With the ever-increasing life expectancies of people, the prevalence of aging-related diseases is also increasing. We studied a unique cohort of people; offspring of nonagenarian siblings with the... Show moreWith the ever-increasing life expectancies of people, the prevalence of aging-related diseases is also increasing. We studied a unique cohort of people; offspring of nonagenarian siblings with the propensity for longevity were also recruited and compared to their partners, representing the general population. The offspring (~60 year old) already show lower prevalence of cardiovascular disease and diabetes. In the thesis __Cellular stress in vitro and longevity on vivo__ it is reported how cells (dermal fibroblasts) derived from these offspring were compared with cells from their partners. It was shown that they react differenly in vitro under both non stressed and under conditions of oxidative stress. Future research is warranted to further elucidate the genes involved in these differences. Show less
Depression involves multiple mental problems, including low mood, inability to experience pleasure and emotional, cognitive and behavioral problems. It has a lifetime prevalence of ~15% in the... Show moreDepression involves multiple mental problems, including low mood, inability to experience pleasure and emotional, cognitive and behavioral problems. It has a lifetime prevalence of ~15% in the Dutch population, striking women twice as often as men. The disorder often comprises persisting disturbances in the neuroendocrine stress system, the hypothalamic- pituitary-adrenal (HPA) axis, including disregulation of its end-hormone cortisol. Cortisol normally stimulates emotional, cognitive and behavioral processes in order to cope with a stressor and promotes recovery, learning and memory. This thesis describes the identification of a specific genetic variant of the mineralocorticoid receptor (MR), one of the two receptors for cortisol, which protects against depression. MR transcript expression was found to be lower in postmortem limbic brain regions of depressed patients compared to non-depressed subjects. In addition, a specific and common MR gene variant was identified that results in higher MR expression in vitro. This same variant was found to associate with personality characteristics that predict the risk of depression later in life and with a lower risk of depression itself. All associations were found only in women and not in men. To conclude, the MR is an important determinant of resilience; increased MR expression seems to be protective against depression. Show less
Schizophrenia is a devastating mental disorder characterized by a hyperactive dopamine system and deregulated stress system. Human studies have suggested that the schizophrenia symptoms precipitate... Show moreSchizophrenia is a devastating mental disorder characterized by a hyperactive dopamine system and deregulated stress system. Human studies have suggested that the schizophrenia symptoms precipitate if a hyperactive dopaminergic genotype interacts with adverse life experiences that activate the stress system. To examine this gene-by-environment interaction, we exposed rats genetically-selected for enhanced apomorphine susceptibility to two stress-provoking life events, poor maternal care early-in-life, and isolation rearing later-in-life. This promoted the development of schizophrenia endophenotypes. Our experiments involved two complementary steps: First, we focused on the immediate endocrine adaptations to maternal separation in common rats. It is known that a single episode of prolonged maternal separation slowly increases corticosterone levels in the neonate rat. We discovered that if the pups had been previously exposed to maternal separation, this rise in corticosterone was abolished, suggesting that the pups had learned to predict the return of the dam. While readily adapting to repeated maternal absence, the pups, surprisingly, stayed alert and displayed a rapid response to an acute stressor. We then investigated whether pup__s stress responsiveness was influenced by the context of maternal separation. It appeared that the experience of being kept in isolation in a novel environment during repeated maternal separation, rather than the maternal absence per se, caused priming of the amygdala fear pathway, with lasting consequences for the responsiveness of the neuroendocrine and behavioral stress system. These endocrine and behavioral alterations, caused by early-life stress experience, consisted of schizophrenia-like phenotypes. Second, we sought to investigate the interplay of such early-life stress experience with schizophrenia genetic predisposition and/or later-life social stress experience. Thus, we were able to test the three-hit (cumulative stress) and the developmental mismatch hypotheses. The former states that exposure to earlylife adversity and later-life psychosocial stressors, superimposed on genetic susceptibility, result in a severe schizophrenia-like