Dimeric ligands for G-protein coupled receptors that are involved in human reproduction, namely the gonadotropin releasing hormone receptor, the luteinizing hormone receptor and the follicle... Show moreDimeric ligands for G-protein coupled receptors that are involved in human reproduction, namely the gonadotropin releasing hormone receptor, the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were synthesized and biologically evaluated. Show less
In this thesis a combined approach is described to investigate the constitutive activity of G protein protein-coupled receptors (GPCRs) using human adenosine A2B receptors and to evaluate disease... Show moreIn this thesis a combined approach is described to investigate the constitutive activity of G protein protein-coupled receptors (GPCRs) using human adenosine A2B receptors and to evaluate disease-related constitutive GPCR activity as a target for treatment. To this end a yeast expression system together with pharmacological and theoretical receptor models have been applied. In Chapter 2 the advantages of yeasts as tools to study GPCRs are reviewed. Adapted yeast cells able to communicate with mammalian GPCRs have become available and provide a very convenient system to express mutated receptors. A major advantage of yeast cells over mammalian cells extends from easy culturing conditions to the characteristic of yeast cells to allow entry of only a single plasmid. This latter property in combination with the robust screening assay based on yeast growth makes them an ideal test system to study randomly as well as site-directed mutated receptors. In chapters 3 to 5 this yeast growth assay is the main experimental tool to evaluate the functional properties of random and site-directed mutant receptors. In chapters 3 and 4 the yeast system is exploited to study inverse agonism of the human adenosine A2B receptor. At first, constitutively active mutant (CAM) human adenosine A2B receptors have been used to discriminate inverse agonists of the adenosine A2B receptor from A2B receptor antagonists. As a result, three inverse agonists ZM241385, DPCPX and MRS1706 were identified and their rank order of efficacy determined. Moreover, an interesting system-dependent phenomenon was noticed, that is the intrinsic activities of the inverse agonists were affected by the level of constitutive activity. It was demonstrated that inverse agonists show the greatest intrinsic activity on receptors displaying a medium level of constitutive activity. To further investigate the relationship between the effectiveness of an inverse agonist and the level of constitutive activity of the receptor, the two-state receptor model was introduced in both Chapter 3 and Chapter 4. According to this two-state model, both the receptor isomerization constant (L) and the intrinsic efficacy (_) of the inverse agonist determines the sensitivity to detect the intrinsic activity of an inverse agonist which is reflected by the observed experimental window. The biggest experimental window can be achieved on receptors with an L value equaling the reciprocal square root of _. Our experiments show that mutant A2B receptors with an intermediate level of constitutive activity possess the greatest experimental window, whereas mutants with a low level of constitutive activity showed small experimental windows and highly constitutively active mutants did not respond to our tested inverse agonists. Based on these findings we conclude that receptors with intermediate levels of constitutive activity should be the most sensitive screening tools for detecting inverse agonists. In Chapter 5 the activation of the human adenosine A2B receptor was investigated. To investigate the role of the NxxxNPxxY motif and the potential salt bridge between TM1 (E14) and TM7 (H280) in receptor activation, site-directed mutagenesis was applied to yield 15 mutant A2B receptors. The mutations were selected based on an adenosine A2B receptor model using the structure of bovine rhodopsin as a template. The expression levels of these mutants were determined by western blot analysis and the activation of the receptors was measured in the presence or absence of the following agonists NECA, CPA, CGS21680, IBMECA and LUF5833. None of the mutant receptors displayed constitutive activity. On the contrary, most mutants had a reduced potency and/or efficacy, e.g. mutants N282Q, N282R, N286A, N286Q, N286R, and Y290F showed impaired activation and mutants Y290N, E14H, H280E, E14H/H280E, I61A, I61D and I61K could not be activated by any of the agonists tested. Among all the mutants constructed, only N282R and N286R receptors behaved similarly to the wild-type receptor. Moreover, mutant N286A reduced receptor expression and H280E and E14H/H280E abolished receptor expression. These results suggest an important role for the NxxxNPxxY motif and the potential salt bridge in receptor expression and activation. The recent publication of the _2-adrenergic receptor structure enabled us to construct a second model of the adenosine A2B receptor. Comparison of the two A2B receptor models based on these two different templates is also described in Chapter 5. The various effects caused by mutations in the NxxxNPxxY motif and the potential salt bridge of different receptors in both our experiments and from literature do suggest that receptor activation is a receptor-specific phenomenon. In Chapter 6 we provide a theoretical