Streptomyces bacteria are a valuable source of natural products, many of which are used in the clinic or in biotechnology. In our search for novel antibiotics we discovered lugdunomycin, a natural... Show moreStreptomyces bacteria are a valuable source of natural products, many of which are used in the clinic or in biotechnology. In our search for novel antibiotics we discovered lugdunomycin, a natural product with a highly complex chemical architecture that is produced by Streptomyces sp. QL37. It is derived from the angucyclines, a well-known class of molecules known for their antibacterial and anticancer activities. Though angucyclines are produced in high quantities under most conditions, lugdunomycin is produced in minimal amounts. This thesis describes novel insights into the transcriptional control of the lugdunomycin biosynthetic gene cluster and into the lugdunomycin biosynthesis pathway. These insights may be applied to improve the yield of lugdunomycin and expand the chemical diversity of angucyclines. Using molecular biology, bioinformatic approaches and omics studies, such as metabolomics and transcriptomics, we have characterized the lugdunomycin biosynthetic gene cluster, the regulatory genes (lugRI–lugRV) required for transcriptional activation of the cluster, and the oxygenase genes (lugOI–lugOV) that play a key role in the different chemical rearrangements of the angucyclines. Furthermore, this thesis contains a detailed review of the regulatory network that controls antibiotic production in Actinobacteria. Show less
The largest genus within the Phyllanthaceae family is a group called Phyllanthus L. Recent studies have shown, that Phyllanthus is paraphyletic with the genera Glochidion, Breynia and Synostemon... Show moreThe largest genus within the Phyllanthaceae family is a group called Phyllanthus L. Recent studies have shown, that Phyllanthus is paraphyletic with the genera Glochidion, Breynia and Synostemon nested within it. In this thesis, I study the evolution and systematics of Phyllanthus and how to solve the apparent paraphyly. First we explore past morphological subgroups to come to a subdivision, which was tested using molecular phylogenetics. Several species of Phyllanthus are also used in traditional medicine and by utilizing the living collections of the Hortus botanicus Leiden, we tested for antibacterial and antifungal bioactivity. The phylogeny resulting from this study is used to study the historical biogeography and diversification of the tribe and to propose a new classification where Phyllanthus is separated into several smaller genera. Show less
Single-Molecule Microscopy (SMM) techniques constitute a group of powerful imaging tools that enable researchers to study the dynamic behavior of individual molecules.In the research described in... Show moreSingle-Molecule Microscopy (SMM) techniques constitute a group of powerful imaging tools that enable researchers to study the dynamic behavior of individual molecules.In the research described in this doctoral thesis, SMM techniques have been developed to image individual proteins inside cells of a living zebrafish embryo model and to study patterns of their mobility.The results of the mobility pattern analyses offer new insights into the dynamics of single molecules diffusing inside cells within the context of an intact vertebrate organism. Show less
Humans and songbirds learn their vocalizations early in life by exposure to the vocalizations of adult conspecifics. Often, better learning outcomes are achieved with live, social, tutors than with... Show moreHumans and songbirds learn their vocalizations early in life by exposure to the vocalizations of adult conspecifics. Often, better learning outcomes are achieved with live, social, tutors than with audio-only exposure to vocalizations. Many researchers argue that this is because social tutor-tutee interactions are important in the vocal learning process. It is unclear, however, whether and to what extent live tutoring also facilitates song learning because it results in multimodal exposure to a tutor, as tutees can both see and hear their tutor, while audio-only tutoring results in unimodal exposure. This thesis investigates whether multi- compared to unimodal exposure affects song learning in zebra finches, the primary experimental animal model for vocal learning studies, by revisiting the zebra finch song learning literature and describing three song tutoring experiments. In these experiments, young birds were provided with audio-only or audio-visual tutor exposure, using one-way mirrors, videos and robotic birds. The results suggest that multi- versus unimodal exposure to a live tutor can affect the timing of vocal development and possibly the amount of vocal learning. Multimodal exposure to artificial tutors made stimulus presentation more salient but did not affect the song learning outcomes assessed in this thesis. Show less
Filamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet,... Show moreFilamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet, the vast majority of the biosynthetic potential of Actinobacteria remains uncharacterised, largely because their biosynthetic gene clusters (BGCs) are poorly expressed in the laboratory, preventing the discovery of the cognate natural products. Additionally, only a narrow band of environments and a few taxonomic groups have been explored for gifted Actinobacteria. In this thesis different approaches are described, wherein we combined drug discovery with ecology, aimed at accessing the full potential of Actinobacteria. Bioactive Actinobacteria were isolated from a faecal sample of a 28,000-year-old-mammoth and their taxonomic and metabolic diversity was analysed. Furthermore, the effect of human stress hormones on the production of antibiotics by Streptomyces was investigated, resulting in the discovery of adrenaline as elicitor of siderophore production. This was later shown to be caused by the adrenaline analog catechol, which is ubiquitous in nature. Catechol also elicited the production of angucycline glycosides, well known for their therapeutic potential as anticancer and antibiotic compounds. Lastly, zebrafish were used as an in vivo model to explore the bioactive and functional potential of Actinobacteria within the animal microbiome. Show less
Vancomycin is a last-resort antibiotic for the treatment of many Gram-positive bacterial infections, while remaining inactive against Gram-negative strains. Resistance to vancomycin in Gram... Show moreVancomycin is a last-resort antibiotic for the treatment of many Gram-positive bacterial infections, while remaining inactive against Gram-negative strains. Resistance to vancomycin in Gram-positive stains continues to develop. This thesis describes the recent developments in semisynthetically modifying glycopeptide antibiotics to improve their antibacterial activity. Furthermore, the development of several semisynthetic glycopeptide antibiotics are described including the guanidino lipoglycopeptides, the vancomyxins, and the vancomycin-sideromycins. The guanidino lipoglycopeptides are readily synthesized from vancomycin and display potent in vitro and in vivo activity against Gram-positive bacteria, including vancomycin-resistant strains. Assessment of the activity, properties, and mechanism of action of the guanidino lipoglycopeptides shows the potential of these novel glycopeptides to become best-in class. The vancomyxins, which consist of covalently conjugated vancomycin and outer membrane disruptor polymyxin nonapeptide, display enhanced activity against Gram-negative bacterial strains compared to vancomycin monotherapy or co-administration of the two components. The vancomycin-sideromycins are also aimed at conferring antibacterial activity against Gram-negative bacteria by exploiting an iron-uptake system. Overall, a variety of semisynthetic vancomycin derivatives, aimed at overcoming vancomycin resistance or sensitizing Gram-negative strains, are developed and assessed on their activity in this work. Show less
Extending our current arsenal of antibiotics is key to staying ahead in the arms race between humans and resistant bacteria. Classes of antibiotics otherwise limited to the treatment of Gram... Show moreExtending our current arsenal of antibiotics is key to staying ahead in the arms race between humans and resistant bacteria. Classes of antibiotics otherwise limited to the treatment of Gram-positive pathogens may be potentiated against Gram-negative bacteria by disruption of their outer membrane. The work described in this thesis focuses on the development of novel synergists designed to selectively disrupt the outer membrane and in doing potentiate the activity of antibiotics that are otherwise inactive against Gram-negative bacteria. Show less
In his dissertation Michiel Hooykaas outlines the results of six empirical research projects focused at biodiversity awareness in the Netherlands, specifically people’s knowledge about animals.... Show moreIn his dissertation Michiel Hooykaas outlines the results of six empirical research projects focused at biodiversity awareness in the Netherlands, specifically people’s knowledge about animals. Providing insight into people’s perception of animal biodiversity is valuable both from a scientific perspective and from the perspective of conservation, as biodiversity loss continues and the relationship between humans and nature is under increasing pressure. The first part of this thesis focuses on species literacy, a newly coined concept that stands for broad and in-depth knowledge about species. Quantitative research projects are described that established the level of species literacy in professionals and laypeople. The second part of this thesis explores the portrayal of animals in cultural products aimed at children. Quantitative content analyses were used to examine the image of animal biodiversity conveyed by two product categories: picture books and