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The vertebrate immune system uses several strategies to combat viral infections. Cellular immune responses evoked by the cytotoxic T cells (CTLs) are initiated after screening host cells for abnormalities. Examples are peptides derived from virus (epitopes) that are presented on the cell surface by major histocompability molecules (MHCs). Viruses therefore face strong selection pressures to evade the pathways signaling such abnormalities to the immune cells. In this thesis we address the co-evolutionof pathogens and hosts of one such a pathway, i.e., the classical Ag presentation pathway. The recent adaptation of HIV-1 to its new human host was studied by enumerating the total number of predicted CTL epitopes and epitope precursors in a HIV-1 sequences gathered over more than 25 years. We investigated the adaptation of HIV-1 by comparing the distribution of CTL epitopes in HIV-1 proteins with other proteins, and found no conclusive evidence for any ongoing accumulation of CTL epitope escape mutations in HIV-1. This was surprising because two important molecules in the pathway (proteasome and TAP) display hardly any genetic variation between individuals, and a rapidly evolving virus like HIV-1 should in 25 years be able to evolve an escape from such monomorphic molecules. We hypothesized that the virus fails to escape from the monomorphic steps of the pathway because the most specific molecule in this pathway, i.e., the MHC, is highly polymorphic (i.e., is genetically diverse in the human population). Upon transmission of virus to a new host, previous proteasome and TAP escape mutations are released from immune selection pressure because the new host's MHC selects another set peptides from HIV-1's proteome than the previous host. This scenario was tested in an individual-based computer simulation model, in which an HIV-1 like virus was allowed to adapt to a human like host population. We showed that different degrees of MHC polymorphism in the host population affected how much the virus was adapting to the polymorphic MHC, or to the monomorphic proteasome and TAP. Finally, we extended the model such that the host population could evolve all components of its antigen presentation pathway in response to multiple endemic pathogens. This allowed us to study whether the current structure of the human antigen presentation pathway could be explained just in terms of host-pathogen co-evolution. Based on our earlier hypothesized mechanism, we expected that only the most specific step of the pathway would evolve a polymorphism. Under the constraint that the individual steps of the pathway remain co-evolved, it was indeed only the most specific step of the Ag presentation pathway that became polymorphic.

The vertebrate immune system uses several strategies to combat viral infections. Cellular immune responses evoked by the cytotoxic T cells (CTLs) are initiated after screening host cells for abnormalities. Examples are peptides derived from virus (epitopes) that are presented on the cell surface by major histocompability molecules (MHCs). Viruses therefore face strong selection pressures to evade the pathways signaling such abnormalities to the immune cells. In this thesis we address the co-evolutionof pathogens and hosts of one such a pathway, i.e., the classical Ag presentation pathway. The recent adaptation of HIV-1 to its new human host was studied by enumerating the total number of predicted CTL epitopes and epitope precursors in a HIV-1 sequences gathered over more than 25 years. We investigated the adaptation of HIV-1 by comparing the distribution of CTL epitopes in HIV-1 proteins with other proteins, and found no conclusive evidence for any ongoing accumulation of CTL epitope escape mutations in HIV-1. This was surprising because two important molecules in the pathway (proteasome and TAP) display hardly any genetic variation between individuals, and a rapidly evolving virus like HIV-1 should in 25 years be able to evolve an escape from such monomorphic molecules. We hypothesized that the virus fails to escape from the monomorphic steps of the pathway because the most specific molecule in this pathway, i.e., the MHC, is highly polymorphic (i.e., is genetically diverse in the human population). Upon transmission of virus to a new host, previous proteasome and TAP escape mutations are released from immune selection pressure because the new host's MHC selects another set peptides from HIV-1's proteome than the previous host. This scenario was tested in an individual-based computer simulation model, in which an HIV-1 like virus was allowed to adapt to a human like host population. We showed that different degrees of MHC polymorphism in the host population affected how much the virus was adapting to the polymorphic MHC, or to the monomorphic proteasome and TAP. Finally, we extended the model such that the host population could evolve all components of its antigen presentation pathway in response to multiple endemic pathogens. This allowed us to study whether the current structure of the human antigen presentation pathway could be explained just in terms of host-pathogen co-evolution. Based on our earlier hypothesized mechanism, we expected that only the most specific step of the pathway would evolve a polymorphism. Under the constraint that the individual steps of the pathway remain co-evolved, it was indeed only the most specific step of the Ag presentation pathway that became polymorphic.

Across vertebrates, spermatogenesis is under the endocrine control of two hormones, follicle-stimulating hormone (FSH) and androgens; the testicular production and secretion of the latter are controlled by luteinizing hormone. In fish, also the strong steroidogenic potency of Fsh should be taken into consideration indicating that a large part of Fsh action in fish spermatogenesis is mediated by androgens. In the studies encompassing this thesis, we aimed at elucidating the mechanisms, by which FSH and especially androgens regulate adult spermatogenesis with particular interest for the spermatogonial phase. Zebrafish was chosen as the experimental animal model. First, our studies identified 11-ketotestosterone (11-KT) as the main end product of the androgen synthesis pathway in zebrafish testis. Together with testosterone, 11-KT binds to and activates the zebrafish Ar at physiologically relevant concentrations. Subsequently, our developed experimental models revealed that all stages of spermatogenesis are dependent of androgens, and that the spermatogonial phase is particularly sensitive. In an in vitro model, an organ culture system for adult zebrafish testis, complete spermatogenesis was supported in the presence of 11-KT. Even more, a higher proportion of type A spermatogonia proliferated and differentiated to commit to the transformation process towards becoming spermatozoa. In contrast, in the absence of 11-KT spermatogenesis in the same testis culture system became depleted. This relates very well to the spontaneous and rapid downfall of the steroidogenic system under basal culture conditions. An in vivo model for oestrogen-induced androgen insufficiency revealed a marked inhibition of spermatogonial proliferation and differentiation, and germ cell depletion became more prominent with their progressive differentiation. The mechanism behind the inhibition of spermatogenesis in this in vivo model is negative oestrogenic feedback on the brain-pituitary system causing a reduction of expression of factors responsible for androgen synthesis, ultimately leading to reduced testicular androgen production. Sertoli cells express functional receptors for both FSH and androgens, in contrast to germ cells. We showed that zebrafish Sertoli cells contacting type A spermatogonia express high levels of ar transcript. It is thought that Sertoli cells mediate androgen or Fsh action by regulating the secretion of paracrine factors, which in turn influence spermatogonial differentiation. We showed that the gonad-specific fish representative of such a group of paracrine factors, the insulin-like growth factors (i.e. igf3), was highly expressed by zebrafish Sertoli cells contacting type A spermatogonia. Furthermore, its mRNA expression was positively modulated by both androgens as well as Fsh, for the latter independent of its steroidogenic potential. Studies on the action of a commercially available recombinant fish Igf protein (i.e. Igf1) on zebrafish spermatogenesis in vitro revealed a stimulatory effect on the proliferation of spermatogonia and their entrance into meiosis, and demonstrated the ability to enhance 11-KT-stimulated spermatogenesis. The present thesis expanded the knowledge on the sites of action and the mechanisms of endocrine control of adult fish spermatogenesis. In addition, novel experimental models to investigate the endocrine control of adult spermatogenesis were developed. Conclusively, zebrafish were proven to be an exciting animal model to investigate endocrine control mechanisms of the spermatogonial phase.

