This thesis describes our search to identify and understand possible regulatory mechanisms of integrin α6β4 in cell-matrix adhesion and intracelular signaling.
In this thesis, we aim to shed light on the diverse and often opposing roles of integrin α3β1 in cancer. Our work highlights that the role of α3β1 in cancer depends on time and place: the nature of... Show moreIn this thesis, we aim to shed light on the diverse and often opposing roles of integrin α3β1 in cancer. Our work highlights that the role of α3β1 in cancer depends on time and place: the nature of the cell environment (such as extracellular matrix composition), type of cancer and its driving mechanism, as well as the stage of the disease. We provide a new insight into the mechanisms behind the role of α3β1 in HER2-driven breast cancer and in DMBA/TPA-induced non-melanoma skin tumorigenesis. Show less
Dissolving microneedles are a subgroup of microneedle types that completely dissolve within the skin. During this dissolving process the vaccine, stored in the needle matrix, is released into the... Show moreDissolving microneedles are a subgroup of microneedle types that completely dissolve within the skin. During this dissolving process the vaccine, stored in the needle matrix, is released into the skin. Dissolving microneedles are made of a water-soluble, inert and safe material, such as hyaluronic acid. The microneedles have been used to vaccinate via the skin, which is a very immune competent organ that holds great potential for vaccine delivery. The minimally invasive and easy nature of the injections can reduce the risk of infections and alleviate the need for trained personnel. Furthermore, they eliminate all sharp waste as the needles dissolve during drug delivery.The goals of this project has been to improve the immunogenicity of vaccines through skin delivery, allowing for increased understanding of skin immunology and the development of pain free and safe vaccine delivery systems. Show less
In this thesis, we used genetically engineered mouse models and a variety of cell-culture based assays to identify genes and pathways that are involved in the development and treatment of invasive... Show moreIn this thesis, we used genetically engineered mouse models and a variety of cell-culture based assays to identify genes and pathways that are involved in the development and treatment of invasive lobular carcinoma (ILC). To identify novel genes and pathways involved in the development of ILCs we employed a Sleeping Beauty (SB)-based insertional mutagenesis screen in conditional Cdh1 knockout mice. We show that active transposon mutagenesis drives ILC formation and analysis of common insertion sites in SB-induced tumors identified a mutually exclusive group of four genes (MYH9, MYPT1/2 and ASPP2), three of which are frequently altered in human ILCs. We then went on to show that these hits not only drive ILC development but also do so through a shared mechanism. We identified that all four hits result in actomyosin relaxation which enables E-cadherin deficient mammary epithelial cells to invade into the mammary stroma and initiate tumor development. In addition, we show that mammary epithelial cells that lose E-cadherin expression can survive in the fibrous stroma directly surrounding the mammary ducts through interactions with components of the basement membrane. Lastly, we used active mobilization of transposons to identify resistance mechanisms to the FGFR inhibitor AZD4547. Show less
Cholesterol influences many pathways, including serving as precursor for adrenal steroidogenesis. Imbalance of cholesterol levels has been implicated in several diseases including cardiovascular... Show moreCholesterol influences many pathways, including serving as precursor for adrenal steroidogenesis. Imbalance of cholesterol levels has been implicated in several diseases including cardiovascular diseases and its underlying pathology, atherosclerosis. Here we concentrate on the role of a) lipid metabolism, especially high-density lipoprotein (HDL), in the development and regression of atherosclerosis and b) apolipoprotein E in adrenal glucocorticoid (GC) synthesis. We showed the importance of HDL size and functionality on atherosclerotic lesion formation in scavenger receptor-BI (SR-BI) knockout mice. Normalisation of the enlarged HDL particle size phenotype in these mice, trough depletion of phospholipid transfer protein, decreased atherosclerotic susceptibility and, contrary, development of a metabolic syndrome like phenotype. Furthermore, we studied the importance of HDL during regression of existing lesions in hypercholesterolemic apolipoprotein-E (ApoE) knockout mice. Normalizing the hypercholesterolemia resulted in regression of lesions and additional HDL depletion impaired the regression.The specific contribution of lipoprotein fractions to steroidogenesis is unknown. We lowered the (very) large-density lipoprotein fraction in ApoE-KO mice, resulting in a decreased GC output. ApoE is also produced within the adrenal where its local role is unclear. By transplanting an ApoE KO adrenal into an adrenalectomized wild-type mouse we revealed that local ApoE does not impact GC synthesis. Show less
