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No description available.

Proefschrift Rijksuniversiteit Groningen. / Auteursnaam op omslag: Daan Thorn Leeson. Datum laatste controle: 22-12-1997. Lit.opg. - Met samenvatting in het Nederlands.

Iron plays a potential role in oxidative stress-mediated injuries and pathologies e.g. rheumatoid arthritis (RA). Four decades ago it was suggested that iron may have a crucial role in the progression of inflammation in RA. Indeed, free radicals generated by iron can cause damage to lipids, proteins, carbohydrates, and DNA. It is this destructive process that is believed to occur in rheumatoid joints. However, none had differentiated between the role of iron in both acute and chronic phases of the disease and the origin of this 'labile' iron. Since RA cells are chronically exposed to oxidative stress, we have therefore chosen Jurkat cells to be our cell model. We used the parental (J16) cell line was used to mimic the acute phase of oxidative stress and the H2O2-resistant (HJ16) cells to mimic the chronic phase. By using hydrogen peroxide (H2O2) as the oxidising agent, we aim to study the role of iron in acute and chronic phase of oxidative stress and to know its origin. In the present study, we found that both antioxidants and H2O2-induced labile iron are modulated when cells are chronically exposed to H2O2. HJ16 cells contain higher total intracellular glutathione levels and glutathione peroxidase activity than J16 cells while the superoxide dismutase and catalase activity are similar. Haem oxygenase-1 (HO-1) was not detectable nor was it induced in these cell lines; HO-2 on the other hand was expressed but not induced. Although they had the same ‘basal’ LIP and L-Ft levels, J16 cells contain more than 7-fold higher H-Ft levels than in HJ16 cells. It was also found that H2O2-induced labile iron is directly correlated with necrotic cell death. These results are consistent with the conclusion that both antioxidant defence mechanism and labile iron status are modulated in cells chronically exposed to H2O2. We have also shown that the ‘basal’ and ‘H2O2-induced’ NFκB activation was higher in the HJ16 cells. We have also provided a link between labile iron release, lysosomal membrane damage and the ensuing necrotic cell death following H2O2 treatment.

Ticks acquire nutrients only by a parasitic nature of feeding on animals, including humans. During this process, a wide array of pathogens is transmitted. Ticks of the Ixodidae family receive exactly one blood meal in each active developmental. Knowing the trophic dependence of tick metabolism on the host blood meal components may enable discovering processes essential for the tick physiology and development. Exploiting a membrane system of tick feeding and whole blood fractionation, we have revealed that adult ticks need to acquire host haemoglobin-derived haem so that they can produce viable larvae, and reproduce. Haem is not further catabolised in ticks, and iron is thus acquired via independent route with the host serum transferrin as a source molecule. Using RNA-seq, we compared transcriptome compositions between guts of blood- and serum-fed ticks. We identified fifteen gut transcripts that change their levels with respect to the presence/absence of dietary red blood cells. Glutathione S-transferase, one of the identified encoded molecules, shows a clear haeminresponsive expression at both transcript and protein levels. Its apparent haem-binding properties suggest that this protein is directly involved in haem homeostasis maintenance within the tick gut. The ultimate goal of such research is to identify and verify targets that, when blocked, would render the acquisition and/or distribution system of haem in ticks nonfunctional. This would represent a novel way of anti-tick interventions in veterinary and human medicine.

