The type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in... Show moreThe type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in Agrobacterium tumefaciens and the conjugative plasmid RP4. A. tumefaciens is able to transfer DNA into plant cells and transform them genetically. We discovered that a novel hairpin structure on the terminus of the transferred DNA allows for earlier expression of its genes in the recipient plant cell. Further, we could describe the translocation signal of the relaxase protein TraI of RP4 responsible for DNA processing and transfer via the T4SS. Additionally, it was shown that the T4SS of RP4 is able to translocate proteins from one cell to another without parallel DNA transfer. Furthermore, we were able to create a hybrid relaxase created consisting of parts from A. tumefaciens (relaxase part) and RP4 (translocation signal) that was shown to be translocated via the T4SS of RP4. Show less
The type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in... Show moreThe type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in Agrobacterium tumefaciens and the conjugative plasmid RP4. A. tumefaciens is able to transfer DNA into plant cells and transform them genetically. We discovered that a novel hairpin structure on the terminus of the transferred DNA allows for earlier expression of its genes in the recipient plant cell. Further, we could describe the translocation signal of the relaxase protein TraI of RP4 responsible for DNA processing and transfer via the T4SS. Additionally, it was shown that the T4SS of RP4 is able to translocate proteins from one cell to another without parallel DNA transfer. Furthermore, we were able to create a hybrid relaxase created consisting of parts from A. tumefaciens (relaxase part) and RP4 (translocation signal) that was shown to be translocated via the T4SS of RP4. Show less
In the thesis the dynamics of strategies is studied from two perspectives.In the first part of the thesis strategies are considered to be opinions present in a community. Firstly, the change of... Show moreIn the thesis the dynamics of strategies is studied from two perspectives.In the first part of the thesis strategies are considered to be opinions present in a community. Firstly, the change of support in time for two opinions is being analysed in case opinion bearers repeatedly meet in groups of size three. An opinion bearer is either an inflexible or a floater. An inflexible always adheres to the supported opinion, whereas a floater changes from opinion if the originally supported opinion has the minority in the group. A second chapter on opinion dynamics allows floaters to show either contrarian or non-contrarian behaviour. Both types of floaters adopt the opinion that has the majority in the group of size three, but a contrarian floater subsequently shifts to the alternative opinion.The second part deals with adaptive dynamics. Adaptive dynamics mathematically models the formation of evolutionary trees. The focus here is on phenotypic strategies present in ecological communities. The interest does not so much lie in the densities these strategies have, but in which strategies are present in the course of evolutionary time. The changes in presence are caused by repeated invasions of a mutant population in which the individuals express a strategy that deviates from those present in the invaded community. Show less
Streptomyces present a valuable platform for natural product discovery. Lugdunomycin is a novel angucycline-derived polyketide from Streptomyces sp QL37, with unprecedented skeleton and... Show moreStreptomyces present a valuable platform for natural product discovery. Lugdunomycin is a novel angucycline-derived polyketide from Streptomyces sp QL37, with unprecedented skeleton and antimicrobial activity. This dissertation covers the research on the biosynthesis of this novel antibiotic and the developmental biology of Streptomyces. The data in this thesis have provided important new insights into the puzzle of lugdunomycin biosynthesis and the sporulation-specific cell division of Streptomyces. By means of molecular biology, structural biology, biochemical, chemical and bioinformatics approaches, we have uncovered the potential functions of the key enzymes, especially those encoded oxygenases (LugOI-LugOV) in lugdunomycin biosynthesis. Furthermore, we extensively studied the role of SsgB in Streptomyces development, that led us to the discovery of longitudinal cell division that support the predominant role of SsgB in the accurate positioning of the division site and the placement of the Z-ring. Show less
Bacteria have the ability to alter their morphology in order to adapt to changing environments. We have investigated the role of the cell envelope in the development and stress-adaptation... Show moreBacteria have the ability to alter their morphology in order to adapt to changing environments. We have investigated the role of the cell envelope in the development and stress-adaptation strategies of Actinobacteria. Here, we demonstrate how cryo-electron microscopy techniques can be used as efficient tools to study cell envelope of various bacteria. An in-depth study on the Streptomyces cell wall with cryo-electron tomography reveals the structural complexity of the Gram-positive cell wall in apically growing bacteria. Additionally, we report the formation of intracellular membrane accumulations in Streptomyces as a result of exposure to a stress-inducing agent. Furthermore, we studied the ability of the filamentous actinomycete Kitasatospora viridifaciens to extrude wall-deficient cells or S-cells upon exposure to hyperosmotic stress. We characterized the structural alterations associated with S-cell formation using cryo-electron microscopy and reveal that S-cell formation requires cytoskeletal protein FilP in K. viridifaciens. To summarize, this thesis provides new insights in the structural complexity and stress-induced alterations of the bacterial cell envelope. Show less
Glucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of... Show moreGlucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of side effects and drug resistance. Therefore, there is still a major need to investigate the molecular and cellular mechanisms underlying the effects of GCs. Zebrafish are increasingly used as an in vivo model system for studying the immune system, in particular the inflammatory response. In Chapter 2, an overview is provided of the available inflammation models in zebrafish, and how they are used to unravel molecular mechanisms underlying the inflammatory response and for testing of potential novel anti-inflammatory drugs, in particular GCs. In this thesis, we have used zebrafish model system to study molecular and cellular mechanisms of GC action on the immune system and to develop a model for in vivo screening of the anti-inflammatory effects as well as possible adverse effects of novel GC therapies. For this purpose, we have studied the effect of GCs on leukocyte migration and differentiation during an inflammatory response (Chapter 3), how GCs modulate the immune response to a mycobacterial infection (Chapter 4), and we have investigated targeting of GCs to inflamed tissue by liposomal delivery (Chapter 5). Show less
By growing in a soil plants change the biotic and abiotic properties of the soil in which they grow. This can influence the performance of plants that grow in the same soil subsequently and is... Show moreBy growing in a soil plants change the biotic and abiotic properties of the soil in which they grow. This can influence the performance of plants that grow in the same soil subsequently and is known as plant soil feedback (PSF). Species largely differ in how they influence the soil while they grow in it and how they react to conditioned soil. So far PSF was mainly shown to influence biomass of plants and certain specific chemical compounds in plants. This thesis demonstrates how the whole metabolome of a range of different plants changes due to different microbiomes in the soil and that the influence of soil on the metabolome of plants can be stronger than the effect of herbivory. Furthermore, this thesis shows how these PSF change over time and on different spatial scales and which methods are most suitable to study the metabolomic response of plants. Show less
Bacterial chemosensory arrays are protein assemblies that are the key structural and functional component for motile bacteria to sense their internal or environmental chemical signals. Cryo... Show moreBacterial chemosensory arrays are protein assemblies that are the key structural and functional component for motile bacteria to sense their internal or environmental chemical signals. Cryo electron tomography is a technique of transmission electron microscopy that allows the visualization of protein complexes in their near-native cellular context. This thesis includes a general introduction of the basics of bacterial chemosensory and the structural diversity of chemosensory arrays, as well as a description of the workflow of using cryo electron tomography to image bacterial chemosensory arrays in situ. This thesis presents the discovery of a different stoichiometry of chemotaxis proteins and a direct visualization of the kinase distribution within the chemosensory arrays in Vibrio cholerae. This highlight that a structural diversity of chemoreceptor arrays does exist, and that is the norm and not an exception. This thesis also includes studies illustrating the conformational dynamics of the Escherichia coli chemoreceptors correlated to its signaling states, which gives crucial insights on how the receptors function in a trimers-of-dimers packing arrangement. In short, studies present here broaden the appreciation of the structural diversity of chemosensory arrays and deepen our understanding of how arrays dynamically functions during chemosensory signal transduction. Show less
Aspergillus niger is an important industrial producer of organic acids and enzymes producing large amounts of spent fungal biomass. In the European Research Area Industrial Biotechnology (ERA-IB)... Show moreAspergillus niger is an important industrial producer of organic acids and enzymes producing large amounts of spent fungal biomass. In the European Research Area Industrial Biotechnology (ERA-IB) funded project, we effectively aimed to improve the composition of post-fermentation fungal biomass for extraction of the value-added product chitosan as a derivative of cell wall chitin (FunChi). As chitin/chitosan is not encountered in plant or human tissue, it often acts as an elicitor to plant and animal immune responses in order to fight off possible impending fungal infections. The application of both chitin and chitosan oligomers have been shown to prime plants against infection. This thesis discusses the identification of genes that are important for chitin deposition in the cell wall of A. niger. In addition, the work described here also investigates the genes that facilitate chitin cross-linking to the cell wall. The relevance of all findings are discussed in relation to both the improvement of chitin extraction from post-fermentation biomass and to the integrity of the fungal cell wall. Show less
