently, the post-glacial recolonization of L. sibirica was not enhanced by northern glacial refugia. In case of sites in extant distribution area of L. gmelinii, the absence of a genetic turn-over point to a continuous population rather than an invasion of southern refugia. The results suggest that climate has a strong influence on the distribution of Larix species and that species may also respond differently to future climate warming. Because species differ in their ecological characteristics, species distribution is also relevant with respect to further feedbacks between vegetation and climate.
With this thesis, I give an overview of present and past larch occurrences and evaluate which factors promote their dominance. Furthermore, I provide the tools to study past Larix species and give first important insights into the glacial history of Larix populations.
Artificial light at night (ALAN) is altering the behaviour of nocturnal animals in a manifold of ways. Nocturnal invertebrates are particularly affected, due to their fatal attraction to ALAN. This selective pressure has the potential to reduce the strength of the flight-to-light response in insects, as shown recently in a moth species. Here we investigated light attraction of ground beetles (Coleoptera: Carabidae).We compared among animals (three genera) from a highly light polluted (HLP) grassland in the centre of Berlin and animals collected at a low-polluted area in a Dark Sky Reserve (DSR), captured using odour bait. In an arena setting tested at night time, HLP beetles (n = 75 across all genera) showed a reduced attraction towards ALAN. Tested during daytime, HLP beetles were less active in an open field test (measured as latency to start moving), compared to DSR (n = 143). However, we did not observe a reduced attraction towards ALAN within the species most common at both sides, Calathus fuscipes (HLP = 37, DSR = 118 individuals) indicating that not all species may be equally affected by ALAN. Reduced attraction to ALAN in urban beetles may either be a result of phenotypic selection in each generation removing HLP individuals that are attracted to light, or an indication for ongoing evolutionary differentiation among city and rural populations in their light response. Reduced attraction to light sources may directly enhance survival and reproductive success of urban individuals. However, decrease in mobility may negatively influence dispersal, reproduction and foraging success, highlighting the selective pressure that light pollution may have on fitness, by shaping and modifying the behaviour of insects.
In plant cells, subcellular transport of cargo proteins relies to a large extent on post-Golgi transport pathways, many of which are mediated by clathrin-coated vesicles (CCVs). Vesicle formation is facilitated by different factors like accessory proteins and adaptor protein complexes (APs), the latter serving as a bridge between cargo proteins and the coat protein clathrin. One type of accessory proteins is defined by a conserved EPSIN N-TERMINAL HOMOLOGY (ENTH) domain and interacts with APs and clathrin via motifs in the C-terminal part. In Arabidopsis thaliana, there are three closely related ENTH domain proteins (EPSIN1, 2 and 3) and one highly conserved but phylogenetically distant outlier, termed MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1). In case of the trans-Golgi network (TGN) located MTV1, clathrin association and a role in vacuolar transport have been shown previously (Sauer et al. 2013). In contrast, for EPSIN1 and EPSIN2 limited functional and localization data were available; and EPSIN3 remained completely uncharacterized prior to this study (Song et al. 2006; Lee et al. 2007). The molecular details of ENTH domain proteins in plants are still unknown. In order to systematically characterize all four ENTH proteins in planta, we first investigated expression and subcellular localization by analysis of stable reporter lines under their endogenous promotors. Although all four genes are ubiquitously expressed, their subcellular distribution differs markedly. EPSIN1 and MTV1 are located at the TGN, whereas EPSIN2 and EPSIN3 are associated with the plasma membrane (PM) and the cell plate. To examine potential functional redundancy, we isolated knockout T-DNA mutant lines and created all higher order mutant combinations. The clearest evidence for functional redundancy was observed in the epsin1 mtv1 double mutant, which is a dwarf displaying overall growth reduction. These findings are in line with the TGN localization of both MTV1 and EPS1. In contrast, loss of EPSIN2 and EPSIN3 does not result in a growth phenotype compared to wild type, however, a triple knockout of EPSIN1, EPSIN2 and EPSIN3 shows partially sterile plants. We focused mainly on the epsin1 mtv1 double mutant and addressed the functional role of these two genes in clathrin-mediated vesicle transport by comprehensive molecular, biochemical, and genetic analyses. Our results demonstrate that EPSIN1 and MTV1 promote vacuolar transport and secretion of a subset of cargo. However, they do not seem to be involved in endocytosis and recycling. Importantly, employing high-resolution imaging, genetic and biochemical experiments probing the relationship of the AP complexes, we found that EPSIN1/AP1 and MTV1/AP4 define two spatially and molecularly distinct subdomains of the TGN. The AP4 complex is essential for MTV1 recruitment to the TGN, whereas EPSIN1 is independent of AP4 but presumably acts in an AP1-dependent framework. Our findings suggest that this ENTH/AP pairing preference is conserved between animals and plants.
