Biological clocks exist across all life forms and serve to coordinate organismal physiology with periodic environmental changes. The underlying mechanism of these clocks is predominantly based on cellular transcription-translation feedback loops in which clock proteins mediate the periodic expression of numerous genes. However, recent studies point to the existence of a conserved timekeeping mechanism independent of cellular transcription and translation, but based on cellular metabolism. These metabolic clocks were concluded based upon the observation of circadian and ultradian oscillations in the level of hyperoxidized peroxiredoxin proteins. Peroxiredoxins are enzymes found almost ubiquitously throughout life. Originally identified as H2O2 scavengers, recent studies show that peroxiredoxins can transfer oxidation to, and thereby regulate, a wide range of cellular proteins. Thus, it is conceivable that peroxiredoxins, using H2O2 as the primary signaling molecule, have the potential to integrate and coordinate much of cellular physiology and behavior with metabolic changes. Nonetheless, it remained unclear if peroxiredoxins are passive reporters of metabolic clock activity or active determinants of cellular timekeeping. Budding yeast possess an ultradian metabolic clock termed the Yeast Metabolic Cycle (YMC). The most obvious feature of the YMC is a high amplitude oscillation in oxygen consumption. Like circadian clocks, the YMC temporally compartmentalizes cellular processes (e.g. metabolism) and coordinates cellular programs such as gene expression and cell division. The YMC also exhibits oscillations in the level of hyperoxidized peroxiredoxin proteins. In this study, I used the YMC clock model to investigate the role of peroxiredoxins in cellular timekeeping, as well as the coordination of cell division with the metabolic clock. I observed that cytosolic 2-Cys peroxiredoxins are essential for robust metabolic clock function. I provide direct evidence for oscillations in cytosolic H2O2 levels, as well as cyclical changes in oxidation state of a peroxiredoxin and a model peroxiredoxin target protein during the YMC. I noted two distinct metabolic states during the YMC: low oxygen consumption (LOC) and high oxygen consumption (HOC). I demonstrate that thiol-disulfide oxidation and reduction are necessary for switching between LOC and HOC. Specifically, a thiol reductant promotes switching to HOC, whilst a thiol oxidant prevents switching to HOC, forcing cells to remain in LOC. Transient peroxiredoxin inactivation triggered rapid and premature switching from LOC to HOC. Furthermore, I show that cell division is normally synchronized with the YMC and that deletion of typical 2-Cys peroxiredoxins leads to complete uncoupling of cell division from metabolic cycling. Moreover, metabolic oscillations are crucial for regulating cell cycle entry and exit. Intriguingly, switching to HOC is crucial for initiating cell cycle entry whilst switching to LOC is crucial for cell cycle completion and exit. Consequently, forcing cells to remain in HOC by application of a thiol reductant leads to multiple rounds of cell cycle entry despite failure to complete the preceding cell cycle. On the other hand, forcing cells to remain in LOC by treating with a thiol oxidant prevents initiation of cell cycle entry. In conclusion, I propose that peroxiredoxins – by controlling metabolic cycles, which are in turn crucial for regulating the progression through cell cycle – play a central role in the coordination of cellular metabolism with cell division. This proposition, thus, positions peroxiredoxins as active players in the cellular timekeeping mechanism.

MsmS is a heme-based redox sensor kinase in Methanosarcina acetivorans consisting of alternating PAS and GAF domains connected to a C-terminal kinase domain. In addition to MsmS, M. acetivorans possesses a second kinase, MA0863 with high sequence similarity. Interestingly, MA0863 possesses an amber codon in its second GAF domain, encoding for the amino acid pyrrolysine. Thus far, no function of this residue has been resolved. In order to examine the heme iron coordination in both proteins, an improved method for the production of heme proteins was established using the Escherichia coli strain Nissle 1917. This method enables the complete reconstitution of a recombinant hemoprotein during protein production, thereby resulting in a native heme coordination. Analysis of the full-length MsmS and MA0863 confirmed a covalently bound heme cofactor, which is connected to one conserved cysteine residue in each protein. In order to identify the coordinating amino acid residues of the heme iron, UV/vis spectra of different variants were measured. These studies revealed His702 in MsmS and the corresponding His666 in MA0863 as the proximal heme ligands. MsmS has previously been described as a heme-based redox sensor. In order to examine whether the same is true for MA0863, redox dependent kinase assays were performed. MA0863 indeed displays redox dependent autophosphorylation activity, which is independent of heme ligands and only observed under oxidizing conditions. Interestingly, autophosphorylation was shown to be independent of the heme cofactor but rather relies on thiol oxidation. Therefore, MA0863 was renamed in RdmS (redox dependent methyltransferase-associated sensor). In order to identify the phosphorylation site of RdmS, thin layer chromatography was performed identifying a tyrosine as the putative phosphorylation site. This observation is in agreement with the lack of a so-called H-box in typical histidine kinases. Due to their genomic localization, MsmS and RdmS were postulated to form two-component systems (TCS) with vicinal encoded regulator proteins MsrG and MsrF. Therefore, protein-protein interaction studies using the bacterial adenylate two hybrid system were performed suggesting an interaction of RdmS and MsmS with the three regulators MsrG/F/C. Due to these multiple interactions these signal transduction pathways should rather be considered multicomponent system instead of two component systems.