investigation of the treatment of disease-related constitutively active receptor mutations. Comparison of the characteristics of allosteric ligands with traditional orthosteric ligands using a two-state allosteric model predicts that allosteric ligands display a more complicated interaction with a receptor/endogenous ligand pair and are able to cooperatively modify receptor binding and function. As a result allosteric modulators may affect the level of constitutive activity without changing the potency of the endogenous ligand. Thus allosteric modulators may provide advantages over orthosteric ligands in the treatment of diseases caused by constitutively active GPCR mutations. Finally in Chapter 7, general conclusions about the research described in this thesis are drawn. This is also supplemented by an outlook on some potential aspects of research to be pursued, based upon the application of receptor models, pharmacology models and functional receptor assays. Show less
Each protein is characterized by its unique sequential order of amino acids, the so-called protein sequence. Biology__s paradigm is that this order of amino acids determines the protein__s... Show moreEach protein is characterized by its unique sequential order of amino acids, the so-called protein sequence. Biology__s paradigm is that this order of amino acids determines the protein__s architecture and function. In this thesis, we introduce novel algorithms to analyze protein sequences. Chapter 1 begins with the introduction of amino acids, proteins and protein families. Then fundamental techniques from computer science related to the thesis are briefly described. Making a multiple sequence alignment (MSA) and constructing a phylogenetic tree are traditional means of sequence analysis. Information entropy, feature selection and sequential pattern mining provide alternative ways to analyze protein sequences and they are all from computer science. In Chapter 2, information entropy was used to measure the conservation on a given position of the alignment. From an alignment which is grouped into subfamilies, two types of information entropy values are calculated for each position in the MSA. One is the average entropy for a given position among the subfamilies, the other is the entropy for the same position in the entire multiple sequence alignment. This so-called two-entropies analysis or TEA in short, yields a scatter-plot in which all positions are represented with their two entropy values as x- and y-coordinates. The different locations of the positions (or dots) in the scatter-plot are indicative of various conservation patterns and may suggest different biological functions. The globally conserved positions show up at the lower left corner of the graph, which suggests that these positions may be essential for the folding or for the main functions of the protein superfamily. In contrast the positions neither conserved between subfamilies nor conserved in each individual subfamily appear at the upper right corner. The positions conserved within each subfamily but divergent among subfamilies are in the upper left corner. They may participate in biological functions that divide subfamilies, such as recognition of an endogenous ligand in G protein-coupled receptors. The TEA method requires a definition of protein subfamilies as an input. However such definition is a challenging problem by itself, particularly because this definition is crucial for the following prediction of specificity positions. In Chapter 3, we automated the TEA method described in Chapter 2 by tracing the evolutionary pressure from the root to the branches of the phylogenetic tree. At each level of the tree, a TEA plot is produced to capture the signal of the evolutionary pressure. A consensus TEA-O plot is composed from the whole series of plots to provide a condensed representation. Positions related to functions that evolved early (conserved) or later (specificity) are close to the lower left or upper left corner of the TEA-O plot, respectively. This novel approach allows an unbiased, user-independent, analysis of residue relevance in a protein family. We tested the TEA-O method on a synthetic dataset as well as on __real__ data, i.e., LacI and GPCR datasets. The ROC plots for the real data showed that TEA-O works perfectly well on all datasets and much better than other considered methods such as evolutionary trace, SDPpred and TreeDet. While positions were treated independently from each other in Chapter 2 and 3 in predicting specificity positions, in Chapter 4 multi-RELIEF considers both sequence similarity and distance in 3D structure in the specificity scoring function. The multi-RELIEF method was developed based on RELIEF, a state-of-the-art Machine-Learning technique for feature weighting. It estimates the expected __local__ functional specificity of residues from an alignment divided in multiple classes. Optionally, 3D structure information is exploited by increasing the weight of residues that have high-weight neighbors. Using ROC curves over a large body of experimental reference data, we showed that multi-RELIEF identifies specificity residues for the seven test sets used. In addition, incorporating structural information improved the prediction for specificity of interaction with small molecules. Comparison of multi-RELIEF with four other state-of-the-art algorithms indicates its robustness and best overall