Across vertebrates, spermatogenesis is under the endocrine control of two hormones, follicle-stimulating hormone (FSH) and androgens; the testicular production and secretion of the latter are controlled by luteinizing hormone. In fish, also the strong steroidogenic potency of Fsh should be taken into consideration indicating that a large part of Fsh action in fish spermatogenesis is mediated by androgens. In the studies encompassing this thesis, we aimed at elucidating the mechanisms, by which FSH and especially androgens regulate adult spermatogenesis with particular interest for the spermatogonial phase. Zebrafish was chosen as the experimental animal model. First, our studies identified 11-ketotestosterone (11-KT) as the main end product of the androgen synthesis pathway in zebrafish testis. Together with testosterone, 11-KT binds to and activates the zebrafish Ar at physiologically relevant concentrations. Subsequently, our developed experimental models revealed that all stages of spermatogenesis are dependent of androgens, and that the spermatogonial phase is particularly sensitive. In an in vitro model, an organ culture system for adult zebrafish testis, complete spermatogenesis was supported in the presence of 11-KT. Even more, a higher proportion of type A spermatogonia proliferated and differentiated to commit to the transformation process towards becoming spermatozoa. In contrast, in the absence of 11-KT spermatogenesis in the same testis culture system became depleted. This relates very well to the spontaneous and rapid downfall of the steroidogenic system under basal culture conditions. An in vivo model for oestrogen-induced androgen insufficiency revealed a marked inhibition of spermatogonial proliferation and differentiation, and germ cell depletion became more prominent with their progressive differentiation. The mechanism behind the inhibition of spermatogenesis in this in vivo model is negative oestrogenic feedback on the brain-pituitary system causing a reduction of expression of factors responsible for androgen synthesis, ultimately leading to reduced testicular androgen production. Sertoli cells express functional receptors for both FSH and androgens, in contrast to germ cells. We showed that zebrafish Sertoli cells contacting type A spermatogonia express high levels of ar transcript. It is thought that Sertoli cells mediate androgen or Fsh action by regulating the secretion of paracrine factors, which in turn influence spermatogonial differentiation. We showed that the gonad-specific fish representative of such a group of paracrine factors, the insulin-like growth factors (i.e. igf3), was highly expressed by zebrafish Sertoli cells contacting type A spermatogonia. Furthermore, its mRNA expression was positively modulated by both androgens as well as Fsh, for the latter independent of its steroidogenic potential. Studies on the action of a commercially available recombinant fish Igf protein (i.e. Igf1) on zebrafish spermatogenesis in vitro revealed a stimulatory effect on the proliferation of spermatogonia and their entrance into meiosis, and demonstrated the ability to enhance 11-KT-stimulated spermatogenesis. The present thesis expanded the knowledge on the sites of action and the mechanisms of endocrine control of adult fish spermatogenesis. In addition, novel experimental models to investigate the endocrine control of adult spermatogenesis were developed. Conclusively, zebrafish were proven to be an exciting animal model to investigate endocrine control mechanisms of the spermatogonial phase.

DNA double strand breaks (DSBs) are induced by ionizing radiation, and during meiotic recombination. DSBs are repaired via two main pathways, homologous recombination (HR) and non homologous end-joining (NHEJ). There are three main types of male germ cells, spermatogonia, spermatocytes and spermatids. Spermatogonia include stem cells and subsequent types of proliferating spermatogonia and express many DNA repair proteins including gamma-H2AX (a marker for DSBs) and p53 binding protein1 (53BP1) but not MDC1 (mediator of DNA damage checkpoint). MDC1 is an important protein that functions in both HR and NHEJ. In the lack of MDC1, spermatogonia differ from proliferating somatic cells, and this may explain why our studies revealed a slow and incomplete repair of DSBs in spermatogonia. In early spermatocytes (leptotene, zygotene and early pachytene stages of the meiotic prophase) during meiotic recombination DSB are induced. Our findings indicate that, the error prone repair via NHEJ does not take place in early spermatocytes, also not to repair radiation induced DSBs. First, these cells do not express 53BP1, an important protein in NHEJ. Second, this notion could be further established by studying the repair of recombination related DSBs and of DSBs induced by irradiation in scid mice (deficient for NHEJ) and in Rad54/Rad54B deficient mice, potentially deficient for HR. Repair of DSBs in early spermatocytes in scid mice was similar to that in wild type mice, however the repair was significantly lower in Rad54/Rad54B deficient mice. Hence, DSB repair in early spermatocytes is exclusively carried out through HR. In late spermatocytes (mid-pachytene through diplotene stages) all important repair proteins were found and using Rad54/Rad54B deficient mice, and scid mice, we could establish that both NHEJ and HR pathways are active in these cells, NHEJ probably being the most important. The meiotic divisions render round spermatids that elongate and transform into spermatozoa. In spermatids, HR is not possible as thy are haploid and earlier results indicate that in these cells DNA-PKcs, a protein indispensable for classical NHEJ, is not expressed or at very low levels. Nevertheless, after irradiation gamma-H2AX foci disappear in these cells indicating that these cells are capable of DSB repair. Using scid mice and mice in which PARP1 protein function was inhibited, we could establish that in spermatids some residual classical NHEJ takes place and that about half of the repair takes place through the recently discovered alternative pathway for NHEJ, involving the PARP1/XRCC1 proteins. Nevertheless, DSB repair in round spermatids is slow and incomplete. We also found that, in contrast to other somatic cells in the testis, Sertoli cells, the supporting somatic cells in the seminiferous epithelium, express several DNA repair proteins and repair DSBs, as shown by the in vivo disappearance of 53BP1 foci after irradiation and the results of the comet assay. This, together with our finding that adult Sertoli cells resume proliferation in culture indicates that adult Sertoli cells can no longer be regarded as a classical example of terminally differentiated cells that are no longer able to proliferate and do not repair DNA DSBs.

In tropical lowland forests, which dominated by trees of Dipterocarpaceae family, trees and fungi are strongly linked, through ectomycorrhiza (EcM) formation. However, as part of rapid deforestation and forest transformation, mixed dipterocarp forests have been replaced by other vegetation and land use types. Considerable areas of the lowland tropical forest in Sumatra have been turned into Rubber Agroforests (RAF). RAF is a land use type in which rubber trees are planted, while allowing spontaneous establishment of forest tree species. The method presented in this thesis includes four main elements. Firstly, vegetation analysis in seven types of land use. Secondly, study in a nursery using dipterocarps species as bait plant to assess the effect of land use change on EcM inoculum and to analyze the effect of soil heating and drying on EcM propagules. Thirdly, an in-situ experiment in RAF with different history was done to assess whether fungal inoculant increased the survival, growth, nutrient uptake and EcM formation on Shorea selanica and Shorea lamellata. Finally, molecular techniques of PCR and sequencing have been applied to identify EcM fungal symbionts on dipterocarps. The study has been conducted in Jambi province, Sumatra, Indonesia. The ex-situ experiment has been conducted in the nursery of the Forest Research and Development Agency, Bogor, Indonesia. Identification of EcM fungi was done in the Fungal Biodiversity Centre (CBS), Utrecht, the Netherlands. The result of vegetation analysis showed that human activity in RAF management has strong effects on species richness. Very few trees dependent on EcM fungi were encountered in RAF. Our study in the nursery shows small differences in EcM colonization between soils derived from a wide range of land use types. It may indicate that survival of spores and colonization potential are not sensitive to the history of the site. Rapid recolonization of the heated soil in the experiment may imply sufficient availability of EcM fungi for dipterocarps under field conditions, where fire as a tool for land clearing is commonly used in Sumatra. The in-situ experiment in the RAF showed that lack of trees that are dependent on EcM fungi in RAF and in non-forested land does not necessarily imply the absence of EcM inoculum belowground in the ecosystem. EcM inoculum persists in the soil after forest was changed to RAF. Molecular identification of EcM fungi showed that Tomentella was the prevalent genus of EcM fungi colonizing dipterocarp seedlings in nursery stage and in the in-situ test 1 year after the seedlings have been planted in the field. None of the fungi was identified as Scleroderma columnare, which was inoculated to S. lamellata and S. selanica seedlings in the nursery stage. This indicated that indigenous EcM fungi dominated on the dipterocarp seedlings planted in the field, regardless of nursery inoculation. The implementation of this study is potentially of great value for reforestation efforts with dipterocarps in RAF in collaboration with rubber farmers. This reforestation approach will ensure the successfulness of the reforestation managed sustainably by the farmer, from which they may benefit in the future.

