The primary goal in repairing a peripheral nerve lesion is to guide the outgrowing axon back to its original target organ, which can be done by bridging the defect with an autograft or, more... Show moreThe primary goal in repairing a peripheral nerve lesion is to guide the outgrowing axon back to its original target organ, which can be done by bridging the defect with an autograft or, more experimentally, a synthetic nerve graft. In this thesis an overview is presented of the evaluation methods that are currently used to assess peripheral nerve regeneration and their expediency is discussed. An in vitro electrophysiological evaluation method that charts the electrophysiological properties of the myelinated Aα- and Aβ-nerve fibres was introduced, and it was demonstrated that small differences between grafted nerves could be discriminated. Moreover, the electrophysiological data could be correlated to the morphometrical data, that was likewise broken up into Aα- and Aβ-components. This correlation especially provided new insight in the changes that occur in regenerating nerve fibres. After applying a panel of evaluation methods it was demonstrated that the presence of pores in and biodegradability of synthetic nerve grafts are beneficial to regeneration, evidenced mainly by preferable values of the electrophysiological parameters. Finally the changes that occurred in reinnervated muscles helped to gain insight into the preferential architecture of a synthetic nerve graft. Show less
This thesis describes very sensitive methods for peptide detection, obtained by the coupling of high efficiency capillary chromatographic techniques to mass spectrometry. The application of novel... Show moreThis thesis describes very sensitive methods for peptide detection, obtained by the coupling of high efficiency capillary chromatographic techniques to mass spectrometry. The application of novel data preprocessing methods to the analysis of such complex MS data generated is also described. The data analysis step was completed by the use of multivariate statistical analysis tools, such as principal component analysis (PCA). The developed platform could be applied to the study of complex peptide mixtures and of proteomic samples. Show less
In the first part of this thesis, polyelectrolyte copolymer micelles are studied to elucidate the role of the micelle concentration on the intermicelle correlation and the extension of the... Show moreIn the first part of this thesis, polyelectrolyte copolymer micelles are studied to elucidate the role of the micelle concentration on the intermicelle correlation and the extension of the polyelectrolyte chains in the coronal layer. With increasing packing fraction the corona shrinks and/or interpenetrates in order to accommodate the micelles in the increasingly crowded volume. It is shown that interpenetration of the polyelectrolyte brushes controls the fluid rheology: the viscosity increases dramatically and the dynamic moduli show the formation of a physical gel. In the second part, we study giant vesicles as a superstructure self-assembled by dsDNA fragments, pUC18 or pEGFP-N1 and oppositely charged cationic diblock copolymer poly(butadiene-b-N-methyl 4-vinyl pyridinium. Under a preparation, the PBd brush collapses and a capsule is formed. The compaction of DNA is shown by the appearance of liquid crystalline textures under crossed polarizers and the increase in fluorescence intensity of labelled DNA. To form vesicles, the capsules are dispersed in aqueous medium supported by an osmotic agent. The integrity of the DNA after encapsulation and subsequent release was confirmed. We demonstrate ``reverse'' transfection of in vitro cultured HeLa cancer cells growing on plasmid-copolymer vesicles by the expressed green fluorescent protein in cultured cells. Show less
The ultimate goal in the transplantation field is the induction and maintenance of donor specific tolerance. Treg cells that control immune responses to alloantigens give opportunities for... Show moreThe ultimate goal in the transplantation field is the induction and maintenance of donor specific tolerance. Treg cells that control immune responses to alloantigens give opportunities for tolerogenic therapies in transplantation. However, it is important to investigate the mechanisms of tolerance induction in order to use the optimal strategy. Therefore, we exploded both natural towards NIMA that can be induced during fetal life and induced tolerance by modulation of DC. Naturrally induced tolerance towards NIMA can have an influence on transplant outcome later in life. In this thesis we explored the influence of NIMA on the alloreactive T cells repetoire in healthy individuals and additionally