performance. In Chapter 2, 3 and 4, we heavily relied on multiple sequence alignment to identify conserved and specificity positions. As mentioned before, the construction of such alignment is not self-evident. Following the principle of sequential pattern mining, in Chapter 5, we proposed a new algorithm that directly identifies frequent biologically meaningful patterns from unaligned sequences. Six algorithms were designed and implemented to mine three different pattern types from either one or two datasets using a pattern growth approach. We compared our approach to PRATT2 and TEIRESIAS in efficiency, completeness and the diversity of pattern types. Compared to PRATT2, our approach is faster, capable of processing large datasets and able to identify the so-called type III patterns. Our approach is comparable to TEIRESIAS in the discovery of the so-called type I patterns but has additional functionality such as mining the so-called type II and type III patterns and finding discriminating patterns between two datasets. From Chapter 2 to 5, we aimed to identify functional residues from either aligned or unaligned protein sequences. In Chapter 6, we introduce an alignment-independent procedure to cluster protein sequences, which may be used to predict protein function. Traditionally phylogeny reconstruction is usually based on multiple sequence alignment. The procedure can be computationally intensive and often requires manual adjustment, which may be particularly difficult for a set of deviating sequences. In cheminformatics, constructing a similarity tree of ligands is usually alignment free. Feature spaces are routine means to convert compounds into binary fingerprints. Then distances among compounds can be obtained and similarity trees are constructed via clustering techniques. We explored building feature spaces for phylogeny reconstruction either using the so-called k-mer method or via sequential pattern mining with additional filtering and combining operations. Satisfying trees were built from both approaches compared with alignment-based methods. We found that when k equals 3, the phylogenetic tree built from the k-mer fingerprints is as good as one of the alignment-based methods, in which PAM and Neighborhood joining are used for computing distance and constructing a tree, respectively (NJ-PAM). As for the sequential pattern mining approach, the quality of the phylogenetic tree is better than one of the alignment-based method (NJ-PAM), if we set the support value to 10% and used maximum patterns only as descriptors. Finally in Chapter 7, general conclusions about the research described in this thesis are drawn. They are supplemented with an outlook on further research lines. We are convinced that the described algorithms can be useful in, e.g., genomic analyses, and provide further ideas for novel algorithms in this respect. Show less
This thesis describes various studies during the long-term follow-up of patients after treatment for pituitary diseases. The focus of this thesis is acromegaly, growth hormone deficiency, sleep and... Show moreThis thesis describes various studies during the long-term follow-up of patients after treatment for pituitary diseases. The focus of this thesis is acromegaly, growth hormone deficiency, sleep and quality of life. Various aspects are described. Show less
Various non-invasive imaging techniques are available for the diagnosis of coronary artery disease (CAD). Multi-slice computed tomography (MSCT) is an upcoming technique that allows direct... Show moreVarious non-invasive imaging techniques are available for the diagnosis of coronary artery disease (CAD). Multi-slice computed tomography (MSCT) is an upcoming technique that allows direct visualization of the coronary arteries. Apart from the assessment of atherosclerosis, MSCT can also provide important information on the global and regional left ventricular (LV) function. However, it should be realized that the hemodynamic consequences of atherosclerosis can not be obtained by MSCT imaging, and that functional testing, with for example nuclear imaging, will remain necessary to determine therapeutic strategy. CAD is the most common cause of chronic heart failure. Recently, cardiac resynchronization therapy (CRT) has become implemented in the treatment of patients with severe heart failure. Although the improvement in systolic function after CRT implantation can be considerable, 20% to 30% of patients do not respond to CRT. It has been shown that the presence of LV dyssynchrony is an important predictor of response to CRT. Gated SPECT allows not only the assessment of myocardial perfusion (scar tissue and viable myocardium) but also the evaluation of LV dyssynchrony. As many patients with heart failure undergo nuclear imaging with SPECT, integrated assessment of LV dyssynchrony, viability and scar tissue may be an attractive option. Show less
