L’un des domaines clés de la recherche en neurosciences modernes consiste à comprendre les interactions complexes entre le stress et la génétique qui conduisent à la manifestation de troubles tels que la dépression, l’anxiété et le dysfonctionnement cognitif. Des preuves de plus en plus nombreuses suggèrent que le système glutamatergique peut être une cible thérapeutique pertinente pour de tels troubles. Le glutamate est le neurotransmetteur utilisé par la grande majorité des synapses excitatrices dans le cerveau. Et les sous-types des récepteurs métabotropique au glutamate (mGluR1 – mGluR8) agissent avant tout comme d’importants régulateurs postsynaptiques de la neurotransmission dans le système nerveux central (SNC), en fournissant un mécanisme par lequel les réponses synaptiques rapides à travers des canaux cationiques dépendants du glutamate peuvent être affinées. Ainsi, les récepteurs mGluR participent à une grande variété de fonctions du système nerveux central. Au sein dela famille des récepteurs métabotropiques au glutamate, le récepteur présynaptique mGluR7 montre la conservation évolutive la plus élevée et on pense qu'il agit comme un régulateur de la libération de neurotransmetteurs. Le récepteur mGluR7 est également le plus largement distribué des récepteurs présynaptiques mGluR, présent sur une large gamme de synapses démontrées comme critiques à la fois dans le fonctionnement normal du système nerveux central, mais également dans une large gamme de troubles psychiatriques et neurologiques. De plus, un nombre croissant de preuves expérimentales suggèrent que le récepteur mGluR7 est non seulement un acteur clé dans l’élaboration de réponses synaptiques au niveau des synapses glutamatergiques, mais qu’il est également un régulateur clé de la transmission GABAergique inhibitrice. Le développement d’outils pharmacologiques et génétiques sélectifs a permis le démantèlement de la fonction du récepteur mGluR7 dans une multitude de processus physiologiques et comportementaux. Ainsi les souris knock-out ont mis en évidence un rôle du récepteur mGluR7 dans l’anxiété, le conditionnement de la peur, l’aversion, l’apprentissage et la mémoire spatiale. De plus, ces souris dépourvues du récepteur métabrotrope mGluR7 démontrent une sensibilité accrue aux crises épileptiques suggérant un rôle unique de ce récepteur dans la régulation de l’excitabilité neuronale. De même, une altération de la plasticité synaptique à court terme dans les souris transgéniques dépourvues du récepteur métabotrope au glutamate mGluR7 démontre que l’absence de récepteurs mGluR7 engendre des altérations de la plasticité synaptique à court terme dans l’hippocampe. En outre, la découverte et la caractérisation récente du premier antagoniste allostérique agissant sur le domaine VFTD de l’extrémité N-terminale du récepteur mGluR7 potentialise définitivement les observations effectuées sur les souris mGluR7 knock-out quant à la fonction de ce récepteur dans l’anxiété et la dépression. Ensemble, ces données suggèrent que le récepteur mGluR7 est un important régulateur de la fonction glutamatergique, de la peur, de l’aversion et de la cognition et donc ce récepteur représente une cible thérapeutique innovante pour les troubles liés au stress à l’interface de la cognition et de l’anxiété. / Metabotropic glutamate receptors (mGluRs) consist of eight different subtypes and exert their effects on second messengers and ion channels via G-proteins. The function of individual mGluR subtypes in the CNS, however, largely remains to be clarified. To study the role of mGluR7 receptors, we used homologous recombination to generate mice lacking this metabotropic receptor subtype (mGluR7). Immunohistochemical and immunoelectron-microscopic analyses showed that mGluR7 is highly expressed in amygdala and preferentially localized at the presynaptic axon terminals of glutamatergic neurons, suggesting strongly that mGluR7 is involved in neural processes subserving amygdala-dependent averse responses. To examine amygdala-dependent behavior, we examined first the fear response of freezing after electric shock in wild-type and mGluR7 (mGluR7-/-) knockout littermates. Wild-type mice displayed freezing immediately after footshock. In comparison, mGluR7 knockout mice showed significantly reduced levels in both immediate postshock and delayed freezing responses. However, the knockout mice exhibited no abnormalities in pain sensitivity and locomotor activity. Secondly, we performed conditioned taste aversion (CTA) experiments. In wild-type mice, the administration of saccharin followed by intraperitoneal injection of the malaise-inducing agent LiCl resulted in an association between saccharin and LiCl. This association caused strong CTA toward saccharin. In contrast, mGluR7 knockout mice failed to associate between the taste and the negative reinforcer in CTA experiments. Again, the knockout mice showed no abnormalities in taste preference and in the sensitivity to LiCl toxicity. These results indicated that mGluR7 deficiency causes an impairment of two distinct amygdala-dependent behavioral paradigms. Because the amygdale function is essential for these two distinct behavioral paradigms, our results suggest that mGluR7 is critical in amygdale function. The amygdale is a brain region that is known to be critical for the manifestation of anxiolysis and antidepressant action and glutamatergic neurotransmission has been strongly implicated in the pathophysiology of affective disorders. To this end we analyzed the behavioral profiles of mGluR7-/- mice in animal models of depression and anxiety. mGluR7-/- mice were compared to wildtype littermates and showed substantially less behavioural immobility in both the forced swim test and the tail suspension test. Both behavioural paradigms are widely used to predict antidepressant-like activity. Further, mGluR7-/- mice displayed anxiolytic activity in four different behavioural tests namely the light-dark box, the elevated plus maze, the staircase test, and the stress-induced hyperthermia test, while their cognitive performance was normal in the passive avoidance paradigm. [...]

