The G protein coupled receptor rhodopsin was characterised by physical chemical methods like solid-state NMR, FTIR and UV/Vis spectroscopy. Goal of the research was to determine the impact of... Show moreThe G protein coupled receptor rhodopsin was characterised by physical chemical methods like solid-state NMR, FTIR and UV/Vis spectroscopy. Goal of the research was to determine the impact of steric and electronic properties of the retinal ligand on the rate and efficiency of the photochemical reaction of this light activated receptor. First the required 13C labelled and chemically modified retinal derivatives were obtained by chemical synthesis. Subsequently, solid-state 13C NMR was used as a tool to characterise the electronic structure of the native ligand bound to rhodopsin, while FTIR difference spectroscopy was applied to determine the effect retinal ligands that were modified in the isomerisation region. It transpires that the combined approach of synthesis and spectroscopic techniques can reveal fundamental aspects of the interplay of the electronic properties and the spatial arrangement of the ligand that may ultimately allow a more profound understanding of the activation of GPCRs, in addition to knowledge about the ultrafast and efficient isomerisation of the retinylidene chromophore in rhodopsin Show less
The introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in... Show moreThe introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in the life science area. In this thesis we explored the possibilities to apply these holistic technologies to investigate the effects of known and potential anti-inflammatory compounds on macrophages. For this purpose we made use of a monocyte-like human histocytic lymphoma cell line U937. U937 cells can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. The two differentiation stages, monocyte and macrophage, were compared by using oligonucleotide microarrays and 2-D gel electrophoresis in combination with principal component analysis (PCA). This differentiation study is described in Chapter 2. The differential expression of three protein biomarkers, gamma interferon inducible lysosomal thiol reductase (GILT), cathepsin D and adipocyte-fatty acid binding protein (A-FABP) were biologically validated by Western blot and real time polymerase chain reaction (real time PCR). GILT and A-FABP were also found to be differentially expressed at the mRNA level as indicated by the results of the microarray experiment. Moreover, the transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. From the results presented in Chapter 2 can be concluded that the U937 cell line is an excellent model system for the blood-derived macrophage and that microarrays and 2-D gel electrophoresis are suitable methods to identify biomarkers for differentiation. Chapter 3 describes the use of a systems biology approach to categorize anti-inflammatory drugs based on their mRNA, protein and lipid expression pattern, as determined by oligonucleotide microarrays, 2-D gel electrophoresis and a LC-MS method for lipids, in combination with principal component discriminant analysis (PC-DA). The results described in this chapter demonstrate that different classes of anti-inflammatory compounds show distinct and characteristic mRNA, protein, and lipid expression patterns, which can be used to categorize known anti-inflammatory drugs, as well as to discover and classify new leads. The latter was exemplified by the categorization of zilpaterol, a poorly characterized ____-agonist. Exposure to zilpaterol gives rise to an almost identical expression pattern as that observed after exposure to the well-characterized __2-agonists clenbuterol and salbutamol, suggesting that zilpaterol is indeed a ____-agonist. In addition, this study revealed potential biomarkers for the different anti-inflammatory drugs under investigation. The categorization of the anti-inflammatory drugs on the basis of proteomics data alone was not successful. The most likely explanation for this is that by the analysis of whole cell lysates, only highly abundant proteins can be visualized, while the low abundant proteins, which are often involved in important metabolic pathways, are not. Therefore, a more focused approach was used to investigate the mechanism of action of zilpaterol, which is described in Chapter 4. In Chapter 4, U937 macrophages were stimulated with LPS to induce an inflammatory response. This