Plants and their pollinators form complex interaction networks. Within these networks, species differ widely in the number of species they interact with. These interaction patterns are important... Show morePlants and their pollinators form complex interaction networks. Within these networks, species differ widely in the number of species they interact with. These interaction patterns are important aspects in pollination ecology, since it influences species coexistence and community stability. An in-depth understanding of the mechanisms behind plant-pollinator interactions is thus important to predict the dependency of species on each other and responses of plant-pollinator communities to (global) changes, such as the introduction of invasive species and climate change. In this study, I investigated whether flower abundance, flower morphology (nectar tube depth and flower display area), nectar production and pollinator foraging efficiency can predict plant-pollinator interaction patterns and the stability of these interactions. This study shows that flower density might be less important for structuring plant-pollinator interactions. Pollinators do not forage randomly. Rather flower morphology, nectar production and pollinator foraging efficiency are important aspects that determine interaction patterns, including species generalization degree and matching between flower nectar tube depth and pollinator proboscis length (size-matching). Although plant species with deeper flowers are more specialized, they are visited by pollinator species which, locally, fluctuate less across years. Thus, specialized plants might be less vulnerable to yearly fluctuations in plant-pollinator communities than often assumed. Show less
My PhD project studies how the temporally sequential Hox gene expression is regulated during head-tail patterning of the frog embryo. This question has not been solved and remains one of the... Show moreMy PhD project studies how the temporally sequential Hox gene expression is regulated during head-tail patterning of the frog embryo. This question has not been solved and remains one of the greatest unsolved mysteries in developmental biology. Currently, a very attractive explanation attributes sequential Hox gene activation to progressive opening of the chromosome, because it parallels the linear arrangement of Hox genes on the chromosome. While promising, this explanation is surrounded by extra complexities that ensure Hox expression is synchronized between different cells. By using gain-of-function and loss-of-function approaches, I discovered that Hox-Hox interactions play a crucial role in the regulation of Hox expression, and for the first time, I dissected different roles for these interactions in axis formation. The findings are likely to resolve the above complexities, and reveal a new facet of the mechanisms underlying Hox gene regulation. Furthermore, by timed modulation of the actions of BMP signaling, I showed that there is a BMP-dependent timing mechanism in the head that could be continued by the Hox temporal sequence in the trunk. The two constitute an integrative timer which can be translated into spatial patterns of gene expression along the whole head-tail axis via a BMP/anti-BMP dependent mechanism. Show less
The name ‘Jambi flora’ refers to fossil plants found as part of a rock formation from the Early Permian (296 million years old), located in the Jambi Province of Sumatra, Indonesia. The flora... Show moreThe name ‘Jambi flora’ refers to fossil plants found as part of a rock formation from the Early Permian (296 million years old), located in the Jambi Province of Sumatra, Indonesia. The flora is characterized by the occurrence of both plant groups known from classic coal swamp floras, established for tens of millions of years, as well as newly emerging groups that in a number of cases are making their first known occurrence there and would play an important role in the vegetations of the Permian era. The latter are part of distinct associations with an ecology different and generally drier than that of the swamp flora species. The large amount of fossil wood collected from the Jambi region allowed for a quantitative analysis larger than anything undertaken before for this early gymnosperm wood type. Although more than 250 different species have been described worldwide for this fossil wood type in the past, the results from a morphological analysis of the Jambi material shows it to be a coherent whole wherein no individual species can be discerned. This throws great doubt on whether it is even possible to distinguish traditional species in this wood type. Show less
Hydrogen peroxide (H2O2) is produced following injury and is required for regeneration. However, how H2O2 coordinates multiple signalling pathways required for regeneration is not known. Protein... Show moreHydrogen peroxide (H2O2) is produced following injury and is required for regeneration. However, how H2O2 coordinates multiple signalling pathways required for regeneration is not known. Protein-tyrosine phosphatases (PTPs) are regulators of signal transduction, reversing the phosphorylation of substrates, and are essential for many cellular processes. PTPs are also highly sensitive to reversible oxidation-mediated inhibition by H2O2.My research identified that some, but not all, PTPs become oxidized following amputation of the zebrafish caudal fin, and implicates PTPs that are oxidized as necessary for the regenerative process. Furthermore, I delineated that the PTPs Shp2 and Pten are required for the regenerative outgrowth phase of regeneration, and that Shp2 and Pten have a role in driving MAPK signalling and balancing PI3K signalling, respectively, during this phase. Show less
