Nicotinamide adenine dinucleotide (NAD+) is the substrate used for the introduction of the ubiquitous and highly dynamic PTM in which either one or multiple adenosine diphosphate ribose (ADPr)... Show moreNicotinamide adenine dinucleotide (NAD+) is the substrate used for the introduction of the ubiquitous and highly dynamic PTM in which either one or multiple adenosine diphosphate ribose (ADPr) moieties are covalently attached to a nucleophilic side chain of an specific amino acid in the target protein to regulate cellular pathways including adipogenesis, DNA damage repair and gene expression. A significant fraction of the nucleophilic amino acid functionalities, most recently histidine and tyrosine, have been identified as ADPr-acceptor sites. In this thesis, new methodologies have been developed to synthesize peptide fragments carrying an ADPr modification to investigate ADP-ribosylation on histidine. Show less
Diacylglycerol lipases (DAGLα and DAGLβ) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain and peripheral tissues. Selective DAGLβ inhibitors... Show moreDiacylglycerol lipases (DAGLα and DAGLβ) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the brain and peripheral tissues. Selective DAGLβ inhibitors have been proposed as a potential treatment for inflammatory diseases with reduced potential for central nervous system (CNS) mediated side effects, but they are currently lacking. To develop DAGLβ selective inhibitors, a fluorescent biochemical assay was optimized and applied in a high-throughput screening (HTS) for DAGLβ. During the HTS, eight hits classified into four distinct chemotypes were identified. Subsequent structure-activity relationship (SAR) studies, focusing on hit 1 and its modifications, revealed a specific group as the modification hotspot crucial for achieving selectivity towards DAGLβ. Through an extensive SAR investigation, focusing on modifying this group, the first-in-class DAGLβ selective inhibitors, LEI-130 and LEI-131, were discovered. Following their discovery, LEI-130 and LEI-131 underwent comprehensive in vitro and in situ profiling studies. These investigations confirmed that LEI-130 and LEI-131 are selective and noncompetitive inhibitors of DAGLβ, effectively reducing inflammation. Show less
In this thesis, I focused on studying the above- and belowground interactions of J. vulgaris from a plant-soil feedback (hereafter, PSF) perspective. I investigated the temporal variation of... Show moreIn this thesis, I focused on studying the above- and belowground interactions of J. vulgaris from a plant-soil feedback (hereafter, PSF) perspective. I investigated the temporal variation of negative PSF and examined the effects of root-associated bacteria on plant performance and aboveground herbivores. Additionally, I tested the role of PSF in relation to plant population structure and the significance of soil legacy effects in natural conditions. The findings reveal that temporal dynamics in PSF are driven by changes in plant sensitivity and in the soil microbiome. Although bacteria isolated from J. vulgaris roots can negatively affect plant performance, they can also affect aboveground herbivores and other plant species. Consequently, these bacteria may not be suitable for biological control of J. vulgaris. Moreover, I discovered that soil nematodes can mediate plant-plant interactions, but often favoring J. vulgaris. In my field work, I detected soil legacy effects, but seedling recruitment spatial patterns of J. vulgaris were not soil-mediated. The insights gained from studying PSF and above- and belowground interactions have the potential to reshape traditional approaches employed in controlling invasive plants. This thesis emphasizes the importance of transitioning PSF experiments from indoor to outdoor settings considering various influencing factors simultaneously. Show less
Human vocal communication and music perception represent advanced cognitive skills, seemingly innate and universal. These faculties encompass a range of perceptual and cognitive abilities. Cross... Show moreHuman vocal communication and music perception represent advanced cognitive skills, seemingly innate and universal. These faculties encompass a range of perceptual and cognitive abilities. Cross-species research sheds light on the origins of musicality by investigating whether these traits are shared by nonhuman species. Songbirds, notably zebra finches, serve as valuable models due to their complex vocalizations and similarities to humans in auditory perception. My thesis explored zebra finches' sensitivity to spectral and temporal sound features. Chapter 2 examines the influence of song duration and spectral characteristics on song discrimination, while Chapter 3 tests song preferences. Chapter 4 investigates sequential and spectral feature recognition. Chapter 5 focuses on melody recognition. Zebra finches demonstrate perceptual flexibility, adapting focus based on stimulus characteristics. These findings underscore the importance of training conditions and stimulus nature in shaping auditory perception. Overall, my thesis enhances understanding of auditory cognition and cognitive flexibility among songbirds. Show less
