Protokolle zur Ovulationssynchronisation dienen dazu, Mängel in der visuellen Brunstbeobachtung zu kompensieren und eine terminorientierte künstliche Besamung zu ermöglichen. Im Ovsynchprotokoll mit einmaliger PGF2α-Gabe an Tag 7 zeigen 13 – 21 % der Kühe eine unvollständige Luteolyse. Eine unvollständige Luteolyse bzw. Konzentrationen von Progesteron > 0,4 ng / ml zum Zeitpunkt der zweiten GnRH-Gabe führen zu schlechteren Besamungserfolgen. Eine besondere Risikogruppe für hohe Konzentrationen zur zweiten GnRH-Gabe stellen Kühe dar, die das Ovsynchprotokoll ohne Gelbkörper bzw. mit niedrigen Konzentrationen von Progesteron beginnen. Um den Anteil an Kühen mit einer vollständigen Luteolyse und dadurch den Besamungserfolg zu erhöhen, wurden modifizierte Ovsynchprotokolle beschrieben, die entweder die PGF2α-Dosis erhöhten, oder eine zusätzliche PGF2α-Gabe integrierten. Das Ziel der vorliegenden Studie war es, den Einfluss einer doppelten PGF2α-Dosis (1.000 µg Cloprostenol) direkt mit dem Einfluss einer zweimaligen PGF2α-Gabe (500 µg Cloprostenol an Tag 7 und Tag 8) während eines 7 d-Ovsynchprotokolls auf die Konzentration von Progesteron zur zweiten GnRH-Gabe und den Besamungserfolg zu untersuchen. Die Hypothesen lauteten, erstens, dass durch beide Modifikationen niedrigere Konzentrationen von Progesteron zur zweiten GnRH-Gabe und höhere Besamungserfolge erreicht werden als durch ein herkömmliches Ovsynchprotokoll und zweitens, dass Tiere, die das Ovsynchprotokoll ohne Gelbkörper beginnen, besonders von der zweimaligen PGF2α-Gabe profitieren. Dazu wurden zwei kontrollierte, randomisierte Feldversuche mit einem vergleichbaren Studiendesign auf insgesamt acht Milchviehbetrieben in Brandenburg durchgeführt. Im wöchentlich rotierenden Rhythmus wurden laktierende Milchkühe in eines von drei Behandlungsprotokollen aufgenommen. 1) Ovsynch (Kontrolle: Tag 0 GnRH – Tag 7 500 µg PGF2α – Tag 9 GnRH). 2) Ovsynch mit einer doppelten Dosis PGF2α (GDPG: Tag 0 GnRH – Tag 7 1.000 µg PGF2α – Tag 9 GnRH). 3) Ovsynch mit einer zweimaligen PGF2α Gabe (GPPG: Tag 0 GnRH – Tag 7 500 µg PGF2α – Tag 8 500 µg PGF2α – Tag 9 GnRH). Alle Kühe wurden 16 h nach der zweiten GnRH-Gabe künstlich besamt. In Experiment 1 wurden 1.979 Kühe von sechs Milchviehbetrieben aufgenommen. Bei jedem Tier wurde an Tag 0 eine transrektale sonographische Untersuchung durchgeführt und die An- oder Abwesenheit eines Gelbkörpers dokumentiert. In Experiment 2 wurden 1.581 Kühe von zwei Milchviehbetrieben aufgenommen. Von einer Subpopulation von Kühen aus Experiment 2 (n = 491) wurden Blutproben zum Zeitpunkt der zweiten GnRH-Gabe entnommen, um die Konzentration von Progesteron zu bestimmen. In Experiment 1 hatte das Behandlungsprotokoll einen signifikanten Einfluss auf den Besamungserfolg (P = 0,03). Der Anteil tragender Tiere war bei den zweimalig mit PGF2α behandelten Tieren (38,0 %) größer als bei den mit der doppelten Dosis behandelten Tieren (31,9 %; P = 0,04) oder den Tieren der Kontrollgruppe (31,2 %; P = 0,02). Der Besamungserfolg bei Tieren der Kontrollgruppe und bei Tieren der GDPG Gruppe unterschied sich nicht signifikant (P = 0,80). Der Besamungserfolg wurde von der Anwesenheit eines Gelbkörpers (P = 0,55) nicht beeinflusst. Auch war keine Interaktion zwischen dem Effekt der Behandlung und der Anwesenheit eines Gelbkörpers zur ersten GnRH-Gabe feststellbar. In Experiment 2 war der Anteil tragender Tiere bei GPPG Tieren (40,6 %; P < 0,01) und bei GDPG Tieren (38,1 %; P = 0,02) größer als bei Tieren der Kontrollgruppe (31,0 %). Zwischen GDPG und GPPG Kühen unterschied sich der Besamungserfolg nicht (P = 0,45). Im Vergleich zur Kontrollgruppe erhöhte sowohl die zweimalige Gabe als auch die doppelte Dosis den Anteil an Kühen mit besonders niedrigen Konzentrationen von Progesteron (0,00 – 0,09 ng / ml) zur zweiten GnRH-Gabe. Die GPPG Behandlung reduzierte zudem den Anteil an Kühen mit besonders hohen Konzentrationen von Progesteron (≥ 0,6 ng / ml) zur zweiten GnRH-Gabe im Vergleich zur Kontrollgruppe. Da die beiden Experimente ein ähnliches Versuchsdesign hatten, wurden die Ergebnisse im Hinblick auf den Besamungserfolg kombiniert. Kühe, die wiederholt mit PGF2α behandelt wurden, hatten einen höheren Besamungserfolg (40,5 %; P < 0,01) als Kühe, die nur eine einfache Dosis PGF2α an Tag 7 erhielten (32,4 %) und hatten tendenziell auch einen höheren Besamungserfolg als Tiere, die eine doppelte Dosis an Tag 7 erhielten (36,3 %; P = 0,05). Tendenziell war der Besamungserfolg bei GDPG Kühen größer als bei Kühen der Kontrollgruppe (P = 0,06). Die Ergebnisse der beiden Experimente zeigten, dass bei einer zweiten PGF2α-Gabe an Tag 8 in einem 7 d-Ovsynchprotokoll der Besamungserfolg im Vergleich zu einem herkömmlichen 7 d-Ovsynchprotokoll gesteigert werden konnte. Die Verwendung der doppelten PGF2α-Dosis an Tag 7 erwies sich als weniger effektiv. Die Anwesenheit eines Gelbkörpers zur ersten GnRH-Gabe hatte keinen Einfluss auf den Behandlungseffekt. Eine Ultraschalluntersuchung an Tag 0 bietet damit keine Entscheidungshilfe im Stall hinsichtlich der Wahl eines der drei Behandlungsprotokolle.