we focused on the NIMA effect in patients transplanted with a NIMA haplotype mismatched kidney graft. In order to actively induce tolerance, we modulated DC to generate Treg cells, since this may be of clinical relevance in the future for patients that are on the waiting list for transplantation. In this thesis we explored the possibility of using modulated DC for the induction of transplantation tolerance in a fully allogeneic setting in mice. Furthermore we describe an in vitro system for the use of human modulated DC to induce Treg cells. We show that two differentially modulated human DC can lead to different types of Treg cells. Finally, we examined the possibility to use in vitro tools to measure a possible tolerant state in patients. Monitoring of e.g. Treg cells and/or cytokines may give an indication which patients are at risk for rejection and which patients are more predisposed to tolerance. We describe the Elispot technique as a possible tool to monitor patients that received a renal allograft. In conclusion , this thesis contributes to the fundamental understanding of both natural and induced tolerance in transplantation and gives a handhold for future research. As donor-specific tolerance is still far away from the clinic, the in vitro monitoring tool described in this thesis may contribute to the optimalization of immunosuppressive therapies in transplant recipients. Show less
Osteoarthritis (OA) refers to a heterogeneous group of conditions. This thesis focuses on OA with a hereditary background; Familial OA at multiple joint sites and radiological hand OA at middle age... Show moreOsteoarthritis (OA) refers to a heterogeneous group of conditions. This thesis focuses on OA with a hereditary background; Familial OA at multiple joint sites and radiological hand OA at middle age. The main objective is to identify risk factors that play a role in the development of OA in order to gain further insight in the aetiology of OA. The secondary objective is to investigate factors that determine the outcome in OA. This thesis provides evidence that familial clustering of symptomatic OA is most prominent for hand and hip OA. In search for genetic risk factors, we present data suggesting that a proportion of the genetic susceptibility for OA at multiple sites is encoded by variation in innate cytokine activity. Further, we find HLA-DR antigens to be associated with radiological hand OA. In addition to genetic risk factors, this thesis demonstrates that other systemic risk factors such as hormonal status and local factors, to be important in the susceptibility of familial OA at multiple sites, underscoring the multicausal etioliology of this phenotype. Finally, this thesis addresses the resulting disability from OA. Using the International Classification of Functioning, Disability and Health as framework, we show illness perceptions and mental health to be important modifying factors in OA in the hands and lower extremities. Show less
Through epidemiological and biochemical studies the role of mucosal HPV types in human cancer has been confirmed and the molecular mechanisms underlying the transformation process have been... Show moreThrough epidemiological and biochemical studies the role of mucosal HPV types in human cancer has been confirmed and the molecular mechanisms underlying the transformation process have been elucidated. However, although research on the cutaneous HPV types is progressing fast, many questions remain unanswered regarding their potential role in human skin carcinogenesis.This thesis describes the characterization of the biological properties of E6 and E7 proteins from mucosal and cutaneous HPV types using novel or previously described assays and models. First, we describe the development and use of an in vitro systems to evaluate and quantify the ability of the oncoprotein E7 to associate with pRb (chapter 2). However, we show that this binding although indicative of a high risk HPV type, does not necessarily correlate with in vivo carcinogenicity (chapter 3) and that other in vitro studies are essential to asses the carcinogenicity of any given HPV type. Next we focused on the cutaneous HPV type 38, which displays in vitro transforming properties and appears to be frequently present in NMSC. We show that this cutaneous HPV type can increase the risk for development of skin cancer (chapter 4)and we describe a novel HPV transformation mechanism (chapter 5). Show less