phenotype. The latter proposes that experiences early-in-life program the developing brain in preparation for the future. In the case of genetically-predisposed apomorphine susceptible rats (schizophrenia-susceptible), we provide strong evidence for the three-hit hypothesis. In the case of the nongenetically selected Wistar rats, the mismatch hypothesis is supported since the outcome of early-life stress often negatively interacted with the pre-puberty social context. In agreement with the three-hit hypothesis of schizophrenia, we conclude from the current experiments that early-life stress experience in interaction with highly reactive dopaminergic alleles, leads to amygdala priming that, together with additional stressors, precipitate schizophrenia. Show less
Recent studies have suggested that the fetus is capable of exhibiting a stress response to intrauterine needling, resulting in alterations in fetal stress hormone levels. Intrauterine transfusions... Show moreRecent studies have suggested that the fetus is capable of exhibiting a stress response to intrauterine needling, resulting in alterations in fetal stress hormone levels. Intrauterine transfusions are performed by inserting a needle either in the umbilical cord root at the placental surface (PCI), or in the intrahepatic portion of the umbilical vein (IHV). Aim of our study was to test the hypothesis that fetal hormonal changes during intrauterine transfusion are more pronounced when the needle is inserted in the fetal abdomen. Furthermore we aimed to evaluate the effect of fetal analgesia with remifentanil on the fetal stress hormone changes. Exploring the hemodynamic changes following a noxious stimulus, we saw no differences in transfusions through the IHV or the PCI. Remifentanil did not influence the stress hormone changes. We concluded that the stress hormone changes are independent of both site of transfusion and the use of remifentanil. Our results do not confirm nor deny that the fetus is capable to react to a potential painful stimulus, or to show signs of stress or even pain. However, previous research has suggested that presumably painful fetal conditions can lead to alterations in stress reactions after birth. This phenomenon is called ‘fetal programming’. Fetal programming could possibly lead to life-long changes in stress responses and even to increased susceptibility for certain diseases. With the current understanding of fetal pain and fetal analgesia we would advocate the following: 1. Fetal analgesia for invasive procedures should be provided from at least 20 weeks gestation onwards 2. All invasive fetal procedures warrant fetal analgesia, but in procedures involving more than just a single puncture with a thin needle it is obligatory. 3. Analgesics should be given intravenously to the mother. The drug of choice should be ultra-short working (like remifentanil) therefore minimising possible undesirable side-effects to both fetus and mother. Show less
The project described in this thesis was designed to test if genetic variation in the mineralocorticoid receptor (MR) gene is a risk factor for developing major depression. First the MR-gene was... Show moreThe project described in this thesis was designed to test if genetic variation in the mineralocorticoid receptor (MR) gene is a risk factor for developing major depression. First the MR-gene was screened for genetic variation. Two selected single nucleotide polymorphisms (SNPs) were tested for in vitro functionality at different levels including: protein and mRNA expression, transactivational capacity and ligand binding. Functionality in vitro was confirmed leading us to test their influence on electrolyte regulation, stress responsiveness and personality. First, in three different cohorts one SNP influenced blood pressure and salt regulation, as could be expected for the MR. Second, the SNPs were associated with the cortisol awaking response (CAR) after dexamethasone administration and with the cortisol and autonomic response following psychosocial stress. This indicates an important role for the MR in the regulation of the stress-response. Third in a relatively small cohort (n=150) the SNPs were not associated with mood and/or anxiety disorders but in the patient group there was an association with the personality trait neuroticism. We hypothesize that genetic variants in the MR-gene are determinants of vulnerability for psychiatric disorders. Show less