response was inhibited by the addition of zilpaterol (LZ) and this inhibition was antagonized by the _2-adrenergic receptor antagonist propranolol (LZP). Two-dimensional difference gel electrophoresis (DIGE) in combination with Student__s t-test and two multivariate data analysis tools (PCA and partial least squares discriminant analysis PLS-DA) were used to examine the secreted proteome induced by the three treatments. This revealed 8 potential protein biomarkers. The protein spots were identified using nano LC-MS-MS. Only two of the identified proteins, namely macrophage inflammatory protein-1_ (MIP-1_) and macrophage inflammatory protein-1_ (MIP-1_) are known to be secreted proteins. The inhibition of MIP-1_ by zilpaterol and the involvement of the _2-AR and cyclic adenosine-3__,5__-cyclic monophosphate (cAMP) were confirmed using a specific immuno-assay. The experiments described in this chapter demonstrate the importance of pre-fractionation of complex protein samples before performing proteomics studies. The categorization of zilpaterol in Chapter 3 as a _2-adrenegic receptor agonist was further explored in Chapter 5. In this chapter we investigated the binding affinity of zilpaterol to the _1- and _2 receptor by using a receptor binding assay. Furthermore, we examined the role of the _1- and _2 adrenoceptor in the inhibition of the LPS induced tumor necrosis factor-alpha (TNF-_) production and the induction of cAMP by U937 macrophages. For this purpose we made use of a selective _1-receptor antagonist (atenolol), a selective _2-antagonist (ICI 118551) and a non-selective _-antagonist (propranolol). Finally, the inhibitory effect of zilpaterol on the TNF-_ production was investigated in LPS-treated male Wistar rats. The results obtained in this way clearly show that zilpaterol is a _2-adrenergic agonist and a inhibitor of the LPS-induced TNF-_ production by macrophages both in vivo and in vitro. The three _2-agonists specific biomarkers, Granulocyte Chemotactic Protein-2 (GCP-2/CXCL6), Oncostatin M (OSM), and Vascular Endothelial Growth Factor (VEGF) that were identified in Chapter 3, were further examined in Chapter 6. The three markers were significantly up-regulated both in U937 macrophages and in blood-derived macrophages exposed to a _2-agonist (clenbuterol and zilpaterol) in the absence or presence of LPS, as determined by a specific enzyme-linked immunosorbent assays (ELISA). Moreover, this up-regulation was also accomplished by other cyclic AMP elevating agents (forskolin, prostaglandins E2, and dibutyryl cAMP), suggesting a role of cAMP in the up-regulation of GCP-2/CXCL6, VEGF and OSM. We hypothesize that these proteins may be involved in some of the adverse effects in the treatment of asthma with _2-adrenergic receptor agonists. In the second part of this thesis we focussed on a multi-component drug, namely Cannabis sativa. In Chapter 7, the immuno-modulating effects of unheated and heated Cannabis extracts were investigated. This study revealed that unheated Cannabis extracts and its major non-psychoactive compound _9-tetrahydrocannabinolic acid (THCa) were able to inhibit the LPS induced TNF-_ production both in U937 macrophages and in blood-derived macrophages. The inhibitory effect on TNF-_ was not mediated by the cannabinoid receptors CB1 and CB2. Furthermore, this study showed that unheated Cannabis extracts and THCa exert their inhibitory effect on the TNF-_ production via a mechanism that is different from that of heated Cannabis extract and its main constituent the psychoactive compound _9-tetrahydrocannabinol (THC). The inhibition of TNF-_ release by unheated Cannabis extract and THCa was prolonged over a relatively long period of time. By contrast, although THC and heated extracts initially inhibit the release of TNF-_, after longer incubation times they seem to increase TNF-_ production to levels that are even higher than in the absence of THC or Cannabis extract. This difference in response of the U937 macrophages to THC and THCa was also observed in an experiment in which we examined the effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibited the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. Finally, we studied the effect of THCa and unheated Cannabis extract in a pilot study using an Experimental Autoimmune Encephalomyelitis (EAE) mouse model. Unheated Cannabis extract and THCa had a favourable effect on the clinical and histological