In tropical lowland forests, which dominated by trees of Dipterocarpaceae family, trees and fungi are strongly linked, through ectomycorrhiza (EcM) formation. However, as part of rapid deforestation and forest transformation, mixed dipterocarp forests have been replaced by other vegetation and land use types. Considerable areas of the lowland tropical forest in Sumatra have been turned into Rubber Agroforests (RAF). RAF is a land use type in which rubber trees are planted, while allowing spontaneous establishment of forest tree species. The method presented in this thesis includes four main elements. Firstly, vegetation analysis in seven types of land use. Secondly, study in a nursery using dipterocarps species as bait plant to assess the effect of land use change on EcM inoculum and to analyze the effect of soil heating and drying on EcM propagules. Thirdly, an in-situ experiment in RAF with different history was done to assess whether fungal inoculant increased the survival, growth, nutrient uptake and EcM formation on Shorea selanica and Shorea lamellata. Finally, molecular techniques of PCR and sequencing have been applied to identify EcM fungal symbionts on dipterocarps. The study has been conducted in Jambi province, Sumatra, Indonesia. The ex-situ experiment has been conducted in the nursery of the Forest Research and Development Agency, Bogor, Indonesia. Identification of EcM fungi was done in the Fungal Biodiversity Centre (CBS), Utrecht, the Netherlands. The result of vegetation analysis showed that human activity in RAF management has strong effects on species richness. Very few trees dependent on EcM fungi were encountered in RAF. Our study in the nursery shows small differences in EcM colonization between soils derived from a wide range of land use types. It may indicate that survival of spores and colonization potential are not sensitive to the history of the site. Rapid recolonization of the heated soil in the experiment may imply sufficient availability of EcM fungi for dipterocarps under field conditions, where fire as a tool for land clearing is commonly used in Sumatra. The in-situ experiment in the RAF showed that lack of trees that are dependent on EcM fungi in RAF and in non-forested land does not necessarily imply the absence of EcM inoculum belowground in the ecosystem. EcM inoculum persists in the soil after forest was changed to RAF. Molecular identification of EcM fungi showed that Tomentella was the prevalent genus of EcM fungi colonizing dipterocarp seedlings in nursery stage and in the in-situ test 1 year after the seedlings have been planted in the field. None of the fungi was identified as Scleroderma columnare, which was inoculated to S. lamellata and S. selanica seedlings in the nursery stage. This indicated that indigenous EcM fungi dominated on the dipterocarp seedlings planted in the field, regardless of nursery inoculation. The implementation of this study is potentially of great value for reforestation efforts with dipterocarps in RAF in collaboration with rubber farmers. This reforestation approach will ensure the successfulness of the reforestation managed sustainably by the farmer, from which they may benefit in the future.

Cryptococcus neoformans is a human pathogen that mainly causes disease in immuno-compromised individuals. The yeast cells or basidiospores spread through dissemination from environmental sources, for example pigeon droppings and dust, and causes a systemic infection, cryptococcosis. A severe clinical manifestation of cryptococcosis is meningo-encephalitis, but also infection of other organs like the lungs, and skin have been reported. Since the increased incidence of AIDS due to the spread of the HIV virus, especially in third world countries, C. neoformans has become one of the most emerging opportunistic pathogens in hospitals. In fact, it is one of the leading causes of death in patients suffering from AIDS. The incidence of cryptococcosis is also increasing in the Western world especially due to use of immunosuppressive drugs and increasing numbers of organ transplantations. The surface of the C. neoformans yeast cell can be surrounded by a thick capsule. This capsule is mainly composed of the polysaccharides GXM and GalXM. GalXM consists of the monosaccharides mannose, xylose and glucuronic acid, whereas GXM contains galactose, mannose and xylose. The capsule is critical for pathogenesis and harbors components that have immuno-modulatory properties. This and the physical properties of the capsule makes it a barrier for the immune system to reach receptors on the cell wall. Mutants have been isolated that have an acapsular phenotype. These mutants of the serotype A strain of C. neoformans are called CAP10, CAP59, CAP60 and CAP64. CAP67 is a serotype D mutant strain. The genes that are affected in these strains have been identified, but the exact role of the CAP genes is not clear. In this Thesis biosynthesis of the capsule and its role in pathogenesis was studied. This was done by investigating the content and the structure of the capsule, by looking at the genes involved in capsule biogenesis and by investigating the effect of mutants affected in capsule biogenesis on human dendritic cells. This is not only interesting from a fundamental point of view, it may also identify targets that can be used to improve treatment of cryptococcosis in the future.

Cryptococcus neoformans is a human pathogen that mainly causes disease in immuno-compromised individuals. The yeast cells or basidiospores spread through dissemination from environmental sources, for example pigeon droppings and dust, and causes a systemic infection, cryptococcosis. A severe clinical manifestation of cryptococcosis is meningo-encephalitis, but also infection of other organs like the lungs, and skin have been reported. Since the increased incidence of AIDS due to the spread of the HIV virus, especially in third world countries, C. neoformans has become one of the most emerging opportunistic pathogens in hospitals. In fact, it is one of the leading causes of death in patients suffering from AIDS. The incidence of cryptococcosis is also increasing in the Western world especially due to use of immunosuppressive drugs and increasing numbers of organ transplantations. The surface of the C. neoformans yeast cell can be surrounded by a thick capsule. This capsule is mainly composed of the polysaccharides GXM and GalXM. GalXM consists of the monosaccharides mannose, xylose and glucuronic acid, whereas GXM contains galactose, mannose and xylose. The capsule is critical for pathogenesis and harbors components that have immuno-modulatory properties. This and the physical properties of the capsule makes it a barrier for the immune system to reach receptors on the cell wall. Mutants have been isolated that have an acapsular phenotype. These mutants of the serotype A strain of C. neoformans are called CAP10, CAP59, CAP60 and CAP64. CAP67 is a serotype D mutant strain. The genes that are affected in these strains have been identified, but the exact role of the CAP genes is not clear. In this Thesis biosynthesis of the capsule and its role in pathogenesis was studied. This was done by investigating the content and the structure of the capsule, by looking at the genes involved in capsule biogenesis and by investigating the effect of mutants affected in capsule biogenesis on human dendritic cells. This is not only interesting from a fundamental point of view, it may also identify targets that can be used to improve treatment of cryptococcosis in the future.

In this thesis we studied various aspects of siRNA mediated silencing. siRNA mediated silencing is initiated by the introduction of dsRNA, transgenes and viral infection. Our first goal was to study the ability of the core pathway of RNA silencing to explain transgene and dsRNA induced silencing. To that extend we developed and studied concise models of the RNA silencing pathway. Secondly, we investigated the efficacy of RNA silencing to reduce viral infections, and added a replicating RNA virus. While we initially studied antiviral silencing within the cell, we extended our study to tissue level dynamics and symptom development in plants. Finally, we added virus encoded silencing suppressors and studied the effect and capability of the different silencing suppressors. We found that the available knowledge about the RNA silencing pathway was not sufficient to explain transgene induced silencing. We proposed mechanisms that could result in transgene induced silencing and recently the existence of these mechanisms has been confirmed experimentally. Our detailed model of both intra- and inter-cellular dynamics gives an explanation for the observed patterns on plant leaves that occur during viral infection. These patterns can have the form of local spots, concentric circles, or resemble mosaic-like patterns. We have shown that models studies are indispensable to understand complex cellular pathways. On the one hand we have shown that the generally accepted pathway of RNA silencing was insufficient to explain the experimental findings on transgene induced silencing it was supposed to explain. On the other hand we have shown that the interaction of RNA silencing and viral growth is indeed able to generate infection patterns for which the link to RNA silencing was still tentative.

In this thesis we studied various aspects of siRNA mediated silencing. siRNA mediated silencing is initiated by the introduction of dsRNA, transgenes and viral infection. Our first goal was to study the ability of the core pathway of RNA silencing to explain transgene and dsRNA induced silencing. To that extend we developed and studied concise models of the RNA silencing pathway. Secondly, we investigated the efficacy of RNA silencing to reduce viral infections, and added a replicating RNA virus. While we initially studied antiviral silencing within the cell, we extended our study to tissue level dynamics and symptom development in plants. Finally, we added virus encoded silencing suppressors and studied the effect and capability of the different silencing suppressors. We found that the available knowledge about the RNA silencing pathway was not sufficient to explain transgene induced silencing. We proposed mechanisms that could result in transgene induced silencing and recently the existence of these mechanisms has been confirmed experimentally. Our detailed model of both intra- and inter-cellular dynamics gives an explanation for the observed patterns on plant leaves that occur during viral infection. These patterns can have the form of local spots, concentric circles, or resemble mosaic-like patterns. We have shown that models studies are indispensable to understand complex cellular pathways. On the one hand we have shown that the generally accepted pathway of RNA silencing was insufficient to explain the experimental findings on transgene induced silencing it was supposed to explain. On the other hand we have shown that the interaction of RNA silencing and viral growth is indeed able to generate infection patterns for which the link to RNA silencing was still tentative.