Pronounced ultradian and circadian rhythms in the hormones of the hypothalamic-pituitary-adrenal (HPA) axis (i.e. glucocorticoids), one of the body__s major neuroendocrine axes, were already... Show morePronounced ultradian and circadian rhythms in the hormones of the hypothalamic-pituitary-adrenal (HPA) axis (i.e. glucocorticoids), one of the body__s major neuroendocrine axes, were already demonstrated several decades ago. Until now, the clinical relevance of the pulsatile nature of glucocorticoids was poorly understood or sometimes even regarded as not important. Its evolutionary conservation across many species however implies biological significance. Indeed, glucocorticoids have been proven to be crucial for a plethora of bodily functions, e.g. emotion, cognition and the central mechanism underlying the adaptation to stress. Furthermore, disturbances in the characteristic temporal pattern of glucocorticoid exposure have often been described in stress-related pathology. However, the significance of glucocorticoids secretory patterns for physiology, stress responsiveness and nuclear receptor signalling is still largely unexplored and is accordingly addressed in this thesis. A new concept in the endocrinology of glucocorticoids has evolved from the data presented here showing that pulsatile release of glucocorticoids is a major determinant in __resilience__ of glucocorticoid signalling in neuronal cells and stress responsiveness. Moreover, we show that particularly the glucocorticoid receptor is affected after disrupting glucocorticoid pulsatility and could thus provide an excellent target for therapy to normalise the downstream effects of disturbances in glucocorticoid rhythms in stress-related disease. Show less
For a majority of people worries about upcoming stressful events are a common experience in daily life. The aim of this thesis was to examine the effects of common worries on somatic health. In... Show moreFor a majority of people worries about upcoming stressful events are a common experience in daily life. The aim of this thesis was to examine the effects of common worries on somatic health. In particular, the effects of worry on somatic health complaints, like headache and back pain, and on cardiac activity after stressful events were tested. Furthermore, the mechanisms by which worry was hypothesized to affect somatic health were investigated. The studies in this thesis add to the accumulating number of studies testing the perseverative cognition hypothesis stating that only perseverative cognitive representations of stressful events (worries) lead to prolonged stress-related physiological activity which, in turn, can lead to somatic disease. Taken together, the studies reported in this thesis provide evidence for the perseverative cognition hypothesis, that is, worry predicted adverse somatic health outcomes. In addition, the results of the studies presented in the second part of this thesis yielded several new and more specific hypotheses concerning the mechanisms behind the health effects of worry and the effects of worry on stress management therapies. All in all, evidence from real life and laboratory studies makes clear that worry might play a substantial role in somatic health. Show less
In this thesis, determinants, risk and protective factors of parental reactions to childhood cancer are described and research areas that are understudied until now have been identified. Chapter 2... Show moreIn this thesis, determinants, risk and protective factors of parental reactions to childhood cancer are described and research areas that are understudied until now have been identified. Chapter 2 contains a review study on stress and adaptation in parents of pediatric cancer patients. Chapter 3 describes the results of a multicenter study among parents of children with cancer with the aim to evaluate the psychometric qualities of the Dutch version of a disease-related instrument measuring parental stress. Chapter 4 is a review article on parental stress and adaptation among parents of children undergoing stem cell transplantation (SCT). Chapter 5 contains the results of a longitudinal study on child- and parent reported health related quality of life and parenting stress in parents of children undergoing SCT. In Chapter 6, the results of a cross-sectional study on parental stress and perceptions of child vulnerability in parents of children who underwent SCT either 5 or 10 years ago are reported. In Chapter 7, cognitive problems, behavior problems and health related quality of life of children with LCH are described. Show less
The main goal of the present thesis was to study the effects of stress and stress hormones on the retrieval of emotional memories in healthy humans. In addition, we were interested in the effects... Show moreThe main goal of the present thesis was to study the effects of stress and stress hormones on the retrieval of emotional memories in healthy humans. In addition, we were interested in the effects of stress hormones on post-retrieval processes like reconsolidation. That is, are there only acute and temporary effects of stress hormones on memory retrieval, or are there also long-term effects? Studying effects of stress hormones can be done in two ways; either by (experimentally) inducing stress in humans, or by exogenously administering doses of stress hormones. In the present thesis both ways were used. Furthermore, when investigating emotional memories, we can make use of memories that are created in a laboratory setting or those that derive from real life experiences, i.e. autobiographical memories. Again, both methods were investigated. We found acute stress and a single cortisol administration to have direct and long-term impairing effects on memory for neutral and emotional information that was learned and reactivated in a controlled laboratory situation. Future studies should shed more light on the generalizability of these findings to real life settings and clinical practice. Show less