signs of EAE. However, these results are preliminary and not clearly significant, therefore further investigation is necessary. Chapter 8 describes the categorization of unheated and heated Cannabis extracts using the same model system as described in Chapter 3. The mRNA patterns obtained from U937 macrophages exposed to LPS in the absence or presence of different anti-inflammatory drugs and unheated and heated Cannabis extracts were analysed using PC-DA. The study revealed that heated and unheated Cannabis extracts give rise to different expression patterns, which is in agreement with the observations made in Chapter 7 that they exert their TNF-_ inhibitory effect via different pathways. Moreover, their expression patterns did not overlap with that of other classes of anti-inflammatory compounds known to inhibit the TNF-_ production. These results suggest that the Cannabis extracts can not be assigned to one of the above mentioned classes of inflammatory inhibitors. Further investigation is necessary to unravel the exact mechanism of action of unheated and heated Cannabis extracts. In conclusion, the studies in this thesis show that the application of systems biology approaches are very useful in the categorization of anti-inflammatory compounds based on their mRNA and lipid expression patterns and to find specific biomarkers for these compounds. The categorization based on the protein expression pattern was less successful. This is most probably due to the fraction of proteins that was analysed on the gel. With proteomics techniques only a small fraction of proteins can be analysed simultaneously. Pre-fractionation, enrichment techniques and different analytical methods are therefore necessary to analyse a wide range of proteins with diverse physiological properties and dynamic range. The datasets obtained by transcriptomics, proteomics and metabolomics were analysed using statistical and pattern recognition tools. The datasets often contained a limited number of samples with respect to the large number of variables. It is therefore important to use these techniques as an explorative tool only and to validate the potential biomarkers found by additional individual measurements. Taken together, the use of systems biology for the investigation of anti-inflammatory drugs yielded very promising results, even though only a small part of the systems biology circle was used. Show less
In a time of terrorist attacks and other crises, governments are faced with the question how to protect the population and the national security. Many states tend to use instruments and take... Show moreIn a time of terrorist attacks and other crises, governments are faced with the question how to protect the population and the national security. Many states tend to use instruments and take measures that infringe upon human rights and individual freedoms in order to protect national security. At the same time, UN Security Council resolutions stress that the 'fight against tarrorism' should take place within the borders set by the rule of law, by international human rights treaties and international humanitarian law. Next to that, international human rights contain a certain 'positive obligation' for states to adequately protect national security and the right to life of the population against terrorist attacks. Therefore, there is a tense and also complicated relation between human rights and national security. The question arises whether there is a way to secure that human rights and national security are compatible entities. This study analyses the relevant articles from human rights treaties and the jurisprudence of international monitoring organs (European Court of Human Rights/UN Human Rights Committee) in order to find out whether international law leaves room for states to limit or even derogate human rights in states of emergency or other situations that threaten national security. The study shows that international law indeed does leave some room, but only limited. In very serious crises that amount to a public emergency threatening the life of the nation, states may derogate from the ordinary level of human right protection, but even during such serious crisis situations there is no 'carte blanche' for states to take any measures they deem necessary. Certain human rights are given absolute protection, even in crisis situations, and emergency measures that interfere with non-absolute human rights should pass a test of strict necessity and proportionality. In this field new norms and concepts have been developed during the last decades. It is, however, clear that the international monitoring of human rights protection during states of emergency and other severe crises needs improvement. Show less
