Cardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and... Show moreCardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and immune cells in the arterial wall leading to a chronic local inflammation and lesion formation. In this thesis, we aimed to (1) validate the use of zebrafish in cholesterol metabolism and atherosclerosis research, (2) study the role of certain classes of scavenger receptors in lipoprotein uptake and cholesterol-based functions, and (3) validated two immune-based potential targets for atherosclerosis. 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
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
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
In this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin... Show moreIn this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin gene on cachexia and diabetes in rodent and zebrafish animal models. 3) how tuberculosis infection and resulting metabolic reprogramming are dependent on leptin signaling in mice and zebrafish larvae. Show less
In this thesis, two potential therapeutic targets for diabetic nephropathy were dentified and investigated. First, we show that glomerular clusterin is upregulated in diabetic nephropathy and... Show moreIn this thesis, two potential therapeutic targets for diabetic nephropathy were dentified and investigated. First, we show that glomerular clusterin is upregulated in diabetic nephropathy and demonstrated that recombinant clusterin protein can protect the podocytes against oxidative stress in vitro. Second, we reveal that hCN1 overexpression accelerated and aggravated diabetic nephropathy in BTBR ob/ob mice. We also studied two novel zebrafish models to investigate chronic kidney disease. We showed that lepb-/- adult zebrafish have the early signs of human diabetic nephropathy, and we demonstrated that ctns mutant adult zebrafish have the kidney pathologic features of human nephropathic cystinosis. Show less
This thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome... Show moreThis thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome scale metabolic reconstruction for zebrafish, that we used to study zebrafish metabolism upon infection with Mycobacterium marinum integrating gene expression data from control and infected zebrafish larvae. The chapters focusing on cell motility in response to the environment, revolve around the question of how the environmental inputs of cell-matrix interactions, cell-sized obstacles and cell-signalling upon wounding shape and guide cell motility. Show less
This thesis describes the possible applicability of mesenchymal stromal cells (MSCs) in the treatment of liver fibrosis. In CCL4 induced animal models for liver fibrosis, we showed that local... Show moreThis thesis describes the possible applicability of mesenchymal stromal cells (MSCs) in the treatment of liver fibrosis. In CCL4 induced animal models for liver fibrosis, we showed that local administration of MSCs after partial hepatectomy, results in a dose‐dependent on‐site amelioration of fibrosis. Furthermore, we compared the pro-regenerative and anti-fibrotic effects of four different subpopulations of MSCs, categorized on Endoglin (CD105) and VCAM (CD106) membrane expression. Our results showed that VCAM-positive subpopulations of MSCs are superior compared to VCAM-negative subpopulations in relation to their anti-fibrotic and pro-regenerative properties. In another study we showed that TAA induce liver fibrogenesis in zebrafish embryos through mechanisms similar to man and mice. In addition, we found that MSCs ameliorate fibrogenesis in this model.CRIPTO-1 is an (onco)foetal protein and is correlated to poor prognosis in HCC. The observations of our HCC study are suggestive for the existence of a more aggressive subgroup of HCCs recognized by their high CRIPTO-1 expression which also seems to be resistant to Sorafenib treatment. Cell survival and cell proliferation are some of the processes stimulated by CRIPTO-1, which are also known to be important during liver regeneration and fibrogenesis. We identified that multiple species show enhanced CRIPTO-1 during fibrogenesis and that elevated CRIPTO-1 plasma levels in humans with cirrhosis normalize after liver transplantation. Show less
Glucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of... Show moreGlucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of side effects and drug resistance. Therefore, there is still a major need to investigate the molecular and cellular mechanisms underlying the effects of GCs. Zebrafish are increasingly used as an in vivo model system for studying the immune system, in particular the inflammatory response. In Chapter 2, an overview is provided of the available inflammation models in zebrafish, and how they are used to unravel molecular mechanisms underlying the inflammatory response and for testing of potential novel anti-inflammatory drugs, in particular GCs. In this thesis, we have used zebrafish model system to study molecular and cellular mechanisms of GC action on the immune system and to develop a model for in vivo screening of the anti-inflammatory effects as well as possible adverse effects of novel GC therapies. For this purpose, we have studied the effect of GCs on leukocyte migration and differentiation during an inflammatory response (Chapter 3), how GCs modulate the immune response to a mycobacterial infection (Chapter 4), and we have investigated targeting of GCs to inflamed tissue by liposomal delivery (Chapter 5). Show less
