This thesis describes different ways of exploring the pharmacological and therapeutic effects of novel GABA-ergic and GABA-like agents in humans. Systematic pharmacodynamic evaluations, using well... Show moreThis thesis describes different ways of exploring the pharmacological and therapeutic effects of novel GABA-ergic and GABA-like agents in humans. Systematic pharmacodynamic evaluations, using well-characterised positive controls, can confirm or refute the unique pharmacological properties of GABAA-subtype selective drugs in healthy volunteers. Such studies can help to predict dosing regimens and therapeutic advantages of these drugs. The distribution of different GABAA-receptor subtypes provides clues for their functional relevance. This knowledge can be used to optimise the desirable and undesirable effect profiles of selective GABA-ergic drugs. Very little is still known about the pathophysiological relevance of GABA-systems in CNS-disorders, although GABA-ergic treatments are in use for a wide range of clinical conditions. The availability of novel compounds with well defined pharmacological characteristics can clarify the involvement of these mechanisms in normal or abnormal physiology. This thesis hopes to show that carefully designed studies, using a range of CNS-measurement that reflect different GABAergic systems, can aid in the development of new GABA-ergic drugs, and help to unravel the role of the different GABA-ergic systems in health and disease. Show less
Despite major improvements in immunosuppressive agents and a reduction in acute rejection episodes, there has been no significant improvement in overall kidney transplant survival beyond the first... Show moreDespite major improvements in immunosuppressive agents and a reduction in acute rejection episodes, there has been no significant improvement in overall kidney transplant survival beyond the first 3 months after transplantation1. Up to sixty percent of all renal allografts are lost within 10 years after transplantation. Apart from death, the main cause of graft loss is chronic allograft nephropathy (CAN)2;3. CAN is characterized by the deterioration of graft function and structure as a consequence of immunological processes (i.e., chronic rejection) and/or a variety of often co-existing non-immunological factors that include: advanced donor age, ischemic injury to the graft during implantation, chronic calcineurin inhibitor induced nephrotoxicity, hypertension, reflux, infection, increased ureteral pressure, and glomerular hyperfiltration. The initial histological characteristics of CAN are the presen ce of tubulointerstitial fibrosis and tubular atrophy (IF/TA)4. Over time, additional features evolve including vasculopathy, glomerulopathy, and glomerulosclerosis. Unfortunately, functional studies significantly underestimate the incidence of graft injury. One of the largest studies with repeated protocol biopsies clearly showed that CAN is a process that develops early after transplantation. Up to 94% of protocol biopsies obtained one year after renal transplantation exhibited IF/TA in patients with stable graft function5. In addition, Solez et al. have shown that 2 years after transplantation two thirds of all kidney allografts exhibit CAN without deterioration of renal function 6. The most significant predictors for the development of CAN were the occurrence of acute rejection episodes, acute calcineurin toxicity, and the initial quality of the transplanted kidney. Often, the first clinical sign of CAN is the progressive decline in renal function as measured by increasing serum creatinine or the development of overt proteinuria2. Although renal function correlates with glomerulosclerosis, unfortunately the clinical tests currently available for renal function are not sensitive enough to detect early lesions associated with CAN. Inulin and iothalamate clearances are more reliable and precise techniques for measuring renal function, but they are expensive, difficult to perform, and time consuming; thus they are unlikely to become routine in daily practice. This situation has given rise to intensified interest in studies using protocol biopsies that may identify surrogate markers and provide insight into the development of CAN. Ideally, a surrogate marker is minimally invasive and amenable to frequent assessment. However, the least invasive markers in blood or urine samples can only identify qualities and quantities of structural lesions i n the renal allograft that typically occur in the advanced stages of disease. Currently, protocol biopsies are considered __the gold standard__ for discovering novel surrogate markers that better predict long-term outcomes for patients. In this thesis, we focused on identifying molecular markers in kidney transplant biopsies that could predict long-term allograft survival. The identification of molecular markers provides a means for superior monitoring of the transplant condition in order to maintain drug efficacy and limit drug-related nephrotoxicity. Moreover, potential new therapeutic targets might be discovered for developing therapies that improve long-term graft survival. Show less
