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
The power of personalized nutrition lies in being able to conduct clinical research on healthy people while capturing metabolic markers sensitive to the impact of environmental and metabolic... Show moreThe power of personalized nutrition lies in being able to conduct clinical research on healthy people while capturing metabolic markers sensitive to the impact of environmental and metabolic stressors (e.g. diet, changing sex hormones and the menstrual cycle). Using clinical biomarkers, metabolomics, and diet interventions with intake analyses, we demonstrated the metabolic impact of vegan and animal diet interventions using fasting plasma analysis after 48 hours and using postprandial plasma analysis after meals and snacks. Sexually dimorphic responses were differentiated using proteomics and pathway analyses in two larger, sex-balanced cohorts. Finally, clinical biomarker and metabolomics analyses identified metabolic subtypes across menstrual cycle phases. Although challenges with integrating –omics technology and nutrition remain, the fundamental information generated from these research studies may provide a foundation for future novel personalized nutrition strategies. Show less
The aim of this thesis is to expedite and ensure the systematic accuracy of clearance scaling from adults to paediatric patients, with a special focus on drugs undergoing hepatic metabolism. A... Show moreThe aim of this thesis is to expedite and ensure the systematic accuracy of clearance scaling from adults to paediatric patients, with a special focus on drugs undergoing hepatic metabolism. A physiologically-based pharmacokinetic simulation workflow was developed to unravel the conditions for accurate scaling of drug clearance from adults to children as young as term neonates of one day for various methods. This disproved the belief that a universal allometric exponent can scale size-related changes in clearance across the paediatric age range, and showed that isoenzyme maturation and drug properties, especially extraction ratio and drug binding to alpha-1-acid glycoprotein, should be accounted for when scaling clearance to young children. Based on these results, a clearance scaling decision tree is proposed, which allows pharmacologists for the first-time to select scaling method(s) that require a minimum but still sufficient amount of information to accurately scale clearance of drugs with known properties to a desired paediatric age-range. Moreover, an analysis framework is provided to assess the feasibility and clinical trial requirements for the estimation of PBPK parameters using population pharmacokinetic modelling, which has the potential to expedite development of PBPK models for understudied paediatric subpopulations. Show less
The research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a... Show moreThe research described in this thesis focused on identifying novel drug targets and synergistic combinations for triple-negative breast cancer (TNBC), a virulent subtype of breast cancer with a dismal prognosis and limited therapeutic options. In particular, the work centred on reversing resistance of TNBC cells to EGFR inhibitors. High-throughput kinase inhibitor library-based screens were utilised to evaluate the potential of novel targeted agents in a panel of TNBC cell lines and subsequently identify TNBC-specific genetic dependencies using siRNA-based screening. The signal transduction pathways perturbed by drug treatment were delineated and subsequently scrutinised using transcriptomic profiling and western blotting. The impact of drug treatment or gene silencing on cell death, proliferation, cell cycle progression and migration was assessed simultaneously. This work demonstrated that TNBC cell lines resistant to both MEK and Akt inhibitors are sensitive to disruption of CDK function. Additionally, it revealed that novel CDK inhibitors with strong activity against P-TEFb/CDK9 are highly effective against TNBC cells as single agents and in combination with multiple targeted therapies. These agents provoked profound down-regulation of multiple oncogenic pro-proliferative pathways, the silencing of which was detrimental to TNBC cell proliferation, thus defining several genes as potential future drug targets. Show less
Growth and development affect the metabolism of drugs administered to neonates, infants, and children. Research in this thesis focused on the metabolism by cytochrome P450 (CYP) 3A enzymes, aiming... Show moreGrowth and development affect the metabolism of drugs administered to neonates, infants, and children. Research in this thesis focused on the metabolism by cytochrome P450 (CYP) 3A enzymes, aiming to predict CYP3A-mediated clearance in neonates, infants, and children, by development of pediatric (physiological) population pharmacokinetic models.CYP3A-mediated systemic metabolism of midazolam in critically ill pediatric patients was found to be impacted by body weight, critical illness, and inflammation. The developed model was subsequently found to accurately predict clearance in postoperative children or critically ill patients. Furthermore, advanced physiological modelling methods were applied to distinguish between first-pass and systemic CYP3A-mediated metabolism to elucidate the role of intestinal and hepatic CYP3A in neonates and children covering the whole pediatric age range. Lastly, it was described when a pediatric covariate function for CYP3A-mediated midazolam clearance could be applied to scale plasma clearance of other CYP3A substrates in the pediatric population.This work will significantly improve CYP3A-mediated clearance predictions in neonates, infants, and children, which will ultimately lead to rational support for pediatric doses of CYP3A substrates in first-in-child studies during drug development and for pediatric dose recommendations for CYP3A substrates in clinical practice. Show less
Discovery and development of Central Nervous System (CNS) drugs is hampered by high attrition rates. One of the reasons is the lack of blood-based biomarkers that represent the interaction between... Show moreDiscovery and development of Central Nervous System (CNS) drugs is hampered by high attrition rates. One of the reasons is the lack of blood-based biomarkers that represent the interaction between the drug and the neurological systems of interest. Here we present a systems-pharmacology approach that combines a multi-biomarker approach (e.g. metabolomics) with pharmacokinetic/pharmacodynamic (PK/PD) modeling to reveal quantitative pharmacological characteristics that are relevant to dopaminergic drug action. Moreover, we set out to identify biomarkers that can be obtained from the blood as non-invasive sampling site. In the first section of this thesis the methodology is introduced in the context of translational CNS drug development. Moreover, a systematic search is performed to available biomarkers of dopaminergic drug action. Then, in the second part, the multi-biomarker PK/PD approach is applied to biomarkers from the neuroendocrine system as connection between brain and blood. In the third section, the methodology is developed using the simultaneous, time-resolved metabolomics response in brain extracellular fluid and plasma. By applying multi-biomarker PK/PD modeling we revealed quantitative pharmacological characteristics of dopaminergic drugs with regard to multiple biological processes. Moreover, we identified potential blood-based biomarkers of dopaminergic effect in the brain. Show less