clothes. The patterns uncovered in cultural portrayals of animals mirrored the knowledge patterns found in the first part of the dissertation. Animal groups well known by people predominated children’s fashion and picture books, while others were portrayed less frequently and in less specific manners. The revealed patterns imply that Dutch laypeople currently miss out on enriching experiences with biodiversity, and they hold important implications for conservation. The third and final part of this thesis explores, from the perspective of biodiversity communicators, the potential to connect people with biodiversity in places that are becoming increasingly urbanized. As such, promising avenues to foster species literacy and engage people with biodiversity are distilled. Show less
Death in all types of melanomas is generally caused by metastasis. Uveal melanoma (UM) is the most common intraocular melanoma, there are currently no (patient-derived) animal models that... Show moreDeath in all types of melanomas is generally caused by metastasis. Uveal melanoma (UM) is the most common intraocular melanoma, there are currently no (patient-derived) animal models that faithfully recapitulate metastatic dissemination of UM. Here we generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma. In doing so we can recapitulate the etiology of cancer in its totality. Subsequently, we developed a patient-derived zebrafish xenograft (zf-PDX) model, using spheroid cultures generated from metastatic and primary UM tissues. Harnessing this versatile model, we reveal high sensitivity of circulating UM cells to ferroptosis induction in vivo by Erastin and RSL3, implicating ferroptosis as a new potential therapy in metastatic UM.Increased melanin levels in cutaneous melanoma are associated with decreased patient survival. Melanin levels in primary uveal melanoma patient cells positively correlate with their metastatic potential in zebrafish. Modulation of melanin levels of pan-melanoma cells results in enhanced/reduced metastatic potential upon increased or decreased melanin levels, respectively. Melanin depletion sensitizes melanoma cells to ferroptosis inducers in zebrafish leading to a decreased metastatic burden. Collectively, our data identify melanin biosynthetic enzymes as potential future target to treat melanoma and show that melanin protects metastasizing melanoma cells from ferroptosis. Show less
The work described in this thesis focuses on the development of linear or cyclized peptide probes against protein N-methyltransferases to characterize their specific binding behavior, providing... Show moreThe work described in this thesis focuses on the development of linear or cyclized peptide probes against protein N-methyltransferases to characterize their specific binding behavior, providing further binding details for inhibitory activity study. The thesis not only describes the extended application to produce peptide-based transition states mimicking PRMT inhibitors but builds an LC-MS/MS method to evaluate CARM1 inhibition and activity. Show less
The experiments described in this thesis employ local lentiviral knockdowns in brain areas of female zebra finches followed by behavioural assays consisting of preference and Go/Nogo tasks.... Show moreThe experiments described in this thesis employ local lentiviral knockdowns in brain areas of female zebra finches followed by behavioural assays consisting of preference and Go/Nogo tasks. Ultimately, the targeted brain areas are extracted for gene expression analyses.The findings suggest that localised reduction of FoxP1 expression in HVC or CMM of female zebra finches does not impair the establishment or maintenance of auditory memories of conspecific song nor the females’ ability to discriminate or categorise auditory stimuli based on spectral or sequential features. Females which received a knockdown of FoxP1 in HVC as adults requested fewer familiar and unfamiliar playbacks and had a lower preference for familiar song than their matched controls. This might suggest that FoxP1 contributes to motivational behaviours in female zebra finches.Gene expression analyses links FoxP1 to pathways that have previously also been associated with FOXP2 in mammals including retinoic acid signalling and the SLIT-ROBO signalling cascade. Altered energy metabolism in different brain areas might also contribute to the observed phenotypes.Ultimately, the results presented in this thesis suggest implications of the transcription factor FoxP1 beyond vocal motor learning which need to be investigated in future studies. Show less
New drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder... Show moreNew drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder disease to treat. This thesis is focused on using the in vivo whole animalzebrafish embryo model for TB to evaluate potential anti-TB host-directed therapeutics (HDTs) arising from in vitro screens. Although in vitro screens for HDTs using cellular models can be performed at high throughput, a limiting step is the validation in whole animal models and translation of results to clinical applications. Due to the complex infection dynamics of mycobacteria, the use of whole animal models is indispensable in research into TB and the zebrafish model has contributed key findings about host-pathogen dynamics during mycobacterial infection. One of the most promising host targets of HDTs is autophagy, which is recognized as an important host-protective pathway. Boosting autophagy levels using HDTs could be a way to overcome the pathogen’s autophagy evasion strategies and could therefore be a promising therapeutic route. For this thesis we took advantage of the possibilities of the zebrafish embryo model for TB and the zebrafish toolkit to study several autophagy-modulating HDTs as potential anti-TB drugs. Show less
The research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and... Show moreThe research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and glucocorticoids.Leptin plays an important role during TB infection and has a huge impact on insulin sensitivity in zebrafish larvae. Similarly to what has been observed in the murine model, leptin deficiency in zebrafish increased the bacterial burden and mortality during the infection, leading to hyperglycemia and the development of insulin resistance. In addition, a novel SHP-1/SHP-2 inhibitor, NSC-87877, was shown to represent a promising anti-diabetic drug that can be used for further DM2 research, as it is able to rescue the phenotype of the leptin-deficient zebrafish and to restore glucose transport to the tissues. In contrast to metformin, NSC-87877 can act at very early developmental stages and inhibits the function of SHP-1 and factors that underlay impaired glucose metabolism, whereas metformin is mostly known to improve insulin sensitivity. Additionally, treatment with the glucocorticoid beclomethasone attenuates the metabolic changes associated with the infection, and transcriptional alterations induced by beclomethasone treatment suggest that genes involved in glucose metabolism, insulin and leptin signaling all play an important role in the modulation of the metabolism.Our data show that zebrafish larvae represent an interesting model system to investigate the complex pathology of TB, and the studies described in this thesis in which this model has been used have provided novel insights into the molecular mechanisms underlying wasting syndrome and the possibilities for adjunctive glucocorticoid therapy to alleviate this metabolic state. Show less
We developed the bitterling as a unique, well-studied model organism in the area of the evolutionary ecology of brood parasitism. The bitterling-mussel relationship, interspecific mussel host... Show moreWe developed the bitterling as a unique, well-studied model organism in the area of the evolutionary ecology of brood parasitism. The bitterling-mussel relationship, interspecific mussel host preference, and mussel gill structure are studied in detail, to help understand the developmental adaptation of bitterling embryos in response to their mussel hosts. Our complete stage series of the bitterling species R. ocellatus in Chapter 2 is a new, character-based systems that are compatible with the widely-used zebrafish staging system. With time-lapse video, we demonstrated the dynamic processes of hatching moment of the rosy bitterling in real time, which indicates the hatching process is mechanical rather than enzymatic. In Chapter 3, we described the neuroanatomy of bitterling for the first time, filling the gaps in the previous embryonic research in various bitterling taxa. Combined with the molecular analysis of brain early development in Chapter 4, brain development in the rosy bitterling is compared with that in the zebrafish. In Chapter 5, we studied the morphogenetic process of blastokinesis in the bitterling embryo, and its possible relation to brood parasitism. Show less
The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included... Show moreThe explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom ormucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes. Show less
We have examined sequences from the ligand-binding domain of the nicotinic acetyl choline receptor (nAChR) in 148 vertebrate species. We are in interested in this receptor because the α-neurotoxins... Show moreWe have examined sequences from the ligand-binding domain of the nicotinic acetyl choline receptor (nAChR) in 148 vertebrate species. We are in interested in this receptor because the α-neurotoxins of many venomous snakes binds to this receptor in its location at the neuromuscular junction in all vertebrates. Furthermore, some animals have evolved resistance to snake venoms and show modifications in the ligand binding domain of the nAChR which inhibit the binding of snake α-neurotoxins. Our analysis has shown that numerous vertebrate species, most of which were not previously known to possess