View lessThe Internet of Things (IoT) interconnects many heterogeneous embedded devices either locally between each other, or globally with the Internet. These things are resource-constrained, e.g., powered by battery, and typically communicate via low-power and lossy wireless links. Communication needs to be secured and relies on crypto-operations that are often resource-intensive and in conflict with the device constraints. These challenging operational conditions on the cheapest hardware possible, the unreliable wireless transmission, and the need for protection against common threats of the inter-network, impose severe challenges to IoT networks. In this thesis, we advance the current state of the art in two dimensions. Part I assesses Information-centric networking (ICN) for the IoT, a network paradigm that promises enhanced reliability for data retrieval in constrained edge networks. ICN lacks a lower layer definition, which, however, is the key to enable device sleep cycles and exclusive wireless media access. This part of the thesis designs and evaluates an effective media access strategy for ICN to reduce the energy consumption and wireless interference on constrained IoT nodes. Part II examines the performance of hardware and software crypto-operations, executed on off-the-shelf IoT platforms. A novel system design enables the accessibility and auto-configuration of crypto-hardware through an operating system. One main focus is the generation of random numbers in the IoT. This part of the thesis further designs and evaluates Physical Unclonable Functions (PUFs) to provide novel randomness sources that generate highly unpredictable secrets, on low-cost devices that lack hardware-based security features. This thesis takes a practical view on the constrained IoT and is accompanied by real-world implementations and measurements. We contribute open source software, automation tools, a simulator, and reproducible measurement results from real IoT deployments using off-the-shelf hardware. The large-scale experiments in an open access testbed provide a direct starting point for future research.
View lessHintergrund: Sarkome treten im Erwachsenenalter sehr selten auf und sind gleichzeitig sehr heterogen in ihrem biologischen Verhalten. Systematische retrospektive Auswertungen sind darum von elementarer Bedeutung für das Erzielen eines besseren Krankheitsverständnisses und Grundlage der Therapieoptimierung bei Weichgewebssarkomen, welche das Thema der vorliegenden Habilitationsschrift ist. Methoden: Es werden fünf Originalarbeiten zusammengefasst und diskutiert, in welchen retrospektiv einfach zu erhebende klinische Charakteristika untersucht, die Ergebnisse multimodaler Therapiestrategien analysiert und die positiven Effekte supportiver Maßnahmen demonstriert wurden. Eine weitere Originalarbeit hat systematisch die verfügbare Evidenz zu hypofraktionierter Bestrahlung ausgewertet. Ergebnisse: Das mediane progressionsfreie Überleben bei älteren Patient:innen mit Tumoren der Ewingsarkom-Familie wird durch Komorbiditäten und das Tumorstadium beeinflusst. Bei intensivmedizinisch betreuten Sarkompatient:innen haben etablierte Sepsis- und Performance-Scores und allgemeine Charakteristika (Geschlecht, Tumorlokalisation, Therapieintention) prognostische Aussagekraft. Die Hinzunahme von Olaratumab zu Doxorubin führt nicht zu einer signifikanten Verbesserung des Überlebens. Durch die kombinierte Radiochemotherapie wird bei Patient:innen mit high grade lokalisierten Weichgewebssarkomen im Vergleich zur alleinigen Strahlentherapie sowohl die lokale Kontrolle als auch die Verhinderung von Fernmetastasen verbessert. Die präoperative hypofraktionierte Radiatio ist der normofaktionierten Bestrahlung nicht unterlegen und somit eine gute Behandlungsoption für Patient:innen mit Weichgewebssarkomen. Eine spezialisierte palliativmedizinische Intervention bei Patient:innen mit fortgeschrittenem Weichgewebssarkom führt zu einer signifikanten Abnahme der Symptomlast (Schmerzen, allgemeine Symptome und Stresslevel). Schlussfolgerungen: Die dargestellten Innovationen der Sarkomtherapie lassen sich drei unterschiedlichen Bereichen zuordnen. Beginnend bei der Identifizierung von in der klinischen Routine einfach zu erhebenden Biomarkern zur präziseren Patient:innenselektion werden Beispiele für die Optimierung der Therapie selbst gegeben: dazu gehören der Einsatz zielgerichteter Substanzen insbesondere im Rahmen multimodaler Strategien und relevante technische Fortschritte in der Strahlentherapie. Abschließend wird am Beispiel der Integration palliativmedizinischer Interventionen in die Tumorbehandlung die Bedeutung des frühzeitigen Einsatzes supportiver Maßnahmen gezeigt.
View lessAufgrund der deutlich ungünstigeren Prognose, die Patient:innen nach dem Erleiden eines Delirs haben, gewinnen Prävention und Therapie des Delirs in der klinischen Versorgung erhebliche Bedeutung. Patient:innen mit erhöhter Vulnerabilität können auf verschiedene Auslöser hin ein klinisches Bild eines Delirs entwickeln. Bei der Ätiologie des Delirs spielen sowohl das cholinerge als auch das adrenerge System sowie eine resultierende Neuroinflammation eine herausragende Rolle. Die vielfältigen Mechanismen, die zu einem Delir führen können, umfassen ein cholinerges Delir mit Acetylcholinmangel sowie ein adrenerges Delir, das durch Stress und Aktivierung des sympathischen Nervensystems verursacht wird. In der vorliegenden Habilitationsschrift wurden verschiedene Aspekte des postoperativen Delirs untersucht. Die CESARO-Studie beschrieb den perioperativen Verlauf der Cholinesteraseaktivitäten. Die Studie zur postoperativen Anämie zeigte, dass postoperative Anämie mit einem höheren Delirrisiko und längeren Krankenhausaufenthalten verbunden sein könnte. Die Untersuchung an Krebspatient:innen deutete darauf hin, dass die Belastung durch langfristige Medikamenteneinnahme von Wirkstoffen mit anticholinergen Eigenschaften ein unabhängiger Risikofaktor für postoperatives Delir sein könnte. Die Auswirkungen von Dexmedetomidin deuteten darauf hin, dass dadurch das Risiko für postoperatives Delir reduziert wird. Die sekundäre Analyse zur cholinergen Dysregulation durch Dexmedetomidin zeigte keine signifikanten Veränderungen der cholinergen Enzyme, legte jedoch nahe, dass Dexmedetomidin möglicherweise die cholinerge Dysregulation bei größeren chirurgischen Eingriffen blockiert. Es besteht die Notwendigkeit weiterer differenzierter Forschung, um die Mechanismen und Faktoren des postoperativen Delirs besser zu verstehen. Die Identifikation von Regulationsmechanismen könnte zu gezielten therapeutischen Ansätzen führen, um das Management des Delirs zu verbessern. Dazu sollten klinische Studien die Rolle des vegetativen Nervensystems im perioperativen Stressmanagement weiter untersuchen, um die Prävention und Behandlung des Delirs zu optimieren.
