We have developed novel fluorescence bio-imaging based automated models to screen for novel candidate targets involved in prostate cancer metastasis. Utilizing these models and adopting a... Show moreWe have developed novel fluorescence bio-imaging based automated models to screen for novel candidate targets involved in prostate cancer metastasis. Utilizing these models and adopting a functional genomics based approach; we identified SYK as a novel regulator of prostate cancer progression. We also identified functional involvement of MST1R in regulating the progression of prostate cancer. For both of these targets, there is supporting human clinical data to validate our results in prostate cancer. Show less
In this thesis, nanochannels as well as nanofluidic phenomena are used to provide new and miniaturized bioanalytical tools for the life sciences. Isotachophoresis performed in nanochannels showed... Show moreIn this thesis, nanochannels as well as nanofluidic phenomena are used to provide new and miniaturized bioanalytical tools for the life sciences. Isotachophoresis performed in nanochannels showed the focusing and separation of analytes in a 0.4 picoliter volume, which is a volume in the order of a sample from a single cell. Depletion zone isotachophoresis (dzITP) is demonstrated which uses a nanofluidic phenomenon, concentration polarization, to enable isotachophoresis in a microchannel while using only a single electrolyte. A concept for surface enhanced Raman spectroscopy (SERS) sensor (SERSOR) is explored; a coating protecting the SERS surface from irreversible adsorption may enable dynamic measurements of biomolecules in solution in minute volumes. Working with nanochannels has led to the discovery of new unexpected fundamental phenomena: the very high surface to volume ratio in nanochannels causes acidification of solutions introduced in them, despite the presence of up to 1 mol/L of buffer. Also, extreme pressures of more than a 1000 bar may be induced in a nanochannel by what we named electrocavitation, an effects shown to impose a limit on further downscaling of ITP. Show less
This thesis focuses on the development of image analysis methods for ultra-high content analysis of high-throughput screens where cellular phenotype responses to various genetic or chemical... Show moreThis thesis focuses on the development of image analysis methods for ultra-high content analysis of high-throughput screens where cellular phenotype responses to various genetic or chemical perturbations that are under investigation. Our primary goal is to deliver efficient and robust image analysis platforms which can 1) automatically detect cellular structures of interest from florescence microscope images and 2) quantify dynamics and organization of multi-cellular systems with phenotypic features. To recover heterogeneity of cellular behavior, we aim to develop single-cell-based image analysis methods so that cell subpopulations can be distinguished and investigated. Furthermore, we intend to develop methods to extract an ultra-high level of phenotypic details from images. This would enable system-level studies of phenotype characterization. Show less
This thesis is dedicated to the empirical study of image analysis in HT/HC screen study. Often a HT/HC screening produces extensive amounts that cannot be manually analyzed. Thus, an automated... Show moreThis thesis is dedicated to the empirical study of image analysis in HT/HC screen study. Often a HT/HC screening produces extensive amounts that cannot be manually analyzed. Thus, an automated image analysis solution is prior to an objective understanding of the raw image data. Compared to general application domain, the efficiency of HT/HC image analysis is highly subjected to image quantity and quality. Accordingly, this thesis will address two major procedures, namely image segmentation and object tracking, in the image analysis step of HT/HC screen study. Moreover, this thesis focuses on expending generic computer science and machine learning theorems into the design of dedicated algorithms for HT/HC image analysis. Additionally, this thesis exemplifies a practical implementation of image analysis and data analysis workflow via empirical case studies with different image modalities and experiment settings. However, the data analysis theorem will be generally illustrated without further expansions. Finally, the thesis will briefly address supplementary infrastructures for end-user interaction and data visualization. Show less
Chronic pain is a significant health problem that greatly impacts the quality of life of individual patients and imparts high costs to society. Despite intense research effort and progress in our... Show moreChronic pain is a significant health problem that greatly impacts the quality of life of individual patients and imparts high costs to society. Despite intense research effort and progress in our understanding of the mechanistic and molecular basis of pain, chronic pain remains a significant clinical problem that has few effective therapies Throughout the various chapters we have highlighted some important conceptual and experimental flaws in the way that pain signalling and pharmacological activity are characterised and translated across species and disease conditions. The common denominator of the work presented here is the requirement for accurate characterisation of exposure-response relationships, without which the dose rationale for the progression of a molecule cannot justified, whether drugs are aimed at