La porphyrie aiguë intermittente (PAI) est la plus fréquente des porphyries hépatiques aiguës. Elle est décrite comme une maladie autosomique dominante dont le trait génétique est estimé à 1/1675 en France avec une pénétrance faible et variable allant de 10% à 50% dans les familles connues de PAI. La PAI est due à des mutations réduisant le niveau d’activité de l’hydroxyméthylbilane-synthase (HMBS). Son déficit entraîne l’accumulation de précurseurs neurotoxiques responsables de la symptomatologie clinique. Dans le foie, la synthèse d’hème est contrôlée par l’enzyme ALA-Synthétase 1 (ALAS1) dont l’activité est régulée par un rétrocontrôle négatif par le produit final : l’hème. Le traitement consiste à freiner l’induction d’ALAS1 induit par la carence en hème, par l’administration d’hème exogène. Ce traitement de la crise aiguë est très efficace mais génère rapidement une dépendance physique avec apparition de crises récurrentes nécessitant l’administration chronique d’hème exogène. L’objectif principal de ce projet a été d’étudier les mécanismes physiopathologiques et génétiques liés à cette pathologie afin de traiter et conseiller au mieux les patients. Une partie du projet a consisté à explorer les facteurs génétiques modulateurs de la pénétrance de la maladie. Tout d’abord, une prévalence minimale du trait génétique dans la population générale a été estimée à 1/1299 permettant d’en déduire une pénétrance de l’ordre de 1% alors que celle dans les familles PAI suivies par le CFP est estimée à 22,9 %. Ensuite, concernant les facteurs pouvant expliquer cette différence, la présence d’une mutation type non-sens est plus fréquemment associée aux formes sévères et à une pénétrance plus élevée. De plus, les études de corrélation et d’héritabilité suggèrent plutôt une transmission de type oligogénique associée à des facteurs épigénétiques modulateurs de la pénétrance dont le facteur environnemental. Une autre partie a consisté à explorer les effets de l’administration d’hème exogène sur les patients et un modèle murin de PAI créé génétiquement. Chez l’homme, le traitement est associé à une augmentation des formes chroniques (1,7 % avant vs 7,5 % après l’introduction du celui-ci). Dans le modèle murin de PAI, les injections intrapéritonéales répétées induisent une augmentation paradoxale d’ALAS1 (3 fois), une augmentation de l’hème oxygénase 1 qui catabolise l’hème (HMOX1, 9 fois) ainsi que des voies de l’inflammation (analyse transcriptomique et protéomique hépatique) et une surcharge en fer. De plus, cette administration induit une altération des complexes de la chaine respiratoire mitochondriale responsable d’anomalies du métabolisme énergétique au niveau hépatique, cérébral et musculaire pouvant expliquer la symptomatologie neuroviscérale. En conclusion, ce travail a permis d’explorer les caractéristiques génétiques de la maladie (prévalence, pénétrance) en remettant en cause le mode de transmission autosomique dominant jusqu’ici admis, et d’explorer les mécanismes physiopathologiques associées à l’administration d’hème exogène faisant de cette thérapeutique un pharmakon / The biosynthesis of porphyrins is one of the most conserved pathways known. By associating different metals, porphyrins give rise to the "pigments of life". The formation of haem is accomplished by a sequence of eight dedicated enzymes encoded by different genes, some being active in ubiquitous as well as in erythroid isoforms. In humans, the genes for each of the haem synthetic enzymes may become the target of mutations that give rise to an impaired cellular enzyme activity called porphyrias. The acute porphyrias are characterized by attacks of neuropsychiatric symptoms, which may be due to a toxic surplus of the porphyrin precursor 5-aminolevulinic acid, or a consequence of a deficit of vital hemoproteins. Mutations of the gene encoding the third enzyme: hydroxymethylbilane synthase, are associated with the most frequent type of acute hepatic porphyria, acute intermittent porphyria. AIP is thought to display autosomal dominant inheritance with incomplete penetrance. In the classical form of AIP, HMBS activity is about 50% lower than normal in all tissues. These levels of activity in basal conditions are not sufficiently low to cause symptoms. However, factors increasing hepatic heme demand, resulting in an upregulation of hepatic aminolevulinate synthase (ALAS1, the first enzyme of the heme biosynthesis pathway), precipitate acute attacks. The treatment of the attack of AIP consists to repress ALAS1 and restores metabolic equilibrium. But this treatment leads side effects and dependency. The pathophysiological mechanism of the disease is partially known and difficult to explore because there is not an AIP model or prediction model of porphyrogenicity. We aimed to obtain further insight into the pathophysiological mechanism of AIP and into the genetic (prevalence and penetrance) of AIP, and the contribution of genetic factors to the variable clinical expression of HMBS mutations.We first calculated the penetrance of HMBS mutations in AIP patients seen at the French reference center for porphyria: 22.9%. We then used the Exome Variant Server (EVS) to estimate the prevalence of deleterious HMBS mutations in the general population: 1/1299; and the penetrance of the AIP genetic trait in France: 1%. Finally, we investigated further the genetic factors underlying the penetrance of AIP by analyzing genotype/phenotype correlations, and the pattern of familial correlations for the symptoms of the acute crises of AIP. Intrafamily correlation studies showed correlations to be strong overall and modulated by kinship and the era in which the person was living, demonstrating strong influences of genetic and environmental modifiers on inheritance suggesting that AIP inheritance does not follow the classical autosomal dominant model. Null alleles were associated with a more severe phenotype and a higher penetrance than for other mutant alleles.On the other hand, we explored the effect of heme administration. In human, the introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. We show that repeated hemin infusions in mice trigger a high level heme oxygenase 1 response, induce a pro-oxidative iron accumulation, a complex pattern of liver inflammation with macrophage infiltration and an alteration of oxidative phosphorylation