The present study comprises a classification and analysis of the syntax of the non-verbal clause in Qumran Hebrew, i.e. the linguistic variety (or varieties) found in the so-called Dead Sea Scrolls... Show moreThe present study comprises a classification and analysis of the syntax of the non-verbal clause in Qumran Hebrew, i.e. the linguistic variety (or varieties) found in the so-called Dead Sea Scrolls. The corpus consists of the non-biblical texts written in Hebrew; biblical texts and texts written in Aramaic are excluded. Chapter 1, ‘Introduction’ discusses methodological problems (approach, description) and provides an overview of non-verbal clause patters in variety of Semitic languages. Chapter 2, ‘A survey of previous research’, summarises the results of earlier research into specifically Biblical, Mishnaic and Qumran Hebrew. Chapter 3, ‘The non-verbal clause in Qumran Hebrew’ is the core of the present work, which offers an analysis of the syntax of the non-verbal clause (including the existential clause) in Qumran Hebrew. Chapter 4 contains the conclusions. A bibliography, a Dutch summary and a Curriculum Vitae are appended. Show less
The research in this thesis comprises two separate topics: single-molecule spectroscopy and resonant Raman spectroscopy. The first part concerns single-molecule (SM) spectroscopy on polyethylene ... Show moreThe research in this thesis comprises two separate topics: single-molecule spectroscopy and resonant Raman spectroscopy. The first part concerns single-molecule (SM) spectroscopy on polyethylene (PE) films. Ultra thin (200 nm) films of pure high density PE were produced by spincoating. By determining the position (accuracy 10 nm) and in-plane orientation (acc. 5 deg.) of single 2.3,8.9-dibenzanthanthrene (DBATT) guest molecules, by means of SM microscopy and spectroscopy respectively, we demonstrated that these thin PE films have a shish-kebab morphology, instead of the spherulitic morphology common in thicker PE films. We have also investigated the alignment process of individual quest chromophores by stretching thicker (50 um), low density PE films. Using SM spectroscopy, we have shown that individual guest chromophores are not better aligned along the stretch direction, as draw ratio increases. Instead alignment occurs suddenly, due to the destruction and (oriented) reformation of local crystalline regions and subsequent adsorption of chromophores. Each chromophore's orientation is determined by specific interactions with the oriented PE crystal surface. The second part of this thesis concerns a quantum-chemical analysis of the resonant Raman spectrum of the carotenoid spheroidene reconstituted in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Our analysis demonstrates that spheroidene can adopt at least two cis forms in the RC. One of these has been conclusively shown to be the 15,15'-cis structure. Show less
The presence of calcium deposits in the vessel wall is indicative of advanced atherosclerosis, and the extent of coronary calcification has been found to add prognostic significance to conventional... Show moreThe presence of calcium deposits in the vessel wall is indicative of advanced atherosclerosis, and the extent of coronary calcification has been found to add prognostic significance to conventional risk factors of coronary artery disease. However, the mechanisms underlying vascular calcification are still obscure. The major objective of the work described in the first part of this thesis was to elucidate the mechanisms involved in atherosclerotic calcification. To study the process of VSMC calcification we developed and characterized an in vitro model of neonatal rat VSMC calcification. To investigate whether pharmacotherapy may affect vascular calcifications, we have studied the effect of a calcium antagonist (amlodipine) and a statin (atorvastatin) and their combination on this process. Inflammation is an important mechanism in the atherosclerotic process, and prospective and cross-sectional clinical and epidemiological studies have shown that CRP is consistently associated with CVD. The causality of CRP in atherosclerosis is discussed. To enable the study of the effect of CRP on atherosclerosis development in vivo, ApoE*3-Leiden/hCRP transgenic mice were generated and studied. The effects of a calcium antagonist (amlodipine), administered either alone or in combination with a statin (atorvastatin), on early atherosclerosis development in ApoE*3-Leiden/hCRP was investigated. Show less