Aspergillus niger is an important industrial enzyme producer. Highly efficient production of enzymes mediated by modulating transcriptional regulation is meaningful. This thesis focuses on the... Show moreAspergillus niger is an important industrial enzyme producer. Highly efficient production of enzymes mediated by modulating transcriptional regulation is meaningful. This thesis focuses on the complex regulation of the expression of pectinolytic genes in A. niger. Chapter 1 gives an introduction of filamentous fungi with a focus on A. niger, and their industrial application. Chapter 2 describes a split marker approach in combination with NHEJ mutants for efficient targeted gene deletion. In Chapter 3, we constructed a set of isogenic auxotrophic strains by recycling pyrG marker. In Chapter 4, we selected five galacturonic acid (GA) induced genes to analyze the regulation of promoter activity of these genes in vivo by constructing promoter_amdS reporter strains. In Chapter 5, we identified the GA responsive transcription activator GaaR of A. niger by homology to BcGaaR of Botrytis cinerea. In Chapter 6 full genome sequencing of five mutants showing constitutive pectinolytic activity revealed allelic mutations in one particular gene gaaX. GaaX is likely to act as a transcription repressor which inhibits GaaR activity under non-inducing conditions. Chapter 7 summarizes and discusses the major conclusions of this thesis and proposes some future directions to study the regulatory mechanism of pectin degradation in A. niger. Show less
Control of infectious diseases poses continuous challenges for human health. Salmonella bacteria are a major cause of gastrointestinal infections and systemic disease like typhoid fever. We used... Show moreControl of infectious diseases poses continuous challenges for human health. Salmonella bacteria are a major cause of gastrointestinal infections and systemic disease like typhoid fever. We used zebrafish-Salmonella infection models to study host immune responses to Salmonella, particularly focusing on the role of the autophagy machinery. Autophagy and a related process known as Lc3-associated phagocytosis (LAP) trap invading microbes in intracellular vesicles and eventually eliminate them through the lysosomal degradation pathway. We show that macrophages and neutrophils trap Salmonella in Lc3-decorated phagosomes by a process dependent on the host factors Rubicon and NADPH oxidase, which generates anti-bacterial reactive oxygen species. This process could be defined as LAP as it requires some but not all components of the autophagy machinery. Genetic inhibition of LAP and ablation of macrophages resulted in hypersusceptibility to infections with both wild type and attenuated strains, revealing that LAP is an essential line of defense against Salmonella during systemic disease, when macrophages are the main carriers of the infection. Our studies also revealed a novel functional link between the autophagy modulator Dram1 and induction of LAP. This work encourages further studies aimed at the identification of autophagy modulating drugs for host-directed therapy of antibiotic-resistant Salmonella infections. Show less
Bacteria are highly complex and diverse organisms that have adapted to survive in ecological niches ranging from the most extreme to the most heterogeneous environments. Actinobacteria, with their... Show moreBacteria are highly complex and diverse organisms that have adapted to survive in ecological niches ranging from the most extreme to the most heterogeneous environments. Actinobacteria, with their beautiful morphologies and complex multicellular life cycle, are a striking example. These filamentous bacteria produce many enzymes and natural products, including two-thirds of all known antibiotics, which makes them highly relevant for medical, biotechnological and industrial applications. In Streptomyces, complex and intertwining regulatory networks, consisting of pathway-specific and global regulators, control development and antibiotic production in response to environmental stimuli and stresses. This thesis explores how the model organism Streptomyces coelicolor senses environmental signals, related to oxygen stress and the availability of aminosugars, and how the bacterium translates these into appropriate responses. Show less