Utilizing the polymeric platform of polypept(o)ides, this thesis describes synthesis and investigation of novel triblock copolymers to obtain carrier systems with multiple compartments for... Show moreUtilizing the polymeric platform of polypept(o)ides, this thesis describes synthesis and investigation of novel triblock copolymers to obtain carrier systems with multiple compartments for efficient siRNA delivery. Although the individual microstructure of nanoparticles differs depending on the polymeric building blocks, desired application and cargo, the final nanoparticles always combine a polysarcosine (pSar) shell with a polypeptide core, providing the ability of siRNA complexation by a polycationic segment. In addition, a third block enabled either covalent cross-linking, hydrophobic / π- π-stacking mediated stabilization or co-encapsulation of small hydrophobic drugs. Broadening the structural variety of such polypept(o)ides, a novel synthetic procedure was introduced to access AA'B- and ABC-type miktoarm star polymers.Investigations have been dedicated to the design of novel polymeric structures based on polypept(o)ides, to improve the delivery of siRNA by Polyion Complex Micelles (PICMs), provide access to different polymeric architectures, and to establish novel synthetic methods for the synthesis of these materials. Covering aspects from the synthesis of novel polymeric species up to advanced drug delivery strategies for siRNA in vivo, developments throughout this thesis extent the accessibility of the polypept(o)ide platform for nucleic acid delivery, highlight their potential in nanomedicine and further elaborate delivery strategies for next-generation nanomedical applications. Show less
The outbreaks of AIDS and COVID-19 showed clearly how infectious viruses can influence people’s lives. Investigating the changes in the host metabolism may provide a paradigm shift to consider... Show moreThe outbreaks of AIDS and COVID-19 showed clearly how infectious viruses can influence people’s lives. Investigating the changes in the host metabolism may provide a paradigm shift to consider immune-metabolic interactions as therapeutic targets. The aim of this thesis is to examine the interplay between the immune system and metabolism during viral infections, such as HIV and coronavirus. These investigations will utilize metabolomic and lipidomic mass spectrometry techniques to gain a comprehensive understanding of the metabolic changes that occur during viral infections. To enhance the coverage of the lipidome, a new method will be developed. Show less
In the current global context, there is a pressing need to address sustainable energy supplies to safeguard our Planet and its ecosystems. The choices made by human society have a significant... Show moreIn the current global context, there is a pressing need to address sustainable energy supplies to safeguard our Planet and its ecosystems. The choices made by human society have a significant impact on genetic evolution and climate. To ensure a better future for all, it is crucial to exercise foresight, foster collaboration across various sectors, and reach agreements based on fair and ethical principles. Science plays a pivotal role in advancing energy conversion, offering the potential for significant scientific breakthroughs that contribute to the protection and respect of our World. Specifically, the development of solar-to-fuel devices holds promise for achieving this transition to green energy. This Ph.D. dissertation centers on the development and functionalization of 2D membranes and materials, which constitute integral components of these conversion devices. The optimization of functionalized 2D materials necessitates a comprehensive computational design approach. This involves the adoption of a multiscale computational framework for the thorough design of these materials and the precise prediction and understanding of molecular processes, encompassing molecular self-assembly, ion transport, and catalytic surface reactions. Show less
Understanding how galaxies form, interact, and evolve comes largely from comparing theory predictions with observational data. Numerical simulations of galaxies provide the most accurate approach... Show moreUnderstanding how galaxies form, interact, and evolve comes largely from comparing theory predictions with observational data. Numerical simulations of galaxies provide the most accurate approach to testing the theory, as they follow the non-linear evolution of gas and dark matter in great detail and incorporate numerous baryonic processes, among which are energy feedback from supernovae (SNe) and Active Galactic Nuclei (AGN). In this thesis, we show the results of the development of the new model COLIBRE for cosmological simulations of galaxy formation that include a cold interstellar medium. First, we present a new SN feedback recipe developed for COLIBRE, whereby SN energy is injected into the gas in thermal and kinetic forms, and the total energy and momentum of the system of gas and stars are exactly conserved. Second, we conduct a detailed comparison of different ways in which SN energy is distributed in the gas environment around young stellar populations. Third, by using our simulation setup originally developed to test COLIBRE’s SN feedback, we show that the radioactive isotope Fe60 that has been detected on Earth is likely of SN origin. Finally, we present the calibration of the SN and AGN feedback of the COLIBRE model using machine learning. Show less