The main objective of this research was to increase our knowledge of the pathological mechanisms underlying LGMD 2B, to better characterize each member of the dysferlin complex and, more... Show moreThe main objective of this research was to increase our knowledge of the pathological mechanisms underlying LGMD 2B, to better characterize each member of the dysferlin complex and, more importantly, to try to identify potential modifier genes that might modulate the clinical course in patients. In chapter 2, by diverse selection methodologies on different truncated forms of recombinant dysferlin, VHH antibody fragments with specificity for two different dysferlin domains were selected from a non-immune phage display library. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation. Using these antibody fragments, we found that CAPN3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.In order to gain functional insight into the molecular mechanisms of dysferlin, we have searched for yet unknown proteins that interact with dysferlin in skeletal muscle. By co-immunoprecipitation coupled to mass spectrometry, we demonstrated that dysferlin interacts with AHNAK. Furthermore, the binding domains in dysferlin and AHNAK were mapped and the AHNAK expression in normal skeletal muscle dysferlinopathy was assessed by immonofluorescence analysis (Chapter 3).Chapter 4 describes the proteolysis of AHNAK by CAPN3. Direct interaction of AHNAK and CAPN3 was studied by GST-pull down assay and specific AHNAK domains that were cleaved by CAPN3 were identified in a cell model. The biological relevance of this cleavage was also studied at cellular level.Finally, in chapter 5 the selection strategies of antibody phage display and the biomedical application of VHH will be discussed. This chapter will mainly focus on the role of dysferlin complex in LGMD 2B and future perspectives will be discussed in this chapter. Show less
Porphyrins are essential in living organisms. E.g. in the red oxygen carrying blood protein hemoglobin oxygen binds to the Fe2+-containing heme and in plants the green pigment chlorophyll is... Show morePorphyrins are essential in living organisms. E.g. in the red oxygen carrying blood protein hemoglobin oxygen binds to the Fe2+-containing heme and in plants the green pigment chlorophyll is responsible for the initial steps in photosynthesis. The absorption of light by a porphyrin, followed by energy transfer to oxygen can lead to the formation of highly toxic but short living singlet oxygen. When such a porphyrin localizes in a tumor it is possible to destroy the tumor locally by the irradiating it with light. This treatment is known as photodynamic therapy and the pigment is called a photosensitizer. However a major drawback of current photosensitizers is that they do not show strong absorption in the far-red part of the spectrum where light penetrates tissue optimal. Besides patients treated with these photosensitizers remain light sensitive for 3-8 weeks. This thesis describes the preparation of new photosensitizers with strong absorption of light at 680-690 nm. The preparation of these porphyrin derivatives takes place via unprecedented chemical reactions and an explanation for the chemistry involved is given. Tests on mice showed that the lifetime of these new porphyrin derivatives in their body is reduced to less then 3 days, which makes them very interesting candidates as new generation photosensitizers for photodynamic therapy Show less
In the group of women with equivocal cytology (Pap II) 15-30% have high-grade CIN or even cervical carcinoma. This high percentage underlines the importance of identifying these women. The... Show moreIn the group of women with equivocal cytology (Pap II) 15-30% have high-grade CIN or even cervical carcinoma. This high percentage underlines the importance of identifying these women. The sensitivity and specificity of cervical cytology are low. There are other methods besides cytology to identify women at risk for high-grade CIN or cervical cancer. This thesis focuses mainly on the prevalence of high-grade CIN in women with equivocal cytology (chapter 2) and on other methods than cytology alone to identify women at risk for high-grade CIN in this category of abnormal cytology. Methods like colposcopy (chapter 2, 3), detection of high-risk HPV (chapter 2, 5), persistence/ clearance of HPV (chapter 4), HPV viral load (chapter 4 and 5), and biomarker Ki-67 (chapter 6) were studied to this means. Show less
Neuropsychiatric systemic lupus erythematosus (NPSLE) is a rheumatologic disorder causing neurologic, psychiatric and/or psychologic symptoms. The most important clinical problems are the aspecific... Show moreNeuropsychiatric systemic lupus erythematosus (NPSLE) is a rheumatologic disorder causing neurologic, psychiatric and/or psychologic symptoms. The most important clinical problems are the aspecific nature of signs and symptoms, the limited knowledge on pathogenesis and the absence of a diagnostic gold standard. Magnetic resonance imaging (MRI) is considered the most important imaging modality of the brain in NPSLE patients. Abnormalities visible on conventional MRI appear anywhere in the brain, and may normalize, stabilize