Adenosine is an endogenous ligand which exerts its action by activating adenosine receptors (ARs), while its circulating levels are controlled via a variety of mechanism and proteins, amongst... Show moreAdenosine is an endogenous ligand which exerts its action by activating adenosine receptors (ARs), while its circulating levels are controlled via a variety of mechanism and proteins, amongst others the equilibrative nucleoside transporters (ENTs). Distortion of the adenosinergic tone is implicated in a multitude of pathophysiological conditions, thus numerous drug discovery efforts have been made to develop drugs targeting ARs and ENTs. Yet, there is only a limited number of drugs targeting these proteins on the market. Therefore, there is a pivotal need to develop novel concepts that allow us to increase our understanding of the mechanism of action at a molecular level, as well as physiologically relevant assays to evaluate drug candidates in early stages of drug discovery. Hence, this thesis focuses on exploring the concept of binding kinetics for two adenosinergic targets, i.e. the A2BAR and ENT1 (SCL29A1), as well as to develop novel kinetic binding and label-free functional assays. Show less
The overarching clinical aim of this thesis was to improve pharmacological pain management by characterizing the pharmacodynamics of analgesics. To achieve this aim, available advanced... Show moreThe overarching clinical aim of this thesis was to improve pharmacological pain management by characterizing the pharmacodynamics of analgesics. To achieve this aim, available advanced pharmacometric modelling approaches are used to maximize the information that can be obtained from the available clinical data. To address methodological gaps for pharmacodynamic analysis, we introduced two new model development tools, as well as two new pharmacodynamics modelling approaches for the characterization of iatrogenic withdrawal syndrome in children.In addition to expanding our pharmacodynamic knowledge, this work can increase the clinical applicability of existing pharmacokinetic models: knowing what drug concentrations will give desirable clinical outcomes, we can use pharmacokinetic models to evaluate if an analgesic dose regimen is likely to produce this concentration. The models developed in this thesis can serve as a starting point for further research into the pharmacodynamics of analgesics by extending these models to include additional patient populations or other analgesics. Moreover, the novel pharmacometric techniques that were introduced in this thesis can support future analyses in a variety of settings: repeated time-to-event data, iatrogenic withdrawal syndrome data, and composite clinical scale data. Show less
This thesis describes the importance of being able to control the selectivity of potential drug candidates. It explains how computational models are employed to predict and rationalize compound... Show moreThis thesis describes the importance of being able to control the selectivity of potential drug candidates. It explains how computational models are employed to predict and rationalize compound-protein binding (affinity) and therewith, selectivity of compounds. Moreover, it shows that selectivity can purposely be tuned to target either a single protein or an entire panel of proteins. The challenges of selectivity modeling are addressed based on case studies in the sodium-dependent glucose co-transporters, G protein-coupled receptors, and kinases. Show less
The objective of this study was to investigate the expression and function of GRHL2 in different breast cancer subtypes. In Chapter 2, we focused on the expression of GRHL2 in human breast cancer... Show moreThe objective of this study was to investigate the expression and function of GRHL2 in different breast cancer subtypes. In Chapter 2, we focused on the expression of GRHL2 in human breast cancer and the distinct effects of GRHL2 knockout on aspects of growth versus migration in basal A and luminal-like subtypes. In Chapter 3, ChIP-seq was used to explore the genomic landscape of GRHL2 binding sites in basal A and luminal-like subtypes of breast cancer and this data was used to predict shared and distinct GRHL2 target genes. In Chapter 4, based on a conditional GRHL2 knockout system, we determined the dynamic changes in genome-wide DNA transcription triggered by loss of GRHL2 in luminal-like breast cancer cells and used the data to predict affected pathways. In Chapter 5, ChIP-seq and BrU-seq data were integrated to identify genes whose transcription is controlled by GRHL2 and establish gene expression networks regulated by GRHL2 in luminal-like breast cancer. Show less
This thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and... Show moreThis thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and atherosclerosis. For each of these treatment strategies, the liposomal formulation was tailored to obtain the desired therapeutic effect. Chapter 2 reviews some of the most important physicochemical properties (size, shape, and rigidity) that determine the immunological effects of liposomes and other nanoparticles. In chapter 3 we present a detailed study on the effect of the rigidity of anionic liposomes, as measured by atomic force microscopy, on antigen-specific regulatory T-cell (Treg) responses. In chapter 4, we show that our optimized anionic liposomes can induce potent antigen-specific Treg responses, and can be used to delay atherosclerosis progression in a mouse model. Chapter 5 also focuses on liposomal treatment of atherosclerosis, but here targeted liposomes were prepared to successfully deliver a small molecule to foam cells in atherosclerotic plaques. In Chapter 6, we used cationic liposomes in combination with an adjuvant for cancer immunotherapy in mice. Finally, we summarize the overall findings in chapter 7 and discuss perspectives of using liposomes for vaccination and targeted drug delivery. Show less
Atherosclerosis is the most important underlying process that drives cardiovascular disease, and is characterized by an accumulation of cholesterol which triggers an inflammatory response in the... Show moreAtherosclerosis is the most important underlying process that drives cardiovascular disease, and is characterized by an accumulation of cholesterol which triggers an inflammatory response in the vessel wall. This results in the recruitment of many types of inflammatory cells towards the plaques that form in the vessel wall, among which are CD8+ T-cells. In this thesis, the role of CD8+ T-cells in the advanced stages of lesion development has been investigated, as this is the most clinically relevant stage of the disease. This thesis demonstrates that CD8+ T-cells exert a protective function. We show that the absence of CD8+ T-cells in a mouse model results in less stable atherosclerotic lesions with increased numbers of inflammatory cells. In a subsequent study, we show that CD8+ T-cells express an enzyme that inhibits the inflammatory process. We also show that injecting a specific subset of CD8+ T-cells is protective against the development of atherosclerotic lesions in mice. Importantly, we show that this data can be translated to atherosclerosis development in humans, as we demonstrate similar results using patient material obtained from endarterectomy surgery. Finally, we show that developing therapies directed towards activating CD8+ T-cells may be of value to inhibit the immune response, and thus reduce the risk of cardiovascular disease. Show less
The major and ultimate aim of metabolomics is to obtain an answer to a specific biological or clinical question. For that, many strategies have been applied in this field, including NMR and MS... Show moreThe major and ultimate aim of metabolomics is to obtain an answer to a specific biological or clinical question. For that, many strategies have been applied in this field, including NMR and MS-based approaches. CE-MS is one of the techniques in this field but remains underappreciated because of issues such as compromised sensitivity and poor reproducibility. In this thesis, we proposed standardized protocols for CE-MS studies using a sheathless interface and applied them in the metabolic analysis of ultra-small biological samples, such as low numbers of HepG2 cells. Another CE-MS technique used in this thesis is the traditional sheath liquid CE-MS, and it was successfully used in the metabolic profiling of plasma samples. We also introduced CE-MS with both interfaces in the field of nanosciences with our efforts in revealing the adsorption characteristics of polar metabolites to the surface of nanomaterials. This work clearly demonstrates that CE-MS can be reliably performed for metabolomics studies with acceptable repeatability. The use of sheathless CE-MS interface enables us to have in-depth profiling for ultra-small samples, and may become a powerful tool in the emerging field of single-cell analysis. 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 molecular mechanisms that instigate a healthy cell to become malignant are fueled by (epi)genetic alterations in so-called driver genes. While the Holy Grail of precision medicine is to... Show moreThe molecular mechanisms that instigate a healthy cell to become malignant are fueled by (epi)genetic alterations in so-called driver genes. While the Holy Grail of precision medicine is to identify these genetic dependencies and to target them with specific compounds in a personalized fashion, this has proven a daunting task, as tumors are exquisitely characterized by genetic instability and a mutator phenotype. Genetically engineered mouse models (GEMMs) are uniquely suited for functional in vivo validation of genotype-phenotype relationships, as they enable in vivo assessment of de novo tumorigenesis in a mammalian organism with intact immune and stromal compartments upon perturbation of (combinations of) oncogenes and/or tumor suppressor genes. Somatic modeling of cancer using CRISPR technology in vivo proved to be a true game-changing tool, allowing for rapid functional validation of candidate cancer genes enrolling from forward genetic screens and catalogs of alterations in human tumors. In this work, I showed how CRISPR approaches were deployed to precisely engineer tumorigenic events in the mouse mammary gland for dissecting oncogenic cascades, unraveling new therapeutic vulnerabilities and mechanisms of therapy resistance in different breast cancer subtypes. Show less