Even with the remarkable results of cardiac resynchronization therapy (CRT) in the large randomized trials, approximately 30-40% of the patients failed to improve after CRT when the established... Show moreEven with the remarkable results of cardiac resynchronization therapy (CRT) in the large randomized trials, approximately 30-40% of the patients failed to improve after CRT when the established selection criteria are used, highlighting the need for improvement of the current criteria. In addition, the exact mechanism and effects of CRT on echocardiographic and clinical parameters such as mitral regurgitation, strain and incidence of ventricular arrhythmias are currently unknown. The aim of the current thesis was to further explore these issues using varying non-invasive imaging techniques such as echocardiography, nuclear imaging, magnetic resonance imaging as well as device-based diagnostics. The results of Part I demonstrate that presence of mechanical dyssynchrony within the LV as measured with echocardiography is an important predictor of response after CRT. Also, other factors may influence response such as the presence and location of scar tissue in the LV as well the position of the LV pacing lead. Part II describes the acute and long-term benefit (LV function, strain, mitral regurgitation, myocardial blood flow, oxidative metabolism), prognosis, interruption of CRT and optimization of device settings. In addition, implantable cardioverter-defibrillator (ICD)-backup is preferred in all CRT candidates since 21% experienced appropriate ICD therapy within 2 year after implantation. Show less
Cannabis sativa L. plants produce a diverse array of secondary metabolites, which have been grouped in cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans; the cannabinoids are... Show moreCannabis sativa L. plants produce a diverse array of secondary metabolites, which have been grouped in cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans; the cannabinoids are the best known group of natural products from this plant. The pharmacological aspects of this secondary metabolite group have been extensively studied and the cannabinoid biosynthetic pathway has been partially elucidated. Although, it is known that the geranyl diphosphate (GPP) and the olivetolic acid are initial precursors in this route the biosynthesis of the olivetolic acid has not been found yet. It has been suggested that the olivetolic acid biosynthesis could be initiated by a polyketide synthase (PKS). This thesis was focused on the characterization of PKSs in cannabis plants. More than 480 compounds have been identified from C. sativa but only 247 are considered as secondary metabolites. These latter are grouped into cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans. However, what do we know about their biosynthesis and role in the plant? Chapter 1 summarizes the natural compounds in cannabis from a biosynthetic view. It seems that enzymes belonging to the polyketide synthase group could be involved in the biosynthesis of the initial precursors from the cannabinoid, flavonoid and stilbenoid biosynthetic pathways. The Polyketide Synthases (PKSs) are condensing enzymes which form a myriad of polyketide compounds. In plants several PKSs have been identified and studied. Aspects such as specificity, reaction mechanisms, structure, as well as evolution are reviewed in Chapter 2. In Chapter 3 polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Differences in activities of chalcone synthase (CHS), stilbene synthase (STS) and olivetol-forming PKS were observed during the development and growth of glandular trichomes on the female flowers. Although, cannabinoid biosynthesis and accumulation take place in glandular trichomes no activity for an olivetolic acid-forming PKS was detected in this tissue. Content analyses of cannabinoids and flavonoids from different tissues revealed differences in their distribution, suggesting a diverse regulatory control on the biosynthetic fluxes of their biosynthetic pathways in the plant. Chapter 4 reports in silicio expression analysis of a PKS gene isolated from glandular trichomes. The deduced amino acid sequence showed 51-72% identity to other CHS/STS type sequences of the PKS family. Further phylogenetic analysis revealed that this PKS (PKSG2) grouped with other non-chalcone and stilbene-producing PKSs. Homology modeling analyses of this cannabis PKS predicts a 3D overall fold similar to alfalfa CHS2 with small steric differences on the residues