or increase with loss of brain parenchyma. Apart from conventional MRI sequences, in this thesis advanced MRI techniques such as magnetization transfer imaging (MTI) were also applied, which are more sensitive to microscopic brain damage invisible to the human eye. Microscopic brain damage in NPSLE appears mostly in the cortical gray matter and is associated with the presence of anticardiolipin antibodies. The final brain damage, as observed using different MRI techniques, consists of neuronal and axonal damage, atrophy, demyelination and gliosis. Changes in the total amount of microscopic brain damage as detected by MTI correlate with changes in clinical status. This thesis contributes to the knowledge on the pathogenesis of NPSLE, and illustrates that advanced and conventional radiological techniques can be helpful in making diagnostic and therapeutic decisions. Show less
Despite elaborate scholarly attention, the necropolis of Assiut, capital of the 13th Upper Egyptian Nome, has never been properly surveyed. Several of the major excavations, mainly undertaken in... Show moreDespite elaborate scholarly attention, the necropolis of Assiut, capital of the 13th Upper Egyptian Nome, has never been properly surveyed. Several of the major excavations, mainly undertaken in the first decades of the last century, still await publication or more detailed edition. These not only include the French excavations by Chassinat and Palanque (1903-1904), but also the work led by Hogarth for the British Museum (1906-1907), and the digs conducted by Schiaparelli (1905, 1908, 1910, 1911-1913). Whereas the historical significance of Assiut during the First Intermediate Period and the Middle Kingdom, and its distinct local style of coffin decoration have long been recognised, the basic requirements for a systematic study of this important site are still absent. The aim of this study is to generate a framework for the social, spatial and chronological development of the necropolis of Assiut, concentrating on the times from the Old Kingdom to the end of the Middle Kingdom. Mainly based on hitherto unpublished material, the available funerary remains are carefully scrutinised to establish a chronological ordering. The approach is multifaceted, among other things using tomb architecture, the morphology of pottery, stone vases, seals, coffin decoration (choice of decoration patterns, textual content, epigraphic and palaeographic features), and their spatial distribution. Their combined analysis results in four distinct (chronological) phases in the funerary culture at Assiut: Late Old Kingdom, First Intermediate Period, Early Middle Kingdom and Advanced - Late Middle Kingdom. The remains and features of each of these phases are discussed, and used as a means to put the history of Assiut into better perspective.A brief survey of contents:Chapter 1 - Introduction, Aims and methodsChapter 2 - Compiles a map of the Necropolis and the tombs of its mayors/nomarchs. With regard to the latter, 7 new candidates are put forward, resulting in a reconsideration of the chronology of the Siutian mayors and nomarchs.Chapter 3 - A full survey of the excavations conducted, their location(s) in the field, results, and problems in analysing the available material and reports.Chapter 4 - Survey of Late Old Kingdom burial grounds identified at AssiutChapter 5 - Survey of First Intermediate Period burial grounds identified at AssiutChapter 6 - Survey of Early Middle Kingdom burial grounds identified at Assiut (I)Chapter 7 - Survey of Early Middle Kingdom burial grounds identified at Assiut (II)Chapter 8 - Survey of Advanced - Late Middle Kingdom burial grounds identified at AssiutChapter 9 - Conclusions Show less
Negotiating Life analyzes the funerals of the Garo, a matrilineal hill society of the Indian State of Meghalaya. Garo funerals serve to dispose of the corpse, and to guide the soul of the deceased... Show moreNegotiating Life analyzes the funerals of the Garo, a matrilineal hill society of the Indian State of Meghalaya. Garo funerals serve to dispose of the corpse, and to guide the soul of the deceased to the afterworld. In addition, the rituals allow for the reorganization of social relationships among people and their Houses. Mortuary rituals are instrumental in the transformation of the dead from social persons into anonymous ancestors. Particularly in the latter sense, the dead are a source of authority and prestige, and play an important role in structuring social relationships among people. The author argues that Garo mortuary rituals derive much of their significance from the transfer of gifts between representatives of the deceased and the people who attend a mortuary ritual. The kind of gifts that can be offered depend on the relationship that people trace to the deceased. The acceptance and rejection of these gifts is decided in processes of negotiation. Consequently, gift exchange plays an important role in defining and (re)constructing social relationships. It is shown that people's participation in rituals of death is of structural importance to Garo society and allows them to reconstruct life in the context of death. Show less