Cardiovascular disease is the number one cause of death worldwide. The most important risk factor for developing this disease is high cholesterol levels in the blood. Other risk factors... Show moreCardiovascular disease is the number one cause of death worldwide. The most important risk factor for developing this disease is high cholesterol levels in the blood. Other risk factors contributing to cardiovascular disease can develop in individuals which are overweight. The clinical consequences of being overweight are clustered in the medical term: metabolic syndrome. Included in the metabolic syndrome are high blood pressure, dyslipidemia and glucose intolerance. At present, most cardiovascular disease patients are treated with statins which lower blood cholesterol levels. However, this treatment is not as effective in all patients and can cause some adverse drug reactions. Therefore, it is essential that novel therapeutic targets for the treatment of cardiovascular disease are identified. In this thesis, potential novel therapeutic targets in cardiovascular disease and metabolic syndrome are validated. In total, three potential targets were investigated: proteoglycan 4, protein arginine methyltransferase 3 and stabilin 1. Our studies showed the involvement of two of these targets in the development of cardiovascular disease and metabolic syndrome. Moreover, our results stress (1) that cardiovascular disease and metabolic syndrome are complex, multifactorial diseases with overlapping mechanisms and (2) that integration of research into both diseases can benefit therapeutic target identification and validation. Show less
In this thesis, we used genetically engineered mouse models to identify genes and pathways that are involved in ILC formation and in the development of resistance to FGFR-targeted therapy. These... Show moreIn this thesis, we used genetically engineered mouse models to identify genes and pathways that are involved in ILC formation and in the development of resistance to FGFR-targeted therapy. These mice carry conditional alleles of Cdh1, which result in the inactivation of the cell-adhesion molecule E-cadherin when Cre-recombinase is expressed. As mice with mammary-specific inactivation of E-cadherin alone were not prone to develop mammary tumors, they were used to investigate the contribution of additional genetic mutations to the development of ILCs using different genetic approaches. Firstly, we used non-germline modeling to study the role of PI3K-AKT signaling in the development of ILCs by performing intraductal injections of high-titer lentiviruses. Secondly, we employed a Sleeping Beauty (SB)-based insertional mutagenesis screen in conditional Cdh1 knockout mice to identify novel genes and pathways involved in the development of ILCs. 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, of which three are frequently aberrated in human ILCs. Lastly, we used active mobilization of transposons in transplanted mouse ILCs to identify genes involved in acquiring resistance to the FGFR inhibitor AZD4547. Show less
In summary, the collective results described in this thesis show that nanoparticulate vaccines can be delivered intradermally by coated and hollow microneedles and evoke antigen-specific immune... Show moreIn summary, the collective results described in this thesis show that nanoparticulate vaccines can be delivered intradermally by coated and hollow microneedles and evoke antigen-specific immune responses. The choice of both the nanoparticles and the microneedle(s) could have important influences on the immune responses. Microneedle arrays coated with antigen loaded and lipid bilayer fused mesoporous silica nanoparticles (MSNs) could be a promising system for convenient and fast intradermal delivery of protein antigen, although our results indicate that the system needs to be improved in order to obtain optimal immune responses. Moreover, antigen and adjuvant loaded nanoparticles can increase IgG2a (Th1) and CD8+ responses after intradermal delivery by hollow microneedles. This effect depends on the type and the physicochemical characteristics of the nanoparticles, in which smaller size and controlled release properties of antigen and adjuvant were found to correlate with the stronger effect. Finally, the combination of separate antigen loaded and adjuvant loaded nanoparticles may be as efficient as the antigen and adjuvant co-encapsulated nanoparticles for modification of the immune responses following intradermal immunization. Show less
The stratum corneum is the outermost skin layer and consists of dead cells embedded in a lipid matrix. The lipid matrix, consisting of ceramides, fatty acids, and cholesterol, is crucial for a... Show moreThe stratum corneum is the outermost skin layer and consists of dead cells embedded in a lipid matrix. The lipid matrix, consisting of ceramides, fatty acids, and cholesterol, is crucial for a proper skin barrier function. In inflammatory skin diseases the lipid composition and ordering is altered contributing to the impaired skin barrier. Vernix caseosa (VC) is the cheesy, white cream that covers the skin of the human fetus. Application of an in house developed synthetic VC enhanced skin barrier repair in mice. Currently, there are no suitable skin models available to study human skin barrier repair after application of a topical formulation. This thesis describes the development of a human skin barrier repair model and evaluates VC based formulations using this model. The results demonstrate that the barrier of this repair model mimics several aspects of inflammatory skin diseases. Additionally, it was shown that the lipid properties in this model were improved when a synthetic VC-based formulation was applied. Based on the outcome, clinical studies were performed. These studies showed that application on a disrupted human skin barrier in vivo enhanced the barrier repair. However, the effects of the formulation are limited when applied on atopic dermatitis skin Show less