The screening of metagenomic datasets led to the identification of new phage-derived members of the heme oxygenase and the ferredoxin-dependent bilin reductase enzyme families. The novel bilin biosynthesis genes were shown to form mini-cassettes on metagenomic scaffolds and further form distinct clusters in phylogenetic analyses (Ledermann et al., 2016). In this project, it was demonstrated that the discovered sequences actually encode for active enzymes. The biochemical characterization of a member of the heme oxygenases (ΦHemO) revealed that it possesses a regiospecificity for the α-methine bridge in the cleavage of the heme macrocycle. The reaction product biliverdin IXα was shown to function as the substrate for the novel ferredoxin-dependent bilin reductases (PcyX reductases), which catalyze its reduction to PEB via the intermediate 15,16-DHBV. While it was demonstrated that ΦPcyX, a phage-derived member of the PcyX reductases, is an active enzyme, it also became clear that the rate of the reaction is highly dependent on the employed redox partner. It turned out that the ferredoxin from the cyanophage P-SSM2 is to date the most suitable redox partner for the reductases of the PcyX group. Furthermore, the solution of the ΦPcyX crystal structure revealed that it adopts an α/β/α-sandwich fold, typical for the FDBR-family. Activity assays and subsequent HPLC analyses with different variants of the ΦPcyX protein demonstrated that, despite their similarity, PcyX and PcyA reductases must act via different reaction mechanisms. Another part of this project focused on the biochemical characterization of the FDBR KflaHY2 from the streptophyte alga Klebsormidium flaccidum. Experiments with recombinant KflaHY2 showed that it is an active FDBR which produces 3(Z)-PCB as the main reaction product, like it can be found in reductases of the PcyA group. Moreover, it was shown that under the employed assay conditions the reaction of BV to PCB proceeds in two different ways: Both 3(Z)-PΦB and 18¹,18²-DHBV occur as intermediates. Activity assays with the purified intermediates yielded PCB. Hence, both compounds are suitable substrates for KflaHY2. The results of this work highlight the importance of the biochemical experiments, as catalytic activity cannot solely be predicted by sequence analysis.

The cytosolic Fe65 adaptor protein family, consisting of Fe65, Fe65L1 and Fe65L2 is involved in many intracellular signaling pathways linking via its three interaction domains a continuously growing list of proteins by facilitating functional interactions. One of the most important binding partners of Fe65 family proteins is the amyloid precursor protein (APP), which plays an important role in Alzheimer Disease. To gain deeper insights in the function of the ubiquitously expressed Fe65 and the brain enriched Fe65L1, the goal of my study was I) to analyze their putative synaptic function in vivo, II) to examine structural analysis focusing on a putative dimeric complex of Fe65, III) to consider the involvement of Fe65 in mediating LRP1 and APP intracellular trafficking in murine hippocampal neurons. By utilizing several behavioral analyses of Fe65 KO, Fe65L1 KO and Fe65/Fe65L1 DKO mice I could demonstrate that the Fe65 protein family is essential for learning and memory as well as grip strength and locomotor activity. Furthermore, immunohistological as well as protein biochemical analysis revealed that the Fe65 protein family is important for neuromuscular junction formation in the peripheral nervous system, which involves binding of APP and acting downstream of the APP signaling pathway. Via Co-immunoprecipitation analysis I could verify that Fe65 is capable to form dimers ex vivo, which exclusively occur in the cytosol and upon APP expression are shifted to membrane compartments forming trimeric complexes. The influence of the loss of Fe65 and/or Fe65L1 on APP and/or LRP1 transport characteristics in axons could not be verified, possibly conditioned by the compensatory effect of Fe65L2. However, I could demonstrate that LRP1 affects the APP transport independently of Fe65 by shifting APP into slower types of vesicles leading to changed processing and endocytosis of APP. The outcome of my thesis advanced our understanding of the Fe65 protein family, especially its interplay with APP physiological function in synapse formation and synaptic plasticity.