symptomatic relief, disease modification or prophylaxis. In addition to a comprehensive review of the mechanisms underlying pain signalling and symptoms, the work developed here focuses on three different aspects of research underpinning the use of pharmacokinetic-pharmacodynamic relationships. First, we have explored the requirements for the characterisation of behavioural measures of pain during the early screening of candidate molecules, shedding light onto the shortcomings of experimental protocols commonly used in preclinical research. Then we introduced the prerequisites for the parameterisation of pain behaviour to ensure accurate translation of the pharmacological properties across species as well as for bridging across different phases of development. Lastly, an attempt was made to model clinical response in chronic inflammatory pain and to establish correlations between symptom improvement and the underlying pharmacological effects using biomarkers. In addition our work showed how clinical trial simulations can be used as a design tool, enabling the evaluation of a variety of scenarios that disentangle the contribution of pharmacology from the confounding effects of placebo and disease dynamics. Show less
This PhD thesis contains the following chapters. The first part, containing chapter 2 and 3 mainly concerns model development. Chapter 2 describes the development of a mathematical modeling... Show moreThis PhD thesis contains the following chapters. The first part, containing chapter 2 and 3 mainly concerns model development. Chapter 2 describes the development of a mathematical modeling framework within which different diagnostic models based on lipoprotein profiles can be developed, and a first validation of a model in this framework using data from a stable__isotope flux study (78). Chapter 3 describes a further development of the model, and the development of a new lipoprotein model__based candidate diagnostic and its validation using a range of stable__isotope flux studies. The second part, containing chapter 4, 5, and 6, concerns model applications. Chapter 4 contains the first application of the model__based candidate diagnostic using HPLC__measured lipoprotein profiles, in the context of a nutritional intervention study conducted at TNO. Chapter 5 contains an exploration of lipoprotein profile data from a population study (the GOLDN study), and examines how subgroups of patients, identified via K__means clustering based on lipoprotein profiles, differ in their lipid response to a fenofibrate intervention. It also examines how the model__based candidate diagnostic behaves in these subgroups. Chapter 6 contains the study that shows that lipoprotein metabolism indicators may have predictive value for cardiovascular risk on top of classical risk markers. Chapter 7 contains a methodological reflection on the development of computational models for clinical diagnostic use based on clinical chemistry data. Finally, chapter 8 contains a general discussion of the research presented in this PhD thesis and suggestions for further research Show less
The aim of the research described in this thesis is to study the chemical mechanisms responsible for protein aggregation induced by metal catalyzed oxidation and to investigate the relationship... Show moreThe aim of the research described in this thesis is to study the chemical mechanisms responsible for protein aggregation induced by metal catalyzed oxidation and to investigate the relationship between protein oxidation, aggregation and immunogenicity. To this end, recombinant human insulin rhIFN_-1a and rhIFN_-2a are used in conjunction with transgenic mice immune tolerant for the respective human endogenous counterparts. The impact of PEGylation on aggregate formation and excipients to prevent metal catalyzed oxidation are also evaluated. Show less
Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer... Show moreCellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted cellular changes, elicited by DNA damage, use of high-throughput techniques and integration with bioinformatics tools is of great value. This thesis summarizes recent advances in the field of systems biology studies of the DNA damage response and furthermore shows integrated approaches of the study of DNA damage response signaling networks in embryonic stem and cancer cells. By integration of transcriptional changes and the phosphorylation and metabolic response of cisplatin-treated embryonic stem cells, with RNAi-based knockdown screens we identify novel DNA damage response signaling networks, linking process such as Wnt signaling, translation arrest or altered metabolic pathways to the cellular response to DNA damage. Furthermore, genes, whose knockdown sensitizes embryonic stem cells to DNA damage-induced killing, are tested in cancer cells of varying genetic backgrounds identifying a small subset of genes, which represent potential drug targets for sensitization of cancer cells. Altogether, our systems approach for studying the DNA damage response identifies novel DNA damage-induced signaling networks and molecules, which modulate survival in the presence of DNA damage, potentially providing new targets for therapeutic intervention or biomarker discovery. Show less