Een emotionele gebeurtenis zoals een auto-ongeluk of eerste kus wordt goed onthouden. Stresshormonen spelen een grote rol bij deze link tussen emotie en cognitie. Onder normale omstandigheden... Show moreEen emotionele gebeurtenis zoals een auto-ongeluk of eerste kus wordt goed onthouden. Stresshormonen spelen een grote rol bij deze link tussen emotie en cognitie. Onder normale omstandigheden worden emotionele en cognitieve processen bevorderd door stresshormonen zoals adrenaline en corticostero_den. Echter, te veel of juist te weinig stresshormonen, of een te lange periode van stress kan emotie en cognitie zo be_nvloeden dat sommige mensen stressgerelateerde ziekten zoals posttraumatische stressstoornis (PTSS) ontwikkelen. Waarom de een wel en de ander niet ziek wordt van stress is niet bekend. Men denkt dat de corticostero_den hiervoor van belang zijn. Vera Brinks richtte zich in haar onderzoek op de rol van corticostero_den in de integratie van emotionele en cognitieve processen, en dus stressgerelateerde fysiologie en psychopathologie. De focus lag hierbij op de rol van de corticostero_d receptoren in de hersenen; de mineralo- (MR) en glucocortico_d receptoren (GR). Dit onderzoek verrichte zij bij muizen. De experimenten lieten zien dat emotie een flinke verbetering van cognitieve prestaties gaf bij de muizen. Hierbij bleek dat activering van de GR - in vergelijking met MR activatie - belangrijk is in de integratie van emotie en cognitie. GR activatie met hoeveelheden van het stress hormoon corticosteron die ook vrij komen bij milde stress, resulteert in een optimale prestatie wanneer het dier ook een emotionele ervaring had. Een te lage of te hoge activatie van deze receptor (de GR) verstoorde de integratie van emotie en cognitie. Die GR werkt dus optimaal binnen nauwe grenzen. Wanneer de MR genetisch wordt 'uitgeschakeld'(knockout), dan worden bepaalde negatieve ervaringen niet uitgedoofd (vergeten). Een belangrijke vinding was ook dat corticostero_dbehandeling de herinnering aan een traumatische gebeurtenis zowel kan verminderen als verbeteren afhankelijk van de genetische achtergrond van de muizen Deze kennis kan gebruikt worden bij de behandeling van het veel te sterke geheugen voor traumatische en angstige PTSS-pati_nten. Bovendien is het een basis om de genetische factoren te onderzoeken die bij kunnen dragen aan het ontstaan en de vermindering van het sterke angstgeheugen bij PTSS-pati_nten. Onze experimenten hebben laten zien dat de MR een uitstekende kandidaat is als target voor een geheel nieuw soort geneesmiddelen. Show less
De relatie tussen psychosociale stress en hart- en vaatziekten is intensief onderzocht. In de afgelopen decennia was het ‘reactivity model’ het belangrijkste theoretische uitgangspunt in de meeste... Show moreDe relatie tussen psychosociale stress en hart- en vaatziekten is intensief onderzocht. In de afgelopen decennia was het ‘reactivity model’ het belangrijkste theoretische uitgangspunt in de meeste onderzoeken. In dit model wordt verondersteld dat een heftige reactie van hart- en vaatstelsel tijdens stress een risicofactor zou zijn voor het ontwikkelen van hart- en vaatziekten. Hoewel het ‘reactivity model’ in het dieronderzoek veelbelovende resultaten boekte, ondervond vanwege resultaten uit humaan onderzoek een aantal fundamentele kritieken. Het brandpunt ligt bij momenten waarop een stressvolle gebeurtenis plaatsvindt, terwijl de periode daarvoor of daarna over het hoofd wordt gezien. Stressoren zijn gewoonlijk kort van duur en doen zich bij de meeste mensen niet frequent genoeg voor om consequenties voor de gezondheid te hebben. Deze factoren kunnen dus niet verklaren waarom chronische veranderingen in de balans van het hart- en vaatsysteem optreden. Een alternatief model dat wij voorstellen in dit proefschrift - het ‘prolonged activation model’- gaat er vanuit dat stressoren alleen kunnen leiden tot hart- en vaatziekten als ze in staat zijn verlengde fysiologische effecten te veroorzaken. Daarnaast is het onduidelijk waarom sommige stressoren wel verlengde effecten tot gevolg hebben en anderen niet. Tot nu toe verklaart het ‘perseverative cognition model’, als enige waarom sommige stressoren leiden tot verlengde effecten en anderen niet. Perseveratieve cognitie is het voortdurend denken aan negatieve gebeurtenissen en omvat fenomenen zoals piekeren of zich zorgen maken. Perseveratieve cognitie zorgt voor het ‘vers’ houden van het mentale beeld van een stressor alsmede de negatieve fysiologische effecten ervan, of deze stressor nu wel of niet plaatsvindt. Daarom bestaat een groot deel van dit proefschrift uit onderzoek naar de effecten van perseveratieve cognitie op hartactiviteit. We concluderen dat de resultaten van dit proefschrift aangeven dat perseveratieve cognitie, een belangrijke factor is in het ‘prolonged activation model’. Het verklaart niet alle verlengde effecten van stressoren, maar heeft daarentegen zelf een verlengd fysiologisch effect, dat zelfs langer lijkt dan dat van stressoren. Deze vondst en de effecten van 'piekergeneigdheid' op hartactiviteit gedurende de slaap suggereren dat onderzoek in de toekomst gericht zou moeten worden op de rol van onbewuste perseveratieve cognitie. Show less