The work presented in this thesis involves the development of new instrumental techniques and analysing tools, combining high spectral resolution with high spatial information, with the aim to... Show moreThe work presented in this thesis involves the development of new instrumental techniques and analysing tools, combining high spectral resolution with high spatial information, with the aim to increase our understanding of the formation and evolution of stars and planets. First, a novel instrumental concept is presented that aims to achieve high spatial and spectral resolution by combining existing Echelle spectrographs with existing optical interferometers. Subsequently, several studies combining high spatial and spectral resolution are presented. We investigate the immediate environment of the massive young stellar object MWC349A, using the VLT-interferometer. New methods are presented to analyse the Rossiter-McLaughlin effect during stellar eclipses. By using this effect it is possible to obtain spatial information on stellar surface scales, something difficult to achieve by other means. Using these new tools we show that the spin axes in both stars of in the DI Herculis system are strongly tilted with respect to the orbital angular momentum. This solves a 20-year-old riddle about the DI Herculis system, involving its apparently slow apsidal motion. In the final chapter of this thesis sodium measurements for the atmosphere of the extrasolar planet HD209458b using ground-based transmission spectroscopy are presented. Show less
In the 21st century, space activities increasingly affect global society, economy, technology, culture, and the environment. Many countries wish to develop space activities in order to obtain space... Show moreIn the 21st century, space activities increasingly affect global society, economy, technology, culture, and the environment. Many countries wish to develop space activities in order to obtain space benefits. However, a lot of them, especially developing countries, face huge obstacles such as lack of human and financial resources, technical support, inadequate information and sustainability of transferred technology. Developing countries believe they do not have equal access to space benefits. This may lead to international conflicts between developing and developed countries. Based on the concept of international cooperation this study examines the legal aspects and feasibility of the creation of a space organization in the Southeast Asian region. It aims at providing an instrument for ASEAN countries to ensure equitable access to outer space and to its benefits. On the basis of a comparison of existing space organizations, this study identifies the key elements needed for the creation of a model for ASEAN space cooperation. The study concludes that if ASEAN countries want to break the cycle of technological dependency, they should combine their efforts to promote sustainable space development by creating an ASEAN Space Organization. Show less
Over the past decades, life expectancy in patients with congenital heart disease has increased dramatically. However, serious complications may develop late after total repair in infancy. These... Show moreOver the past decades, life expectancy in patients with congenital heart disease has increased dramatically. However, serious complications may develop late after total repair in infancy. These complications are usually the result of longstanding pulmonary regurgitation which leads tot dilatation of the right ventricle and an increased risk for severe arrhythmias. Therefore lifelong follow-up in these patients is required. Cardiac magnetic resonance imaging is the current imaging tool of choice because it offers superior imaging quality and enables accurate quantification of functional parameters such as flow volumes and systolic and diastolic performance. Pulmonary valve replacement is often performed in Tetralogy of Fallot patients later in life due to pulmonary regurgitation with or without severe right ventricular failure. However, the optimal timing of pulmonary valve replacement has not yet been elucidated. Therefore, the current study focuses on the optimal timing of pulmonary valve replacement in patient late after total repair of Tetralogy of Fallot. Show less
The European natural gas market is in a state of flux. In order to better secure the public service obligations supply security, competitiveness and sustainability this market is currently being... Show moreThe European natural gas market is in a state of flux. In order to better secure the public service obligations supply security, competitiveness and sustainability this market is currently being liberalized. This requires significant structural and regulatory reform. This reform process is not yet finished; the European gas market finds itself in a transition phase from the traditional managed market structure towards an internal European gas market which is governed by competitive forces. In such a transition phase, problems and tensions are inevitable. This study assesses the adequacy of the current approach towards structural and regulatory reform of the European gas market in light of the changes that are taking place on this market. These changes create a new context for gas regulation. Given the vital role of investments in securing any of the public service obligations, the analysis is conducted from the perspective of ensuring sufficient investments. The theoretical part criticizes the current neoclassical approach towards reform and argues that the transaction cost perspective will result in better outcomes in terms of market behavior in the new regulatory context. The empirical part undertakes case studies on two recent amendments to gas regulation the possibility for certain investments to be exempted from the provisions to provide third party access, and the enlarged scope for concluding downstream long-term gas supply contracts in order to assess whether the theoretical critique is also valid in practice. Both case studies confirm the theoretical critique. The current regulatory approach appears to be stuck between the neoclassical and the transaction cost perspective. Recommendations are provided in order to better align current regulation with the insights provided by Transaction Cost Economics. Show less
