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
Inactivating mutations in BRCA1 or BRCA2 genes predispose to several types of cancer. Owing to their roles in maintaining genomic stability, lack of BRCA1/2 results in DNA damage repair defects, a... Show moreInactivating mutations in BRCA1 or BRCA2 genes predispose to several types of cancer. Owing to their roles in maintaining genomic stability, lack of BRCA1/2 results in DNA damage repair defects, a vulnerability that can be exploited therapeutically by the inhibition of poly(ADPribose) polymerase 1 (PARP1). Unfortunately, clinical benefit of PARPi therapy is often limited by emerging drug resistance. Identification of PARPi resistance mechanisms is therefore crucial to improve the clinical outcome and design strategies that would ultimately prevent or target resistant tumors.The use of genetically engineered mouse models (GEMMs) of BRCA1/2-associated breastcancer in this work has allowed us to model PARPi resistance in vivo in well-defined genetic contexts. By combining high-throughput genetic screens, multiple omics analyses and functional assays, we identified several factors of PARPi resistance and explained their role in therapy failure. Moreover, we established a new tumor-derived organoid system thatenables robust in vivo validation of putative drug resistance factors. Finally, work described in this thesis has advanced our understanding of basic biological processes involved in DNA damage signaling and repair. Show less
Triple-negative breast cancer (TNBC) constitutes a small subtype (~15%) of breast cancer, but causes the majority of breast cancer-related deaths. As defined by the absence of ER and PR expression... Show moreTriple-negative breast cancer (TNBC) constitutes a small subtype (~15%) of breast cancer, but causes the majority of breast cancer-related deaths. As defined by the absence of ER and PR expression and HER2 overexpression, TNBC is not curable by hormone receptor or HER2-targeted therapies. Furthermore, TNBC is highly heterogeneous and most aggressive. To date, cytotoxic chemotherapy remains the mainstay in the management of TNBC. Despite the initial response to the standard-of-care chemotherapy, TNBC often exhibits intrinsic or acquired drug resistance, and subsequently, recurs in local and distal organs. Targeted therapies have long been pursued for the treatment of TNBC, but rarely demonstrate satisfactory clinical outcomes. Therefore, improved understanding of the intricate biological basis underlying TNBC insensitivity to targeted agents and defining new therapeutic opportunities are of the upmost importance. The aim of the studies presented in this thesis was to systematically identify gene/kinase susceptibilities of refractory TNBC cells, and reveal novel potent targeted therapies for TNBC as monotherapy or in combination with approved kinase drugs. Show less
Traditionally, vaccines are administered intramuscularly using conventional hypodermic needles, which cause pain and distress. Microneedles are very short needles (smaller than 1 mm) that are... Show moreTraditionally, vaccines are administered intramuscularly using conventional hypodermic needles, which cause pain and distress. Microneedles are very short needles (smaller than 1 mm) that are practically invisible to the naked eye. Microneedles may administer vaccine into the skin in a pain-free manner. By using the intradermal route for immunization, a high number of immune cells in the skin are targeted, which may result in more efficient immunization. In this thesis, several aspects of intradermal immunization were investigated by using hollow microneedles. As differently specialized immune cells reside at different depths in skin, immunization outcomes as function of injection depth in skin were investigated to determine effects of injection depth. Furthermore, as prolonged antigen exposure may enhance immune responses, immunization outcomes resulting from repeated fractional intradermal dosing schedules were compared to those resulting from bolus dosing schedules. Finally, a layer-by-layer coating approach was developed for solid microneedle arrays. In the layer-by-layer coating approach, alternating layers of antigen and polymer are coated onto the surface of microneedle arrays. This approach enabled coating and intradermal delivery of a precise amount of antigen. Intradermal immunization using layer-by-layer coated microneedle arrays resulted in effective immunization. In conclusion, these studies provide important insights towards controlled microneedle-mediated intradermal immunization. Show less
Cardiovascular disease is a major global burden and atherosclerosis is the main underlying pathological process. Despite better management of cholesterol levels, there remains a significant... Show moreCardiovascular disease is a major global burden and atherosclerosis is the main underlying pathological process. Despite better management of cholesterol levels, there remains a significant residual risk of developing atherosclerosis and cardiovascular events. Hence, novel pathways and targets should be identified to optimize atherosclerosis therapy. Despite dyslipidemia, the immune system is also heavily involved in the pathophysiology of atherosclerosis. Protective immune responses in the acute setting of increased cholesterol levels eventually turn into debilitating responses when the immune system is chronically stimulated. Hence, we aimed to identify new therapeutic targets to dampen the immune response in atherosclerosis. More specifically, we focused our efforts on modulating the B lymphocyte response, for which there was a scarcity of data. In this thesis we describe novel ways to modulate the B cell response in atherosclerosis. We have found that there are specific B cell subsets that have different effects on the progress of atherosclerosis. For instance, removal of TIM-1+ B cells resulted in increased atherosclerosis, while removal of BTLA+ follicular B cells reduced atherosclerosis. In conclusion, this thesis provides promising immunological targets for the treatment of atherosclerosis. Show less