that shape the active site of the cannabis PKSG2. Cannabis sativa cell culture induction has been reported for several purposes. However, cannabinoids have not been detected in cell cultures so far. Although, elicitation has been employed in the cell cultures for inducing and/or improving secondary metabolites there are no reports concerning elicitation effect on secondary metabolite production in C. sativa cell cultures. In Chapter 5 the effect of elicitation on secondary metabolism of the plant cell cultures is reported. Metabolic profiles analyzed by 1H-NMR spectroscopy and principal component analyses (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in both control and elicited cannabis cell cultures. THCA synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway. Show less
The work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1... Show moreThe work presented in this thesis has focused on the role of Mitogen Activated Protein Kinases (MAPKs) and their major downstream targets, the AP-1 transcription factors, in particular the AP-1 components ATF3, Fra1, c-Jun, ATF-2 and c-Fos. Chapter II provides information on the signaling pathways involved in the activation of ATF-2 and ATF3 in the response of primary human fibroblasts to ionizing radiation. In chapter III c-Jun and ATF3, the MAPK JNK and the MAPK-phosphatase MKP-1 are identified as important sensors of UV-induced-DNA damage in transcribed genes. Chapter IV shows that ATF3 acts as an antiapoptotic JNK target in T98G glioblastoma cells, whereas Fra1 seems to act as a proapoptotic effector of both JNK and ERK. In addition, it is shown that ATF3 and Fra1 have opposite effects on cisplatin-induced S phase arrest. Chapter V shows that Fra1 also can exhibit a pro-apoptotic function in UV-irradiated fibroblasts. Furthermore, this chapter reports an as yet unknown function of JNK: repression of the transactivating activity of c-Jun/Fos(- like) dimers, mediated via hyper-phosphorylation of the c-Jun transactivation domain. The data further emphasizes that c-Jun/Fos(-like) and c-Jun/ATF dimers and their respective target genes can exhibit opposite functions in DNA damage responses. Show less
In dit proefschrift worden verschillende manieren onderzocht om semi-gestructureerde gegevens te analyseren, bijv. HTML bestanden. HTML bestanden hebben een structuur/opbouw, maar waar en hoe vaak... Show moreIn dit proefschrift worden verschillende manieren onderzocht om semi-gestructureerde gegevens te analyseren, bijv. HTML bestanden. HTML bestanden hebben een structuur/opbouw, maar waar en hoe vaak je een tekst bold of italic maakt varieert voor elke HTML. Er is gekeken naar verschillende manieren om de voorkomens van een patroon (bijvoorbeeld alle moleculen in onze dataset bevatten een bepaalde set van atomen en verbindingen) te tellen om zo interessante patronen te vinden. Het juist presenteren van de resultaten aan de gebruiker is ook van belang. Dit proefschrift behandelt de visuele weergave van resultaten van de analyse (mining) van semi-gestructureerde gegevens, zodat de gebruiker eenvoudiger interessante patronen kan vinden. De conclusies zijn moeilijk kort samen te vatten. Echter het blijkt dat sommige patronen interessanter waren wanneer zij heel vlak achter elkaar voorkwamen en andere wanneer zij bijvoorbeeld wekelijks voorkwamen. Om nog meer interessante patronen te vinden is het aan te raden rekening te houden met dit element van tijd. Verder blijkt het dat visualisaties nodig zijn om de grote hoeveelheid patronen effectief te presenteren, bijvoorbeeld de gebruiker ziet in __n oog opslag substructuren van moleculen die voorkomen. Het onderzoek in dit proefschrift is belangrijk voor de analyse van data. Denk bijvoorbeeld aan de analyse van het gedrag van klanten. Het is interessant voor bedrijven om te weten dat klanten bepaalde producten aanschaffen bijvoorbeeld elke maandag. Dit is vernieuwend omdat wij subgroepen van producten ontdekken, maar wij tellen subgroepen met de juiste eigenschappen voor tijd zwaarder dan subgroepen die gewoon zomaar voorkomen. De visualisatie van samen voorkomende molecuul substructuren kan de analyse van deze versnellen en deze manier van plotten is nieuw. Show less