Light harvesting (LH) lies at the basis of photosynthesis, the process in which energy from the sun is stored by a photochemical reaction. The photophysics of light absorption and energy transfer... Show moreLight harvesting (LH) lies at the basis of photosynthesis, the process in which energy from the sun is stored by a photochemical reaction. The photophysics of light absorption and energy transfer is the key to a detailed understanding of the first steps in this process. This thesis describes the investigation of four types of LH systems that are expressed by bacteria, depending on light conditions. All systems have been studied by optical single-particle spectroscopy in order to eliminate the averaging effects that are typical for ensemble measurements. In this way, the spectral details of the individual systems are reveiled, allowing for a better understanding of the photodynamics that are associated with their spatial structures. Show less
Bot is een dynamisch weefsel en wordt voortdurend afgebroken door osteoclasten, de botafbrekende cellen, terwijl de osteoblasten (botvormende cellen) vervolgens weer voor botaanmaak zorgen.... Show moreBot is een dynamisch weefsel en wordt voortdurend afgebroken door osteoclasten, de botafbrekende cellen, terwijl de osteoblasten (botvormende cellen) vervolgens weer voor botaanmaak zorgen. Osteoporose, ook wel botontkalking genoemd, is een skeletziekte die wordt gekarakteriseerd door een lage botmassa en het verlies van botstructuur. Dit leidt tot een verhoogde kans op botbreuken. Osteoporose wordt veroorzaakt doordat de botafbraak door de osteoclasten groter is dan de botaanmaak door osteoblasten Tot nu toe zijn medicijnen tegen osteoporose vooral gericht op het remmen van de botafbraak (zoals bijvoorbeeld bisphosphonaten). Al eerder verloren gegaan bot kan nog niet worden hersteld. Daarom wordt nu gezocht naar medicijnen die de botvorming kunnen stimuleren. Een van de potenti_le kandidaten is het parathyroid hormoon (PTH) en het gerelateerde eiwit (PTHrP). PTH is een bekende stimulator van botafbraak, maar als PTHrP of PTH in lage doseringen met tussenpozen wordt gegeven dan stimuleert het juist de botvorming. Hoe deze tegenstrijdige effecten van PTHrP en PTH tot stand komen is nog niet bekend. Om het werkingsmechanisme van PTHrP en PTH te onderzoeken en om andere nieuwe anabole medicijnen te ontwikkelen is een betere kennis van de botvormende cel, de osteoblast noodzakelijk. Doel van het onderzoek Het doel van het onderzoek beschreven in dit proefschrift was om verschillende aspecten van osteoblast differentiatie te bestuderen. Osteoblast differentiatie is het ontwikkelingsproces van voorlopercellen, de mesenchymale stamcellen, tot osteoblasten. In dit proefschrift is als model de KS483 cellijn gebruikt. KS483 cellen kunnen differentiëren van een voorlopercel tot een osteoblast, een vetcel (adipocyte) of een kraakbeencel (chondrocyte) afhankelijk van het gebruikte medium en de kweekomstandigheden. De differentiatie van KS483 cellen naar osteoblasten duurt 18 dagen en is verdeeld in verschillende fases. Eerst vermenigvuldigen de cellen zich (proliferatie fase), daarna wordt er een botmatrix gemaakt (matrix productie en maturatie fase) waarna deze matrix verkalkt (mineralisatie fase) Tijdens het differentiatieproces van voorlopercel naar gespecialiseerde cel heeft de voorlopercel signalen nodig om te weten hoe hij zich moet ontwikkelen (figuur 2). Voor de differentiatie naar osteoblast zijn er verschillende signaalroutes belangrijk waaronder de BMP, Hedgehog (Hh) en Wnt/b-catenine routes. Daarnaast zijn RunX2 en osterix essenti_le transcriptiefactoren voor osteoblast differentiatie. Zonder deze transcriptiefactoren is er geen differentiatie naar osteoblasten mogelijk. In dit proefschrift hebben we de effecten en de regulatie van deze belangrijke signaalroutes tijdens osteoblast differentiatie geanalyseerd. Daarnaast hebben we ook gekeken naar de interactie van deze routes met PTHrP en PTH. BMP signaalroute In hoofdstuk 2 zijn de effecten van de BMP signaalroute op osteoblast differentiatie beschreven. Tijdens alle differentiatie fases komen verschillende