Filamentous fungi form networks (mycelia) of long, tubular structures (hyphae) that extend at their tip (apex) and branch subapically (behind the tip). Hyphae are compartmentalized by cross walls (septa) that contain a central pore which is covered by a septal pore cap (SPC) in fungi belonging to the Basidiomycetes. In this thesis I studied the composition and function of the SPC of S. commune. SPCs were purified and their composition, proteins and corresponding genes were identified. Analysis of genes of the SPC was combined with improvement of methods for transformation and gene inactivation. Finally, I developed a method to study septa in the mycelium under stress. SPCs of S. commune were purified by separating homogenized mycelium containing 1 % Triton-X-100 on two discontinuous sucrose gradients, followed by filtration over a membrane. The SPC is composed of a proteinaceous core surrounded by membranous material. We show that this core determines the shape of the SPC and consists of two abundant proteins; SPC14 and SPC33. The SPC14 gene encodes a protein of 86 amino acids without known signal or signature sequences. Expressed sequence tag (EST) analysis indicates that SPC33 encodes a 239 and 340 amino acid variant that share a predicted signal anchor, a putative RXR ER localization signal, and a transmembrane region. SPC14 and SPC33 are only found in basidiomycetes with a SPC. Presence of SPC14 and SPC33 in the SPC was confirmed by immuno-localization. Previously, S. commune was transformed to phleomycin resistance using the ble gene of Streptoalloteychus hindustanus. I show that selective concentrations of phleomycin are mutagenic to resistant strains. Therefore, a nourseothricin resistance cassette was constructed with the nat1 gene of Streptomyces noursei. Transformation efficiency with nourseothricin was ten-fold lower, but could be restored by adding phleomycin which was accompanied by an increase of single copy integrations. The nourseothricin and phleomycin cassette were used to construct a vector for rapid screening of homologous integrations and was successfully applied for gene deletions in our lab. However, out of 800 transformants none showed inactivation of the SPC14 or SPC33 gene. Therefore, they might be essential, which is supported by the fact that RNA interference of the SPC14 gene was unstable. Reduced SPC14 mRNA levels, seemingly correlating with reduced growth in monokaryons and abnormal fruiting in dikaryons, was lost upon transfer to fresh medium. Using laser dissection it was assessed whether septa in a mycelium are open or closed. I showed that the apical septum is always open, the second septum is sometimes closed and the third septum is in most cases closed. Upon exposure to antibiotic, heat stress, or hypertonic conditions the first septum closes. The observed plugging mechanism seems to be an emergency response since arrest of growth and vacuolization of cytoplasm precede septal closure. Heat-induced closure was reversible, which indicates that septa are dynamic structures. Apical compartments were found to be able to reinitiate growth after separation from the mycelium. The fact that the third septum is mostly closed challenges the general assumption that mycelia are continuous systems.

Filamentous fungi form networks (mycelia) of long, tubular structures (hyphae) that extend at their tip (apex) and branch subapically (behind the tip). Hyphae are compartmentalized by cross walls (septa) that contain a central pore which is covered by a septal pore cap (SPC) in fungi belonging to the Basidiomycetes. In this thesis I studied the composition and function of the SPC of S. commune. SPCs were purified and their composition, proteins and corresponding genes were identified. Analysis of genes of the SPC was combined with improvement of methods for transformation and gene inactivation. Finally, I developed a method to study septa in the mycelium under stress. SPCs of S. commune were purified by separating homogenized mycelium containing 1 % Triton-X-100 on two discontinuous sucrose gradients, followed by filtration over a membrane. The SPC is composed of a proteinaceous core surrounded by membranous material. We show that this core determines the shape of the SPC and consists of two abundant proteins; SPC14 and SPC33. The SPC14 gene encodes a protein of 86 amino acids without known signal or signature sequences. Expressed sequence tag (EST) analysis indicates that SPC33 encodes a 239 and 340 amino acid variant that share a predicted signal anchor, a putative RXR ER localization signal, and a transmembrane region. SPC14 and SPC33 are only found in basidiomycetes with a SPC. Presence of SPC14 and SPC33 in the SPC was confirmed by immuno-localization. Previously, S. commune was transformed to phleomycin resistance using the ble gene of Streptoalloteychus hindustanus. I show that selective concentrations of phleomycin are mutagenic to resistant strains. Therefore, a nourseothricin resistance cassette was constructed with the nat1 gene of Streptomyces noursei. Transformation efficiency with nourseothricin was ten-fold lower, but could be restored by adding phleomycin which was accompanied by an increase of single copy integrations. The nourseothricin and phleomycin cassette were used to construct a vector for rapid screening of homologous integrations and was successfully applied for gene deletions in our lab. However, out of 800 transformants none showed inactivation of the SPC14 or SPC33 gene. Therefore, they might be essential, which is supported by the fact that RNA interference of the SPC14 gene was unstable. Reduced SPC14 mRNA levels, seemingly correlating with reduced growth in monokaryons and abnormal fruiting in dikaryons, was lost upon transfer to fresh medium. Using laser dissection it was assessed whether septa in a mycelium are open or closed. I showed that the apical septum is always open, the second septum is sometimes closed and the third septum is in most cases closed. Upon exposure to antibiotic, heat stress, or hypertonic conditions the first septum closes. The observed plugging mechanism seems to be an emergency response since arrest of growth and vacuolization of cytoplasm precede septal closure. Heat-induced closure was reversible, which indicates that septa are dynamic structures. Apical compartments were found to be able to reinitiate growth after separation from the mycelium. The fact that the third septum is mostly closed challenges the general assumption that mycelia are continuous systems.

Ambiguous images are images that have multiple plausible perceptual interpretations. A human observer viewing an ambiguous image may perceive alternate interpretations in turn. One factor that affects perception of ambiguous images is prior exposure to identical or similar visual input. This thesis reports investigations into the influence of prior visual experience and perception on perception of ambiguous images.

Ambiguous images are images that have multiple plausible perceptual interpretations. A human observer viewing an ambiguous image may perceive alternate interpretations in turn. One factor that affects perception of ambiguous images is prior exposure to identical or similar visual input. This thesis reports investigations into the influence of prior visual experience and perception on perception of ambiguous images.

Since two decades it has been suggested that the plasma membrane is organized into lipid-separated domains called lipid rafts. A number of functions have been attributed to these domains, including spatially separating or combining functionally linked proteins. Proteins such as the Epidermal Growth Factor Receptor (EGFR) are found to be localized in membrane domains, as well as effector molecules downstream in the signal transduction cascade. The investigation of these structures has mainly been performed by highly invasive techniques such as biochemical analysis, but lacks data on the situation in intact cells. Chapter 2 of this thesis describes the use of Förster Resonance Energy Transfer (FRET) to study the presence and dynamics of membrane domains containing EGFR. To study this receptor we developed fluorescent nanobodies, monovalent domains from cameloid heavy-chain only antibodies. FRET-FLIM analysis revealed that EGFR resides in a subclass of nanodomains including the ganglioside GM1, and is absent in domains composed of GM1 and GPI-linked GFP. Activation of EGFR results in the coalescence of the two domains, suggesting the formation of signaling platforms. Lipid domains are often suggested to promote the local clustering of their constituents. Therefore, a part of this work describes the development of a novel approach to study the oligomerization state of the domain components in intact cells. This technique, confocal time-resolved fluorescence anisotropy imaging microscopy (CTRFAIM), is based on FRET between identical fluorophores (homo-FRET). The anisotropy, defined as the degree of polarization of the fluorescence, is directly related to the degree of oligomerization. In this thesis, a simplification of this approach is described by applying time-gated data acquisition (chapter 3 and 4). An inducible FKBP dimerization and oligomerization system was developed to correlate the anisotropy value to the oligomerization state. Chapter 4 describes an evaluation of different modes of data acquisition. When compared to steady-state anisotropy, CTR-FAIM shows an improved dynamic range of anisotropy values. A further improvement can be obtained with two-photon excitation, although this improvement is diminished by significant higher photobleaching (chapter 4). When subjected to CTRFAIM analysis, the domain components in our study were indeed found to be clustered. By controlled photobleaching, the lipid raft probe GPI-GFP was found to form small nanoclusters of 1-5 molecules. Also EGFR was found to organize into oligomers, depending on the activation state of the receptor. The CTRFAIM data reveal pre-existing dimers of EGFR in the plasma membrane of resting cells. After stimulation with EGF, the receptor oligomerizes in a kinase- and phosphotyrosine-dependent manner, forming clusters of 3 or more receptors (chapter 5). Furthermore, induced receptor clustering enhances receptor internalization speed, which suggests a stimulatory role for EGFR oligomerization in the internalization process. In conclusion, these data suggest the formation of larger signaling platforms in the plasma membrane upon EGFR activation.