Recently, there has been a renewed interest in theoretical aspects of flows of viscoelastic fluids (such as dilute polymer solutions). This thesis addresses two distinct issues related to such... Show moreRecently, there has been a renewed interest in theoretical aspects of flows of viscoelastic fluids (such as dilute polymer solutions). This thesis addresses two distinct issues related to such flows. Motivated by the possible occurrence of subcritical (finite-amplitude) instabilities in parallel flows - instabilities that cannot be captured by the usual linear stability analyses - I present and evaluate a method to describe these subcritical transitions by means of a direct expansion in the amplitude of the linearly least stable mode. A second issue is the behaviour of viscoelastic fluids in steady elongational flow. Here, singular solutions have recently been found for flows involving a stagnation point. These solutions appear to be the mathematical structures underlying the birefringent strands that have been observed experimentally in these flows. In this thesis, explicit approximate solutions are found for idealized extensional flow geometries and simple constitutive equations. Asymptotic results are derived for the width of the strand and other typical parameters. It appears that non-analytical solutions are a general feature of elongational viscoelastic flows, which should also occur for more realistic flows and models. Show less
Psychologists have shown that reference-point reasoning is a ubiquitous cognitive phenomenon intrinsic to perception, categorisation, spatial orientation, social, organisational and marketing... Show morePsychologists have shown that reference-point reasoning is a ubiquitous cognitive phenomenon intrinsic to perception, categorisation, spatial orientation, social, organisational and marketing behaviour of human beings. Various cognitive tasks involve evoking a salient entity – called cognitive reference point – for establishing mental contact with less salient items. It is then reasonable to assume that language use also involves a lot of reference-point reasoning. However, linguistic aspects of this phenomenon have hardly been investigated. A welcome exception is Langacker’s reference-point model dealing with grammatical constructions and metonymy. This thesis elaborates the reference-point model by applying it to lexical semantics. The only reference point that has been quite intensely studied in lexical semantics is a prototype. By focusing on two adjectival groups (colour and size), the author demonstrates that a whole panoply of cognitive reference points are used to anchor conceptual specifications of lexical items, prototypes being only a special case of the reference-point mechanism. An important finding is that a word may trigger more than one reference point at a time. For example, dimensional adjectives may be interpreted vis-à-vis an average value, endpoints of the scale, prototypes and dimensions of the human body. Contextual variability is claimed to be related to various combinations of reference points, their relative salience and patterns of interaction. Show less
Cardiovascular diseases are the major cause of morbidity and mortality in western societies. The most common clinical manifestations are stroke and acute myocardial infarction and in both ailments... Show moreCardiovascular diseases are the major cause of morbidity and mortality in western societies. The most common clinical manifestations are stroke and acute myocardial infarction and in both ailments atherosclerosis is the underlying culprit. Atherosclerosis is a lipid-mediated chronic inflammatory disease, which is accompanied by leukocyte infiltration into the vessel wall. The migration of leukocytes from the circulation to the vessel wall is directed by a specific class of proteins, the chemokines. Therefore it is likely that chemokines have a distinctive role in leukocyte homeostasis at specific stages of atherosclerotic disease progression and during ischemia-reperfusion injury. This thesis encompasses of number of human and experimental mouse studies on the role of chemokines in cardiovascular diseases and atherosclerosis, from which several new targets were identified. For instance it is evident that patients with high levels of the chemokine CCL3 are 10 times more likely to develop severe cardiovascular events in the future. In conclusion, the research described in this thesis provided novel candidates that might be of value for the early prediction of high risk patients. Moreover, the identified candidates may also represent valuable targets for modulation of leukocyte homeostasis in the plaque that could improve atherosclerotic plaque progression and stability. Show less
This thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact... Show moreThis thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact that the absorption spectrum of the protein__s active site varies upon changing its redox state. This change can be translated into a change in the fluorescence intensity of a label that is covalently linked to the protein on the basis of F_rster Resonance Energy Transfer (FRET). With our method we could show that different redox proteins and enzymes can be studied down to the single molecule level. This exciting finding opens the door to the study of various redox enzymes and to monitor specific substances such as for example nitrite. Depending on the function of the enzyme under investigation a wide range of substrates can be monitored. Another example is the development of an oxygen sensor by employing proteins that are capable of binding oxygen. The findings presented in this thesis might be significant for applications in oxygen sensing and, more generally, in the fast growing field of biosensing Show less