View lessThe aim of this study was to assess important management factors for calves. A) The effects of transdermal flunixin meglumine in combination with local anesthesia during disbudding on 1) plasma cortisol and 2) behavioral changes were evaluated. B) Various analytical methods (RID, ELISA) and on-site measurement methods (digital Brix refractometer, colostrometer, outflow funnel, semi-quantitative immunochromatography) were examined to determine colostrum quality. Additionally, the timing of disbudding was utilized to perform cortisol measurements in different media.
Group allocation: 1) Control group (CON, n = 27), 2) 1-Flunixin group (1-FLU, n = 26) with a single administration of transdermal flunixin meglumine during disbudding, and 3) 2-Flunixin group (2-FLU, n = 24) with two administrations of transdermal flunixin meglumine (during disbudding and a second dose 6 hours later). Sham disbudding was performed one week before disbudding using a cold cautery iron.
During disbudding, the average plasma cortisol concentration was 5.16 + 2.8 ng/mL (range: 0.2–26.4 ng/mL). Plasma cortisol concentration was influenced by treatment (P < 0.01) and sampling time (P < 0.01). Compared to CON, single treatment (1-FLU) showed a significant effect (P < 0.01; -1.29 ng/mL; 95% CI -2.15 to -0.44), as did the second dose (2-FLU; P < 0.01; -1.29 ng/mL; 95% CI: -2.14 to -0.43). The two groups did not differ significantly from each other (P = 0.99; 0.01 ng/mL; 95% CI: -0.84 to 0.84). Treatment influenced total lying time and lying duration per lying period during sham disbudding (P = 0.01) but had no effect during disbudding (P = 0.31).
Additionally, measurements of cortisol in different media (plasma, saliva, feces) were conducted. Cortisol and metabolites were compared. Saliva and fecal samples were collected concurrently with blood samples. The correlation between plasma cortisol and saliva in dairy calves was good (r = 0.73; P < 0.01). The correlation between plasma cortisol and FGM concentration was weak (6 hours: r = 0.24).
For colostrum assessment, radial immunodiffusion (RID) was considered the reference method. Colostrum was defined as high-quality if the IgG content of RID measurement was >50 mg IgG/mL. The average IgG concentration (±SD) was 101.3 ± 45.9 mg/mL. The correlation between RID was: r = 0.78 (ELISA), r = 0.79 (digital Brix refractometer), r = 0.58 (colostrometer: specific gravity), r = 0.61 (colostrometer: temperature-corrected), r = 0.26 (outflow funnel), and r = 0.43 (lateral flow assay). The optimal threshold for identifying high-quality colostrum with ELISA was 50.8 mg/mL, with a sensitivity of 91.3%, specificity of 92.3%, and an AUC of 0.94. The sensitivity of on-site tests varied from 95.7% (Brix refractometer) to 60.9% (lateral flow assay).
In conclusion, this study shows that 1) transdermal flunixin meglumine can decrease plasma cortisol concentration after disbudding, but 2) has no effect on average daily weight gain and total lying time. 3) ELISA is accurate and 4) the digital Brix refractometer and colostrometer were suitable for assessing colostrum quality.
View lessComputer simulations can give essential insights into the dynamics of biomolecular systems but raise significant big data challenges still to be sorted out. To overcome the challenge of large data sets combined with the complexity of biomolecular interactions, I implemented a set of robust algorithms, as part of this doctoral thesis, inspired by graph theory that allows us to use large data sets from atomistic molecular dynamics (MD) simulations and derive simple graphical representations of the hydrogen bond (H-bond) networks of lipid membrane models, proteins in different intermediate states, and of the response of the proteins to mutations. These representations are valuable for the interpretation of data from experiments and computations. Our algorithms facilitate highly efficient analyses of dynamic H-bond networks at the lipid membrane interface. We introduce the implementation of a Connected Components algorithm to cluster lipid molecules and a Depth First Search (DFS) algorithm that allows us to characterize the topology of dynamic H-bond clusters sampled by lipid headgroups in MD simulations. With the algorithm we developed, we identify the transient sampling of four main types of lipid H-bond clusters: linear, star, circular and extensive networks combining these topologies. Water bridges between lipid headgroups are dynamic with lifetimes lasting for a few picoseconds. Our algorithms are further extended to study conformational dynamics in proteins. An example is SecA, a protein motor that couples Adenosine triphosphate (ATP) binding and hydrolysis with the pre-protein substrate's translocation through the membrane embedded SecYEG protein translocon. However, the exact mechanism of SecA’s conformational coupling remains unclear. We present a methodology of applying graph-based approaches to characterize the dynamics of the SecA protein motor by computing long-distance H-bond pathways that inter-connect the nucleotide-binding pocket and the pre-protein binding site, shortest-distance routes and centrality measures that reveal amino acids with a central role in the total connectivity of the protein graph. A key finding enabled by the graph-based approach developed as part of this doctoral thesis is that mutations near the nucleotide-binding site associate with modified dynamics at the pre-protein binding domain. Water molecules participate in extended H-bonded water chains contributing to long-distance conformational coupling. Our methodologies are also applied to protein VASA, a DEAD-box enzyme involved in the cell cycle with ATP and Ribonucleic Acid (RNA) binding sites and explore the conformational coupling between the two binding sites and Channelrhodopsin’s C1C2 lipid-protein H-bond molecular dynamics. Lastly, our algorithms are applied to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2) protein S crystal structures. Protein S undergoes conformational changes and symmetry loss of core H-bonded clusters as it transitions from the closed to the pre-fusion conformation. Our study has identified N501 as a central residue of the H-bond network that interconnects the spike protein S to Angiotensin-Converting Enzyme 2 (ACE2), and that subsequently became mutated into TYR in a new COVID-19 variant.