The book offers a detailed presentation of the ceramic sequence excavated at Tell Sabi Abyad, a late Neolithic archaeological site in northern Syria. This sequence documents the transformation from... Show moreThe book offers a detailed presentation of the ceramic sequence excavated at Tell Sabi Abyad, a late Neolithic archaeological site in northern Syria. This sequence documents the transformation from a Pre-Halaf ceramic assemblage to an Early Halaf ceramic assemblage. The author discusses and critiques existing models for explaining the rise of the Halaf pottery style, and offers an alternative based on the twin concepts of emulation and feasting. Show less
In this thesis the reproducibility of new methods using MRI for measuring total cerebral blood flow and cerebrovascular reserve capacity was assessed. We used these methods to show that NO is an... Show moreIn this thesis the reproducibility of new methods using MRI for measuring total cerebral blood flow and cerebrovascular reserve capacity was assessed. We used these methods to show that NO is an important factor in the hypoxia induced vasodilatation of vessels in the brain. It was shown that basal total cerebral blood flow in the elderly, in contrary to the young, is dependent on the NO pathway. White matter hyperintensities are seen in almost every elderly subject. We showed that besides the white matter hyperintensities there are also change because of aging in the normal appearing white matter. This was demonstrated with the use of magnetization transfer imaging. The appearance of the white matter hyperintensities seems to be independent of the changes in the normal appearing white matter which gives rise to the idea that both entities have different aetiologies. Using this technique we also showed that not all white matter hyperintensities are the same but that differences exists between white matter hyperintensities periventricular and in the deep white matter. Also, differences between frontal and occipital located periventricular white matter hyperintensities exists. Finally, we found that cognitive decline in the elderly is associated with a diminished total cerebral blood flow. Show less
The thesis "Light-induced molecular processes on ice" deals with two main issues: first, the interaction of water with a platinum surface, under very well-defined conditions (at liquid nitrogen... Show moreThe thesis "Light-induced molecular processes on ice" deals with two main issues: first, the interaction of water with a platinum surface, under very well-defined conditions (at liquid nitrogen temperature in a very low-pressure environment (Ultra-High Vacuum: pressure 2 x 10-11 mbar)), and second, the photochemistry of small, naturally occurring, organic molecules such as bromoform (CHBr3) molecules on ice surfaces. The first topic is of relevance for electrochemistry, where water-metal interactions are crucial in determining the system's reactivity. The second topic is relevant for our understanding of processes encountered in the Earth's atmosphere. In particular, for atmospheric chemistry, the fundamental steps of the photochemical reaction of bromoform on ice surfaces induced by UV light, are elucidated. This reaction constitutes an important step in the ozone depletion cycle, which greatly affects our atmosphere. Photodissociation studies reveal a rich UV-induced photochemistry of bromoform on ice: various direct fragmentation pathways, as well as formation of new, ice-mediated C—C and C—O bond containing chemical species. Given the previously reported detection of bromoform in the stratosphere, these observations may have significant implications for current models describing stratospheric ozone depletion. Show less
The thesis contains work on the catalytic mechanism, and then especially on the reversibility of the reaction and the order of substrate addition and reduction of the catalytically active type-2... Show moreThe thesis contains work on the catalytic mechanism, and then especially on the reversibility of the reaction and the order of substrate addition and reduction of the catalytically active type-2 copper site. Furthermore, protein engineering of the type-1 copper site is reported and a study into the reorganizational energy of this site. Show less