Prostate cancer (PCa) is one of the most prevalent cancer in males. Although the majority of the patients can benefit from the present clinical treatments, 20%-30% of the patients who originally... Show moreProstate cancer (PCa) is one of the most prevalent cancer in males. Although the majority of the patients can benefit from the present clinical treatments, 20%-30% of the patients who originally respond to the therapy still develop incurable, castration-resistance bone metastases, which is a main cause of death in PCa . In this thesis, I combined an advanced zebrafish xenograft model with in vitro cellular approaches and mice xenografts to study the early stage of PCa metastasis. Using this comprehensive esearch platform, I identified multiple key signaling pathways that play essential roles in promoting the onset of PCa metastatis. The pathways I discovered include Cripto-associated EMT plasticity, CDC-42-N-Wasp-Cortactin associated mechanosensing and mechanotransduction, microenvironment dependent NF-ĸB-Activin A signaling pathway, and AMPK-Autophagy dependent metabolic stress coping pathway. Show less
In this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration... Show moreIn this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration modalities and anti-cancer efficacies of newly-developed PDT and PACT compounds, and test a light-triggered liposomal system for targeted drug delivery specifically to cancer cells in vivo. In chapter 2, we investigate the role of macrophages in tumour-induced angiogenesis. We show that macrophage-dependent angiogenesis is driven by macrophage recruitment to lactic acid secreted by glycolytic B16 melanoma cells. Chemical inhibition of macrophages and glycolysis blocks the initiation of angiogenesis in these models, suggesting that macrophages attracted to glycolytic melanoma cells contribute to the tumour-induced angiogenesis process.In chapters 3 and 4, we explore novel PDT and PACT compounds, respectively, for treatment of conjunctival melanoma in zebrafish. We inject conjunctival melanoma cells into the retro-orbital site to establish an orthotopic model and into the Duct of Cuvier to generate an ectopic model. Our results prove that zebrafish provides a fast vertebrate cancer model to test the optimal administration regimen of drugs, conditions of light irradiation, host toxicity and anti-cancer efficacy of PDT and PACT drugs against conjunctival melanoma.In chapter 5, we focus on modifying liposomes to be light triggered in order to deliver drugs specifically to cancer cells. We inject MDA231 breast cancer cells into the Duct of Cuvier at 2 days post fertilization (dpf) to initiate metastasis to the CHT. We successfully demonstrate that light-triggered, cell-specific delivery of liposome-encapsulated doxorubicin reduces the xenograft cancer cell burden without enhanced cytotoxicity of the zebrafish embryos. In chapter 6, we summarize the novel anti-cancer strategies, which we have developed using zebrafish xenograft models. In the same chapter, we frame our findings in the current scientific landscape and discuss future perspectives. Show less
This thesis elucidated the possibility of manipulating BMP/TGFβ signaling to achieve inhibition of breast cancer metastasis, including boosting BMP signaling via blockade of the BMP antagonist... Show moreThis thesis elucidated the possibility of manipulating BMP/TGFβ signaling to achieve inhibition of breast cancer metastasis, including boosting BMP signaling via blockade of the BMP antagonist Grem1 extracellularly or via stimulation of small-molecule compounds intracellularly, preventing TGFβ signaling to allow accumulation of pro-oncogenic stimuli. We also highlight the importance of selecting appropriate cancer types when adopting dual inhibition of PD-L1 and TGFβ signaling. I hope my research will aid in more efficient clinical cancer therapies. Show less
The zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the... Show moreThe zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the underlying pharmacological and (patho)physiological processes. In this thesis, we therefore developed and integrated innovative experimental and computational methods for the successful quantification of 1) the internal exposure over time after waterborne drug treatment, 2) disease dynamics and drug-induced changes therein, and 3) between-species differences in disease mechanisms. The state-of-the-art methods that we developed included nanoscale blood sampling, sensitive LC-MS/MS methods for drugs and their isomers and metabolites, and three-dimensional microscopy, integrated with non-linear mixed effects modelling to quantify the pharmacological processes in this small vertebrate. This multidisciplinarity enabled quantification of internal drug exposure-response relationships, contributed to positioning the zebrafish in the preclinical drug development pipeline, and inspired continuous collaborations between experimental and computational scientists. Show less
The work in this thesis describes the fundamental role of Lkb1 as a conductor of metabolism-related processes in zebrafish larvae. We show that Lkb1 is essential for the regulation of glucose... Show moreThe work in this thesis describes the fundamental role of Lkb1 as a conductor of metabolism-related processes in zebrafish larvae. We show that Lkb1 is essential for the regulation of glucose metabolism, the activation of autophagy, and hematopoiesis under conditions of metabolic stress. Furthermore, we also uncovered gene transcription profiles and hematological characteristics that are specific to lkb1 larvae, and independent of metabolic stress. Finally, we illustrate and highlight the potential of lkb1 larvae as screening platform in research related to metabolism, hematopoiesis, and tumors bearing LKB1 mutations. Overall, we have strengthened the value of lkb1 zebrafish larvae as a model to study the effects of Lkb1-inactivation on various metabolism-related processes Show less