The research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study.... Show moreThe research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study. Enzymes inherently embody a delicate balance between activity and stability, and the acquisition of new enzymatic functions is often accompanied by trade-offs, such as decreased stability or reduction of the original activity. Probing evolutionary adaptability of BlaC with laboratory evolution in combination with structural characterization can provide information about the mechanisms of rapid adaptations observed for β-lactamases in the clinic. The role of temperature as a conventional selection pressure in such evolutionary adaptation is unclear. The cooperative nature of enzyme unfolding over a narrow temperature trajectory raises the question whether evolution at temperatures well below the melting point is influenced by temperature. The approach used in this work to answer these questions is by simulating evolution under different selection pressures and characterize the variant enzymes in terms of activity, structure, dynamics and melting temperature. The research makes clear how enzyme kinetics and dynamics vary with different selection pressures and maps the evolutionary path that enzymes may take. The underlying structural mechanisms are established to provide a rationale for the observed effects. Show less
This work describes the use of click-to-release chemistry to get spatiotemporal control over immunocytokine activity. Until now, immunocytokines (cytokines coupled to a tumor-targeting-moiety)... Show moreThis work describes the use of click-to-release chemistry to get spatiotemporal control over immunocytokine activity. Until now, immunocytokines (cytokines coupled to a tumor-targeting-moiety) remained active throughout the body, being able to bind their respective receptors, causing mild to severe side-effects in cancer patients. Attempts have been made to improve the specific action of these immunocytokines, but these solutions remained very cytokine-specific and toxicity was not reduced significantly. Click-to-release chemistry allows us to inactivate a cytokine by blocking its free amines, present in lysines. This prevents the cytokine, IL-1β and TNF-α in particular, from binding its receptor. Removal of the blocking agent using a tetrazine restores the native amine and for IL-1β also its activity. By coupling the blocked cytokine to a targeting moiety allows for transport to the target, the tumor(-environment) upon which the unblocking or decaging can take place. This blocking-unblocking or caging-decaging was assessed using various cell-based assay. This technique can provide new opportunities in the immunocytokine field, as it is not cytokine-specific, and thereby opportunities in cancer therapy development. Show less
The plant hormone auxin regulates plant growth and development through polar cell-to-cell transport-generated maxima and minima. PIN FORMED (PIN) auxin efflux carriers determine the direction of... Show moreThe plant hormone auxin regulates plant growth and development through polar cell-to-cell transport-generated maxima and minima. PIN FORMED (PIN) auxin efflux carriers determine the direction of this auxin flow through their asymmetric placement on the plasma membrane (PM). In Arabidopsis thaliana (Arabidopsis), the PM-associated protein kinase PINOID (PID) regulates PIN localisation and thereby auxin transport polarity by phosphorylating these carriers in their central cytoplasmic loop. PID in turn interacts with the calmodulin-like protein TOUCH3 (TCH3) in response to elevated cytosolic calcium, which dissociates PID from the PM and inhibits its kinase activity. In this thesis, we show that PID also interacts with 10 other CALMODULIN/CALMODULIN-LIKE proteins, which together with TCH3 form a confined clade in the CaM/CML family. The CaM/CML binding domain in PID was found to map to an amphipathic alpha-helix inserted in the catalytic kinase core. Disruption of this alpha-helix did not affect PID kinase activity, but impaired both its PM association and the interaction with the CaM/CMLs, making the kinase “untouchable”. Expression of “untouchable” PID versions in the pid mutant background revealed that proper calcium-CaM/CML-PID signalling is essential to maintain the robust spiral phyllotaxis that is typical for the Arabidopsis inflorescence. Show less
Biological cells, the basic building blocks of all life forms, are surrounded by a lipid membrane. More than half of the membrane is occupied by membrane proteins, which can regulate the cell... Show moreBiological cells, the basic building blocks of all life forms, are surrounded by a lipid membrane. More than half of the membrane is occupied by membrane proteins, which can regulate the cell functionality through specific arrangements. To regulate the arrangements several proteins have to work together. In addition to direct forces, there exists an indirect force between the proteins, which stems from their deformation of the membrane and contributes to their self-organization. Since the actual membrane is very crowded and proteins are too tiny and complex to measure this interaction, in this thesis we used a model system consisting of lipid membranes and solid particles to study the deformation-mediated interaction. We experimentally confirmed for the first time that, unlike many known forces, this deformation-mediated interaction is not additive, i.e. the strength and range of three (or more) deformations cannot be obtained by simple addition of the interactions between pairs of deformations. We found that the interaction weakens with increasing number of membrane-deforming particles and that the particle become less ordered. We investigated deformations in both directions of the membrane and found that the interaction can be both repulsive and attractive, and furthermore depends on the shape of the deformation. This thesis helps to better understand the organization of proteins that deform cellular membranes. Show less