Atherosclerose wordt veroorzaakt door een combinatie van verhoogde cholesterolniveaus en een chronische ontstekingsreactie. Deze ontstekingsreactie is het gevolg van een verstoorde balans tussen... Show moreAtherosclerose wordt veroorzaakt door een combinatie van verhoogde cholesterolniveaus en een chronische ontstekingsreactie. Deze ontstekingsreactie is het gevolg van een verstoorde balans tussen slechte, agressieve en goede, beschermende ontstekingscellen. In dit proefschrift wordt onderzocht hoe deze verstoorde balans in atherosclerose hersteld kan worden. Het onderzoek richt zich hierbij enerzijds op het remmen van de slechte ontstekingscellen en anderzijds op het stimuleren van de goede ontstekingscellen. Dit kan bereikt worden door de werking van costimulatoire en coinhibitoire eiwitten te be_nvloeden. Deze eiwitten zijn aanwezig op het celoppervlak van heel veel verschillende ontstekingscellen en bepalen of een ontstekingscel agressief of beschermend is. Costimulatoire eiwitten zorgen voor de activatie van een ontstekingscel, terwijl coinhibitoire eiwitten ontstekingscellen remmen. Blokkade van de costimulatoire eiwitten OX40L en CD30L remt atherosclerose, terwijl blokkade van het coinhibitoire eiwit Tim-3 atherosclerose verergert. Stimulatie van het coinhibitoire eiwit TIGIT vermindert de functie van T cellen. Een andere manier om de balans tussen goede en slechte ontstekingscellen te herstellen is door het aantal goede ontstekingscellen, zoals regulatoire T cellen en myeloid derived suppressor cellen, te laten toenemen. Eliminatie van regulatoire T cellen tot meer atherosclerose, terwijl een enorme expansie van regulatoire T cellen en myeloid derived suppressor cellen beschermend is. Show less
The skin barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which consists of dead corneocytes embedded in a highly organized extracellular lipid matrix.... Show moreThe skin barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which consists of dead corneocytes embedded in a highly organized extracellular lipid matrix. The lipids are thought to play a crucial role in the skin barrier function. This lipid matrix consists mainly of ceramides, cholesterol and free fatty acids in an approximately equimolar ratio. Atopic eczema (AE) is a chronic relapsing inflammatory skin disease that is characterized by dryness, erythema and pruritus. AE patients have a decreased skin barrier function as monitored with transepidermal water loss. The main objective of this thesis is to determine the SC lipid composition, lipid organization and lipid/protein ratio in AE patients and control subjects and to determine how these changes are associated with the impaired skin barrier function and disease severity of AE patients. The studies demonstrate that there is an altered lipid composition and lipid/protein ratio in non-lesional as well as lesional SC of AE patients. The changes in lipid composition result in an altered lipid organization that is associated with an impaired skin barrier function in AE patients Show less
Drug-induced organ toxicity is a major concern for pharmaceutical industry, due to removal of a high number of drugs from the market, as well as for public health, due to numerous hospitalizations... Show moreDrug-induced organ toxicity is a major concern for pharmaceutical industry, due to removal of a high number of drugs from the market, as well as for public health, due to numerous hospitalizations and patient death. The organs that are the primary target for such toxicities are the liver and the kidneys since both organs are continuously exposed to high concentrations of drugs and have high metabolic capacities. The immune system has been shown to be involved in the toxicity of several drugs- inducing liver and kidney injury. In particular, the cytokine tumor necrosis factor _ (TNF-_) has been shown to be the main constituent of the inflammatory processes responsible for the aggravation of drug-induced liver and kidney injuries. In this thesis, the role of TNF-_ in drug-induced organ injury was investigated. The main question was to assess the possible synergy between TNF-_ and druginduced cytotoxicities, and to unravel the underlying molecular mechanisms of such a synergy. Show less
Over the last decades several disciplines relevant to medicinal chemistry and preclinical drug discovery have made gigantic leaps; this includes chemistry, biology and measurement of bioactivity.... Show moreOver the last decades several disciplines relevant to medicinal chemistry and preclinical drug discovery have made gigantic leaps; this includes chemistry, biology and measurement of bioactivity. Better techniques have led to massive amounts of data. Moreover, sources of chemical and bioactivity data have become available in the public domain. Hence there is a need for new techniques combining and mining these data sources. This thesis focuses on computational methods combining data from these disciplines and demonstrates that the sum of these methods leads to better quality predictions than models using the individual data sources. One of the techniques central in this thesis is proteochemometric modeling, a machine learning approach linking chemical descriptors and protein descriptors to a biologically relevant output variable. This output variable describes the activity of molecules on biological macromolecules and hence proteochemometric models can make relevant predictions for both unseen molecules and unseen macromolecules (e.g. novel viral mutants). Secondly we present a novel technique that is able to combine information from multiple crystal structures in such a way that shared and unique pharmacophoric features can be isolated and visualized. Approaches presented here have been validated prospectively and have been shown to be widely applicable. Show less