The effective treatment of tuberculosis (TB) remains a major challenge to global health. Drug-resistant Mycobacterium tuberculosis (Mtb) strains and co-infection with HIV further increase the... Show moreThe effective treatment of tuberculosis (TB) remains a major challenge to global health. Drug-resistant Mycobacterium tuberculosis (Mtb) strains and co-infection with HIV further increase the difficulty of controlling TB. Thus, under the current situation, it is essential to develop effective treatment strategies for Mtb infections. Autophagy is a lysosomal degradation process and substantial experimental evidence has demonstrated that autophagy is an important host immune defense mechanism against mycobacterial infection. However, the development of effective therapies requires a better understanding of the interaction between the host and invading pathogens to identify host processes that can be targeted. A useful tool for such studies is the zebrafish model for TB. Zebrafish can be infected with Mycobacterium marinum (Mm), which is closely related to Mtb and causes similar disease characteristics. Taking advantage of the zebrafish TB model, this thesis presents new in vivo evidence for the important function of autophagy to inhibit mycobacterial proliferation inside macrophages. Furthermore, this study supports that stimulating the innate host defense processes that are dependent on the autophagy modulator, Dram1, and the selective autophagy receptors, p62 and Optineurin, could be a useful strategy to explore for adjunctive treatment of antibiotic-resistant TB infections. Show less
This thesis describes the introduction of the zebrafish animal model to the field of proteomcis. The work presented provided the research community with the first zebrafish spectral library, which... Show moreThis thesis describes the introduction of the zebrafish animal model to the field of proteomcis. The work presented provided the research community with the first zebrafish spectral library, which is an important first step to introduce zebrafish to proteomics research. This thesis also provides proteomics resourses to automate and simplify the creation of spectral libraries. Furthermore, it describes the use of the software programme CompareMS2 to provide information on similarities across proteomics databases. In addition, the programme has shown to be applicable as a quick first analysis step on large proteomics datasets. Besides, the thesis demonstrates the advantages and applicability of a multidisciplinary approach, addressing multiple omics, exemplified by using two closeley related model species (zebrafish and common carp). Finally, an overview is given on the advantages and disadvantages of the zebrafish animal model within the field of proteomics. The practical approach to zebrafish models in proteomics described in this thesis resulted in experimental protocols, a standard spectral library and data analysis tools, and significantly contributed to maturing of the zebrafish animal model system for proteomics research. Show less
Hydrogen peroxide (H2O2) is produced following injury and is required for regeneration. However, how H2O2 coordinates multiple signalling pathways required for regeneration is not known. Protein... Show moreHydrogen peroxide (H2O2) is produced following injury and is required for regeneration. However, how H2O2 coordinates multiple signalling pathways required for regeneration is not known. Protein-tyrosine phosphatases (PTPs) are regulators of signal transduction, reversing the phosphorylation of substrates, and are essential for many cellular processes. PTPs are also highly sensitive to reversible oxidation-mediated inhibition by H2O2.My research identified that some, but not all, PTPs become oxidized following amputation of the zebrafish caudal fin, and implicates PTPs that are oxidized as necessary for the regenerative process. Furthermore, I delineated that the PTPs Shp2 and Pten are required for the regenerative outgrowth phase of regeneration, and that Shp2 and Pten have a role in driving MAPK signalling and balancing PI3K signalling, respectively, during this phase. Show less
Control of infectious diseases poses continuous challenges for human health. Salmonella bacteria are a major cause of gastrointestinal infections and systemic disease like typhoid fever. We used... Show moreControl of infectious diseases poses continuous challenges for human health. Salmonella bacteria are a major cause of gastrointestinal infections and systemic disease like typhoid fever. We used zebrafish-Salmonella infection models to study host immune responses to Salmonella, particularly focusing on the role of the autophagy machinery. Autophagy and a related process known as Lc3-associated phagocytosis (LAP) trap invading microbes in intracellular vesicles and eventually eliminate them through the lysosomal degradation pathway. We show that macrophages and neutrophils trap Salmonella in Lc3-decorated phagosomes by a process dependent on the host factors Rubicon and NADPH oxidase, which generates anti-bacterial reactive oxygen species. This process could be defined as LAP as it requires some but not all components of the autophagy machinery. Genetic inhibition of LAP and ablation of macrophages resulted in hypersusceptibility to infections with both wild type and attenuated strains, revealing that LAP is an essential line of defense against Salmonella during systemic disease, when macrophages are the main carriers of the infection. Our studies also revealed a novel functional link between the autophagy modulator Dram1 and induction of LAP. This work encourages further studies aimed at the identification of autophagy modulating drugs for host-directed therapy of antibiotic-resistant Salmonella infections. Show less
One of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem.... Show moreOne of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem. The zebrafish is a promising model for in vitro vasculogenesis and angiogenesis studies, as a replacement for currently used mammalian models. However, the culture of endothelial cells from this species is not well characterized. Here, we test different culture strategies, medium supplementations and culture substrates for their effect on the generation of putative endothelial (fli:GFP+ and kdrl:GFP+) cells and vascular morphogenesis in zebrafish blastocyst cell derived embryoid body culture. we have also developed a perfused culture model, using microfluidic technology, to culture zebrafish vascular networks. This study is a step forward to the development of zebrafish vascular networks in vitro. Show less