Natural vanilla flavor is one of the most important in the world. However, the cost of this flavor is expensive. Production of this flavor by alternative methods, could reduce the cost. Towards... Show moreNatural vanilla flavor is one of the most important in the world. However, the cost of this flavor is expensive. Production of this flavor by alternative methods, could reduce the cost. Towards this end, using microbiological methods, fungal endophytes were isolated and identified, by morphological characters and PCR-based methods, from vanilla pods, the site of vanilla flavor. The fungal endophytes were tested for their involvement in the synthesis of vanilla flavor. In this way, three vanilla pod endophytes Hypoxylon investiens, Pestalotiopsis microspora, Diaporthe phaseolorum were found to modify the amounts of vanilla flavor and aroma metabolites vanillin, vanillyl alcohol, p-hydroxybenzoic acid, p-coumaric acid, p-xylene, α-phellandrene, 3-carene, α-terpineol, p-hydroxybenzaldehyde, α-cubebene, β-caryophyllene in Vanilla material containing media (in vitro) as well as in living Vanilla plant material (in vivo - acclimatized plants and calli). These metabolites were synthesized either de novo or through biotransformation reactions of precursors. That the latter describes the involvement of several metabolites related to vanilla flavor, implies complex flavor notes, typical of natural vanilla flavor, as opposed to the synthetic version. The analytical methods used include GC-MS, HPLC, 1H and 2D NMR. The results support the hypothesis that endophytes play a role in vanilla flavor metabolite biosynthesis. Show less
This thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an... Show moreThis thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an atypical and a conventional chemokine receptor during chemotaxis in macrophages, the role of chemotactic signaling in cell polarization and explores an in vivo screening workflow for human anti-inflammatory drugs using zebrafish. Show less
In this thesis, we have studied the potential of the zebrafish larval model in studying the ECS, as a complementary model to the existing rodent models. More specifically, we have looked at the... Show moreIn this thesis, we have studied the potential of the zebrafish larval model in studying the ECS, as a complementary model to the existing rodent models. More specifically, we have looked at the role of the ECS in regulating locomotion and anxiety, and its interaction with the hypothalamic-pituitary-interrenal (HPI) axis, or stress axis. This study has provided us with an interesting animal model which allows for pharmacological screening of Cnr1 agonists, and their involvement in the CNS, as shown by a change in locomotion, anxiety-like behavior and HPI axis activity. The zebrafish larval model can be used as a complementary model to the existing rodent animal models, to study the ECS. The zebrafish larval model brings several interesting features, such as optical transparency and possibilities for high-throughput screening. Furthermore, a complete ECS is present, there is lack of endogenous activity, allowing for exogenous compound screening, and zebrafish data is generally in line with rodent literature. Since the ECS is involved in many diseases, more research of this system may result in the discovery of novel drugs and drug targets. Show less
Plants are colonized by an astounding number of microorganisms that can provide different life-support functions, including nutrient acquisition and protection against (a)biotic stresses like... Show morePlants are colonized by an astounding number of microorganisms that can provide different life-support functions, including nutrient acquisition and protection against (a)biotic stresses like drought or pathogen attack Here, the diversity of bacteria living inside plant root tissue was explored with a focus on Actinobacteria, and in particular Streptomyces. Streptomycetes are filamentous bacteria that are commonly found in soil. They were brought into the laboratory for their ability to produce a large diversity of natural products, including many different antibiotics. In plant-associated environments, Streptomyces can be found in the rhizosphere, the endosphere and the phyllosphere. In these niches, they receive nutrients from the plants, feasting on various biopolymers and exudates. In return, the plant may benefit from their presence by enhanced nutrient acquisition, pathogen antagonism and induced systemic resistance To date, however, plant-Streptomyces interactions are not well understood and the mechanisms underlying plant colonization and invasion by Streptomyces remain largely elusive. Also, the chemistry of plant-Streptomyces interactions is yet underexplored, leaving us with a reservoir of untapped natural products that may contribute to solving the problem of emerging antibiotic resistance. Therefore, this research was focused on the aforementioned topics. Show less