Many legislative decisions of the Council of the EuropeanUnion are de facto made in preparatory bodies by national officials anddiplomats. Ministers rubber-stamp these committee decisions without... Show moreMany legislative decisions of the Council of the EuropeanUnion are de facto made in preparatory bodies by national officials anddiplomats. Ministers rubber-stamp these committee decisions without discussion.Drawing on statistical as well as case study evidence, the study investigatesthe extent to which this actually occurs. Based on a formal-theoreticaldiscussion of possible explanations, the study also examines the causes for whycertain decisions are made by committee members and others by ministers. Ingeneral, the empirical findings alleviate concerns about the democraticlegitimacy of Council decisions. Show less
Stars form as a result of gravitational collapse of an interstellar molecular cloud. In the process, a circumstellar disk, often referred to as a protoplanetary disk, is formed as well as a result... Show moreStars form as a result of gravitational collapse of an interstellar molecular cloud. In the process, a circumstellar disk, often referred to as a protoplanetary disk, is formed as well as a result of the net angular momentum of the parental cloud. This thesis addresses several questions about the formation of this disk and, in particular, the evolution of the velocity field surrounding the young star. The composition of the velocity field (i.e., the ratio of infall to rotation) can be use to trace the evolution of young stellar objects. This method of characterizing the evolutionary stage of protostars is applied to two objects, NGC1333-IRAS2A and L1489 IRS, the first of which we find to be a very young object while the latter is significantly more evolved. In addition, one chapter of this thesis presents a new radiation transfer code which has been developed by the author. Show less
Equine arteritis virus (EAV) is the prototypic virus of the family Arteriviridae. The EAV genome is a positive-sense single-stranded RNA molecule in which two open reading frames (ORFs) encode the... Show moreEquine arteritis virus (EAV) is the prototypic virus of the family Arteriviridae. The EAV genome is a positive-sense single-stranded RNA molecule in which two open reading frames (ORFs) encode the large replicase polyproteins pp1a and pp1ab. Processing of pp1a and pp1ab is mediated by three viral proteases of which a predicted chymotrypsin-like protease residing in nsp4 releases most non-structural proteins from the replicase polyproteins. To obtain more insight in the biochemical properties of this protease, and viral chymotrypsin-like proteases in general, the three-dimensional structure of nsp4 was determined by X-ray crystallography. The nsp4 three-dimensional structure revealed that the enzyme adopts a chymotrypsin-like fold and possesses an additional C-terminal domain (CTD) not found in most other chymotrypsin-like proteases. The structure revealed also that a connecting stretch of amino acids might facilitate movement of the CTD relative to the rest of the molecule. A site-directed mutagenesis study showed that the nsp4 CTD played a crucial role in EAV replicase processing, but that it was not required for proteloytic activity of the protease per se. Furthermore, the formation of an ion pair between Asp-119 and either Arg-4 in the N-terminus or Arg-203 in the C-terminus was suggested, which could play a role in alternate nsp4 conformations needed for e.g. different cis and trans cleavage activities. Mutations targeting the residues involved in these interactions affected the proteolytic activity of nsp4, but the data were inconclusive regarding the importance of ion pair formation. Processing of the C-terminal half of pp1a (the nsp3-8 region) by nsp4 can proceed following either of two alternative proteolytic pathways. To address the importance of both pathways, various cleavage site mutations were engineered, which were expected to block cleavage by nsp4. The experiments showed that all mutations that blocked processing of the corresponding site in the nsp3-8 precursor also blocked or severely inhibited EAV RNA synthesis. Moreover, evidence was obtained for the presence of a novel, internal nsp4 cleavage site in nsp7, which appears to be conserved among arteriviruses. The theoretical chapters in this thesis introduce the reader to (nido)viruses and nidovirus replicase maturation in particular. Show less