View lessAllergic contact dermatitis (ACD) is one of the most common immunotoxicological disorders of the skin. ACD is provoked by exogenous contact allergens that are mistakenly identified as a danger by the immune system and orchestrated by a T cell-mediated adaptive immune response. However, before a symptomatic response occurs (elicitation), a clinically silent sensitization phase precedes. The sensitization phase is characterized by complex interactions of different cell types with dendritic cells (DC) playing a central role. DCs may take up complexes of chemically-modified self-proteins, process and present them on their surface for T cell recognition. Subsequently, they undergo a maturation process and migrate to lymph nodes to initiate the priming and proliferation of allergen-specific T cells. The DCs are thus of crucial importance for the development of contact allergy. Despite this central role, the exact cellular signaling pathways leading to allergen-induced maturation of dendritic cells are not yet fully understood. As the current knowledge is mainly based on transcriptome data, state of the art mass spectrometry-based proteomics are applied in this thesis to complement and extend existing data to enhance the understanding of ACD pathomechanisms. In this work, it was shown for the first time that proteomic methods are suitable to distinguish activation of monocyte-derived dendritic cells (MoDC) triggered by the contact allergen NiSO4 from the bacterial activator lipopolysaccharide (LPS). NiSO4 caused not only a different MoDC phenotype but also the activation of divergent signaling pathways. Both treatments induced metabolic reprogramming towards aerobic glycolysis, which, however, manifested over a longer period in the case of NiSO4 and eventually activated hypoxia-like signaling pathways such as hypoxia-inducible factor (HIF) 1α upregulation. NiSO4 treatment further elicited a pronounced nuclear factor erythroid 2-related factor (Nrf) 2-dependent stress response in MoDCs. Most striking was the selective upregulation of cholesterol biosynthesis after treatment with NiSO4 which was absent upon stimulation with LPS. Concomitantly, the cells exhibited a significant decrease in cellular cholesterol levels. Thus, this signaling pathway was identified as a promising approach in the search for predictive biomarker signatures for sensitization with contact allergens. The second study was designed to verify the above-mentioned results in another cell model. Additional allergens were included to reveal similarities or differences in the activation of DCs by the allergens. THP-1 cells, which are standardly used for the prediction of contact allergens in vitro, were chosen as the cell model. The allergens NiSO4, p-dinitrochlorobenzene (DNCB), p-benzoquinone (BQ), and p-nitrobenzyl bromide (NBB) induced comparable proteomic changes and differed mainly in magnitude of induced proteomic changes. DNCB and NBB were the most potent activators of the cells. In direct comparison with MoDCs, THP-1 cells showed a much more pronounced Nrf2-mediated stress response. Based on these results, the Nrf2-mediated stress response should also be implemented into future in vitro DC test strategies for the prediction of contact allergens. Allergen-treated THP-1 cells developed a proinflammatory phenotype, which was supported by significant upregulation of the citrate cycle. Cholesterol biosynthesis was also significantly regulated in these cells - but in opposing direction to the findings from MoDCs. Overall, the comparison of MoDCs and THP-1 cells revealed relevant differences in the regulation of many proteins and signaling pathways between the cell models, but still resulted in the activation of similar superordinate systemic endpoints. THP-1 cells are therefore limited in their representation of MoDCs as primary DCs model in vitro. The further development of already validated in vitro methods in THP-1 cells is should thus focus on proteins that are differentially regulated by contact allergens in both cell types.
View lessHarvestmen (Arachnida: Opiliones) represent the third most diverse order of arachnids following spiders (Araneae) and mites (Acari). Despite their modern species richness and importance for various ecosystems, fossils of this group are largely understudied. This also includes their evolutionary history, palaeobiogeography and palaeoecology. 156 harvestmen fossils were available for this thesis, which are mostly preserved as inclusions in Baltic (Late Eocene, Priabonian) and Burmese amber (mid-Cretaceous, Cenomanian-Albian). Besides the description of new species, the main focus was to answer two palaeobiogeographical hypotheses: Is the Burmese fauna of Laurasian or Gondwanan origin? Does the European amber complex sample a Holarctic fauna? The fossils were mainly examined with classical light microscopy but also with the help of micro-CT scans and then compared with their living relatives available in the literature and the collection of the Museum für Naturkunde, Berlin. 11 new fossil harvestmen species and 12 additional specimens from all four suborders (Laniatores, Eupnoi, Dyspnoi, Cyphophthalmi) are described in this thesis. Of particular interest is the discovery and description of Sirocellus iunctus (Cyphophthalmi) from Burmese amber, the first dwarf harvestmen which combines characters from the families Sironidae and Stylocellidae, and thus represents a “missing link” between these two families. Based on this fossil, previous hypotheses regarding a Gondwanan origin of the Burmese amber fauna could be reconstructed and confirmed. Tyrannobunus aculeus (Eupnoi) is the first fossil of its suborder from Burmese amber and stands out because of its unusual body characters. Large eyes, a small but strongly armed body, spined legs and pedipalps and an undifferentiated penis indicate an early origin in the Eupnoi tree of life. Its very thin cuticle shows additionally an adaptation to humid and warm tropical climates. Balticolasma wunderlichi represents the first fossil from the subfamily Ortholasmatinae (Dyspnoi) and was described as a male from the Baltic and as a female from the Ukrainian Rovno amber. These ortholasmatine harvestmen only occur in North-Central America and in parts of Asia these days. This record shows once again that some groups of harvestmen were once widespread throughout the Holarctic during the Eocene and subsequently became extinct due to decreasing temperatures, at least in Europe. The harvestmen fossils could be placed in the appropriate palaeoenvironmental context based on known reconstructions of the palaeoenvironment for the Burmese and European amber forests and on analogues to their extant relatives. The described fossil harvestmen species fit well in the proposed warm-temperate Baltic amber forests with a number of swamp and open habitats, and in the very humid and warm tropical Burmese amber forests.
All newly described fossils represent an important foundation for future phylogenetic studies, especially in the form of calibration points, which allows to calculate the origin of the individual groups more precisely. This in combination with the description of new fossils from other unexplored amber sources (e.g. Lebanese amber, African amber, Spanish amber) will further increase our knowledge of the evolution and palaeobiogeography of these animals.