Unfairness increases noise annoyance. Noise annoyance increases due to unfair sound management. Fair sound management reduces annoyance, however only when the sound pressure level is high,... Show moreUnfairness increases noise annoyance. Noise annoyance increases due to unfair sound management. Fair sound management reduces annoyance, however only when the sound pressure level is high, concludes Eveline Maris based on two laboratory experiments.Being exposed to man-made sound is more than mere exposure: it is a social experience, too: You expose Me. This social hypothesis of noise annoyance has been confirmed in two laboratory experiments. Participants were exposed to aircraft noise (sound pressure level (SPL): 50 or 70 dB A) and treated either in a neutral, fair, or unfair manner. The results show that besides SPL, also the fairness of the procedure determines the level of noise annoyance. The first experiment shows an interaction effect of procedural fairness and SPL: annoyance ratings are significantly lower in the fair than in the neutral condition, but the effect is found only when SPL is 70 dB. The second experiment shows a main effect of procedural unfairness on noise annoyance: annoyance ratings are significantly higher in the unfair than in the neutral conditions, regardless of SPL.The findings imply that, in addition to noise reduction engineering, application of knowledge on the social side of noise annoyance can help reduce future noise annoyance levels. Show less
Many decisions in the legal system are based on eyewitness evidence. It seems to be a matter of common sense that the level of confidence expressed by a witness can be used as a diagnostic tool to... Show moreMany decisions in the legal system are based on eyewitness evidence. It seems to be a matter of common sense that the level of confidence expressed by a witness can be used as a diagnostic tool to discriminate between accurate and inaccurate memories. Contrary to this general belief, the bulk of empirical evidence collected in laboratory and field experiments over the past 25 years indicates that the relationship between confidence and accuracy is far from perfect. The central focus of this dissertation is on the accuracy of eyewitness memory, and especially on the relationship between accuracy and confidence (i.e., the subjective judgment of accuracy). To enhance the ecological validity of the laboratory studies, we used a method that allowed us to determine accuracy and confidence scores for the recall of details of complex naturalistic events. The first three chapters are experiments from the laboratory testing the effect of repeated retrieval, retention interval and repeated misleading questioning. The fourth chapter is a case study in which real live witnesses of a robbery on a supermarket were interviewed. Our findings show a clear and consistent relationship between confidence judgements and the actual accuracy of memories. With some caution, confidence may be used as a partial indicator of accuracy, especially during the early stages of an investigation. Unfortunately, however, there always remain incorrect items that are given the maximum confidence score. That is the reason why no single witness statement can be accepted as certainly correct, based on confidence alone. Show less
Following allograft transplantation, the immune system is triggered to induce an immunogenic response against the non-self organ. To prevent the induction of this immunogenic response, recipients... Show moreFollowing allograft transplantation, the immune system is triggered to induce an immunogenic response against the non-self organ. To prevent the induction of this immunogenic response, recipients are treated with immunosuppressive medication. The majority of these medications target T cells, which play a key role in the rejection process, and thereby prevent acute rejection in most of the recipients. Non-specific targeting of these T cells not only prevents acute rejection, it also prevents responses against pathogens or tumor growth. In addition, long-term use of immunosuppressive agents may cause organ failure due to toxic effects on the organ [1]. Therefore, the ultimate goal is to develop a therapy, which targets alloreactive T cells, allowing a normal response against pathogens and tumors, in the absence of chronic use of immunosuppressive agents. Various strategies have been employed to induce such a donor-specific tolerance, amongst which treatment with immature DC [2]. These immature DC have, in contrast to mature DC, the capacity to induce tolerogenic responses and are therefore an attractive candidate for cellular therapy. The studies presented in this thesis demonstrate that in fully mismatched kidney transplantation models, administration of modulated donor-derived DC to recipient__s results in regulation of recipient__s immune response. Both the donor-specific hyporesponsiveness of recipient T cells and the reduced influx of CD8+ T cells into the graft of LPS-DexDC treated recipients indicate a positive effect of this treatment. However, optimization of this treatment is necessary, since no prolonged allograft survival was induced. Several mechanisms, which are not regulated by LPS-DexDC, may be responsible for the observed rejection, amongst which the preformed alloantibodies, increased levels of C3 in the graft and the increased influx of NK cells. Additional studies are required to explore the modulating effects of antibodies which block co-stimulation and/or short courses of immunosuppressive drugs as a co-treatment in these settings. Show less