Wild bee species are important pollinators in agricultural landscapes. However, population decline was reported over the last decades and is still ongoing. While agricultural intensification is a major driver of the rapid loss of pollinating species, transition zones between arable fields and forest or grassland patches, i.e., agricultural buffer zones, are frequently mentioned as suitable mitigation measures to support wild bee populations and other pollinator species. Despite the reported general positive effect, it remains unclear which amount of buffer zones is needed to ensure a sustainable and permanent impact for enhancing bee diversity and abundance. To address this question at a pollinator community level, we implemented a process-based, spatially explicit simulation model of functional bee diversity dynamics in an agricultural landscape. More specifically, we introduced a variable amount of agricultural buffer zones (ABZs) at the transition of arable to grassland, or arable to forest patches to analyze the impact on bee functional diversity and functional richness. We focused our study on solitary bees in a typical agricultural area in the Northeast of Germany. Our results showed positive effects with at least 25% of virtually implemented agricultural buffer zones. However, higher amounts of ABZs of at least 75% should be considered to ensure a sufficient increase in Shannon diversity and decrease in quasi-extinction risks. These high amounts of ABZs represent effective conservation measures to safeguard the stability of pollination services provided by solitary bee species. As the model structure can be easily adapted to other mobile species in agricultural landscapes, our community approach offers the chance to compare the effectiveness of conservation measures also for other pollinator communities in future.
Phenotypic plasticity can increase individual fitness when environmental conditions change over time. Inducible defences are a striking example, allowing species to react to fluctuating predation pressure by only expressing their costly defended phenotype under high predation risk. Previous theoretical investigations have focused on how this affects predator–prey dynamics, but the impact on competitive outcomes and broader community dynamics has received less attention. Here we use a small food web model, consisting of two competing plastic autotrophic species exploited by a shared consumer, to study how the speed of inducible defences across three trade-off constellations affects autotroph coexistence, biomasses across trophic levels, and temporal variability. Contrary to the intuitive idea that faster adaptation increases autotroph fitness, we found that higher switching rates reduced individual fitness as it consistently provoked more maladaptive switching towards undefended phenotypes under high predation pressure. This had an unexpected positive impact on the consumer, increasing consumer biomass and lowering total autotroph biomass. Additionally, maladaptive switching strongly reduced autotroph coexistence through an emerging source-sink dynamic between defended and undefended phenotypes. The striking impact of maladaptive switching on species and food web dynamics indicates that this mechanism may be of more critical importance than previously recognized.
Extreme habitats often harbor specific communities that differ substantially from non-extreme habitats. In many cases, these communities are characterized by archaea, bacteria and protists, whereas the number of species of metazoa and higher plants is relatively low. In extremely acidic habitats, mostly prokaryotes and protists thrive, and only very few metazoa thrive, for example, rotifers. Since many studies have investigated the physiology and ecology of individual species, there is still a gap in research on direct, trophic interactions among extremophiles. To fill this gap, we experimentally studied the trophic interactions between a predatory protist (Actinophrys sol, Heliozoa) and its prey, the rotifers Elosa woralli and Cephalodella sp., the ciliate Urosomoida sp. and the mixotrophic protist Chlamydomonas acidophila (a green phytoflagellate, Chlorophyta). We found substantial predation pressure on all animal prey. High densities of Chlamydomonas acidophila reduced the predation impact on the rotifers by interfering with the feeding behaviour of A. sol. These trophic relations represent a natural case of intraguild predation, with Chlamydomonas acidophila being the common prey and the rotifers/ciliate and A. sol being the intraguild prey and predator, respectively. We further studied this intraguild predation along a resource gradient using Cephalodella sp. as the intraguild prey. The interactions among the three species led to an increase in relative rotifer abundance with increasing resource (Chlamydomonas) densities. By applying a series of laboratory experiments, we revealed the complexity of trophic interactions within a natural extremophilic community.