View lessThe ability to flexibly adapt behavior in the light of changing contextual demands is crucial for successful goal pursuit. With age, children become increasingly able to flexibly switch between tasks but show poorer switching performance even in late childhood. Given the crucial role of cognitive flexibility in daily life, such as the ability to shift to a new strategy to solve a problem when the previous one did not work, studies have aimed to improve cognitive flexibility with training in children. A key question for the effectiveness of these interventions is understanding why children show lower cognitive flexibility than adults. In this dissertation, I shed new light on this question, by investigating 8–11-year-old children using behavioral measures in combination with measures of univariate activation, multivariate decoding, and task-related connectivity based on data from functional magnetic resonance imaging (fMRI). In Papers 1 & 2, I first addressed the question of which neural processes support the development of cognitive flexibility. In Paper 3, I examined how these neural processes change with training. I have prefaced the three papers with a synopsis in which I outline the theoretical framework of the dissertation and summarize the current empirical findings. Finally, I summarize the results of the empirical part of this dissertation and discuss their contribution to our understanding of the development of cognitive flexibility. Flexibly switching between tasks comes at a cost, evident in decreased accuracy and increased response times. Specifically, compared to performing single tasks in isolation, task switching poses greater demands on maintaining and managing multiple task sets, thus eliciting so-called mixing costs. Additionally, a switch to a different task compared to a repetition of the same one requires the inhibition of the previously relevant task set and updating the newly relevant one, resulting in so-called switch costs. Previous research has demonstrated that mixing and switch costs show different patterns of age differences with switch costs approaching adult levels earlier. The present dissertation builds on these findings to examine the neurocognitive processes contributing to the presumably protracted development of mixing costs. In Paper 1, comparing children (8–11 years) and adults (20–30 years), I examined how neural processes supporting sustained and transient control processes support age-related decreases in mixing and switch costs, respectively. I showed that while evident for both, age differences were greater for sustained activation and mixing costs than for transient activation and switch costs. Additionally, the results of Paper 1 outline a potential alternative mechanism via which children can address increased sustained control demands: children that showed a less adult-like sustained activation pattern but greater increases in connectivity performed better. Taken together, Paper 1 demonstrated that children managed increased sustained control demands during task switching in at least two ways, (1) increased activation in the brain regions also recruited by adults, or (2) increased connectivity with additional brain regions in the lateral prefrontal cortex (lPFC), thus potentially relying on additional metacontrol processes. One factor proposed to contribute to age-related improvements in task switching is an increasing ability to represent multiple rules and effectively update these when necessary. Using the same sample of children and adults, Paper 2 investigated this hypothesis using multivariate pattern analysis to elucidate the role of neural task-set representations on age differences in task switching. Results demonstrated that neural activation patterns on switch trials held less information regarding the currently relevant task than on repeat trials. Intriguingly, this switch-related reduction of task-set distinctiveness did not differ between children and adults, showing a striking level of maturity in the neural representation of task sets and raising the question which other mechanisms contributed to greater switch costs in late childhood. Building on the insights from Papers 1 and 2, Paper 3 explored how the neural and cognitive processes supporting the development of task switching changed in children that either trained intensive single tasking or intensive task switching over nine weeks. Using drift-diffusion models and fMRI data, I investigated how cognitive and neural processes during task switching changed with training. Faster accumulation of evidence as indicated by increased drift rates, along with decreased activation in the lPFC suggested more efficient rule processing with intensive task-switching training. The accompanying changes in boundary separation further suggested strategy changes, such that children may have allocated cognitive control resources differently, potentially because of improvements in monitoring task demands, enabling them to match their performance accordingly. Taken together, the empirical findings of this dissertation converge to reveal a consistent picture of increasingly more refined recruitment of frontoparietal brain regions, in particular the lPFC, both with age and with intensive training during childhood. They thus raise questions on the role of hierarchical cognitive control and metacontrol processes for developmental improvements of cognitive flexibility. The novel insights into task switching presented in this dissertation thus further our understanding of development and learning in cognitive control, and cognition more generally.
View lessNanoelectromechanical systems (NEMS) based on two-dimensional (2D) materials represent the ultimate – atomicscale – size limit for the miniaturization of mechanical devices. When employed as resonators, these devices bring a range of remarkable features. Due to their small effective mass, they oscillate at very high frequencies (up to gigahertz). Furthermore, it is easy to drive them in their non-linear regime. Additionally, they come with the exceptional ability to broadly tune their resonance frequencies. In this thesis, I realize two unique systems capitalizing on these properties. First, I conceptualize a tunable phononic crystal made from graphene. By periodically patterning a suspended graphene membrane, I transform it into the world’s thinnest possible phononic crystal. This device features a phononic band gap in the megahertz range, which we can broadly tune using an electrostatic gating approach. Under electrostatic pressure up to 30 kPa, we observe a frequency upshift of the entire phononic system by 350 %. In the next step, we demonstrate full control of the band gap size. By carefully tension engineering our suspended phononic crystal, we are able to dynamically reduce the size of the phononic band gap – down to completely closing it. This change in hierarchy/topology in the phononic band structure can be seen as the mechanical simulation of a metal-insulator transition (mMIT). Upon placing an artificial irregularity in our phononic lattice, we can spatially localize a mechanical ‘defect mode’. This mode is mechanically isolated from its environment, which makes it a highly coherent mechanical oscillator and a potential reservoir for storing quantum information. By taking advantage of our tunable system, we can now control the frequency and the degree of localization of the defect mode, which allows us to dynamically couple it to external excitations. Finally, we make use of the mMIT by constructing and studying a phononic transistor analog, which is controlled by simply applying a gate voltage and has an on/off ratio of 10^5 (100 dB). Second, I develop a platform for nanomechanical spectroscopy (NMS) of 2D materials. Specifically, I show that a purely mechanical measurement can be used as an ultrasensitive spectroscopic probe for transition metal dichalcogenides (TMDs) and plasmonic nanostructures. We extract the optical absorption of a 2D material from frequency shifts of a hybrid NEMS resonator vs. the wavelength of incoming light. In combination with optical reflectivity data, we derive — without any further assumptions—the full dielectric function of the material under study. Our measurement is fast (𝜏_rise = 135 ns), sensitive (noise-equivalent power: 𝑁𝐸𝑃 = 890 fW/ √ Hz) and we can characterize 2D materials in a broad spectral range of 1.2 – 3.1 eV. This method is a direct measurement of absorption, which has practical advantages compared to common optical spectroscopy approaches. Furthermore, while being comparable in performance, it overcomes limitations inherent to conventional optical methods, such as the complications associated with measurements at high magnetic fields and low temperatures. Summarizing, I developed a concept to add tunability to the field of phononics and an approach to spectroscopically characterize 2D materials based on mechanical measurements. These are powerful tools to realize tunable condensed matter physics analogs in phononic systems and to observe hitherto undetected phenomena in 2D materials.
View lessIn der vorliegenden Habilitationsschrift wurden spinopelvine Interaktionen unter dem Aspekt der funktionellen Implantatpositionierung und aktivitätenspezifische Implantatbelastungen untersucht. Übergeordnetes Ziel der Arbeit war es, durch ein grundlegendes Verständnis der Implantatbelastung und der spinopelvinen Wechselwirkung auf die Hüfttotalendoprothese präventive Strategien zur Reduktion von aseptischen Lockerungen und Protheseninstabilitäten zu erarbeiten.