The emergence of complex diseases resulting from abnormal cell-cell signaling and the spread of infectious diseases caused by pathogens are significant threats to humanity. Unraveling the dynamic... Show moreThe emergence of complex diseases resulting from abnormal cell-cell signaling and the spread of infectious diseases caused by pathogens are significant threats to humanity. Unraveling the dynamic mechanisms underlying cell-cell signaling and infectious disease spreading is crucial for effective disease prevention and treatment. As science and technology advance, the availability and diversity of observational and experimental data related to these biological processes continue to grow. In this thesis, we integrate multisource data with dynamic modeling to investigate the biological mechanisms of Notch signaling in biological development and to develop prevention and control strategies for infectious diseases. Show less
Given the accelerating appearance of antimicrobial resistance, there is an urgent need for more fundamental research into novel antibiotic strategies. The work in this thesis helps to address this... Show moreGiven the accelerating appearance of antimicrobial resistance, there is an urgent need for more fundamental research into novel antibiotic strategies. The work in this thesis helps to address this global problem by developing new antibiotic compounds, inspired by the antibacterial mechanisms of the natural antibiotic bacitracin. By unravelling the unique mechanism of action that bacitracin employs, we discovered that the inclusion of a small hydrophobic group in key locations of the molecule results in a dramatic enhancement of antibacterial activity, in some cases more than 100 times more potent than bacitracin. Crucially we found that the most potent analogues are particularly active against antibiotic-resistant bacteria including those bearing clinically challenging resistance genes. In doing so we have developed potent next-generation variants of this classic antibiotic and have taken important steps in the fight against antimicrobial resistance. Show less
Detecting nanoscopic objects plays an important role in nanoscience in particular, in the rapidly growing field of nanobiology. The forebear to modern super-resolution microscopy for single... Show moreDetecting nanoscopic objects plays an important role in nanoscience in particular, in the rapidly growing field of nanobiology. The forebear to modern super-resolution microscopy for single molecule investigation, is fluorescence microscopy. Fluorescence as a contrast mechanism, however, brings several restrictions. These include (1) the use of the label itself, which may introduce artifacts to the interpretation, (2) the limited photoemission caused by photobleaching and photoblinking as well as (3) low bandwidth of the emission. Fluorescence-free alternatives are thus highly desirable to overcome these limitations. Optical detection of individual proteins with high bandwidth holds great promise for understanding important biological processes on the nanoscale. In this thesis, we investigate label-free optoplasmonic detection of single proteins and particles in motion. Analysing the data provide information about the hydrodynamic volume of the diffuser and interaction such as binding events. Show less
This doctoral thesis is an effort to understand how lipid phase-separation induced by diacylglycerol analogues in lipid-based nanoparticles affects their in vivo behavior, leading to specific... Show moreThis doctoral thesis is an effort to understand how lipid phase-separation induced by diacylglycerol analogues in lipid-based nanoparticles affects their in vivo behavior, leading to specific nanoparticle-protein communications and selective cell targeting. By studying how lipid composition affects morphology and this in turn affects the nano-bio interface, a comprehensive picture and prediction of nanoparticle behavior and cell selectivity is provided. More specifically, liposomes containing diacylglycerol analogues are found to phase separate and to be able to specifically target subsets of endothelial cells in zebrafish embryos. The mechanism behind this selective targeting is the result of a triglyceride lipase mediated mechanism due to phase separation and lipid composition, and is conserved in higher organisms (mice). Moreover, mRNA-based lipid nanoparticles that contain diacylglycerol analogues exhibit the same selectivity which leads to cell-specific mRNA delivery and transfection. Show less