The recently introduced nature-originated deep eutectic solvents, so-called natural deep eutectic solvents (NADES) are considered as truly green solvents, which composed of natural ingredients... Show moreThe recently introduced nature-originated deep eutectic solvents, so-called natural deep eutectic solvents (NADES) are considered as truly green solvents, which composed of natural ingredients found abundantly in organisms. NADES has been used in diverse applications, including extraction of bioactive molecules, enzymatic processes, agricultural and pharmaceutical fields, and protein stabilization. Despite the various applications, their physiological roles in organisms still remain unclear. In the thesis, the possible roles of NADES were investigated using a model plant (Drosera species) and for macromolecules. The chemical analysis of Drosera exudates indicated that the exudates contained sugars, amino acids, and organic acids which might be NADES components, and they could be involved in the biological roles of exudates. Several NADES are also proven to be optimum media to improve the stability and activity of enzymes. Additionally, NADES protected the enzymes from microorganism contamination. Besides enzymes, DNA and RNA were well protected from degradation by NADES. Based on the results of the thesis, a new biological role of NADES associated to macromolecules were deduced. Show less
In this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration... Show moreIn this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration modalities and anti-cancer efficacies of newly-developed PDT and PACT compounds, and test a light-triggered liposomal system for targeted drug delivery specifically to cancer cells in vivo. In chapter 2, we investigate the role of macrophages in tumour-induced angiogenesis. We show that macrophage-dependent angiogenesis is driven by macrophage recruitment to lactic acid secreted by glycolytic B16 melanoma cells. Chemical inhibition of macrophages and glycolysis blocks the initiation of angiogenesis in these models, suggesting that macrophages attracted to glycolytic melanoma cells contribute to the tumour-induced angiogenesis process.In chapters 3 and 4, we explore novel PDT and PACT compounds, respectively, for treatment of conjunctival melanoma in zebrafish. We inject conjunctival melanoma cells into the retro-orbital site to establish an orthotopic model and into the Duct of Cuvier to generate an ectopic model. Our results prove that zebrafish provides a fast vertebrate cancer model to test the optimal administration regimen of drugs, conditions of light irradiation, host toxicity and anti-cancer efficacy of PDT and PACT drugs against conjunctival melanoma.In chapter 5, we focus on modifying liposomes to be light triggered in order to deliver drugs specifically to cancer cells. We inject MDA231 breast cancer cells into the Duct of Cuvier at 2 days post fertilization (dpf) to initiate metastasis to the CHT. We successfully demonstrate that light-triggered, cell-specific delivery of liposome-encapsulated doxorubicin reduces the xenograft cancer cell burden without enhanced cytotoxicity of the zebrafish embryos. In chapter 6, we summarize the novel anti-cancer strategies, which we have developed using zebrafish xenograft models. In the same chapter, we frame our findings in the current scientific landscape and discuss future perspectives. Show less
Plant architecture has distinct forms in different plant species, but also within a species the finalarchitecture of a plant is determined by its gradual development and changes therein induced by... Show morePlant architecture has distinct forms in different plant species, but also within a species the finalarchitecture of a plant is determined by its gradual development and changes therein induced by environmental conditions during the plant’s life cycle. Higher plants have the ability tocontinually produce new organs and tissues during their lifetime. This unique ability is provided by pools of stem cells organized in so-called meristems. Primary meristems located at apices, of the plant body are involved in elongation of the main plant axes, whereas secondary meristems in lateral positions play a role in branching and axis thickening. First chapter is reviewing the current advances in our understanding of the molecular mechanisms that regulate plant meristems and their developmental phase transitions in Arabidopsis thaliana. AT-HOOK MOTIF NUCLEAR LOCALIZED (AHL) gene family is introduced in chapter 1,and that of family member AHL15 described and studied more in detail in chapter 2. In chapter 3 we identified a new branch of the ageing pathway in Arabidopsis, in which the longevity-promoting AHL15 protein represses ageing independent of miR156/157. Chapter 4 of this thesis focuses on AHL15's effect on Arabidopsis vascular cambium activity and secondary growth. Analysis showed that in an ahl15 loss-of-function mutant, interfascicular secondary growth is significantly reduced. Show less