View lessThis manuscript represents a comprehensive analysis of non-finite causal constructions in English, German, Dutch, and Czech. Based on a corpus of social media posts, the study provides an analysis of the formal and functional aspects of these constructions, their development, and their cross-linguistic similarities and differences. The study follows the principles of (Diasystematic) Construction Grammar. Formally, these constructions differ from both causal clauses and causal prepositional constructions. In contrast to the former, the complement of non-finite causal constructions must be non-finite. In contrast to the latter, however, the complement slot can be filled by a wider range of elements than just noun phrases. Elliptical clauses, non-elliptical noun phrases, or non-elliptical non-noun phrases can fill the complement slot of non-finite causal constructions. Functionally, non-finite causal constructions express a causal link between a matrix clause, which they follow, and the element in their complement slot. In this regard, these constructions overlap with both causal clauses and prepositional constructions. However, non-finite causal constructions can also serve to express a comment about the causal link. The development of non-finite causal constructions cross-linguistically follows a uniform spiral pathway. Elliptical non-finite causal constructions develop in the first step out of non-elliptical causal clauses. Elliptical non-finite causal constructions subsequently give rise to their non-elliptical variants. Against the backdrop of these empirical observations, the study draws theoretical conclusions regarding the relationship between linguistic data and their intepretation, linguistic categories and categorisation, and questions of language contact.
View lessErgebnisse zu Pathomechanismen, klinischem Management und Therapie von mastzell-vermittelten Erkrankungen der Haut und deren Differentialdiagnosen werden in vorliegender Arbeit dargestellt und diskutiert.
The central aim of this thesis was to visualize changes in cellular architecture induced by environmental factors such as drug treatment or viral infection. High-resolution microscopic analysis of such alterations is crucial to elucidate the mechanisms of disease, as well as to develop potential therapies. The importance thereof was clearly underscored by the COVID 19 pandemic, whose causative agent, the novel severe acute respiratory syndrome coronavirus 2 (SARS CoV 2), infected and killed millions of people across the globe. By using high-resolution 3D imaging by cryo soft X ray tomography (cryo SXT), rapid changes in the cellular ultrastructure upon infection with SARS CoV 2 or feline infectious peritonitis virus (FIPV) could be visualized. The changes induced by viral infection were affected by treatment with FDA-approved drugs, which had previously emerged as candidates from drug-repurposing screens to combat SARS CoV 2. Cryo SXT analysis revealed formation of lysosome-associated dark-rimmed vesicles (DRVs), which were demonstrated to be multilamellar lipid deposits by transmission electron microscopy. The observed striking interplay of drug- and virus-induced alterations on the level of size and number of lysosome-associated DRVs suggests involvement of lysosomal function in the inhibition of the viruses by the drugs. It is likely that this inhibition is either due to impairment of lysosomal escape or due to reduced lipid availability for membrane remodeling essential for virus replication. To comprehensively test these models, complementary screening tools are required, which allow ultrastructural analysis at a higher throughput than cryo SXT. For that, the recently developed ultrastructure expansion microscopy (U ExM) is a promising technique. Comparing the structural preservation of cell organelles after the U ExM sample preparation to that of cells acquired under near-native conditions in cryo SXT, it emerged that the morphology of large organelles such as nuclei and plasma membrane can be visualized efficiently by U ExM, while very small or lipid-dense structures were more difficult to preserve accurately. These results highlight the benefits of cryo SXT, but also the potential of U ExM to complement cryo SXT with its higher throughput of ultrastructure visualization, even if its resolution is lower than that of cryo SXT. Therefore, U ExM can be used for experimental characterization for cryo SXT, as one step towards more efficiency of cryo SXT. Another step is to automate the segmentation of cryo SXT tomograms. To that end, a deep learning platform was trained and validated on a large pool of cryo SXT data acquired in this work. This convoluted neural network performs full annotation of cellular features in cryo SXT tomograms within less than half an hour on consumer-grade GPUs, thereby significantly reducing the time required for data analysis. Taken together, these results illustrate how cryo SXT, U ExM and deep learning can be used complementarily to address highly relevant biological questions.
View lessStudien zu Robinet Testard – Zusammenfassung:
Robinet Testard ist ein Buchmaler des ausgehenden 15. Jahrhunderts, der von der Forschung im Westen Frankreichs angesiedelt wird und der einen großen Teil seiner Werke im Rang eines „Valet de chambre“ für Charles d’Angoulême und dessen Frau Louise de Savoie illuminiert hat. Aufgrund charakteristischer Stilmerkmale können ihm aktuell 33 Handschriften eindeutig zugeordnet werden. Neben Büchern zum liturgischen Gebrauch hat er ein breites Spektrum an profanen Werken gestaltet, die in der Regel zwischen 1470 und 1500 datiert werden, obwohl er als Person bis zum Jahr 1531 dokumentiert ist. Ausgehend von Testards Hauptwerk, dem Stundenbuch des Charles d'Angoulême (Paris, BnF, Ms. lat. 1173), wurde in der vorliegenden Arbeit mit Blick auf das Gesamtwerk eine Auswahl seiner illuminierten Handschriften unter stilkritischen Gesichtspunkten untersucht. Eine wiederkehrende Fragestellung befasste sich mit der Verwendung druckgrafischer Quellen, die zu dieser Zeit in Frankreich nur sehr selten zu finden ist. Es konnte belegt werden, dass Testard neben deutschen und niederländischen Kupferstichen ausgiebig Gebrauch von Italienischen Vorlagen macht und dass er dieses Material in weitaus größerem Umfang nutzt, als bisher angenommen. Zahlreiche Kunstwerke anderer Gattungen dienen ihm ebenfalls als Vorlage für seine Kompositionen. Die Analyse werkimmanenter Qualitätsunterschiede, die in manchen Arbeiten deutlich zutage treten, lässt auf Testards verantwortliche Stellung in einer eigenen Werkstatt schließen. Mit Blick auf seine Position am Fürstenhof ist es wahrscheinlich, dass er nach dem Tod von Charles d'Angoulême für Louise de Savoie auch in Amboise tätig war, sein Schaffenszeitraum lässt sich bis um 1515 erweitern. Vor seiner Anstellung als Hofmaler hat er an mehreren Handschriften in Poitiers mitgewirkt, seine Hand lässt sich deutlich von den anderen Buchmalern, dem Maler von Walters 222 und dem Maler des Yvon du Fou, unterscheiden. An der Zuweisung der Bildthemen läßt sich eine steigende Wertschätzung seiner Fähigkeiten ablesen. Verschiedentlich deuten sich Bezüge zur Werkstatt des Malers der Échevinage de Rouen an. Im Hinblick auf die Frage nach Testards künstlerischem Ursprung wurden einige seiner frühen Handschriften behandelt. Die bislang angenommene Beziehung zum Maler von Poitiers 30 kann im Vergleich nicht untermauert werden. Dagegen lassen sich zahlreiche Verbindungen zum Maler des Genfer Boccaccio aufzeigen. Unter zeitlichen und regionalen Aspekten ließen sich Gruppierungen der Handschriften vornehmen, die eine differenziertere Sicht auf Testards Stil und sein künstlerisches Bewußtsein eröffnen. Außerdem konnten dem Künstler im Verlauf der Untersuchung zwei weitere Handschriften zugeschrieben werden. Auf der Grundlage dieser Ergebnisse werden eine Neuordnung seines Werkes und Ansätze für die weitere Forschung vorgeschlagen.