This thesis pioneers diatom molecular identification and quantification through genome-scale methods, with four key aims: (i) reviewing DNA/RNA sequencing methods in aquatic biomonitoring to... Show moreThis thesis pioneers diatom molecular identification and quantification through genome-scale methods, with four key aims: (i) reviewing DNA/RNA sequencing methods in aquatic biomonitoring to highlight their strengths and limitations; (ii) unraveling the evolutionary history of Nitzschia palea and investigating species delimitation within the species complex; (iii) identifying silica genes in N. palea for insights into ecology and evolution; and (iv) assessing a genome-scale quantification method for diatom biomonitoring to improve accuracy and scalability in estimating abundances. The review (Chapter 2) emphasizes disparities between molecular and morphology-based approaches and introduces the challenges in accurately estimating species abundances. Chapter 3 explores N. palea's evolutionary history using transcriptome data and reveals reticulate evolutionary patterns resulting in a putative hybrid between populations with different morphological characteristics. Chapter 4 pinpoints silica genes in N. palea and reveals variations among different populations that may lead to differences in silica metabolism. Chapter 5 introduces a genome-scale quantification approach that provides a promising alternative for molecular diatom biomonitoring due to its improved taxonomic resolution and quantification accuracy. In summary, this thesis underscores that genome-scale methods' have a critical role in diatom identification and quantification, and in advancing our understanding of microalgal taxonomy, ecology, and evolution. Show less
Viral hemorrhagic fever (VHF) is a group of acute diseases caused by highly infectious viruses including Ebola, Lassa, Dengue viruses. Its high mortality rate poses high risk to public health,... Show moreViral hemorrhagic fever (VHF) is a group of acute diseases caused by highly infectious viruses including Ebola, Lassa, Dengue viruses. Its high mortality rate poses high risk to public health, however, studies on VHF have been hampered due to the non-availability of proper models and incomplete knowledge on its mechanism. In order to fill this gap, this thesis presented new bioanalytical, lab-on-chip and single-cell assays to investigate changes in vascular biology and macrophage immunometabolism induced by VHF viruses. Firstly, an organ chip was developed to mimic the hemorrhagic shock syndrome caused by VHF viruses in vitro and test experimental drug candidates. In addition, acoustic force spectroscopy was applied to investigate the effect of Dengue on the cellular viscoelastic properties of endothelial cells at single-cell level. Then, metabolic profiling of endothelial cells and macrophages upon Ebola viral protein exposure was performed on bulk-level. Finally, the immunometabolism of human macrophages upon polarization was investigated by live single-cell metabolomics, setting the stage for future host-pathogen studies at single-cell level. Overall, this thesis will facilitate the understanding of VHF viruses and the development of treatment strategies. More importantly, the technologies developed here expectedly open up opportunities to combat the viruses that threaten global society. Show less
Neuraminidases are enzymes that cleave glycosidic linkages of sialic acid. These enzymes are involved in influenza infections as well as in many cellular processes in mammals and micro-organisms.... Show moreNeuraminidases are enzymes that cleave glycosidic linkages of sialic acid. These enzymes are involved in influenza infections as well as in many cellular processes in mammals and micro-organisms. Development of molecules that irreversibly inhibit these enzymes, as well as molecules that can covalently bind and also label these enzymes, is described in this thesis. These newly developed molecular tools can be used to study neuraminidsases. Better understanding of these enzymes could for example contribute to the timely invervention if new influenza strains emerge. Show less
A single self-assembled semiconductor quantum dot in a high-finesse optical microcavity - the subject of this thesis - is an interesting quantum-mechanical system for future quantum applications.... Show moreA single self-assembled semiconductor quantum dot in a high-finesse optical microcavity - the subject of this thesis - is an interesting quantum-mechanical system for future quantum applications. For instance, this system allows trapping of an extra electron and thus can serve as a spin quantum memory, or enables high-fidelity and high-rate single-photon production. We investigate several aspects in this thesis:First, the operation and manipulation of the system is achieved using resonant laser spectroscopy. This requires filtering out of the relatively strong excitation laser, which is often done using the cross-polarization technique. This approach, however, is complicated in optical setups by spin-orbit coupling of light at the beamsplitter. We experimentally firstly explore this effect in a cryogenic optical microscope and demonstrate its importance for quantum dot based single photon sources. Next, we develop a unique setup with a cold permanent magnet and firstly realise trapping of a single electron in our particular quantum dot - cavity devices and show spin control. Then we show how true single photons from our device can be used to create novel quantum states of light. First, we investigate theoretically single photon addition to coherent laser light including several experimental imperfections - we find an universal behaviour of the photon correlation function. Finally, we demonstrate entanglement of several consecutive photons by repeatedly using Hong-Ou-Mandel quantum interference of single photons with a photon quantum memory in the form of an optical delay loop. We show that this results in quantum states of light that have Poissonian photon statistics like laser light - therefore we call them artificial coherent states - but also that they are more complicated than ordinary coherent states and contain multi-photon quantum entanglement in the form of linear cluster states, a potential resource for universal quantum computing. Show less