Currently, the raising awareness of the role of glucocorticoids in the onset of numerous (neuro)-pathologies constitutes the increasing necessity of understanding the mechanisms of action of... Show moreCurrently, the raising awareness of the role of glucocorticoids in the onset of numerous (neuro)-pathologies constitutes the increasing necessity of understanding the mechanisms of action of glucocorticoids in bodily processes and brain functioning. Glucocorticoids mediate their effects by binding to intracellular receptors which act as transcription factors. A remarkable and yet unexplained phenomenon described more than two decades ago, is the cell-specific effects glucocorticoids bring about on gene expression in brain. For example, while glucocorticoids suppress corticotrophin-releasing hormone (CRH) synthesis in the hypothalamus, production of CRH in the central nucleus of the amygdala (CeA) is stimulated by increased hormone levels. Inasmuch as the neuroanatomical distribution of the corticosteroid receptors does not satisfactorily explain these effects, it is of interest to decipher the role of recently discovered coregu lator proteins that modulate the direction and the magnitude of steroid receptor-driven transcription. Therefore, in the current thesis the expression and function of central coregulators was studied: the coactivators SRC1a and SRC1e along with the corepressors N-CoR and SMRT were found to be expressed in brain and involved in regulation of CRH gene expression. Finally, a method that allows detection of coregulator recruitment by steroid receptors in brain tissue was developed. Show less
Migraine is a severe headache syndrome, affecting approximately 33% of females and 13% of males. Patients suffer from recurring headache episodes in combination with nausea, vomiting, phono and... Show moreMigraine is a severe headache syndrome, affecting approximately 33% of females and 13% of males. Patients suffer from recurring headache episodes in combination with nausea, vomiting, phono and photophobia. It is a paroxysmal disorder for which several several trigger factors have been identified by patients. This thesis has focused on the relation between i) psychosocial stress, ii) nitroglycerin and iii) hypoxia and the occurrence of a migraine attack, as well as the mechanism of action. The first conclusion is that, in contrast to previous studies, we could not detect a correlation between psychosocial stress and migraine. The second conclusion is that normobaric hypoxia might be a trigger factor for migraine possibly through the development of mild cerebral edema. The third conclusion is that migraine can be triggered by nitroglycerin through a vascular effect without any indication of vasodilation in cerebral or meningeal arteries during the delayed provoked attack. Show less
Not everyone who experiments with cocaine acquires compulsive drug use. The mechanism underlying this individual difference in susceptibility to addiction is poorly understood. Recent studies have... Show moreNot everyone who experiments with cocaine acquires compulsive drug use. The mechanism underlying this individual difference in susceptibility to addiction is poorly understood. Recent studies have identified genes and adverse life events (stress) as risk factors. The objective of this thesis is to investigate the contribution of the adrenal stress hormones glucocorticoids and epinephrine to the psychostimulant effects of cocaine in the inbred DBA/2 and C57BL/6 mouse strains. Behavioural sensitisation, measured as an enhanced locomotor response to repeated cocaine exposure, was used as a model for the long-term neural adaptations underlying aspects of drug addiction. The results demonstrate that adrenal hormones play a critical role in cocaine sensitivity, which depends on genetic background because surgical removal of the adrenals (__adrenalectomy__) prevented cocaine sensitisation in DBA/2, but not C57BL/6 mice. The impact of genetic background was further emphasised by strain-specific changes in the midbrain dopamine system that mediates the rewarding effects of drugs. The effects of adrenalectomy could only be fully reversed by co-administration of glucocorticoids and epinephrine. These findings show that, depending on genetic background, adrenal stress hormones are important risk factors for vulnerability to cocaine, suggesting that pharmacological intervention in stress hormone action has therapeutic potential in drug addiction. Show less