The last decade has witnessed an increased occurrence of bacterial resistance against antibiotics. The first part of this thesis describes the discovery of a novel target, protein kinase B / Akt1,... Show moreThe last decade has witnessed an increased occurrence of bacterial resistance against antibiotics. The first part of this thesis describes the discovery of a novel target, protein kinase B / Akt1, that may be used to combat infection with pathogenic bacteria like Salmonella typhimurium. Inhibitors of this enzyme are currently being developed as anti-tumor agents. These molecules can now also be seen as potential antibiotics. The synthesis and biological evaluation of several series of new inhibitors of this enzyme are described. This resulted in the identification of a compound with sufficient potency and selectivity to allow in vivo inhibition of Salmonella outgrowth. The second part describes the optimization of an existing antibiotic, Gramicidin S. This is a naturally occurring decapeptide that disrupts the cellular membrane of bacteria. This results in leakage of intracellular contents and bacterial death. Unfortunately, Gramicidin S is toxic for red blood cells since their membrane is also destroyed. As part of an ongoing project to reduce the toxicity against red blood cells, part of the original decapeptide is replaced by synthetic molecules. One compound showed similar activity against bacteria and reduced toxicity against red blood cells. Further research is needed to further improve this antibiotic. Show less
Bacteria belonging to the genus Collimonas consist of soil bacteria that can grow at expense of living fungal hyphae i.e. they are mycophagous. This PhD studies deals with the ecology of... Show moreBacteria belonging to the genus Collimonas consist of soil bacteria that can grow at expense of living fungal hyphae i.e. they are mycophagous. This PhD studies deals with the ecology of mycophagous bacteria in soil using collimonads as model organisms. Collimonads were found to be widely distributed in different types of soils albeit at low densities. Highest numbers were present in fungal-rich grassland soils and lowest numbers in fungal-poor arable soils. Yet, no significant positive correlation between numbers of collimonads and fungal biomass densities was obtained when all soils were included in the analysis. Actual growth of indigenous collimonads in soils upon invasion by fungal hyphae was demonstrated using a newly, developed Collimonas-specific qPCR assay. The fungal-induced increase in numbers of collimonads was moderate and did not result in an increased turn-over of fungal biomass. In contrast to the effects on fungal-biomass turn-over, effects of presence of collimonads on soil fungal community composition were strong. This implies that collimonads can change the competitive relationships between soil fungal species. Hence, our conclusion is that a small component of the soil microbial community can have a huge impact on this community. Show less
The study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and _-glucosidase 2 that... Show moreThe study described in this thesis was conducted with the aim of developing lipophilic iminosugars as selective inhibitors for glucosylceramide synthase, glucocerbrosidase and _-glucosidase 2 that are enzymes involved in glucosylceramide metabolism. The study has resulted in many novel inhibitors of these three enzymes among which several that improve upon the inhibition profile of the lead compound in this study. The successful use of lipophilic iminosugars in type 2 diabetes models and the partial elucidation of their mechanism of action therein provide prospects for their development towards therapeutics for diabetes type 2. Show less
The findings of the present thesis can be summarized in the following conclusions: 1. In Leiden, the so-called ‘founder effect’ resulted in genetic clustering with an extremely high prevalence of... Show moreThe findings of the present thesis can be summarized in the following conclusions: 1. In Leiden, the so-called ‘founder effect’ resulted in genetic clustering with an extremely high prevalence of one single SDHD mutation, the SDHD-c.274G>T (p.Asp92Tyr) mutation. 2. Patients with head-and-neck paragangliomas have a considerable impairment of quality of life. 3. Patients with head-and-neck paragangliomas have serious subjective sleep complaints. 4. Patients with SDHD-associated head-and-neck paragangliomas have an increased risk for development of pheochromocytomas and paragangliomas at other locations and life long, repetitive follow up with protocolized clinical, biochemical and radiological screening is therefore mandatory. 5. The use of [123I]-MIBG for detection of intra- and extra-adrenal paragangliomas combined, revealed a sensitivity and specificity of only 80% and 75%, respectively. The sensitivity is dependent on tumor localization and tumor behavior, with an increase in sensitivity to 92% if (intra-adrenal) pheochromocytomas are investigated separately. 