Since two decades it has been suggested that the plasma membrane is organized into lipid-separated domains called lipid rafts. A number of functions have been attributed to these domains, including spatially separating or combining functionally linked proteins. Proteins such as the Epidermal Growth Factor Receptor (EGFR) are found to be localized in membrane domains, as well as effector molecules downstream in the signal transduction cascade. The investigation of these structures has mainly been performed by highly invasive techniques such as biochemical analysis, but lacks data on the situation in intact cells. Chapter 2 of this thesis describes the use of Förster Resonance Energy Transfer (FRET) to study the presence and dynamics of membrane domains containing EGFR. To study this receptor we developed fluorescent nanobodies, monovalent domains from cameloid heavy-chain only antibodies. FRET-FLIM analysis revealed that EGFR resides in a subclass of nanodomains including the ganglioside GM1, and is absent in domains composed of GM1 and GPI-linked GFP. Activation of EGFR results in the coalescence of the two domains, suggesting the formation of signaling platforms. Lipid domains are often suggested to promote the local clustering of their constituents. Therefore, a part of this work describes the development of a novel approach to study the oligomerization state of the domain components in intact cells. This technique, confocal time-resolved fluorescence anisotropy imaging microscopy (CTRFAIM), is based on FRET between identical fluorophores (homo-FRET). The anisotropy, defined as the degree of polarization of the fluorescence, is directly related to the degree of oligomerization. In this thesis, a simplification of this approach is described by applying time-gated data acquisition (chapter 3 and 4). An inducible FKBP dimerization and oligomerization system was developed to correlate the anisotropy value to the oligomerization state. Chapter 4 describes an evaluation of different modes of data acquisition. When compared to steady-state anisotropy, CTR-FAIM shows an improved dynamic range of anisotropy values. A further improvement can be obtained with two-photon excitation, although this improvement is diminished by significant higher photobleaching (chapter 4). When subjected to CTRFAIM analysis, the domain components in our study were indeed found to be clustered. By controlled photobleaching, the lipid raft probe GPI-GFP was found to form small nanoclusters of 1-5 molecules. Also EGFR was found to organize into oligomers, depending on the activation state of the receptor. The CTRFAIM data reveal pre-existing dimers of EGFR in the plasma membrane of resting cells. After stimulation with EGF, the receptor oligomerizes in a kinase- and phosphotyrosine-dependent manner, forming clusters of 3 or more receptors (chapter 5). Furthermore, induced receptor clustering enhances receptor internalization speed, which suggests a stimulatory role for EGFR oligomerization in the internalization process. In conclusion, these data suggest the formation of larger signaling platforms in the plasma membrane upon EGFR activation.

Type II secretion systems (T2SS) of Gram-negative bacteria are multi-component apparatuses that secrete proteins into the extracellular milieu. These systems have homology to type IV pili, competence systems of Gram-positive bacteria and archaeal flagella. Although the systems are only involved in the translocation step over the outer membrane, most components are located in or attached to the inner membrane. Here we describe our studies into the main T2SS of Pseudomonas aeruginosa, the Xcp system. This system consists of 12 proteins, named XcpA and XcpP to XcpZ. Of these, only XcpQ is located in the outer membrane. It is thought to form a pore in this membrane through which other proteins are secreted. The components XcpT to XcpX, named pseudopilins, are believed to form a pilus-like structure, called pseudopilus, that pushes the secreted proteins through the pore. The pseudopilins are matured by the prepilin peptidase XcpA. The pseudopilus is believed to be assembled on top an inner membrane platform, consisting of the inner membrane proteins XcpS, XcpY and XcpZ and the cytoplasmic protein XcpR. Of these, XcpR presumably generates the energy required for the secretion process. XcpY and XcpZ mutually stabilise each other, and XcpY docks XcpR to the inner membrane. A last protein, XcpP, is believed to link XcpQ in the outer membrane to the inner membrane platform. We have studied the inner membrane protein XcpS. This is a highly conserved protein, of which the function is still unknown. XcpS has three membrane spanning domains. Its N terminus is located in the cytoplasm, whereas the C terminus is located in the periplasm. Investigations into its multimeric state showed that it preferentially trimerises. Furthermore, indications for higher oligomeric forms were obtained. Purified negatively stained XcpS was visualised with electron microscopy as ring-like structures, which seemed to contain a central cavity. XcpS was also demonstrated to interact with the major pseudopilin XcpT. When these two proteins were simultaneously introduced in an artificial lipid bilayer, evidence was found for pore-formation by XcpS. When XcpS and XcpT were co-produced in Escherichia coli, this induced a growth defect and a Psp response; both indications for stress. This further demonstrated the putative pore-forming capacities of XcpS upon interaction with XcpT. Additional experiments indicated that an XcpR-XcpY-XcpZ complex can block the XcpS pore, which explains how pore formation by XcpS does normally not lead to toxic effects in vivo. Other experiments showed that XcpS is N-terminally processed. This procedure was found essential for the functioning of the T2SS. A region in the cytoplasmic N-terminal domain important for this event was identified. This domain is also found in many homologues of XcpS, suggesting that processing of XcpS-like proteins occurs in all T2SSs. A last experimental chapter provides evidence for the polar localisation of the Xcp system and type II secretion. Altogether these results have been integrated into a new model for type II secretion, which basically explains the reason for pore-formation by multimeric XcpS. Thus, our results provide novel insights in the functioning of T2SSs.

Type II secretion systems (T2SS) of Gram-negative bacteria are multi-component apparatuses that secrete proteins into the extracellular milieu. These systems have homology to type IV pili, competence systems of Gram-positive bacteria and archaeal flagella. Although the systems are only involved in the translocation step over the outer membrane, most components are located in or attached to the inner membrane. Here we describe our studies into the main T2SS of Pseudomonas aeruginosa, the Xcp system. This system consists of 12 proteins, named XcpA and XcpP to XcpZ. Of these, only XcpQ is located in the outer membrane. It is thought to form a pore in this membrane through which other proteins are secreted. The components XcpT to XcpX, named pseudopilins, are believed to form a pilus-like structure, called pseudopilus, that pushes the secreted proteins through the pore. The pseudopilins are matured by the prepilin peptidase XcpA. The pseudopilus is believed to be assembled on top an inner membrane platform, consisting of the inner membrane proteins XcpS, XcpY and XcpZ and the cytoplasmic protein XcpR. Of these, XcpR presumably generates the energy required for the secretion process. XcpY and XcpZ mutually stabilise each other, and XcpY docks XcpR to the inner membrane. A last protein, XcpP, is believed to link XcpQ in the outer membrane to the inner membrane platform. We have studied the inner membrane protein XcpS. This is a highly conserved protein, of which the function is still unknown. XcpS has three membrane spanning domains. Its N terminus is located in the cytoplasm, whereas the C terminus is located in the periplasm. Investigations into its multimeric state showed that it preferentially trimerises. Furthermore, indications for higher oligomeric forms were obtained. Purified negatively stained XcpS was visualised with electron microscopy as ring-like structures, which seemed to contain a central cavity. XcpS was also demonstrated to interact with the major pseudopilin XcpT. When these two proteins were simultaneously introduced in an artificial lipid bilayer, evidence was found for pore-formation by XcpS. When XcpS and XcpT were co-produced in Escherichia coli, this induced a growth defect and a Psp response; both indications for stress. This further demonstrated the putative pore-forming capacities of XcpS upon interaction with XcpT. Additional experiments indicated that an XcpR-XcpY-XcpZ complex can block the XcpS pore, which explains how pore formation by XcpS does normally not lead to toxic effects in vivo. Other experiments showed that XcpS is N-terminally processed. This procedure was found essential for the functioning of the T2SS. A region in the cytoplasmic N-terminal domain important for this event was identified. This domain is also found in many homologues of XcpS, suggesting that processing of XcpS-like proteins occurs in all T2SSs. A last experimental chapter provides evidence for the polar localisation of the Xcp system and type II secretion. Altogether these results have been integrated into a new model for type II secretion, which basically explains the reason for pore-formation by multimeric XcpS. Thus, our results provide novel insights in the functioning of T2SSs.

It is generally thought that asymmetric competition for light and asymmetric growth, determines the course of succession in a regenerating secondary tropical forest, but it is hardly ever quantified. In this study a shrub and three woody tree species were monitored in time during the first five years of succession. Light competition and light-use were related to the success of species. It appeared that in a slow growing vegetation the importance of asymmetric competition for light and growth can be much less than often assumed. The species composition that was present from the very early beginning of succession remained for at least the first five years. Nitrogen-use efficiency may partly determine a plant’s success during forest regeneration. Whole canopy nitrogen-use efficiency and its underlying traits were compared among pioneer species over the first five years of succession. Nitrogen-use efficiency was largely determined by leaf life span and resorption and differed twofold among species. It was however not related to growth rates and only partly to species height. Nitrogen-use efficiency was slightly different within a species between successional stands that differed in height, leaf area index and resource availability, but an increase in competitive pressure did not result in major changes in the use of nitrogen. Forest regeneration is often slow or stagnates due to the excessive growth of non-woody plants and shrubs. One often used method to accelerate succession, called liberation, is opening up the vegetation canopy around young target trees which increases their growth. A 3D-model is used which enables us to examine how stature, crown structure and physiological traits of target trees and characteristics of the surrounding vegetation together determine the growth of trees. The model was applied to a liberation experiment that was conducted on pioneer species in a young secondary forest. Species responded differently to the treatment depending on their height, crown structure and their light demands. The responses were also dependent on the height and density of the vegetation and the gap radius from which it was removed. Another method to enhance regeneration is by planting tree species in the existing vegetation. Lines are cut and overstory thinning is applied to increase light levels. The line width and the degree of overstory thinning affect growth of target species differently depending on the characteristics of the surrounding vegetation and the light demands of the target trees themselves. The same model approach was used to test for the effects of line width, the degree of overstory thinning, planting trees in different successional stands of the same forest and planting trees in a forest stand with different light levels than the stand in which they were originally grown. It was shown that the optimal stand for each species, the optimal line width in a stand and the optimal degree of overstory thinning, can be predicted. With the model approach presented here it is possible to simulate different management activities aimed at improving forest regeneration and it has therefore a large potential in forest restoration ecology.