<p>The genetic basis for behavioural traits is largely unknown. The overall aim of this thesis was to find genes with importance for behavioural traits related to fear and anxiety. Microarray analysis was used to screen expression profiles of brain regions important for emotional behaviour in dogs, wolves, foxes and mice. In a first experiment, dogs and their wild ancestors the wolves were compared. Our results suggested that directed selection for behaviour might have resulted in expression changes in few genes acting on several brain functions, possibly affecting behaviour. However, the observed expressional differences were confounded with environmental effects. This was addressed in a second study on domesticated silver foxes. By correlating behaviour and brain gene expression in foxes selected for tameness to non-selected foxes raised in the same environment, we found large behavioural differences but only few genes with differential expression in the brain. Fifteen of the 40 genes showing evidence of expression difference were related to haem or haemoglobins. Further studies showed an additive genetic effect on brain gene expression, similar to the additive genetic inheritance of behaviour, indicating an involvement in domestication. Transcriptional profiling was also used for finding genes involved with the sleep disorder narcolepsy. Narcoleptic Doberman pinschers homozygous for the canarc-1 mutation were compared to their unaffected heterozygots revealing reduced expression of three genes, TAC1, PENK and SOCS2, with relevance to the narcoleptic phenotype. Finally gene expression was investigated in relation to anxiety-related traits in a mouse model. Surprisingly, as in the fox study, genes coding for haemoglobins indicated differential expression in the brain between animals with different anxiety levels. Our combined results suggest that genes like haemoglobins, best known for their function in oxygen transport in blood, may also participate in brain functions related to decreased anxiety in domestic animals. </p>

The genetic basis for behavioural traits is largely unknown. The overall aim of this thesis was to find genes with importance for behavioural traits related to fear and anxiety. Microarray analysis was used to screen expression profiles of brain regions important for emotional behaviour in dogs, wolves, foxes and mice. In a first experiment, dogs and their wild ancestors the wolves were compared. Our results suggested that directed selection for behaviour might have resulted in expression changes in few genes acting on several brain functions, possibly affecting behaviour. However, the observed expressional differences were confounded with environmental effects. This was addressed in a second study on domesticated silver foxes. By correlating behaviour and brain gene expression in foxes selected for tameness to non-selected foxes raised in the same environment, we found large behavioural differences but only few genes with differential expression in the brain. Fifteen of the 40 genes showing evidence of expression difference were related to haem or haemoglobins. Further studies showed an additive genetic effect on brain gene expression, similar to the additive genetic inheritance of behaviour, indicating an involvement in domestication. Transcriptional profiling was also used for finding genes involved with the sleep disorder narcolepsy. Narcoleptic Doberman pinschers homozygous for the canarc-1 mutation were compared to their unaffected heterozygots revealing reduced expression of three genes, TAC1, PENK and SOCS2, with relevance to the narcoleptic phenotype. Finally gene expression was investigated in relation to anxiety-related traits in a mouse model. Surprisingly, as in the fox study, genes coding for haemoglobins indicated differential expression in the brain between animals with different anxiety levels. Our combined results suggest that genes like haemoglobins, best known for their function in oxygen transport in blood, may also participate in brain functions related to decreased anxiety in domestic animals.