View lessMajor Depression Disorder (MDD) is often accompanied by cognitive impairments, including concentration problems and attention deficits. These issues are related to the construct of working memory (WM). Additionally, a reduction in hippocampal volume is frequently observed in Major Depression. There is substantial evidence suggesting that physical exercise training can have positive effects on depressive and cognitive symptoms in MDD. This dissertation aims to integrate these areas of study to investigate the positive effects of physical fitness and exercise on WM in MDD patients, leading to a better understanding of the pathophysiology of the disease and its treatment through physical exercise training. The dissertation comprises three empirical studies that are part of the SPeED study (Sport/Exercise Therapy and Psychotherapy – evaluating treatment Effects in Depressive patients). In Study I (Heinzel et al. 2022), we examine whether a prior exercise intervention enhances the success of subsequent cognitive-behavioral therapy (CBT) and whether this effect is associated with specific physiological changes. Study II (Schwefel et al. 2023) analyzes neural activity and physical fitness in depressive patients during a WM task. Study III (Schwefel et al., sub) focuses on functional and structural neural changes following physical exercise training, with particular emphasis on the hippocampus. The n-back paradigm was used to measure WM function during functional magnetic resonance imaging (fMRI). The physical exercise intervention lasted 12 weeks and was supervised by sports therapists. The results indicate that physical fitness can be improved through training, and surprisingly, depressive symptoms improved in all groups. However, high exercise intensity did not lead to a general boosting effect for CBT. Nonetheless, regression analyses revealed that improvement of individual fitness predicted the success of CBT. MDD patients exhibited a specific activation pattern in frontoparietal brain regions, associated with longer reaction times and poorer performance during high demands in WM tasks compared to healthy controls. Additionally, a parietal fitness correlate was identified in the depressive sample. Improved performance and shorter reaction times were observed after the training intervention, particularly during high demands in the WM tasks. Furthermore, in patients who underwent intensive training, an increased activation of the hippocampus was observed as a result of the training. No structural changes in hippocampal volume were detected. The findings suggest that physical training holds promise as a treatment option for improving WM function in MDD patients. These insights may serve as a foundation for future research on the effects of physical fitness and exercise on mental health and cognition, offering valuable supplements to optimized physical exercise therapies for the treatment of MDD.
View lessFreshwater ecosystems, including rivers, lakes and wetlands, provide critical habitats for diverse species and support essential ecosystem services for human well-being. Meanwhile, they are under growing threats from various sources, including overexploitation, habitat degradation, flow modification, water pollution and invasive species. Consequently, monitored populations of freshwater vertebrates showed an average decline of 83% from 1970 to 2016. Freshwater megafauna (i.e., animals that spend a crucial part of their life cycle in freshwater or brackish ecosystems and have a maximum reported body mass of 30 kg) are particularly susceptible to anthropogenic impacts. Previous studies mainly focus on the impacts of overexploitation and dam construction on these large animals. The impacts of alien species on freshwater megafauna have been largely overlooked. While impacts of alien species on native freshwater megafauna are documented, many freshwater megafauna species, such as sturgeons, Asian carps, American beaver, hippo, crocodilians and the Chinese giant salamander, have been introduced outside of their native distributions. However, their impacts on native species and human well-being in the introduced regions have yet to be systematically investigated at a global scale. This thesis aims to provide a comprehensive understanding of alien species impacts related to freshwater megafauna. I used Environmental Impact Classification for Alien Taxa (EICAT) framework to assess the environmental impacts of alien species on native freshwater megafauna. Then I took a different angle and focused on environmental impacts of alien freshwater megafauna, using megafish as an example and considering both negative and positive aspects with EICAT and EICAT+ frameworks. Moreover, I adapted the approach of nature’s contributions to people (NCP) and assessed the beneficial and detrimental impacts on humans by alien freshwater megafauna. Freshwater megafauna have been affected by a wide array of alien species group from freshwater and terrestrial ecosystems, including both vertebrates and invertebrates. Alien species impact native freshwater megafauna through mechanisms such as predation, competition and hybridization, leading to declines in individual performance and population abundance, or even local extinction of native freshwater megafauna. In addition, native freshwater megafauna showed distinct susceptibility to alien-species impacts between life-cycle stages. Meanwhile, almost half of the 134 extant freshwater megafish species have been introduced to new freshwater ecosystems, with almost 70% of the introduced species established self-sustaining alien populations. These alien megafish caused negative impacts through nine different mechanisms. Predation is the most frequently reported mechanism, followed by herbivory and competition. More than half of the alien megafish species that have sufficient data for assessing impact magnitudes caused population declines of native species, or even species extirpation. A broad range of beneficial NCP categories have been documented for 59 alien freshwater megafauna species in 430 records, with food supply being the most frequently reported category (58%), followed by physical and psychological experiences (20%) and materials and companionship (12%). Much fewer records (154) were identified for detrimental NCP associated with 25 alien freshwater megafauna species, covering four categories including reduced food resources, damage to properties, reduced physical and psychological experiences and risk to health and safety. This thesis emphasizes the vulnerability of native freshwater megafauna to alien species and demonstrates the profound environmental and socio-economic impacts of alien freshwater megafauna. Additionally, it highlights gaps in long-term monitoring and bias in geographical and taxonomic coverage. Long-term monitoring studies are deemed critical for a comprehensive assessment of alien species impacts, given that short-term studies may underestimate the potentially severe, population-level effects. Furthermore, there is an urgent need for monitoring the introduction and assessing impacts of alien species in the Global South. Considering the high economic values of freshwater megafauna due to their use in aquaculture, recreational fishing and pet trade, it is anticipated that more species will be introduced and established outside of their native ranges. Strict biosecurity requirements, mandatory risk assessments and management plans should be implemented when introducing alien freshwater megafauna for activities These measures will help reduce the risk of escapes or releases into natural waterbodies and help safeguard freshwater biodiversity and human well-being.