This thesis describes the functional analysis of the Arabidopsis AHL15 gene. We showed that AHL15 and plays major roles in directing plant cell totipotency. Furthermore, our data show that... Show moreThis thesis describes the functional analysis of the Arabidopsis AHL15 gene. We showed that AHL15 and plays major roles in directing plant cell totipotency. Furthermore, our data show that AHL15 has a role in opening of chromatin, leading to endomitosis and polyploidy in embryonic cells and that AHL15 overexpression can lead to polyploid SEs and plants, probable by endomitotic events caused by incomplete chromatin condensation during cell division. More detailed analyses revealed that AHL15 is not specifically involved in the embryogenesis program, but that, surprisingly, the protein rather is involved in post embryonic development. Our analyses in Arabidopsis and tobacco showed that AHL15act as suppressors of developmental phase changes. In Arabidopsis, reduced AHL15 expression coincided with a faster progression from the vegetative to the reproductive phase. By contrast, AHL15 overexpression delayed the vegetative to reproductive phase change in both Arabidopsis and tobacco, causing some axillary meristems to be maintained in the vegetative phase, thereby allowing polycarpic development in these monocarpic annuals. By using RNA sequencing analysis, an extensive reprogramming of the transcriptome detected after AHL15 activation. Rapid decondensation of heterochromatin was observed after AHL15 activation, indicating that the global reprogramming of the transcriptome by AHL15 might at least in part be caused by extensive modulation of the chromatin configuration. Show less
The plant hormone auxin plays a central role in the growth and development of plants. Auxin acts in a concentration dependent manner and polar cell-to-cell transport of this hormone determines its... Show moreThe plant hormone auxin plays a central role in the growth and development of plants. Auxin acts in a concentration dependent manner and polar cell-to-cell transport of this hormone determines its distribution in the tissues of plants. This polar auxin transport is mediated by several families of auxin transporters, including the PIN FORMED (PIN) auxin efflux carriers that determine the direction of transport by their polar localization at the plasma membrane. The plasma membrane abundance and polarity of PINs (and thereby of polar auxin transport) is regulated by their post-translational modification, of which phosphorylation is best studied. PIN proteins in Arabidopsis consist of two transmembrane domains separated by a ‘long’ (PIN1,2,3,4,6,7) or by a ‘short’ (PIN5,8) hydrophilic loop. Phosphorylation of ‘long’ PINs in their central hydrophilic loop by the AGC3 kinases PINOID, WAG1 and WAG2 triggers shootward (apical) or outer-lateral polarity. The AGC1- type D6 kinases also phosphorylate the PIN hydrophilic loop, however this was reported to result in auxin transport activation rather than subcellular polarity establishment. Here we investigate the conservation and phylogeny of AGC3 and D6 kinases and their phosphorylation sites in PINs from the earliest land plants to flowering plants. In early land plants, many of the same proteins and conserved motifs can be found, however it is in monocots and dicots that conservation of PIN phosphorylation by AGC3 and D6 kinases is strongest. The expansion and increased conservation of AGC3 and D6 kinases and PINs in later lineages such as monocot and dicot flowering plants, is in line with their important role in the formation of reproductive organs and in the tropic growth responses that allow plants to adapt to changes in their environment. Show less
One of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem.... Show moreOne of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem. The zebrafish is a promising model for in vitro vasculogenesis and angiogenesis studies, as a replacement for currently used mammalian models. However, the culture of endothelial cells from this species is not well characterized. Here, we test different culture strategies, medium supplementations and culture substrates for their effect on the generation of putative endothelial (fli:GFP+ and kdrl:GFP+) cells and vascular morphogenesis in zebrafish blastocyst cell derived embryoid body culture. we have also developed a perfused culture model, using microfluidic technology, to culture zebrafish vascular networks. This study is a step forward to the development of zebrafish vascular networks in vitro. Show less
Biological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical... Show moreBiological molecules, especially the proteins, have a special and important function. We study their structure to understand their functions, and further make application, like the medical research. The routine method is diffraction, but not work for molecules which cannot grow into crystal and molecules which their crystal are too small. Cryo-EM technique provides another way to solve their structures through their images, it does not need crystals. Meanwhile, electron diffraction can work small crystals (micro- and nano-crystals) after the sample preparation was improved. Hence, we try to build a method that can restore the structure from the crystal’s image. We collect images of protein nano-crystal, and these images were processed to enhance their contrast. The key step is to find the orientations of these images in the procedure of reconstruction, therefore, we create a method that calculates these orientations. Nano-crystals, which cannot be used in diffraction method, then can be used in this method. Show less