This thesis introduces the posterior heart field contributing to the venous pole of the heart by epithelial-mesenchymal-transformation of the coelomic epithelium. Based on studying of podoplanin... Show moreThis thesis introduces the posterior heart field contributing to the venous pole of the heart by epithelial-mesenchymal-transformation of the coelomic epithelium. Based on studying of podoplanin and Sp3 (novel genes in cardiogenesis) wildtype and knockout mouse embryos between stages 9.5-18.5, we postulate that the posterior heart field contributes through mesenchymal and myocardial cell populations. The mesenchymal population is involved in the formation of the proepicardial organ, epicardium and epicardium-derived cells. The hypoplastic proepicardial organ and impaired epicardial-myocardial interaction result from altered mesenchymal contribution of the posterior heart field by lack of podoplanin and SP3 leading to hypoplasia of the chamber myocardium and coronary arterial vascular wall as well as (atrioventricular) septal defects. Myocardial contribution concerns myocardium of the sinus venosus including the sinoatrial node, venous valves, primary atrial septum and the left atrial dorsal wall as well as the wall of the pulmonary and cardinal veins. Development of smooth-muscle-cells of the wall of the pulmonary vein is also related to the posterior heart field. Moreover, we have reported formation of a transient left-sided sinoatrial node which persists during development in 10% of the cases. Podoplanin mutants show cardiac malformations including a hypoplastic sinoatrial node. This thesis contributes to the understanding of the mechanism underlying the mentioned cardiac malformations and arrhythmias originating in the sinus venosus region. Show less
Although the influence of magnetic fields on the rates and product yields of a host of chemical reactions are well documented and can be understood in the framework of the Radical Pair Mechanism ... Show moreAlthough the influence of magnetic fields on the rates and product yields of a host of chemical reactions are well documented and can be understood in the framework of the Radical Pair Mechanism (RPM), it has so far proved impossible to demonstrate convincingly a biological RPM effect. In this work we present proof that biological systems, i.e. photosynthetic bacteria, for which it is known that the RPM is operative, can generate toxic products in amounts that depend on the presence of an applied magnetic field. This work, in turn, illustrates that magnetic fields influence the extent of photoinhibition in such a biological system. We show, to our knowledge, the first observation of magnetic field dependent singlet oxygen production in biological system. These measurements could in principle explain beneficial and adverse effects for low and high magnetic fields, respectively. Show less
This thesis deals with emotional and social influences on childhood somatic complaints. Strong support is provided for the idea that negative affect contributes to the development of somatic... Show moreThis thesis deals with emotional and social influences on childhood somatic complaints. Strong support is provided for the idea that negative affect contributes to the development of somatic complaints in childhood. In addition, the studies described give information about the type and levels of negative affect associated with somatic complaints in childhood. When appraisal is characterized by the experience of little control over negative situations and emotions, this is likely to directly cause more feelings of negative affect and also to contribute to a more negative processing of emotions: children who feel little control evidently use more maladaptive emotion regulation strategies. This negative emotional processing further contributes to negative affect and subsequent somatic complaints. These relationships between negative affect and somatic complaints are even present at subclinical levels of emotional problems. With regard to social problems, it is found that they have an effect on somatic complaints that is fully mediated by aspects of children’s emotional functioning: self-perceived social problems indicate lower feelings of control and more maladaptive emotion regulation. Show less
Consistentie in de straftoemeting bekeken vanuit een analyse van de constitutionele en institutionele context waarbinnen de strafbeslissing wordt genomen, alsmede een systenatische analyse van het... Show moreConsistentie in de straftoemeting bekeken vanuit een analyse van de constitutionele en institutionele context waarbinnen de strafbeslissing wordt genomen, alsmede een systenatische analyse van het wettelijk kader van de straftoemeting. In dat verband wordt een visie geboden op het straftoemetingsrecht en de wijze waarop die beslissing totstandkomt: de strafvorming. Show less