It is generally thought that asymmetric competition for light and asymmetric growth, determines the course of succession in a regenerating secondary tropical forest, but it is hardly ever quantified. In this study a shrub and three woody tree species were monitored in time during the first five years of succession. Light competition and light-use were related to the success of species. It appeared that in a slow growing vegetation the importance of asymmetric competition for light and growth can be much less than often assumed. The species composition that was present from the very early beginning of succession remained for at least the first five years. Nitrogen-use efficiency may partly determine a plant’s success during forest regeneration. Whole canopy nitrogen-use efficiency and its underlying traits were compared among pioneer species over the first five years of succession. Nitrogen-use efficiency was largely determined by leaf life span and resorption and differed twofold among species. It was however not related to growth rates and only partly to species height. Nitrogen-use efficiency was slightly different within a species between successional stands that differed in height, leaf area index and resource availability, but an increase in competitive pressure did not result in major changes in the use of nitrogen. Forest regeneration is often slow or stagnates due to the excessive growth of non-woody plants and shrubs. One often used method to accelerate succession, called liberation, is opening up the vegetation canopy around young target trees which increases their growth. A 3D-model is used which enables us to examine how stature, crown structure and physiological traits of target trees and characteristics of the surrounding vegetation together determine the growth of trees. The model was applied to a liberation experiment that was conducted on pioneer species in a young secondary forest. Species responded differently to the treatment depending on their height, crown structure and their light demands. The responses were also dependent on the height and density of the vegetation and the gap radius from which it was removed. Another method to enhance regeneration is by planting tree species in the existing vegetation. Lines are cut and overstory thinning is applied to increase light levels. The line width and the degree of overstory thinning affect growth of target species differently depending on the characteristics of the surrounding vegetation and the light demands of the target trees themselves. The same model approach was used to test for the effects of line width, the degree of overstory thinning, planting trees in different successional stands of the same forest and planting trees in a forest stand with different light levels than the stand in which they were originally grown. It was shown that the optimal stand for each species, the optimal line width in a stand and the optimal degree of overstory thinning, can be predicted. With the model approach presented here it is possible to simulate different management activities aimed at improving forest regeneration and it has therefore a large potential in forest restoration ecology.

Stem cells replenish the cells present in an organism throughout its lifetime and sustain growth. They have unique characteristics: the capability to self-renew and the potential to differentiate into several cell types. Recently, it has become clear that chromatin factors support these unique features in mammalian stem cells (Chapter 1). The role of chromatin factors in plant stem cell control is just starting to be revealed. In this thesis, we describe investigations on the roles of chromatin factors in Arabidopsis root stem cell maintenance. We performed a genome-wide survey of chromatin factor to identify which classes of chromatin factors could be genetically linked to stem cell action. In addition, we investigated the relationship of these chromatin factors with existing stem cell regulatory pathways. We find that several classes of chromatin factors (histone acetyltransferase GCN5, the CAF-1 complex, histone deacetylases (HDACs), PRC2 Polycomb proteins) regulate root stem cell niche maintenance. We show that GCN5 affects root stem cell niche maintenance mainly through expression regulation of stem cell transcription factor PLETHORA (PLT) (Chapter 2). The GCN5 associated factor ADA2b is not involved in stem cell niche maintenance, but mediates proliferation of the transit amplifying cells directly. Furthermore, we show that the chromatin assembly factor 1 (CAF-1) complex and RETINOBLASTOMA RELATED (RBR) repress synergistically root stem cell proliferation through regulation of stem cell niche transcription factors and promote correct differentiation (Chapter 3). The CAF-1 complex acts in the SHORTROOT (SHR)/ SCARECROW (SCR) pathway. We provide evidence that the CAF-1 complex and RBR may ensure propagation of repressive epigenetic marks at overlapping target genes together with HDACs and in this way play an important role in epigenetic stem cell control. In addition, we show that PRC2 Polycomb proteins SWINGER (SWN) and CURLY LEAF (CLF) are redundantly required to maintain the stem cells and expression of the root stem cell transcription factor SCR (Chapter 4). Our data reveal a novel function for RBR: it is redundantly required with SWN and CLF to stimulate proliferation or prevent differentiation of the transit amplifying cells. Finally, the results described in this thesis are discussed in light of recent insights into the specialized chromatin state in stem cells (Chapter 5). In conclusion, the research performed in this thesis reveals that several classes of chromatin factors are required for root stem cell maintenance and play a role in both parallel pathways known to define the root stem cells. In mammals, pluripotent stem cell transcription factors probably regulate chromatin factors to obtain a special chromatin state in ES cells. Similarly, in Arabidopsis, stem cell transcription factors are sufficient to induce root or shoot stem cells and it is an interesting possibility that they regulate chromatin factors, whereas it is already evident that chromatin factors regulate stem cell transcription factors.

Stem cells replenish the cells present in an organism throughout its lifetime and sustain growth. They have unique characteristics: the capability to self-renew and the potential to differentiate into several cell types. Recently, it has become clear that chromatin factors support these unique features in mammalian stem cells (Chapter 1). The role of chromatin factors in plant stem cell control is just starting to be revealed. In this thesis, we describe investigations on the roles of chromatin factors in Arabidopsis root stem cell maintenance. We performed a genome-wide survey of chromatin factor to identify which classes of chromatin factors could be genetically linked to stem cell action. In addition, we investigated the relationship of these chromatin factors with existing stem cell regulatory pathways. We find that several classes of chromatin factors (histone acetyltransferase GCN5, the CAF-1 complex, histone deacetylases (HDACs), PRC2 Polycomb proteins) regulate root stem cell niche maintenance. We show that GCN5 affects root stem cell niche maintenance mainly through expression regulation of stem cell transcription factor PLETHORA (PLT) (Chapter 2). The GCN5 associated factor ADA2b is not involved in stem cell niche maintenance, but mediates proliferation of the transit amplifying cells directly. Furthermore, we show that the chromatin assembly factor 1 (CAF-1) complex and RETINOBLASTOMA RELATED (RBR) repress synergistically root stem cell proliferation through regulation of stem cell niche transcription factors and promote correct differentiation (Chapter 3). The CAF-1 complex acts in the SHORTROOT (SHR)/ SCARECROW (SCR) pathway. We provide evidence that the CAF-1 complex and RBR may ensure propagation of repressive epigenetic marks at overlapping target genes together with HDACs and in this way play an important role in epigenetic stem cell control. In addition, we show that PRC2 Polycomb proteins SWINGER (SWN) and CURLY LEAF (CLF) are redundantly required to maintain the stem cells and expression of the root stem cell transcription factor SCR (Chapter 4). Our data reveal a novel function for RBR: it is redundantly required with SWN and CLF to stimulate proliferation or prevent differentiation of the transit amplifying cells. Finally, the results described in this thesis are discussed in light of recent insights into the specialized chromatin state in stem cells (Chapter 5). In conclusion, the research performed in this thesis reveals that several classes of chromatin factors are required for root stem cell maintenance and play a role in both parallel pathways known to define the root stem cells. In mammals, pluripotent stem cell transcription factors probably regulate chromatin factors to obtain a special chromatin state in ES cells. Similarly, in Arabidopsis, stem cell transcription factors are sufficient to induce root or shoot stem cells and it is an interesting possibility that they regulate chromatin factors, whereas it is already evident that chromatin factors regulate stem cell transcription factors.