An adverse early life event is considered a risk factor for stress-related psychiatric disorders in genetically predisposed individuals, probably because of its lasting effect on susceptibility to... Show moreAn adverse early life event is considered a risk factor for stress-related psychiatric disorders in genetically predisposed individuals, probably because of its lasting effect on susceptibility to stress. The objective of this thesis research was to examine in the mouse CD1 strain the immediate and permanent effects of an adverse early experience on the neuroendocrine stress system. For this purpose the hypothalamic-pituitary-adrenal (HPA) axis was examined of mouse pups that were refrained from maternal care, a laboratory model for neglect mimicking aspects of abuse. The data show that the infants__ stress response system readily adapts to daily repeated 8 hours of maternal separation, but that it continues to respond to a novelty stressor. The rapid adaptation to repeated maternal absence seems rather due to the ability to predict return of the mother than to adjust metabolism to episodic food deprivation. If maternal separation was extended to a single episode of 24 hours the immediate outcome was more profound but transient, although subtle effects on stress reactions and cognitive performance did persist. The findings demonstrate the amazing plasticity of the newborn brain and provide a basis to study the mechanistic underpinning of vulnerability or resilience to psychopathology. Show less
A general overview of regulation of secondary metabolism in Pseudomonas species is given in Chapter 1. Several approaches were combined to identify novel genes involved in the regulation of PCN... Show moreA general overview of regulation of secondary metabolism in Pseudomonas species is given in Chapter 1. Several approaches were combined to identify novel genes involved in the regulation of PCN synthesis and to study their interactions with other regulators. Site-directed mutagenesis was used to test the hypothesis that rpoS is a regulatory gene of PCN synthesis (Chapter 2). To discover additional genes in the regulatory cascade, which already contains psrA and rpoS, a random DNA-fragment microarray of the PCL1391 genome was constructed and used for transcriptomics of the psrA and rpoS mutants (Chapter 3). A random mutagenesis approach resulted in the identification of pip, a novel gene that stimulates PCN production in PCL1391 (Chapter 4). Analyses on the role of Pip as a switch of PCN production depending on environmental conditions are described in Chapter 5. The results described in this thesis are summarized in Chapter 6, where in addition the regulatory network of PCN synthesis in P. chlororaphis PCL1391 is compared to regulatory networks of secondary metabolism in other Pseudomonas species. Show less
This study describes the effects of prominence and boundary on the temporal and melodic structures of two Indonesian languages, viz. Toba Batak, a stress language and Betawi Malay, a non-stress... Show moreThis study describes the effects of prominence and boundary on the temporal and melodic structures of two Indonesian languages, viz. Toba Batak, a stress language and Betawi Malay, a non-stress language. Experimental evidence shows that lengthening effects were more than twice as strong in the non-stress Betawi Malay as in Toba Batak. Durational prominence effects in Toba Batak are comparable to those found in western stress language at the word level, but not at the lower levels: stress syllables, especially consonants are hardly affected. Arguably, the use of duration as a stress correlate is restricted by the phonemic consonant contrast in the language. To compensate for this, prominence-related pitch movements, which are connected to the stressed syllable, occur in non-prominent as well as prominent words in Toba Batak. In Betawi Malay, prominence-related pitch movements are larger but vary considerably in shape and position; they serve to cue accents and boundaries, but not stress position. To determine the audible consequences of the native language for the production of Dutch stress, three perception experiments were run. The native language clearly affects the prosody of second-language speech. Toba Batak speakers of Dutch sound more acceptable to Dutch listeners than Betawi Malay speakers do. Show less
