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
There is a fundamental difference between mammals and fish in how hypoxia affects the lipid metabolism by means of the stress hormone noradrenaline. In mammals, hypoxia induces an increase in lipid... Show moreThere is a fundamental difference between mammals and fish in how hypoxia affects the lipid metabolism by means of the stress hormone noradrenaline. In mammals, hypoxia induces an increase in lipid metabolism, which can eventually lead to tissue damage due to elevated plasma fatty acid levels, e.g. in case of a heart attack. However, hypoxia is not a normally occurring situation in healthy mammals as opposed to many fish species, because water is a relative poor source of oxygen. In hypoxic fish therefore, noradrenaline mediates a decrease in lipid metabolism, and we believe that this is a general protection mechanism in fish against lipid poisoning. There is a clear difference in the mode of breathing between mammals and fish, namely air- vs. water-breathing. Hence, we hypothesise that this difference is the cause for the opposing effects of noradrenaline. Therefore, we studied the effects of hypoxia on an air-breathing fish, the African catfish. However, physiologically this species reacted the same as other water-breathing fish, namely by means of a reduced lipid metabolism. Additionally, we demonstrated that, despite the opposing effects on the lipid metabolism, the transduction pathways in fish and mammals are very alike, and thus only a minor change has occurred in the course of evolution. This research has aided in a better understanding of the evolutionary changes in lipid metabolism.|Er is een fundamenteel verschil tussen zoogdieren en vissen in hoe het vetmetabolisme onder hypoxie (=zuurstoftekort) wordt veranderd door het stresshormoon noradrenaline. In zoogdieren leidt hypoxie tot een verhoogd vetmetabolisme, wat uiteindelijk zelfs weefselschade kan veroorzaken door te hoge vetzuurgehaltes, b.v. bij een hartaanval. Gezonde zoogdieren komen normaliter niet in zuurstofnood, in tegenstelling tot veel vissoorten, omdat water een relatief arme zuurstofbron is. In hypoxische vissen daalt dan ook het vetmetabolisme door noradrenaline, en wij denken dat dit een beschermingsmechanisme is tegen een vetzuurvergiftiging. Er is een duidelijk verschil in de manier van ademhalen van zoogdieren en vissen, respectievelijk lucht- en waterademhaling, en dit verschil ligt mogelijkerwijs ten grondslag aan deze verschillende effecten van noradrenaline; dit is de centrale hypothese van het onderzoek. Daarom is gekeken naar het effect van zuurstoftekort bij een luchtademhalende vissoort, de Afrikaanse meerval. Deze vis bleek echter fysiologisch hetzelfde te reageren als waterademhalende vissen, namelijk een verlaagd vetmetabolisme. Daarnaast is aangetoond dat, ondanks tegenovergestelde effecten op het vetmetabolisme, de aansturingmechanismen in vissen en zoogdieren vergelijkbaar zijn, en dat er dus maar een minimale verandering in de evolutie heeft plaatsgevonden. Dit onderzoek heeft bijgedragen tot een beter begrip van de evolutionaire veranderingen in het vetmetabolisme. 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