Functional traits determine biomass dynamics, coexistence and energetics in plankton food webs
(2022)
Plankton food webs are the basis of marine and limnetic ecosystems. Especially aquatic ecosystems of high biodiversity provide important ecosystem services for humankind as providers of food, coastal protection, climate regulation, and tourism. Understanding the dynamics of biomass and coexistence in these food webs is a first step to understanding the ecosystems. It also lays the foundation for the development of management strategies for the maintenance of the marine and freshwater biodiversity despite anthropogenic influences.
Natural food webs are highly complex, and thus often equally complex methods are needed to analyse and understand them well. Models can help to do so as they depict simplified parts of reality. In the attempt to get a broader understanding of the complex food webs, diverse methods are used to investigate different questions.
In my first project, we compared the energetics of a food chain in two versions of an allometric trophic network model. In particular, we solved the problem of unrealistically high trophic transfer efficiencies (up to 70%) by accounting for both basal respiration and activity respiration, which decreased the trophic transfer efficiency to realistic values of ≤30%. Next in my second project I turned to plankton food webs and especially phytoplankton traits. Investigating a long-term data set from Lake Constance we found evidence for a trade-off between defence and growth rate in this natural phytoplankton community. I continued working with this data set in my third project focusing on ciliates, the main grazer of phytoplankton in spring. Boosted regression trees revealed that temperature and predators have the highest influence on net growth rates of ciliates. We finally investigated in my fourth project a food web model inspired by ciliates to explore the coexistence of plastic competitors and to study the new concept of maladaptive switching, which revealed some drawbacks of plasticity: faster adaptation led to higher maladaptive switching towards undefended phenotypes which reduced autotroph biomass and coexistence and increased consumer biomass.
It became obvious that even well-established models should be critically questioned as it is important not to forget reality on the way to a simplistic model. The results showed furthermore that long-term data sets are necessary as they can help to disentangle complex natural processes. Last, one should keep in mind that the interplay between models and experiments/ field data can deliver fruitful insights about our complex world.
Das Centrosom von Dictyostelium ist acentriolär aufgebaut, misst ca. 500 nm und besteht aus einer dreischichten Core-Struktur mit umgebender Corona, an der Mikrotubuli nukleieren. In dieser Arbeit wurden das centrosomale Protein Cep192 und mögliche Interaktionspartner am Centrosom eingehend untersucht. Die einleitende Lokalisationsuntersuchung von Cep192 ergab, dass es während der gesamten Mitose an den Spindelpolen lokalisiert und im Vergleich zu den anderen Strukturproteinen der Core-Struktur am stärksten exprimiert ist. Die dauerhafte Lokalisation an den Spindelpolen während der Mitose wird für Proteine angenommen, die in den beiden identisch aufgebauten äußeren Core-Schichten lokalisieren, die das mitotische Centrosom formen. Ein Knockdown von Cep192 führte zur Ausbildung von überzähligen Mikrotubuli-organisierenden Zentren (MTOC) sowie zu einer leicht erhöhten Ploidie. Deshalb wird eine Destabilisierung des Centrosoms durch die verminderte Cep192-Expression angenommen. An Cep192 wurden zwei kleine Tags, der SpotH6- und BioH6-Tag, etabliert, die mit kleinen fluoreszierenden Nachweiskonjugaten markiert werden konnten. Mit den so getagten Proteinen konnte die hochauflösende Expansion Microscopy für das Centrosom optimiert werden und die Core-Struktur erstmals proteinspezifisch in der Fluoreszenzmikroskopie dargestellt werden. Cep192 lokalisiert dabei in den äußeren Core-Schichten. Die kombinierte Markierung von Cep192 und den centrosomalen Proteinen CP39 und CP91 in der Expansion Microscopy erlaubte die Darstellung des dreischichtigen Aufbaus der centrosomalen Core-Struktur, wobei CP39 und CP91 zwischen Cep192 in der inneren Core-Schicht lokalisieren. Auch die Corona wurde in der Expansion Microscopy untersucht: Das Corona-Protein CDK5RAP2 lokalisiert in räumlicher Nähe zu Cep192 in der inneren Corona. Ein Vergleich der Corona-Proteine CDK5RAP2, CP148 und CP224 in der Expansion Microscopy ergab unterscheidbare Sublokalisationen der Proteine innerhalb der Corona und relativ zur Core-Struktur. In Biotinylierungsassays mit den centrosomalen Core-Proteinen CP39 und CP91 sowie des Corona-Proteins CDK5RAP2 konnte Cep192 als möglicher Interaktionspartner identifiziert werden.