In this thesis, we aimed to better understand the underlying mechanisms involved in TNBC progression and metastasis formation and discover new targets to reduce breast cancer related deaths. We... Show moreIn this thesis, we aimed to better understand the underlying mechanisms involved in TNBC progression and metastasis formation and discover new targets to reduce breast cancer related deaths. We performed an imaging-based RNAi phenotypic cell migration screen in two highly motile TNBC cancer cell lines to provide a repository of signaling determinants that functionally drive TNBC cell motility. Interestingly, two modulators essential for cancer cell migration were known to be involved in RNA splicing, making us decide to focus on the role of RNA splicing in breast cancer progression. We next summarized the current knowledge about splicing factors in breast cancer development and progression and identified co-regulated splicing factors that were associated with aggressive breast cancer phenotypes and metastasis formation that was not only restricted to breast cancer, increasing the global understanding of the role of the spliceosome in cancer development and progression. Moreover, the role of splicing factors in two major processes in cancer progression, cell migration and proliferation, was examined. Finally, using RNA sequencing, we systematically compared the transcriptomes of 14 breast cancer cell lines cultured both in 2D and 3D conditions to unravel the reprogramming that is associated with the invasive phenotype of basal B TNBC. Show less
Cardiovascular diseases are the primary cause of death in the world with atherosclerosis as primary underlying cause. Atherosclerosis is characterized by cholesterol accumulation in the vessel wall... Show moreCardiovascular diseases are the primary cause of death in the world with atherosclerosis as primary underlying cause. Atherosclerosis is characterized by cholesterol accumulation in the vessel wall and inflammation of the vessel wall of medium to large size arteries. Both cholesterol accumulation and inflammation are pathogenic in the context of atherosclerosis. Current treatment regimens are tailored to reduce cholesterol levels in the blood. However, even a successful lowering of cholesterol is in many patients not sufficient to prevent a major cardiovascular event due to unresolved inflammation. Therefore, the immune system provides an interesting therapeutic target for the treatment of atherosclerosis. In this thesis we have explored the effect on atherosclerosis of several immunomodulatory strategies in pre-clinical models.As cholesterol is not soluble in water, cholesterol is transported in the bloodstream in particles called lipoproteins. The low-density lipoprotein (LDL) carries the highest concentration of cholesterol and accumulates in the vessel wall where a pathogenic specific immune response against LDL is instigated. In this thesis we have used several strategies to modulate the specific immune response against LDL, inducing LDL-specific regulatory T cells, antibodies, and cytotoxic T cells. Through immunoproteasomal inhibition we assessed the effect of general immune inhibition on atherosclerosis. Show less
Most small-molecule drugs are designed to interact with their biological targets under equilibrium binding conditions, whereby the desired drug-protein interaction is a rapid and reversible (non... Show moreMost small-molecule drugs are designed to interact with their biological targets under equilibrium binding conditions, whereby the desired drug-protein interaction is a rapid and reversible (non-covalent) process. As an extension to maximizing the strength of these noncovalent molecular interactions, a less conventional strategy termed ‘covalent interactions’ has recently gained reputation in the field of drug discovery. In this thesis a covalent strategy is applied and shown to be compatible with a target-directed, structure-guided discovery paradigm, with a focus on adenosine receptors as drug targets. The development and application of chemical tools and strategies are described to study three subtypes of ARs, A1R, A2AR and A3R. We set up a work flow of in vitro pharmacological assays as a robust tool for measuring and quantifying covalent modulation. Besides, we developed affinity-based probes, which allow monitoring of GPCR expression in cell fragments. Combined, this research approach may ultimately aid in the discovery and development of novel adenosine receptor-based therapeutics that lack potential side effects as much as possible. Show less