Childhood obesity is an increasing health issue. In the first part of this thesis comorbidities in children with obesity were studied, concerning the diagnostic process and dosing regimens. In... Show moreChildhood obesity is an increasing health issue. In the first part of this thesis comorbidities in children with obesity were studied, concerning the diagnostic process and dosing regimens. In children with obesity and respiratory symptoms the diagnosis of asthma was studied and in children with ADHD dosing regimens. Overtreatment as a consequence of overdiagnosis was frequently observed in children with obesity and asthma and undertreatment due to relative underdosing in the ADHD population with obesity. This highlights the necessity for accurate diagnostic processes alongside dosing regimens based on pharmacokinetic changes caused by obesity. The focus in the second part of this thesis was on screening for complications of obesity namely insulin resistance and cardiovascular diseases. Given the high prevalence of insulin resistance and the observed changes of cardiovascular parameters, screening on cardiometabolic complications is warranted in all children with obesity. Pharmacological treatment with metformin in addition to lifestyle intervention was studied in the last part of this thesis. Given the favorable effect on BMI in children and adults and the maintenance of weight loss and reduction in progression towards T2DM in adults, metformin can be considered in children with obesity and insulin resistance in addition to lifestyle intervention. Show less
To improve cancer treatments, personalized medicine approaches have aimed to identify exactly which mutations are driving tumor development in a given patient and specifically target these... Show moreTo improve cancer treatments, personalized medicine approaches have aimed to identify exactly which mutations are driving tumor development in a given patient and specifically target these mutations using precision therapies. However, one of the main challenges of this approach is identifying which mutations are true drivers, as tumors typically contain many additional passenger mutations that do not actually contribute to tumor development. Besides this, many patients often relapse after prolonged treatment due to the emergence of acquired resistance, limiting the clinical effectiveness of targeted treatments. In this thesis, we focussed on using genetically engineered mouse models to identify candidate cancer genes and therapy resistance mechanisms in two different breast cancers: invasive lobular carcinoma (ILC) and triple-negative breast cancer (TNBC). For ILC, we used transposon-based insertional mutagenesis (TIM) to uncover several novel cancer genes driving ILC development. Besides this, we also developed a novel computational approach (IM-Fusion) for improving the discovery of cancer genes from TIM screens and explored mechanisms of resistance in Fgfr2-driven ILC. For TNBC, we used CRISPR-based iterative mouse modeling combined with comparative oncogenomics to identify novel drivers of BRCA1-deficient TNBC. Finally, using combined in-vivo/in-vitro screens, we identified Parg as a driver of treatment resistance in BRCA2-deficient TNBC. Show less
Cardiovascular disease is the leading cause of death worldwide. The primary underlying pathology of cardiovascular disease is atherosclerosis. Atherosclerosis is a chronic, multifactorial disease... Show moreCardiovascular disease is the leading cause of death worldwide. The primary underlying pathology of cardiovascular disease is atherosclerosis. Atherosclerosis is a chronic, multifactorial disease in which lipid accumulates in the arterial wall, leading to a local inflammatory reaction and atherosclerotic plaque formation. Atherosclerotic disease develops largely asymptomatic over a lifetime. However, plaque rupture or erosion can cause the formation of a superimposed thrombus, blocking the flow of blood, and cause acute cardiovascular events such as myocardial infarction or ischemic stroke. Defects in cholesterol metabolism and hypercholesterolemia, which are major risk factors for atherosclerosis, have been shown to affect hematopoiesis, immune cell production and platelet counts and reactivity. Therefore, bone marrow cholesterol handling is an interesting target in the battle against cardiovascular disease, and acute cardiovascular events in particular. This thesis describes novel interactions between cholesterol metabolism and the production of immune cells and platelets, and its effects on atherosclerosis and atherothrombosis development. Show less
Atherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the... Show moreAtherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the vessel wall. Over time, this accumulation of lipids and immune cells induce morphological abnormalities in the vessel wall which cause the vessel lumen to narrow. This narrowing of the lumen (stenosis) causes ischemia in the downstream tissue. Prolonged ischemia causes myocardial ischemia and/or stroke. The research described in my thesis examines a well-recognized risk factor of atherosclerosis, being dyslipidemia, from an entirely new perspective. More specifically, it describes how dyslipidemia affects intrinsic metabolic processes in T cells, the conductors of the immune response characterizing atherosclerosis, and how this affects their function. My research has contributed to knowledge on the pathophysiology of atherosclerosis and might one day pave the way for the development of novel therapeutic approaches to treat cardiovascular disease. Show less