Fungi are a very successful species and are distributed worldwide. However, the presence of fungi is not always desired. Filamentous fungi can grow on living or dead organic material and even... Show moreFungi are a very successful species and are distributed worldwide. However, the presence of fungi is not always desired. Filamentous fungi can grow on living or dead organic material and even inside the host. Current methods to prevent fungal growth are insufficient, causing fatality after fungal infections or loss of crops. The cell wall of a fungus is an intriguing component. It protects the cell from the harsh environment and determines the shape of the cell. Hence the cell wall is an essential component to the cell and provides an attractive target for antifungals. Additionally, the cell wall contains components only found in fungi, and the target is a desirable target as it is exposed on the outside of the cell. Currently, little is known about the cell wall of filamentous fungi. In order to design new or improved antifungal compounds, a better understanding of the fungal cell wall and of its adaptation to various conditions is required. In this thesis, we have used Aspergillus niger as a model filamentous fungus to study the biosynthesis of the fungal cell wall. The cell wall is a highly dynamic structure and able to adapt to various changes, either developmental (e.g. mating, growth, budding, branching and sporulation), environmental (e.g. heat, pH, osmolarity, chemical compounds), or genetic (e.g. mutations in cell-wall related genes). In chapter 1 an overview is presented of the current state of knowledge about the fungal cell wall. The architecture, biosynthesis and the remodeling are discussed in this chapter. The response of our model fungus A. niger to chemical induced cell wall stress is described in chapter 2. The fluorescent brightener Calcofluor White (CFW) was used to induce cell wall stress. We show that A. niger, like Saccharomyces cerevisiae, responds to cell wall stress by an increase of chitin deposition in the cell walls. This increase in chitin, a structural cell wall polymer, was accompanied by an increased transcription level of gfaA. It was also shown that this mechanism is not only limited to A. niger but is also observed in other filamentous fungi like the plant pathogenic fungus Fusarium oxysporum and the food spoilage fungus Penicillium chrysogenum. It is further shown that gfaA is an essential gene and the deletion strain can be rescued by addition of glucosamine. In chapter 3, a family of five 1,3-__-D-glucan synthase encoding genes is described. The expression of these genes during various types of cell wall stress was monitored and it was found that the expression of agsA and agsE was induced. The induction of an 1,3-__-D-glucan synthase encoding gene after cell wall stress was also observed in P. chrysogenum. The deletion of agsA led to an increased sensitivity towards CFW. While in chapter 2 and 3 changes in expression levels of genes encoding proteins involved in cell wall biosynthesis are described, the mechanism behind the induction of cell wall stress responsive genes is described in chapter 4. A promoter deletion study combined with an in silico analysis indicated that the induction of agsA in response to cell wall stress is dependent on a putative Rlm1p binding site in its promoter. Therefore a gene, named rlmA encoding for a MADS-box transcription factor was isolated from A. niger after database searches. The role of this gene in the induction of agsA and gfaA after CFW stress was investigated. A deletion of the rlmA gene was constructed and this resulted in an increased sensitivity towards cell wall disturbing compounds. In S. cerevisiae an important part of the response towards cell wall threatening conditions is the up-regulation of GPI-anchored cell wall proteins. In chapter 5 the isolation and characterisation of an HF-extractable cell wall protein from A. niger, named CwpA, is described. It was shown by simple fractionation experiments that the protein was mainly present in the cell wall fraction. Deletion of cwpA resulted in an increased sensitivity towards CFW suggesting a structural role for CwpA. Chapter 6 describes a novel method for the identification of cell wall mutants. The mutants are first selected based on their compensatory reaction (induction of agsA) and subsequenly subjected to various secondary screens, to confirm an altered cell wall integrity. Four out of 240 mutants with induced agsA expression levels, named miaA-D, were selected for complementation. All four mutants were complemented by cosmids. Further subcloning experiments are underway to identify the mutated genes. In chapter 7 a GFP-based reporter system is described. The system allows the rapid screening of compounds to see if they trigger the cell wall integrity pathway and thereby induce the PagsA(-H2B)-GFP reporter. The method has been evaluated towards various putative antifungal compounds and is a promising tool for the identification of new cell wall related antifungal compounds. In conclusion, this thesis provides evidence for the existence of a cell wall remodeling mechanism in filamentous fungi and in particular A. niger. Also, signal transduction pathway components were identified by which cell wall weakening is sensed and transduced into a transcriptional response. Additionally, a cell wall stress reporter system was developed to identify new cell wall related antifungal targets and to identify cell wall related antifungal compounds. Show less