Die Ergebnisse dieser Arbeit zeigen die wichtige Funktion des Proteins Cep192 im Dictyostelium-Centrosom und ermöglichen durch die Kombination aus Biotinylierungsassays und Expansion Microscopy der untersuchten Proteine ein verbessertes Verständnis der Topologie des Centrosoms.
More than a century ago the phenomenon of non-Mendelian inheritance (NMI), defined as any type of inheritance pattern in which traits do not segregate in accordance with Mendel’s laws, was first reported. In the plant kingdom three genomic compartments, the nucleus, chloroplast, and mitochondrion, can participate in such a phenomenon. High-throughput sequencing (HTS) proved to be a key technology to investigate NMI phenomena by assembling and/or resequencing entire genomes. However, generation, analysis and interpretation of such datasets remain challenging by the multi-layered biological complexity. To advance our knowledge in the field of NMI, I conducted three studies involving different HTS technologies and implemented two new algorithms to analyze them.
In the first study I implemented a novel post-assembly pipeline, called Semi-Automated Graph-Based Assembly Curator (SAGBAC), which visualizes non-graph-based assemblies as graphs, identifies recombinogenic repeat pairs (RRPs), and reconstructs plant mitochondrial genomes (PMG) in a semiautomated workflow. We applied this pipeline to assemblies of three Oenothera species resulting in a spatially folded and circularized model. This model was confirmed by PCR and Southern blot analyses and was used to predict a defined set of 70 PMG isoforms. With Illumina Mate Pair and PacBio RSII data, the stoichiometry of the RRPs was determined quantitatively differing up to three-fold.
In the second study I developed a post-multiple sequence alignment algorithm, called correlation mapping (CM), which correlates segment-wise numbers of nucleotide changes to a numeric ascertainable phenotype. We applied this algorithm to 14 wild type and 18 mutagenized plastome assemblies within the Oenothera genus and identified two genes, accD and ycf2 that may cause the competitive behavior of plastid genotypes as plastids can be biparental inherited in Oenothera. Moreover, lipid composition of the plastid envelope membrane is affected by polymorphisms within these two genes.
For the third study, I programmed a pipeline to investigate a NMI phenomenon, known as paramutation, in tomato by analyzing DNA and bisulfite sequencing data as well as microarray data. We identified the responsible gene (Solyc02g0005200) and were able to fully repress its caused phenotype by heterologous complementation with a paramutation insensitive transgene of the Arabidopsis thaliana orthologue. Additionally, a suppressor mutant shows a globally altered DNA methylation pattern and carries a large deletion leading to a gene fusion involving a histone deacetylase.
In conclusion, my developed and implemented algorithms and data analysis pipelines are suitable to investigate NMI and led to novel insights about such phenomena by reconstructing PMGs (SAGBAC) as a requirement to study mitochondria-associated phenotypes, by identifying genes (CM) causing interplastidial competition as well by applying a DNA/Bisulfite-seq analysis pipeline to shed light in a transgenerational epigenetic inheritance phenomenon.
Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
Aldehyde oxidases (AOXs) (E.C. 1.2.3.1) are molybdoflavo-enzymes belonging to the xanthine oxidase (XO) family. AOXs in mammals contain one molybdenum cofactor (Moco), one flavin adenine dinucleotide (FAD) and two [2Fe-2S] clusters, the presence of which is essential for the activity of the enzyme. Human aldehyde oxidase (hAOX1) is a cytosolic enzyme mainly expressed in the liver. hAOX1is involved in the metabolism of xenobiotics. It oxidizes aldehydes to their corresponding carboxylic acids and hydroxylates N-heterocyclic compounds. Since these functional groups are widely present in therapeutics, understanding the behaviour of hAOX1 has important implications in medicine. During the catalytic cycle of hAOX1, the substrate is oxidized at Moco and electrons are internally transferred to FAD via the FeS clusters. An electron acceptor juxtaposed to the FAD receives the electrons and re-oxidizes the enzyme for the next catalytic cycle. Molecular oxygen is the endogenous electron acceptor of hAOX1 and in doing so it is reduced and produces reactive oxygen species (ROS) including hydrogen peroxide (H2O2) and superoxide (O2.-). The production of ROS has patho-physiological importance, as ROS can have a wide range of effects on cell components including the enzyme itself.