Complete submergence is one of the most severe abiotic stresses worldwide, both in natural and man-made ecosystems. A limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris, a model for studying plant responses to flooding, manages to outgrow the water, and thus restore contact with the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment and mimics largely this morphological submergence response. The complexity of ethylene-driven growth in R. palustris upon submergence suggests the involvement of many genes under tight regulation. A comprehensive understanding of the changes in the expression of genes responsive to ethylene is crucial to elucidate the signal transduction networks that lead to hyponastic growth and under-water elongation in this plant. By applying cDNA subtractive hybridisation and cDNA-AFLP (RNA fingerprinting) we identified 119 differentially expressed genes during ethylene-induced hyponastic growth and petiole elongation. From those, 21 have very good homology with Arabidopsis genes. The expression kinetics of these genes were monitored in response to ethylene and submergence using Real Time RTPCR and the putative involvement of candidate genes in (differential) growth during ethylene and submergence treatment was confirmed. Furthermore, we applied whole genome profiling by hybridising mRNA from R. palustris to a CATMA spotted array containing Arabidopsis gene specific tags. This resulted in more than 5000 genes that were differentially regulated in one or both traits. Additionally, the presence of transcription factor binding motifs in the promoters of the differentially expressed genes were examined and demonstrated an over-representation of Abscisic acid responsive elements. Aanalysing the function of putative R. palustris orthologous genes in Arabidopsis during ethylene-induced hyponastic growth revealed that a subset of these genes is likely to control the initial angle of the petiole since, mutations in these genes resulted in constitutive elevated initial petiole angles in Arabidopsis mutant plants compared to the wild type Columbia-0. The others have an effect on either the angle of the petiole or leaf blade angle or both. Mutant analysis revealed a role for PSI-H subunit of photosystem I, glyceraldehyde-3-phosphate dehydrogenase and glutamate synthase in hyponastic growth of Arabidopsis. Furthermore, the effect of auxin response factor ARF8 on hyponastic growth was demonstrated. Reversible protein phosphorylation is a fundamental strategy used by eukaryotes to regulate basic cellular function. Upon a variety of stimuli, kinases phosphorylate downstream kinases usually until the target transcription factor is phosphorylated. Moreover, for many metabolic processes reversible phosphorylation of proteins is a key regulatory mechanism. We examined changes in the kinome profile of R. palustris in response to enhanced levels of ethylene and we discovered differential activity of many kinases. From these experiments, however, we cannot conclude the exact protein kinases involved in ethylene-induced (differential) growth. Our results do show, however, that the kinase regulatory system is strongly regulated during ethylene enriched conditions that induce hyponastic growth and petiole elongation in R. palustris.

Complete submergence is one of the most severe abiotic stresses worldwide, both in natural and man-made ecosystems. A limited number of species have evolved mechanisms to deal with these conditions. Rumex palustris, a model for studying plant responses to flooding, manages to outgrow the water, and thus restore contact with the atmosphere, through upward leaf growth (hyponasty) followed by strongly enhanced petiole elongation. These responses are initiated by the gaseous plant hormone ethylene, which accumulates inside plants due to physical entrapment and mimics largely this morphological submergence response. The complexity of ethylene-driven growth in R. palustris upon submergence suggests the involvement of many genes under tight regulation. A comprehensive understanding of the changes in the expression of genes responsive to ethylene is crucial to elucidate the signal transduction networks that lead to hyponastic growth and under-water elongation in this plant. By applying cDNA subtractive hybridisation and cDNA-AFLP (RNA fingerprinting) we identified 119 differentially expressed genes during ethylene-induced hyponastic growth and petiole elongation. From those, 21 have very good homology with Arabidopsis genes. The expression kinetics of these genes were monitored in response to ethylene and submergence using Real Time RTPCR and the putative involvement of candidate genes in (differential) growth during ethylene and submergence treatment was confirmed. Furthermore, we applied whole genome profiling by hybridising mRNA from R. palustris to a CATMA spotted array containing Arabidopsis gene specific tags. This resulted in more than 5000 genes that were differentially regulated in one or both traits. Additionally, the presence of transcription factor binding motifs in the promoters of the differentially expressed genes were examined and demonstrated an over-representation of Abscisic acid responsive elements. Aanalysing the function of putative R. palustris orthologous genes in Arabidopsis during ethylene-induced hyponastic growth revealed that a subset of these genes is likely to control the initial angle of the petiole since, mutations in these genes resulted in constitutive elevated initial petiole angles in Arabidopsis mutant plants compared to the wild type Columbia-0. The others have an effect on either the angle of the petiole or leaf blade angle or both. Mutant analysis revealed a role for PSI-H subunit of photosystem I, glyceraldehyde-3-phosphate dehydrogenase and glutamate synthase in hyponastic growth of Arabidopsis. Furthermore, the effect of auxin response factor ARF8 on hyponastic growth was demonstrated. Reversible protein phosphorylation is a fundamental strategy used by eukaryotes to regulate basic cellular function. Upon a variety of stimuli, kinases phosphorylate downstream kinases usually until the target transcription factor is phosphorylated. Moreover, for many metabolic processes reversible phosphorylation of proteins is a key regulatory mechanism. We examined changes in the kinome profile of R. palustris in response to enhanced levels of ethylene and we discovered differential activity of many kinases. From these experiments, however, we cannot conclude the exact protein kinases involved in ethylene-induced (differential) growth. Our results do show, however, that the kinase regulatory system is strongly regulated during ethylene enriched conditions that induce hyponastic growth and petiole elongation in R. palustris.

Plants are equipped with an array of defense mechanisms to protect themselves against herbivorous insects and microbial pathogens. Some are pre-existing while others are only activated upon insect attack or pathogen invasion. However, induced defense responses entail fitness costs. Therefore, plants possess elaborate regulatory mechanisms that efficiently coordinate activation of attacker-specific defenses. A major focus in plant defense signaling research is to uncover key mechanisms by which plants tailor their responses to different attackers, and to investigate how plants cope with simultaneous interactions with multiple aggressors. The phytohormones salicylic acid (SA) and jasmonic acid (JA) play a major role in the regulation of induced defense mechanisms against biotrophic pathogens and insect herbivores, respectively. Cross-talk between SA and JA defense signaling pathways is thought to allow fine-tuning of the defense response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PLANT DEFENSIN1.2 (PDF1.2), indicating that the SA pathway can be prioritized over the JA pathway. This antagonistic effect of SA on JA signaling is a robust phenomenon: It is conserved among Arabidopsis accessions, and JA-responsive gene expression is readily suppressed by SA for several days, even when triggered by very low doses of SA. Time interval studies revealed that SA has a window of opportunity to suppress MeJA-responsive gene expression, and that this time interval correlates with the SA-induced redox change in the plant tissue. Thus, redox modulation is likely to play a central role in the regulatory mechanism underlying SA/JA cross-talk. Analysis of the Arabidopsis mutant npr1, impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for suppression of JA-responsive genes, indicating that cross-talk between SA and JA is modulated through a function of NPR1 in the cytosol. TGA transcription factors are similarly required for both SA-dependent gene expression and SA-mediated suppression of JA signaling. However, TGA factors are unlikely to exert their effect through direct interaction with the promoter of JA-responsive genes such as PDF1.2, as removal of the TGA binding site in this promoter did not affect cross-talk. Promoter analysis of SA-suppressed, MeJA-inducible cross-talk genes revealed enrichment of the I box and the GCC box motif. Site-directed mutagenesis of the I box motif in the PDF1.2 promoter did not affect suppression of this gene by SA, indicating that the I box is not essential in mediating cross-talk. The role of the GCC box in cross-talk regulation should be further elucidated. This motif is required for MeJA-responsiveness in the PDF1.2 promoter, and thus provides an attractive target for SA-mediated suppression. Collectively, this work provides novel insight into how plants regulate their defense response upon attack by multiple aggressors, and may prove valuable for the development of novel strategies for crop protection.