6. The practical clinical implications of non-secreting intra-adrenal paragangliomas are currently uncertain. 7. Patients with SDHD mutations have malignant disease in at least ~2.5% of the cases. 8. Mediastinal paragangliomas are associated with mutations in the succinate dehydrogenase genes (either SDHB or SDHD) and aggressive behavior and might be more prevalent than hitherto appreciated. 9. Although the consequences of missing non-secreting and mediastinal paragangliomas in an SDHD-associated population are currently unclear, their possible presence should be taken into consideration in the development of future screening protocols. 10. In the future, [18F]-FDOPA and/or [18F]-FDA are expected to (at least partly) replace MIBG in diagnostic imaging for pheochromocytoma and paraganglioma. Show less
Replicative ageing of fibroblasts has often been used as a model for organismal ageing. The general assumption that the ageing process is mirrored by cellular senescence in vitro is based on lower... Show moreReplicative ageing of fibroblasts has often been used as a model for organismal ageing. The general assumption that the ageing process is mirrored by cellular senescence in vitro is based on lower replicative capacity of human fibroblasts from donors of higher chronological age, but these inverse relations have not been reported unequivocally. The relation between chronological age and fibroblast growth characteristics was assessed in nonagenarian subjects of the Leiden 85+ Study. A high remaining replicative capacity impressively showed that even in the very elderly a crucial number of cells with high mitotic capacity are left to give rise to fibroblast strains with the capacity for more than 100 population doublings. During the course of fibroblast growth in vitro, beta-galactosidase activity has been shown to be a reliable biomarker for replicative senescence. In myoblast cultures the relation between mixed cultures and clonal cultures was studied, showing marked heterogeneity between clonal cultures that all had a significantly lower replicative capacity when compared to mixed cultures, indicating heterogeneity of cells within one tissue compartment in their in vivo history. In a formal review on the replicative capacity of fibroblasts from patients suffering from accelerated ageing syndromes, age related diseases and donor age it was found that except for premature ageing syndromes, the replicative capacity of fibroblasts in vitro does not mirror key characteristics of human life histories. Show less
This thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron... Show moreThis thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron diffraction for structural studies is the predictable availability of tiny crystals. A method for growing such crystals using heterogeneous nucleation is demonstrated. The heterogeneous nucleant (in this case hair fibers) was serendipitously selected. Four different proteins (lysozyme, glucose isomerase, a Fab fragment and potato protease inhibitor) were shown to nucleate preferentially on the selected substrate and sub-micro crystals were grown. Further studies on the mechanism of heterogeneous nucleation using lysozyme as a test protein and different imaging techniques such as atomic force and fluorescent microscopy are also discussed. Sub-micron crystals of potato protease inhibitor and lysozyme were subject of electron diffraction studies. A detailed description of the diffraction experiments is presented. A special focus is given on the sample preparation procedure and in particular the vitrification and the cryo-preservation of the crystals. The preliminary results showed that the heterogeneously grown nano-crystals are well ordered and suitable for electron diffraction. The high beam sensitivity of the protein nano-crystals appeared to be the rate limiting step in the data collection, not allowing orientation of the crystals (a technique used in electron diffraction studies of inorganic crystals) or a 3D data collection of a single crystal (a technique used in X-ray protein crystallography). This suggested that new approaches for data collection and data analysis needed to be developed. Optimization of the diffraction data collection, as is described in this thesis allowed high diffraction resolution (up to 2.1_) to be obtained from vitrified lysozyme crystals. An algorithm for unit cell determination of randomly oriented diffraction patterns of different crystals is presented. The method was used for the analysis of the electron diffraction data acquired from lysozyme nano-crystals. The methods for collecting and analyzing electron diffraction data from lysozyme crystals were also confirmed in the case of penicilline type nano-crystals. The motivation behind these studies and the results obtained are discussed. In this case a crystalline powder sample was subjected to electron diffraction studies. Resolutions up to 0.8_ were obtained from oxacillin crystals and up to 1_ from penicillin G crystals. The unit cell parameters found from the analysis of electron diffraction data with the algorithm presented in the previous chapter were consistent with the unit cell parameters obtained by X-ray crystallography on the same two types of penicillin. Show less