Human forest modification is among the largest global drivers of terrestrial degradation of biodiversity, species interactions, and ecosystem functioning. One of the most pertinent components, forest fragmentation, has a long history in ecological research across the globe, particularly in lower latitudes. However, we still know little how fragmentation shapes temperate ecosystems, irrespective of the ancient status quo of European deforestation. Furthermore, its interaction with another pivotal component of European forests, silvicultural management, are practically unexplored. Hence, answering the question how anthropogenic modification of temperate forests affects fundamental components of forest ecosystems is essential basic research that has been neglected thus far. Most basal ecosystem elements are plants and their insect herbivores, as they form the energetic basis of the tropic pyramid. Furthermore, their respective biodiversity, functional traits, and the networks of interactions they establish are key for a multitude of ecosystem functions, not least ecosystem stability. Hence, the thesis at hand aimed to disentangle this complex system of interdependencies of human impacts, biodiversity, species traits and inter-species interactions. The first step lay in understanding how woody plant assemblages are shaped by human forest modification. For this purpose, field investigations in 57 plots in the hyperfragmented cultural landscape of the Northern Palatinate highlands (SW Germany) were conducted, censusing > 4,000 tree/shrub individuals from 34 species. Use of novel, integrative indices for different types of land-use allowed an accurate quantification of biotic responses. Intriguingly, woody tree/shrub communities reacted strikingly positive to forest fragmentation, with increases in alpha and beta diversity, as well as proliferation of heat/drought/light adapted pioneer species. Contrarily, managed interior forests were homogenized/constrained in biodiversity, with dominance of shade/cold adapted commercial tree species. Comparisons with recently unmanaged stands (> 40 a) revealed first indications for nascent conversion to oldgrowth conditions, with larger variability in light conditions and subsequent community composition. Reactions to microclimatic conditions, the relationship between associated species traits and the corresponding species pool, as well as facilitative/constraining effects by foresters were discussed as underlying mechanisms. Reactions of herbivore assemblages to forest fragmentation and the subsequent changes in host plant communities were assessed by comprehensive sampling of > 1,000 live herbivores from 134 species in the forest understory. Diversity was – similarly to plant communities - higher in fragmentation affected habitats, particularly in edges of continuous control forests. Furthermore, average trophic specialization showed an identical pattern. Mechanistically, benefits from microclimatic conditions, host availability, as well as pronounced niche differentiation are deemed responsible. While communities were heterogeneous, with no segregation across habitats, (smallforest fragments, edges, and interior of control forests), vegetation diversity, herbivore diversity, as well as trophic specialization were identified to shape community composition. This probably reflected a gradient from generalistic/species poor vs. specialist/species rich herbivore assemblages. Insect studies conducted in forest systems are doomed to incompleteness without considering ‘the last biological frontier’, the tree canopies. To access their biodiversity, relationship to edge effects, and their conservational value, the arboricolous arthropod fauna of 24 beech (Fagus sylvatica) canopies was sampled via insecticidal knockdown (‘fogging’). This resulted in an exhaustive collection of > 46,000 specimens from 24 major taxonomic/functional groups. Abundance distributions were markedly negative exponential, indicating high abundance variability in tree crowns. Individuals of six pertinent orders were identified to species level, returning > 3,100 individuals from 175 species and 52 families. This high diversity did marginally differ across habitats, with slightly higher species richness in edge canopies. However, communities in edge crowns were noticeably more heterogeneous than those in the forest interior, possibly due to higher variability in environmental edge conditions. In total, 49 species with protective value were identified, of which only one showed habitat preferences (for near-natural interior forests). Among them, six species (all beetles, Coleoptera) were classified as ‘priority species’ for conservation efforts. Hence, beech canopies of the Northern Palatinate highlands can be considered strongholds of insect biodiversity, incorporating many species of particular protective value. The intricacy of plant-herbivore interaction networks and their relationship to forest fragmentation is largely unexplored, particularly in Central Europe. Illumination of this matter is all the more important, as ecological networks are highly relevant for ecosystem stability, particularly in the face of additional anthropogenic disturbances, such as climate change. Hence, plant-herbivore interaction networks (PHNs) were constructed from woody plants and their associated herbivores, sampled alive in the understory. Herbivory verification was achieved using no-choice-feeding assays, as well as literature references. In total, networks across small forest fragments, edges, and the forest interior consisted of 696 interactions. Network complexity and trophic niche redundancy were compared across habitats using a rarefaction-like resampling procedure. PHNs in fragmentation affected forest habitats were significantly more complex, as well as more redundant in their realized niches, despite being composed of relatively more specialist species. Furthermore, network robustness to climate change was quantified utilizing four different scenarios for climate change susceptibility of involved plants. In this procedure, remaining herbivores in the network were measured upon successive loss of their host plant species. Consistently, PHNs in edges (and to a smaller degree in small fragments) withstood primary extinction of plant species longer, making them more robust. This was attributed to the high prevalence of heat/drought-adapted species, as well as to beneficial effects of network topography (complexity and redundancy). Consequently, strong correlative relationships were found between realized niche redundancy and climate change robustness of PHNs. This was both the first time that biologically realistic extinctions (instead of e.g.random extinctions) were used to measure network robustness, and that topographical network parameters were identified as potential indicators for network robustness against climate change. In synthesis, in the light of global biotic degradation due to human forest modification, the necessity to differentiate must be claimed. Ecosystems react differently to anthropogenic disturbances, and it seems the particular features present in Central European forests (ancient deforestation, extensive management, and, most importantly, high richness in open-forest plant species) cause partly opposed patterns to other biomes. Lenient microclimates and diverse plant communities facilitate equally diverse herbivore assemblages, and hence complex and robust networks, opposed to the forest interior. Therefore, in the reality of extensively used cultural landscapes, fragmentation affected forest ecosystems, particularly forest edges, can be perceived as reservoir for biodiversity, and ecosystem functionality. Nevertheless, as practically all forest habitats considered in this thesis are under human cultivation, recommendations for ecological enhancement of all forest habitats are discussed.