BMPs, BMP receptoren, intracellulaire signaalmoleculen (Smads) en BMP antagonisten tot expressie. Om na te gaan wat de mogelijke rol van BMP signalering tijdens osteoblast differentiatie is, werden BMPs weggevangen door BMP antagonisten toe te dienen. De BMP antagonisten blokkeerden osteoblast differentiatie, wat werd bepaald door de activiteit van alkalische fosfatase (ALP), een osteoblast marker, en de hoeveelheid mineraal in de extracellulaire matrix te meten. Uit deze experimenten bleek dat de productie van BMPs door de osteoblast noodzakelijk is tijdens het gehele differentiatieproces. De behandeling van KS483 cellen met BMPs resulteerde in stimulatie van ALP activiteit en de hoeveelheid mineraal tijdens alle fases van osteoblast differentiatie, wat suggereert dat BMPs niet alleen belangrijk zijn voor de initiatie van de differentiatie, maar ook voor de matrix productie en mineralisatie. Hedgehog signaalroute Een rol voor de Hedgehog (Hh) signaalroute tijdens osteoblast differentiatie was eerder aangetoond in een studie waarin Hh de initiatie van osteoblast differentiatie in de botkoker stimuleerde. In hoofdstuk 3 wordt de rol van Hedgehog tijdens matrix productie en mineralisatie en het werkingsmechanisme beschreven. Eerst is de expressie (hoeveelheid mRNA) van Indian Hh (IHh), de botspecifieke Hh, de Hh receptoren (Ptc1 en Smo) en de intracellulaire signaalmoleculen (Gli__s) bestudeerd. De expressie van IHh, Gli1 en Ptc1 was verhoogd tijdens de matrix maturatie fase, terwijl de expressie van de andere moleculen gelijk bleef. IHh expressie werd ook gevonden in de osteoblasten in de humerus van een groeiend humaan skelet. Behandeling van de KS483 cellen met Hh eiwit resulteerde in een matig verhoogde ALP activiteit, terwijl de mineralisatie sterk werd gestimuleerd. Deze effecten werden geremd door de Hh antagonist cyclopamine, dat op zichzelf geen effect had op de differentiatie. Vervolgens werd bepaald tijdens welke fase van osteoblast differentiatie de Hh signaleringsroute belangrijk is. Uit deze experimenten bleek dat voorlopercellen reageerden op Hh, terwijl osteoblasten niet konden worden gestimuleerd. De Hh ge_nduceerde differentiatie werd compleet geremd door toedienen van BMP antagonisten, wat suggereert dat Hh signalering afhankelijk is van functionele BMP signalering. Toedienen van Hh en BMPs aan KS483 cellen resulteerde in synergistisch verhoogde ALP activiteit en mineralisatie. Experimenten toonden aan dat deze synergie op het niveau van de vroege Hh signaal transductie lag, en niet op het niveau van vroege BMP signalering. Hh stimuleerde niet alleen de osteoblast differentiatie, maar remde ook de differentiatie naar adipocyte. Deze data suggereren dat Hh signalering de ontwikkeling van voorlopercellen naar osteoblast stimuleert ten koste van de vetcelvorming. Een andere rol van Hh is de stimulatie van maturatie van vroege osteoblasten. Wnt/b-catenine signaalroute Een belangrijke rol voor de Wnt/b-catenine signaalroute in botvorming is recent aangetoond. Een aantal studies hebben laten zien dat Wnt/b-catenine signalering de proliferatie en differentiatie van osteoblasten stimuleert. In hoofdstuk 5, hebben we de rol van Wnt/b-catenine signalering tijdens matrix vorming en mineralisatie nader bestudeerd. Het toedienen van Wnt3A of LiCl, een stimulator van de Wnt/b-catenine route, aan KS483 cellen had geen enkel effect op de initiatie van de differentiatie, wat suggereert dat de Wnt/b-catenine signalering al maximaal aanstaat in deze cellen. Dit bleek ook uit experimenten met de Wnt antagonist Dkk-1, waarin toediening van Dkk-1 de initiatie van osteoblast differentiatie remde. Behandeling van KS483 cellen met Wnt3A of LiCl resulteerde in een remming van matrix mineralisatie. LiCl remde ook de mineralisatie van muizen beenmerg cellen. Opvallend was dat Wnt3A alleen in vroege osteoblasten de Wnt/b-catenine signaalroute kon aanzetten, terwijl LiCl dit kon in alle stadia van differentiatie. Dit zou kunnen komen door verlaagde expressie van Wnt receptoren, of door verhoogde expressie van Wnt antagonisten in volwassen osteoblasten. Experimenten toonden aan dat de expressie van Wnt receptoren niet veranderde tijdens osteoblast differentiatie, terwijl de expressie van de Wnt antagonisten juist werd ge_nduceerd. Dit zou kunnen betekenen dat de Wnt antagonisten Dkk-1 en Dkk-2 Wnt/b-catenine signalering remmen, wat noodzakelijk is voor mineralisatie van de matrix. Om dit te