View lessCompartmentalization is one of the decisive characteristics of cellular life, enabling the cell to build a complex network of enzymatic reactions and metabolic pathways. Drivers of this cellular management are organelles and the vesicles and tubular structures that connect them, all of which are separated from the cytoplasm by a lipid membrane. Intracellular transport, especially secretion to the extracellular space or to the cell membrane, is a highly regulated and organized process. In this work, we focused on the secretion of big protein cargo like chylomicrons and collagens. We aimed to analyze the role of alternative splicing as a regulation mechanism for the secretion of large cargo. Alternative splicing is a mechanism in gene expression where different combinations of exons are included or excluded from mRNA transcripts, leading to the production of multiple protein isoforms from a single gene. We comprehensively analyzed RNA sequencing data from human tissues as well as B-lymphocytes. We found a previously uncharacterized exon in the gene SEC31A that codes for the outer layer of coat protein complex II (COPII), a complex responsible for the transport from the endoplasmic reticulum (ER) to the Golgi-apparatus. Using a correlation analysis with the inclusion levels of the exon and gene expression throughout the human tissue data, we found leads to the functionality and regulation of this alternatively spliced exon. We could show that the inclusion of the exon enhances the transport of lipids in polar differentiated Caco-2 cells. Mini gene experiments demonstrated that the inclusion is regulated by the splicing factor RBM47. Finally, AlphaFold structure prediction with the resulting alternative protein isoforms revealed a change in interaction with COPII partner SEC23A. We also investigated a previously characterized alternative splice site in SEC31A, which leads to a shortened exon. This shortened exon decreased the secretion efficiency of glycosylphosphatidylinositol (GPI)-anchored and e-cadherin cargo in the RUSH assay but enhanced collagen secretion. In a fluorescent recovery after photobleaching (FRAP) experiment, we observed a reduced mobile fraction of COPII puncta with the shortened exon. Lastly, we analyzed RNA sequencing data of differentiating B-lymphocytes as a model for the regulatory effect of the transcriptome on intracellular trafficking and secretion. B-lymphocytes show rather extensive changes in gene expression during their change from memory cells, to plasmablasts, and plasma cells, but alternative splicing changes are most abundant in memory cells. We found multiple splice events, which are unique to memory cells, particularly in PICALM and KLHL12, which could potentially have a significant impact on the function of the proteins. In summary, we were able to showcase the mechanism by which the structure and function of COPII is altered via alternative splicing of SEC31A, and we demonstrated similar mechanisms in B-lymhocytes. With these results, we depended our knowledge of the role that alternative splicing has on secretory specificity and regulation of large cargo secretion.
View lessThe artificial tick feeding systems (ATFS) offer a promising alternative to animal experiments. They can be used for studies of tick biology and physiology, for experiments investigating the tick microbiome, novel tick control strategies and the tick-(microbiome)-pathogen interface as well as to facilitate tick rearing. At a time where globalization and global warming is affecting the prevalence, distribution and host encounters of ticks, research regarding ticks and tick-borne diseases is particularly important. The junior research group “Tick-borne Zoonoses” was founded to develop innovative and practical approaches to studying the vector biology of Europe’s most common hard tick species, Ixodes ricinus. This PhD project specifically focused on the use of ATFS for feeding of I. ricinus ticks. Chapter 1 provides an introduction to the biology and relevance of I. ricinus in Europe, as well as delving into an explanation of the different components and applications of ATFS. In Chapter 2, results from our first study are used to compare and contrast tick feeding of multiple consecutive life stages of I. ricinus between the in vitro feeding by ATFS and in vivo on live cattle. Findings showed that artificially fed ticks were generally inferior to ticks fed on live cattle and all life stages showed significantly longer feeding durations. However, in larvae, higher engorgement and molting proportions indicated that the ATFS was more effective than in vivo feeding. Further, the feeding and fecundity parameters for F1 adults improved after B vitamin supplementation and a water bath system. The prolonged feeding durations commonly observed in the ATFS are associated with an increased risk of contamination, even if the blood meal is supplemented with the antibiotic gentamicin. However, both bacterial contamination and antibiotic treatment have been linked to negative effects on feeding success. To further investigate the effects of using gentamicin in the ATFS on the tick microbiome, ticks were consecutively fed on cattle blood with and without gentamicin in an ATFS. A multimethod approach involving amplicon sequencing of 16S rRNA and further quantification of common bacteria using qPCR was used. Chapter 3 describes the results and compares the findings to ticks fed on live cattle. Despite facing challenges around the extraction and sequencing of DNA from individual ticks, we were able to show that in female ticks fed on live cattle the dominant symbiont was Candidatus Midichloria mitochondrii. In female ticks fed in the ATFS using a gentamicin-treated blood meal, the most abundant bacterium was Rickettsia helvetica. Hence, we were able to deduce that both fecundity and microbial composition are likely to be influenced by the ATFS. These findings should be taken into account for future studies using the ATFS.
View lessFluorine is almost excluded from the biotic world. Although largely ignored by nature, a life based on fluorine is both an interesting concept and an absolutely conceivable scenario. Fluorine-containing building blocks have been extensively used to investigate and modify proteins and their interactions. However, the adaptation of living organisms represents a step forward in exploring and understanding the consequences of global fluorination. Hence, a reliable platform and methodologies for the experimental evolution of novel organisms with fluorine as a bioelement were established, created by mimicking natural selection in the laboratory. In this study, adaptive laboratory evolution (ALE) was used to force Tryptophan-auxotrophic Escherichia coli bacteria to fully adapt metabolically to grow and proliferate indefinitely on 6-fluoroindole (6Fi) and 7-fluoroindole (7Fi) as substitutes for indole (Ind). During long-term cultivation, fluorinated indole precursors were supplied, in situ converted into the corresponding fluorinated amino acids, and 6-fluorotryptophan (6FTrp) and 7-fluorotryptophan (7FTrp) were incorporated into the proteome in response to the UGG codon. As consequence of the imposed selection pressure, the cells relinquished their dependence on canonical Trp and instead acquired the ability to use either of the fluorinated counterparts as an integrated part of their metabolism. In the course of this process, five independent descendant E. coli evolved that have converted their entire lifestyle to exist on these unnatural molecules. 6FTrp adaptation revealed to be superior to 7FTrp adaption. While in 6FTrp even tendencies of an indole-rejecting phenotype were observed, on the contrary, the basic adaptation to 7FTrp required extraordinary perseverance. The effect of extensive fluorination and how living organisms cope with it was investigated through comprehensive analyses of genomic, proteomic, and metabolomic levels. One major strategy found involves mitigation of the stress response system, ignoring detrimental effects caused by global fluorine integration. This study is a further step and establishes a strong foundation for further exploration of the mechanisms underlying fluorine-based life and how a former stressor (fluorinated indole) becomes a vital nutrient.
View less