Plant clonal propagation can be achieved through shoot- and root regeneration. Alternatively, it can be induced by somatic embryogenesis (SE). In Arabidopsis thaliana, somatic embryos are... Show morePlant clonal propagation can be achieved through shoot- and root regeneration. Alternatively, it can be induced by somatic embryogenesis (SE). In Arabidopsis thaliana, somatic embryos are efficiently induced by incubating immature zygotic embryos (IZEs) on SE induction medium (SEIM) containing high concentrations of the auxin analogue 2,4-dichlorophenoxyacetic acid (2,4-D). During SE somatic cells are reprogrammed to become totipotent cells, from which a new embryo cell fate is acquired in order to develop somatic embryos. The genetic and molecular mechanism by which 2,4-D induces SE initiation has not been elucidated yet and the aim of this PhD thesis was to identify which components of the auxin response pathway are involved in this developmental process. It is proposed that lateral root initiation is similar to SE initiation in that an auxin maximum followed by a minimum is required for both processes, and that the auxin responses in both systems are modulated by the same SLR/IAA14 module. Substantial work is still required to outline the details of downstream responses. They possibly include the regulation of auxin biosynthesis through TAA1/TAR and the coordinated expression of AUX1/LAX proteins to establish the dynamic auxin response leading to SE initiation. Show less
The development of methods for the genetic modification of plants a few decades ago has provided a tremendous boost for molecular plant science. Crop plants have been generated that are... Show moreThe development of methods for the genetic modification of plants a few decades ago has provided a tremendous boost for molecular plant science. Crop plants have been generated that are resistant to insects or herbicides, or that produce useful sugars or healthy nutrients. Although the ban on growing GM crops in Europe has considerably limited the application of GM technologies, they have still contributed considerably to fundamental plant science. Especially by using the natural and very efficient mechanism of DNA transfer by the soil born bacterium Agrobacterium tumefaciens, many collections of mutant lines of model plant species such as Arabidopsis and rice have been generated, in which genes are disrupted or overexpressed by the insertion of an Agrobacterium transfer DNA (T-DNA) construct. These collections have been used in forward or reverse genetics studies to unravel the function of a gene or a family of genes in plant defense or development, and to identify the key regulators in these processes. The study described in this thesis focused on the use of one of these key regulators, the Arabidopsis AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 15/REJUVENATOR (AHL15/RJV), to alter developmental processes such as flowering, senescence and regeneration. Show less
The T-cell mediated immune response to Aspergillus fumigatus was studied in healthy individuals and in several patient groups. In peripheral blood of healthy individuals low frequencies of... Show moreThe T-cell mediated immune response to Aspergillus fumigatus was studied in healthy individuals and in several patient groups. In peripheral blood of healthy individuals low frequencies of Aspergillus-specific CD4+ T-cells with a Thelper 1 profile were present. In patients with invasive aspergillosis after allogeneic stem cell transplantation Aspergillus-specific T-cells with a Thelper 1 phenotype increased in peripheral blood at the moment of improvement of aspergillus lesions. On the other hand, in patients with allergic bronchopulmonary aspergillosis Aspergillus-specific T-cells with a Thelper 2 phenotype were present, these were directed both to the classical described Aspergillus allergens Aspf1, Aspf2, Aspf3 and Aspf4, as well as to other Aspergillus antigens Crf1 and Catalase1. In COPD patients lung-derived Aspergillus-specific T-cells were characterized and showed a Thelper17 phenotype, in contrast to the Thelper1 phenotype that was present in peripheral blood. Finally, the interplay between different immune cells was studied in an in vitro model. CD4+ T-cells improved the phagocytosis capacity of neutrophil granulocytes, but not of monocytes. Show less
Double-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence,... Show moreDouble-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence, trigger various downstream events and finally bring about repair. Two main pathways are used for DNA DSB repair: Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ). Both of them function together to maintain genome integrity. At least two NHEJ pathways have been identified: the classic NHEJ pathway (c-NHEJ) and the backup-NHEJ pathway (b-NHEJ) also called alternative-NHEJ (a-NHEJ) or microhomology-mediated end-joining (MMEJ). Agrobacterium tumefaciens is widely used as a vector to produce genetically modified plants. Agrobacterium-mediated genetic transformation involves the transfer of T-DNA from its tumor-inducing plasmid to the host cell nucleus, where it integrates into the plant genome. However, the molecular mechanism of T-DNA integration is still unclear. T-DNAs can integrate at artificially induced DSBs, which suggests that DSB repair mechanisms are probably involved in T-DNA integration in plants. Arabidopsis NHEJ mutants have subsequently been studied for T-DNA integration. However, the results obtained by different research groups were variable and revealed either no or limited negative effects. Show less