Acidobacteria is a widely distributed phylum but their functional roles in ecosystem processes are still largely elusive. The Granulicella genus belongs to the class Acidobacteriia and is known to... Show moreAcidobacteria is a widely distributed phylum but their functional roles in ecosystem processes are still largely elusive. The Granulicella genus belongs to the class Acidobacteriia and is known to produce copious amount of Extracellular Polymeric Substances (EPS), which are fundamental for microbial life. The major goal of my thesis was to investigate and understand the metabolism of Granulicella sp. strains WH15 and 5B5, and the functions and environmental fate of EPS of Granulicella sp. WH15 (WH15EPS). Optimization of carbon concentration and manganese in culture medium allowed our strains, especially WH15, to grow faster in laboratory conditions, producing extractable amounts of EPS. Using the Stable Isotope Probing technique, we observed the incorporation of WH15EPS by Singulisphaera and its connections to other Planctomycetes and Acidobacteria, which were not reported before. In addition, our results showed, in the genomes of the microbes which incorporated WH15EPS, a large diversity of glycoside hydrolase with biotechnological potential and a high number of unclassified microorganisms that could be targeted for future studies. The data presented in this thesis establish a solid fundamental basis for more mechanistic studies of Acidobacteria and other uncultivated microbes. Show less
Plant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations... Show morePlant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations modulate plant metabolism. Factorial combinations of different plant species, including Arabidopsis thaliana (model plant), Brassica oleracea var. italica (crop) and Artemisia annua (medicinal plant), and phylogenetically distinct rhizobacterial species, including Pseudomonas fluorescens SS101 (Pf SS101), Microbacterium and three Paraburkholderia species, were used as study model systems in this thesis. Untargeted metabolomics was used to assess the impact of these rhizobacteria on the shoot chemistry of the host plant species. This study revealed that root treatment of different plant species with rhizobacteria altered 18-78% of the detected plant secondary metabolites in the shoot. We also assessed the impact of a known bacterial trait on plant phenotype and chemistry. cysH mutation of Pf SS101 affected the chain elongation of aliphatic glucosinolate biosynthesis in Arabidopsis whereas it led to an accumulation of indolic glucosinolates and flavonoids in Broccoli. To further discover the bacterial traits affected during the interaction with Broccoli roots, genome wide transcriptome analysis was carried out, resulting in upregulation of genes involved in flagellar assembly, chemotaxis, and motility together with nutrient uptake and (an)ion transporter in Paraburkholderia species. Show less
Narcissus is an economically important crop with a large number of species, hybrids and/or varieties in cultivation. It is one of the most popular ornamental plants. The Netherlands is among the... Show moreNarcissus is an economically important crop with a large number of species, hybrids and/or varieties in cultivation. It is one of the most popular ornamental plants. The Netherlands is among the top producers of Narcissus in the world. The ornamental value of Narcissus is quite well known but they can also be a valuable resource of natural compounds. Among these compounds, galanthamine has been successfully commercialized as medicine for the treatment of early to mild Alzheimer’s Disease (AD). The characteristic symptoms of this disease are loss of memory and impairment of multiple cognitive and emotional functions. The objective was to find out how galanthamine levels could be enhanced to convert this floricultural crop into a medicinal crop. There are different factors that influence the production of galanthamine and other metabolites in the bulbs. Effect of various factors such as preanalytical methods, bulb location and age along with effect of biotic and abiotic factors on the growth and yield were studied. From the results, it is evident that factors have detrimental effect on the total bulb yield and galanthamine levels. Show less
Plant exudates appeared long time ago in the history line of life in the early Devonian. They are typical saps produced by specialized cells. They can be naturally excreted outside or right after... Show morePlant exudates appeared long time ago in the history line of life in the early Devonian. They are typical saps produced by specialized cells. They can be naturally excreted outside or right after plants are damaged or infected. Through time, diverse cultures recognized them as valuable sources of natural medicines. Of various types of plant exudates, the most outstanding are gums, mucilage, essential oils, oleoresins, resins, phloem like saps, and latexes. Regardless of their commercial importance, in the aspect of biology, plant exudates are believed to be involved in plant defense mechanisms, but their roles still remain unclear. In this thesis, based on diverse experiments, many features of plant exudates were unveiled. Interestingly, even if plant exudates possess a broad chemical diversity, their specialized biological functions resulted in less variation of their metabolomes as compared with their bearing tissues. This lower variation indicates that plant exudates might partake of a general-broad defense against herbivores and pathogens in their bearing tissues. This defense involves both mechanical and chemical traits which might complement and modulate each other, eventually resulting in a chemo-mechanical defense layer. Of the diverse chemicals in plant exudates, terpenes are one of the most common metabolites in these saps. Show less