This thesis explores different avenues to develop insurmountable antagonists for CC Chemokine Receptors, such as CCR1, CCR2 and CCR5. These receptors, which belong to the large family of G protein... Show moreThis thesis explores different avenues to develop insurmountable antagonists for CC Chemokine Receptors, such as CCR1, CCR2 and CCR5. These receptors, which belong to the large family of G protein-coupled receptors (GPCRs), are implicated in a variety of inflammatory and immune diseases, including atherosclerosis, rheumatoid arthritis and cancer. Thus, numerous drug candidates have been developed over the years to target them. Despite promising preclinical data, most of these candidates have failed in clinical trials due to lack of efficacy, making necessary the development of novel tools and concepts to better study and target these receptors. Thus, throughout this thesis we have explored different mechanisms to achieve insurmountable inhibition, which include intracellular allosteric modulation, covalent inhibition and long residence time. Moreover, the crystal structure presented in this thesis provides a new template for the rational design of future antagonists. Finally, with the identification of several selective or multitarget intracellular ligands for CCR1, CCR2 and CCR5, we are expanding the toolbox to further modulate chemokine receptors. Overall, the results of this thesis may contribute to the development of novel chemokine receptor antagonists, and GPCRs in general, with improved in vivo efficacy. Show less
Growth hormone profiles are pulsatile and highly variable between individuals, limiting the implementation of mathematical models to quantify an individual's secretion.In this thesis, five key... Show moreGrowth hormone profiles are pulsatile and highly variable between individuals, limiting the implementation of mathematical models to quantify an individual's secretion.In this thesis, five key topics regarding the quantification of growth hormone (GH) in literature and the application in (future) clinical trials were addressed consecutively:1. The current standards in reporting clinical trial outcomes in acromegaly patients were assessed and recommendations for future reporting were provided 2. A new deconvolution-informed population pharmacodynamic model wasdeveloped and validated for the quantification of drug effects on pulsatile profiles 3. Population pharmacokinetic/pharmacodynamic models were developed to better understand the clinical pharmacological properties of BIM23B065 to support decision making and future clinical trial design 4. A population model for GH secretion based on physiological information,including a GHRH pulse generator, was developed based on data from differentexperiments to be used for the simulation of pulsatile GH profiles in healthy controls, active acromegaly patients and acromegaly patient after surgery. 5. The impact of different sampling protocols, ranging from a single sample to a 24h GH profile, on the study power and classification of responders in GH research were quantified and implementation of the research methodology in new scenarios was stimulated. Show less
Cell-based in vitro developed human skin equivalents facilitate screenings of compounds for therapeutic potential or toxicity and enable scientific research expanding knowledge on skin physiology... Show moreCell-based in vitro developed human skin equivalents facilitate screenings of compounds for therapeutic potential or toxicity and enable scientific research expanding knowledge on skin physiology and pathophysiology. Human skin equivalents resemble key features of native human skin, including the dermal and epidermal architecture. However, a limitation of human skin equivalents is the altered lipid barrier formation, which leads to a decreased barrier functionality. This could be induced by suboptimal cell culture conditions or the different cell microenvironment. The primary aim of this dissertational research was to enhance the morphogenesis and barrier formation of human skin equivalents to better mimic that of native human skin. The results indicate that modification of the dermal extracellular matrix by the biopolymer chitosan enhanced epidermal morphogenesis and barrier formation. Furthermore, by better resembling native skin conditions in vitro, primarily through a reduction in oxygen level, the epidermal morphogenesis and lipid barrier formation was improved. Finally, using a combinatory approach of optimized cell culture conditions and enhanced cell culture medium, the epidermal morphogenesis and barrier formation of human skin equivalents resembled that of native human skin more closely. Show less