onderzoeken, werden stabiele KS483 cellijnen gemaakt waarin Dkk-1 of Dkk-2 expressie sterk werd verminderd (Dkk-1si en Dkk-2si). Differentiatie en cel proliferatie waren geremd in de Dkk-2si, terwijl mineralisatie was geremd in Dkk-1si en Dkk-2si. Samenvattend: remming van de Wnt/b-catenine signalering door inductie van de expressie van de antagonisten Dkk-1 en Dkk-2 is essentieel voor matrix mineralisatie. Werkingsmechanisme van PTH en PTHrP In hoofdstuk 6 laten we zien dat PTHrP en PTH de differentiatie van voorlopercel naar osteoblast remt, onafhankelijk van de dosering, tijd van toevoegen of de differentiatie status van de cel. Deze remming zou kunnen plaatsvinden doordat PTHrP belangrijke signaalroutes voor osteoblast differentiatie remt of doordat PTHrP osteoblast specifieke transcriptiefactoren remt. Deze remming kan plaatsvinden op allerlei niveaus, zoals (1) remming van de expressie van factoren in de BMP of Hh signaalroutes, (2) remming van de signalering van deze signaalroutes of (3) remming van de expressie of activiteit van de transcriptiefactoren RunX2 en Osterix. Om na te gaan of PTHrP osteoblast differentiatie kon remmen door de BMP route te blokkeren hebben we BMPs en PTHrP toegediend aan KS483 cellen. PTHrP kon inderdaad de BMP ge_nduceerde osteoblast differentiatie remmen en omgekeerd. Uit andere experimenten bleek echter dat deze remming waarschijnlijk indirect is. PTHrP zou ook een remmend effect kunnen hebben op osteoblast differentiatie door de Hh route te onderdrukken. Interacties tussen PTHrP en de Hh route waren al eerder beschreven in de groeischijf. PTHrP kon de Hh ge_nduceerde osteoblast differentiatie remmen en omgekeerd kon hedgehog het effect van PTHrP opheffen. Een van de oorzaken hiervan kan zijn dat in ongedifferentieerde cellen PTHrP de expressie van componenten van de Hh signaal route kon remmen. KSFrt model systeem In hoofdstuk 4 hebben we een model gemaakt waarbij KS483 cellen kunnen worden gebruikt om gemakkelijk en reproduceerbaar stabiele cellijnen te maken door middel van plaats specifieke homologe recombinatie. Daarvoor werd een unieke FRT site ge_ntroduceerd in het genoom (DNA) van de KS483 cellen, waardoor de KSFrt cellijnen ontstonden. Deze FRT site werd vervolgens gebruikt om genen te introduceren in het genoom in combinatie met positieve selectie door middel van een verandering in resistentie tegen antibiotica. Het voordeel van dit systeem is, dat er altijd __n kopie van het gen terechtkomt op dezelfde plek in het genoom. In de KSFrt cellen kunnen bepaalde genen worden ge_ntroduceerd en kan de expressie van genen worden verhoogd. De expressie van genen kan ook sterk worden verminderd door middel van RNA interference (RNAi). Daarvoor is een nieuwe RNAi vector gemaakt waarbij met de introductie van een kopie van de RNAi vector de expressie van een gen voldoende kan worden geremd. Dit model kan worden gebruikt om allerlei (onbekende) genen te introduceren, of juist de expressie van bepaalde genen te verminderen, en vervolgens de effecten te bekijken op de differentiatie naar osteoblast, adipocyte of chondrocyte. Deze studies kunnen als uitgangspunt worden gebruikt voor de ontwikkeling van medicijnen die specifiek op de osteoblast zijn gericht Show less
Since the inception of programming, composition of algorithms has served as the driving force behind software composition. The models and techniques that have emerged out of this focus do not... Show moreSince the inception of programming, composition of algorithms has served as the driving force behind software composition. The models and techniques that have emerged out of this focus do not adequately meet our modern requirements, such as third-party composition of black-box components, or dynamic composition of the behavior of independent distributed subsystems and services. Decades of theoretical and practical work in the field of concurrency has culminated in substantial experience with various aspects of interaction and protocols. Curiously, however, interaction has not been considered as a first-class concept in a constructive model of computation. I believe the inadequacy of our contemporary composition techniques and our neglect to treat interaction as a first-class concept are intertwined. In this lecture, I describe some of my work and ideas on a compositional model for construction of complex systems out of simpler parts, using interaction as the only first-class concept. Show less