The ability to adjust the phenotype (e.g. plasticity) is thought to be beneficial for the performance of a plant, because it prevents overinvestment in support structures when plant density is low, and allows a plant to position its leaves high in the canopy when density is high. However, as the plastic responses differ within species, exclusion of less responsive genotypes from dense vegetation is likely to occur. A 5 year old competition experiment between 10 genotypes of the stoloniferous plant Potentilla reptans was used to study the consequences of the genotypic differences in plasticity, and the interplay between these genotypes, in order to gain more insights in the mechanisms behind the competition for light Within the competition experiment, leaves that were positioned higher in the canopy had higher light interception and higher photosynthetic rates per unit invested biomass than lower places leaves. Placing laminas at the top of the canopy is thus of great importance to the performance of a genotype. Using artificially created light gradients, it was also shown that the speed of height increase determines which genotypes can place their leaves at the top. Height increase of the canopy differed between mono-genotypic stands, and thus depends on the genotypes that form the canopy. A canopy model of the competition experiment showed that the dominant genotype had rather shade tolerant photosynthetic characteristics, which allowed it to have positive carbon gain at the bottom of the vegetation. Therefore, low leaf turnover may have contributed to the abundance of this genotype. Coexistence could than occur because other genotypes have a higher carbon gain in the top layers of the canopy. A game theoretical analysis of the canopy model showed that for all genotypes the total lamina area that would maximize the photosynthetic rate of the genotype itself was higher than total the lamina area that would maximize the photosynthetic rate of the vegetation as whole. Selection would thus favor the former strategy, leading to lower biomass production of the whole canopy. Finally, the analyses indicate that a decrease of the total lamina area of the dominant genotypes would increase its own photosynthetic rate, but that it would increase the performance of other genotypes more. Yet although a mutation in the lamina area of the dominant genotype would not increase its performance, the vegetation is not evolutionarily stable, as a mutant in the lamina area of the other genotypes would lead to better performance of these mutants. Overall the results suggest that the performance of an individual is not only determined by its own characteristics, but also by that of its competitors. In addition to the ability to reach the top of canopy, relatively shade tolerant characteristics may be selected for if plant density is high. The findings also demonstrate that is important to know at what level of organization selection operates. The understanding of competitive exclusion would greatly benefit if plastic response were studied in relation to photosynthetic traits, and to temporal changes that occur during the growing season.

The ability to adjust the phenotype (e.g. plasticity) is thought to be beneficial for the performance of a plant, because it prevents overinvestment in support structures when plant density is low, and allows a plant to position its leaves high in the canopy when density is high. However, as the plastic responses differ within species, exclusion of less responsive genotypes from dense vegetation is likely to occur. A 5 year old competition experiment between 10 genotypes of the stoloniferous plant Potentilla reptans was used to study the consequences of the genotypic differences in plasticity, and the interplay between these genotypes, in order to gain more insights in the mechanisms behind the competition for light Within the competition experiment, leaves that were positioned higher in the canopy had higher light interception and higher photosynthetic rates per unit invested biomass than lower places leaves. Placing laminas at the top of the canopy is thus of great importance to the performance of a genotype. Using artificially created light gradients, it was also shown that the speed of height increase determines which genotypes can place their leaves at the top. Height increase of the canopy differed between mono-genotypic stands, and thus depends on the genotypes that form the canopy. A canopy model of the competition experiment showed that the dominant genotype had rather shade tolerant photosynthetic characteristics, which allowed it to have positive carbon gain at the bottom of the vegetation. Therefore, low leaf turnover may have contributed to the abundance of this genotype. Coexistence could than occur because other genotypes have a higher carbon gain in the top layers of the canopy. A game theoretical analysis of the canopy model showed that for all genotypes the total lamina area that would maximize the photosynthetic rate of the genotype itself was higher than total the lamina area that would maximize the photosynthetic rate of the vegetation as whole. Selection would thus favor the former strategy, leading to lower biomass production of the whole canopy. Finally, the analyses indicate that a decrease of the total lamina area of the dominant genotypes would increase its own photosynthetic rate, but that it would increase the performance of other genotypes more. Yet although a mutation in the lamina area of the dominant genotype would not increase its performance, the vegetation is not evolutionarily stable, as a mutant in the lamina area of the other genotypes would lead to better performance of these mutants. Overall the results suggest that the performance of an individual is not only determined by its own characteristics, but also by that of its competitors. In addition to the ability to reach the top of canopy, relatively shade tolerant characteristics may be selected for if plant density is high. The findings also demonstrate that is important to know at what level of organization selection operates. The understanding of competitive exclusion would greatly benefit if plastic response were studied in relation to photosynthetic traits, and to temporal changes that occur during the growing season.

This thesis describes developmental research, aiming at a useful approach for modern secondary ecology education. The research question is: What are the characteristics of a valid, feasible and effective learning and teaching strategy about ecosystem behaviour using modelling and systems thinking in authentic practices? Knowledge from and insight in complexity and dynamics of an ecosystem has societal relevance. Because of lack of such knowledge and insight, secondary students do not have the opportunity to develop ‘ecological literacy’. By making use of systems thinking and modelling, complexity and dynamics can become transparent for students who perform these activities themselves. In the Netherlands there has been a shift from transmission of knowledge towards construction of knowledge, where the teacher gets the role of a coach, more than as the transmitter of knowledge. The cultural historical approach is interested in the teacher’s role as coach as well as ‘mediator’ between students and culture. Here, learning requires a practice that invites students to participate in activities. The authenticity of the practice could provide students with motives for learning. A concept may have different meanings in different practices (or contexts). Students have to adapt this concept when they use it in another practice: re-contextualisation. For education to be effective, students should be willing and enabled to take an active role in the learning activities. The use of authentic practices will not be sufficient. The problem posing approach aims to actively involve students in the learning process. Therefore the concept-context-approach is linked with this approach. After raising modern scientific ideas of the concept ‘ecosystem’, the present situation in secondary education is investigated. Ecology education uses static models. In examination syllabus, schoolbooks and lessons there are hardly any traces of modern ecological views. Ecologists and teachers differ substantially in their views on the concept ‘ecosystem’. After determining learning aims derived from the modern concept ‘ecosystem’, design criteria are formulated for a learning and teaching strategy. Next, a selection is made of three authentic practices. The developed strategy has been tested in classroom to provide an answer to the research question. This strategy appears to be valid, in terms of introducing adequate ecosystem-related practices where dynamics and complexity are important sub-concepts, enabling students to understand ecosystem behaviour. It is feasible, in terms of the characteristics of a realistic, clear and relevant use of practices, with a transparent role for systems thinking; where there is a problem that elicits student’s ideas; and where the strategy is clear to the students. However, in terms of the characteristics of the systems character of an ecosystem, the modelling activities and the plausibility of the sequence of the practices, the strategy is not (completely) feasible. In some aspects the strategy is effective. Most students performed well on discriminating levels of organization, exploring models and getting an idea of complexity and dynamics. Most of the learning outcomes have been realised. However, three outcomes were certainly not (completely) attained, where ‘exemplify the relation between complexity, dynamics, stability and diversity’ is a very important outcome.

This thesis describes developmental research, aiming at a useful approach for modern secondary ecology education. The research question is: What are the characteristics of a valid, feasible and effective learning and teaching strategy about ecosystem behaviour using modelling and systems thinking in authentic practices? Knowledge from and insight in complexity and dynamics of an ecosystem has societal relevance. Because of lack of such knowledge and insight, secondary students do not have the opportunity to develop ‘ecological literacy’. By making use of systems thinking and modelling, complexity and dynamics can become transparent for students who perform these activities themselves. In the Netherlands there has been a shift from transmission of knowledge towards construction of knowledge, where the teacher gets the role of a coach, more than as the transmitter of knowledge. The cultural historical approach is interested in the teacher’s role as coach as well as ‘mediator’ between students and culture. Here, learning requires a practice that invites students to participate in activities. The authenticity of the practice could provide students with motives for learning. A concept may have different meanings in different practices (or contexts). Students have to adapt this concept when they use it in another practice: re-contextualisation. For education to be effective, students should be willing and enabled to take an active role in the learning activities. The use of authentic practices will not be sufficient. The problem posing approach aims to actively involve students in the learning process. Therefore the concept-context-approach is linked with this approach. After raising modern scientific ideas of the concept ‘ecosystem’, the present situation in secondary education is investigated. Ecology education uses static models. In examination syllabus, schoolbooks and lessons there are hardly any traces of modern ecological views. Ecologists and teachers differ substantially in their views on the concept ‘ecosystem’. After determining learning aims derived from the modern concept ‘ecosystem’, design criteria are formulated for a learning and teaching strategy. Next, a selection is made of three authentic practices. The developed strategy has been tested in classroom to provide an answer to the research question. This strategy appears to be valid, in terms of introducing adequate ecosystem-related practices where dynamics and complexity are important sub-concepts, enabling students to understand ecosystem behaviour. It is feasible, in terms of the characteristics of a realistic, clear and relevant use of practices, with a transparent role for systems thinking; where there is a problem that elicits student’s ideas; and where the strategy is clear to the students. However, in terms of the characteristics of the systems character of an ecosystem, the modelling activities and the plausibility of the sequence of the practices, the strategy is not (completely) feasible. In some aspects the strategy is effective. Most students performed well on discriminating levels of organization, exploring models and getting an idea of complexity and dynamics. Most of the learning outcomes have been realised. However, three outcomes were certainly not (completely) attained, where ‘exemplify the relation between complexity, dynamics, stability and diversity’ is a very important outcome.