In this thesis, we have shown that hAOX1 loses its activity over multiple cycles of catalysis due to endogenous ROS production and have identified a cysteine rich motif that protects hAOX1 from the ROS damaging effects. We have also shown that a sulfido ligand, which is bound at Moco and is essential for the catalytic activity of the enzyme, is vulnerable during turnover. The ROS produced during the course of the reaction are also able to remove this sulfido ligand from Moco. ROS, in addition, oxidize particular cysteine residues. The combined effects of ROS on the sulfido ligand and on specific cysteine residues in the enzyme result in its inactivation. Furthermore, we report that small reducing agents containing reactive sulfhydryl groups, in a selective manner, inactivate some of the mammalian AOXs by modifying the sulfido ligand at Moco. The mechanism of ROS production by hAOX1 is another scope that has been investigated as part of the work in this thesis. We have shown that the ratio of type of ROS, i.e. hydrogen peroxide (H2O2) and superoxide (O2.-), produced by hAOX1 is determined by a particular position on a flexible loop that locates in close proximity of FAD. The size of the cavity at the ROS producing site, i.e. the N5 position of the FAD isoalloxazine ring, kinetically affects the amount of each type of ROS generated by hAOX1. Taken together, hAOX1 is an enzyme with emerging importance in pharmacological and medical studies, not only due to its involvement in drug metabolism, but also due to ROS production which has physiological and pathological implications.
High-throughput proteomics approaches have resulted in large-scale protein–protein interaction (PPI) networks that have been employed for the prediction of protein complexes. However, PPI networks contain false-positive as well as false-negative PPIs that affect the protein complex prediction algorithms. To address this issue, here we propose an algorithm called CUBCO+ that: (1) employs GO semantic similarity to retain only biologically relevant interactions with a high similarity score, (2) based on link prediction approaches, scores the false-negative edges, and (3) incorporates the resulting scores to predict protein complexes. Through comprehensive analyses with PPIs from Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens, we show that CUBCO+ performs as well as the approaches that predict protein complexes based on recently introduced graph partitions into biclique spanned subgraphs and outperforms the other state-of-the-art approaches. Moreover, we illustrate that in combination with GO semantic similarity, CUBCO+ enables us to predict more accurate protein complexes in 36% of the cases in comparison to CUBCO as its predecessor.
An important goal in biotechnology and (bio-) medical research is the isolation of single cells from a heterogeneous cell population. These specialised cells are of great interest for bioproduction, diagnostics, drug development, (cancer) therapy and research. To tackle emerging questions, an ever finer differentiation between target cells and non-target cells is required. This precise differentiation is a challenge for a growing number of available methods.
Since the physiological properties of the cells are closely linked to their morphology, it is beneficial to include their appearance in the sorting decision. For established methods, this represents a non addressable parameter, requiring new methods for the identification and isolation of target cells. Consequently, a variety of new flow-based methods have been developed and presented in recent years utilising 2D imaging data to identify target cells within a sample. As these methods aim for high throughput, the devices developed typically require highly complex fluid handling techniques, making them expensive while offering limited image quality.
In this work, a new continuous flow system for image-based cell sorting was developed that uses dielectrophoresis to precisely handle cells in a microchannel. Dielectrophoretic forces are exerted by inhomogeneous alternating electric fields on polarisable particles (here: cells). In the present system, the electric fields can be switched on and off precisely and quickly by a signal generator. In addition to the resulting simple and effective cell handling, the system is characterised by the outstanding quality of the image data generated and its compatibility with standard microscopes. These aspects result in low complexity, making it both affordable and user-friendly.
With the developed cell sorting system, cells could be sorted reliably and efficiently according to their cytosolic staining as well as morphological properties at different optical magnifications. The achieved purity of the target cell population was up to 95% and about 85% of the sorted cells could be recovered from the system. Good agreement was achieved between the results obtained and theoretical considerations. The achieved throughput of the system was up to 12,000 cells per hour. Cell viability studies indicated a high biocompatibility of the system.
The results presented demonstrate the potential of image-based cell sorting using dielectrophoresis. The outstanding image quality and highly precise yet gentle handling of the cells set the system apart from other technologies. This results in enormous potential for processing valuable and sensitive cell samples.
Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set
(2022)
A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.