α-neurotoxin resistance, do actually contain resistance-related modifications. These modifications are present in most of the taxa in our dataset, with the unexpected exclusion of the birds. It was particularly surprising to us that the snake-specialist predatory birds Circaetus pectoralis (black-chested snake eagle) and Sagittarius serpentarius (secretary bird) did not possess resistance modifications. There were also relatively few resistance-related mutations within the mammals. By contrast, there were multiple convergent evolutions of the well-characterised N-glycosylation motif within the squamate reptiles—particularly the snakes. We also identified a number of sites under positive selection, such as mutations to the proline subsite. Future functional testing will be needed to validate that these modifications do indeed confer resistance. To provide functional confirmation that resistance-related modifications do indeed reduce susceptibility to toxins, we used developmental bioassays. These assays showed that two species possessing resistance-related modifications of the nAChR (stickleback and bearded dragon) were less susceptible to the toxic effects of cobra venom than two species that lacked such modifications (zebrafish and chicken). In summary, we demonstrate that the range of mechanisms along with the phylogenetic distribution of resistance to snake α-neurotoxin appears to be more extensive than was previously appreciated. It also shows strong evidence of the convergent evolution of the same resistance mutations in independent linages. Our findings also support the notion that the mutations we have identified in this thesis may represent adaptive change in response to selective pressures exerted by α-neurotoxic snake venoms in an evolutionary arms race. Thus, we conclude that the evolutionary arms race between predator and prey appears to be a pervasive feature of the trophic interactions surrounding venomous snakes, which is shaping the molecular evolution of the nAChR in the vertebrates. Show less
Fungal food spoilage often starts with a contamination with spores. Experimental data strongly indicate the existence of subpopulations of spores with different levels of resistance to preservation... Show moreFungal food spoilage often starts with a contamination with spores. Experimental data strongly indicate the existence of subpopulations of spores with different levels of resistance to preservation methods. In this thesis, the extent of this heterogeneity and the underlying mechanisms using fungal model systems is studied. The role of the genetic background, environmental conditions and the developmental state of the spores were studied, using quantitative imaging, genome and RNA/protein sequencing as well as functional gene analysis. The role of transcription factors in weak acid stress resistance of Aspergillus niger is described. Next, heat resistance of fungal spores of three food spoilage species was quantified and compared. The genomes of Aspergillus niger strains were sequenced and compared revealing the existence of a possible sexual cycle. Melanin of fungal spores impacts UV-C resistance, but not heat resistance and a functional CRISPR/Cas9 genome editing system for Paecilomyces variotii and Penicillium roqueforti is described. Older spores are more heat resistant than younger spores, which can be contributed to differences in compatible solute composition. Additionally, a high cultivation temperature results in fungal spores with high heat resistance, possibly due to heat shock proteins. Show less
Cryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo... Show moreCryogenic electron microscopy (cryo-EM) is a powerful technique used to visualize the inside of cells and to study specific protein complexes. Within this thesis, I describe the use of various cryo-EM techniques to gain insight into the structural changes of the human pathogen, Vibrio cholerae, as it transitions between different environments. A combination of established and novel techniques is used to prepare the individual cells for cryogenic electron tomography (cryo-ET). For example, I designed a manual plunge freezing apparatus to prepare cryo-EM samples off site and subsequently image them with cryo-ET. Furthermore, I used light microscopy and serial block face scanning EM imaging to visualize changes to the cells’ morphology and structure when transitioning from the environment, into the natural host, the zebrafish (Danio rerio), and back into the environment. In addition, this thesis demonstrates how ultraviolet-C radiation of cryo-EM samples of V. cholerae and the ICP1 bacteriophage can be used to inactivate the pathogen while retaining their ultrastructural details. Lastly, this thesis outlines current and novel methods for processing of larger, more complex samples for cryo-ET. These techniques, together with new models of host-pathogen interactions, offer new tools for exploring microbial interactions with their environments. Show less