Cadmium (Cd), is a non-essential metal with no known physiological function. It is known to alter redox state by disrupting the mitochondrial electron transport chain, as well as inactivating protein and non-protein thiols. It is thus believed that oxidative stress may comprise an important part of the mechanism of Cd toxicity. Accordingly, the initial cellular response to acute Cd exposure is defensive, where various anti-oxidant defence systems are triggered. One of the induced systems is the haem oxygenase-1 (HO-1). Its activation is mediated by the transcription factor Nrf2, which is the general regulator of cellular defence against oxidative stress. The protective effects of HO-1 are mediated, in part, through the generation of potent anti-oxidant bilirubin (BR) and its metabolites, which exploit the intrinsic antioxidant properties of these species at a cellular level. The oxidative metabolism of BR is an important route of detoxification in addition to glucuronidation. However, the major enzyme(s) involved in this oxidative degradation are not known. This thesis presents evidence for a major role of the hepatic cytochrome P450 2a5 (Cyp2a5) in BR degradation during Cd intoxication, where the BR levels are elevated following induction of HO-1. Treatment of DBA/2J male mice with CdCl2 induced both the Cyp2a5 and HO-1, and increased the microsomal BR degradation activity. By way of contrast, the total cytochrome P450 (CYP) content and the expression of Cyp1a2 were down-regulated by the treatment. The induction of the HO-1 and Cyp2a5 was significant at the mRNA, protein and enzyme activity levels. In each case, the up-regulation of the HO-1 preceded that of the Cyp2a5 with a 5-10 hr interval. In addition, BR totally inhibited the microsomal coumarin hydroxylase activity (a Cyp2a5-catalysed reaction) with an IC50 approximately equal to the substrate concentration. The MROD activity, catalysed mainly by the Cyp1a2, was inhibited up to 36% by BR. The microsomal BR degradation was inhibited by coumarin and by a monoclonal antibody against the Cyp2a5 by about 90%. In addition, 7-methoxyresorufin, a substrate for Cyp1a2, inhibited BR degradation activity by approximately 20%. A study using Nrf2 null mutant mice suggests that Cd-mediated induction of Cyp2a5 is dependent on the transcription factor Nrf2. Additionally, acute exposure to Cd activated localisation of Nrf2 from the cytoplasm to the nucleus. Furthermore, electrophoretic mobility shift assay (EMSA) analysis suggests that Cd induced sequence-specific binding of various species of the StRE-binding proteins on the 5’-flanking region of the Cyp2a5 gene. Collectively, these observations strongly suggest that BR may act as a substrate for the hepatic Cyp2a5, a major catalyst for BR degradation under conditions of substantial elevation of BR levels following induction of HO-1 by Cd. Secondly, the concurrent up-regulation of the HO-1 and Cyp2a5 during Cd-mediated injury implicates a coordinated regulation of two enzyme systems in the maintenance of balancing BR production and elimination. Finally, StRE-binding proteins, in particular Nrf2, may be involved in the regulation of the Cyp2a5 gene, which leads to the oxidation of BR. However, the respective roles of these factors in the regulation of the Cyp2a5 gene, as well as the coordinated regulation of ho-1 and Cyp2a5 genes remain an open question, requiring further investigations.