Avian vocalizations function in mate attraction and territorial defence. Vocalizations can act as behavioural barriers and play an important role in speciation processes. Hybrid zones illustrate... Show moreAvian vocalizations function in mate attraction and territorial defence. Vocalizations can act as behavioural barriers and play an important role in speciation processes. Hybrid zones illustrate behavioural barriers are not always impermeable and provide a natural laboratory to examine the role of vocalizations in the causes and consequences of hybridization. This thesis examines a hybrid zone between two species of African doves: the vinaceous (Streptopelia vinacea) and ring-necked dove (S.capicola) by investigating its composition and history with molecular techniques, the mechanisms underlying the variation in dove coos and the response to hybrid signals within and outside the hybrid zone. The variation in hybrid signals and the possibility of learning which vocalizations to respond to can play an important role in facilitating further introgression between the two species. Show less
Self-tolerance to p53 is a major potential limitation for the activation of the endogenous T-cell repertoire. So far, p53 specific CD8+ and CD4+ T-cell immunity has been described in cancer... Show moreSelf-tolerance to p53 is a major potential limitation for the activation of the endogenous T-cell repertoire. So far, p53 specific CD8+ and CD4+ T-cell immunity has been described in cancer patients and healthy individuals. However, the restrictions of tolerance on the recruitment of p53 specific T cells have thus far not been completely elucidated. In this thesis we have studied several basic mechanisms that underlie the availability of p53 specific T-cell immunity and how this repertoire can be employed for immunotherapy against tumors. In chapter 2 we have compared the presence and quality of the p53 specific T-helper cell repertoire in p53 deficient (p53-/-) mice and p53 wt (p53+/+) mice. By direct vaccination with p53, in a viral vector and as 30-mer peptides, we were able to determine the specificity and avidity of T-helper cell populations in the two mouse strains. We show that, unlike the repertoire to other tumor specific antigens such as CEA (88), the p53 specific T-helper cell repertoire in these mouse strains is not blunted by self-tolerance and therefore completely available for immunotherapy of cancer. In chapter 3 we studied the correlation between intracellular accumulation and immunogenicity of several well-described frameshifted colon tumor antigens. Accumulation alone is not a sufficiently indicative marker for the immunogenic properties of a tumor antigen. By comparing intracellular accumulation, direct presentation and cross-presentation in vivo we were able to categorize several tumor antigens and to predict their potency as antigen targets for T helper cells or CTL. In chapter 4 we analyzed the potential beneficial and harmful effects of p53 specific CD8+ T cells in detail. We generated a p53¹⁵⁸⁻¹⁶⁶ specific TCR transgenic mouse and studied the effects of self-tolerance on p53 specific CD8+ T cells in chapter 4.1. We show that thymic selection has a drastic effect on the differentiation of p53 specific CD8+ T cells in p53 +/+ and p53 +/- mice. Education of p53 specific CD8+ T cells in the absence of p53 (in p53-/- mice) leads to a potent population of peripheral p53 specific CD8+ T cells. We studied the in vivo effects of these p53 specific CD8+ T cells in chapter 4.2. In an supplemental chapter 4.3 we show the expression and capacities cells expressing the p53 TCR after retroviral gene transfer. Our data in chapter 4 indicate that immunotherapy with p53 specific CD8+ T cells can lead to acute and severe hematopoietic ablation. These data stress the importance of meticulous pre-clinical research when targeting ubiquitously expressed self-antigens, before starting clinical application. Successful cancer immunotherapy in the absence of any toxicity could nevertheless be achieved by combination of p53 CD8+ T cell transfer and hematopoietic reconstitution with non-sensitive (allogeneic) bone marrow stem cells. A general discussion of this thesis in chapter 5 describes the caveats, challenges and pitfalls of the findings portrayed in this thesis and places them in the context of recent literature. The past decade our understanding of molecular and cellular mechanisms of T-cell mediated anti-tumor responses has taken a leap. Nevertheless, due to improved TCR engineering techniques, the diversity of tumor antigens in (pre) clinical trials is increasing. Despite the differences between models or species the results in the results in this thesis urge for targeting of well-chosen tumor antigens and great caution when targeting ubiquitously expressed self-antigens. Show less