Abstract Collagen molecules consist of three polypeptide chains that are coiled around each other to form a triple-helical structure. The formation of stable collagen triple helices requires the hydroxylation of proline residues catalyzed by collagen prolyl 4-hydroxylases (C-P4H). Vertebrate C-P4H is an ER-resident enzyme that consists of two catalytically active α subunits and two β subunits. Production of recombinant human collagen and gelatin could have numerous medical and industrial applications, but most recombinant systems lack the C-P4H activity. The yeast Pichia pastoris has been successfully engineered to produce stable human collagens and gelatins by co-expression of the collagen polypeptide chains with the two C-P4H subunits. This study examined the effect of deletion of the C-propeptide, or its replacement by a trimerizing foldon domain, on the assembly of type I and III collagen triple helices in P. pastoris. It was observed that the absence of the C-propeptide leads to inefficient collagen chain assembly whereas the replacement of C-propeptide with a foldon domain increased the assembly up to 3-fold. Moreover, the co-expression of α1(I) and α2(I) chains fused with foldon yielded heterotrimeric type I collagen molecules with a typical chain ratio of 2:1. As the foldon domain contains no information for collagen chain recognition, the present data indicate that the chain assembly is defined not only by the C-propeptides but also by other determinants present in the α chains. Another aspect studied here was the expression and secretion of gelatin fragments of varying size and conformation in P. pastoris. It was discovered that gelatin fragment size affects its secretion as the 90 kDa fragment was less efficiently secreted than the 45 kDa fragment. Secretion was also dependent on the fragment conformation as induction of the triple helix formation by either C-propeptide or foldon led to the accumulation of the fragments inside the yeast cells despite the presence of an efficient secretory signal. C-P4H was long assumed to exist as one type only but the cloning of several C-P4H α subunits raised questions concerning the specific roles of the C-P4H isoenzymes. The generation of mice lacking the type I C-P4H, which is regarded as the major C-P4H isoenzyme, indicated that this isoenzyme is essential for the embryonic development of the mouse. The embryos lacking type I C-P4H died at an early stage of their development due to the disruption of basement membranes. It was found that the basement membranes of the homozygous null embryos lacked type IV collagen whereas the fibrillar collagens were synthesized, although with altered morphology. The data reported here also demonstrate that the other C-P4H isoenzymes cannot compensate for the lack of type I isoenzyme.

Abstract Fatty acids are one of the most essential categories of biological lipids and their synthesis and degradation are vital for all organisms. Severely compromised phenotypes of yeast mutants and human patients, which have defective components in their degradative or synthetic processes for fatty acid metabolism, have highlighted the importance of these processes for overall metabolism. Most fatty acids are degraded by β-oxidation, which occurs in mitochondria and peroxisomes in mammals, whereas synthesis is catalyzed by cytosolic multifunctional peptides, although a synthesis system involving individual enzymes in mitochondria has been also proposed. In this study a novel mitochondrial 2-enoyl thioester reductase Etr1p from the yeast Candida tropicalis, its homolog Mrf1p from Saccharomyces cerevisiae, and their mammalian ortholog were identified and characterized. Observations indicating that mitochondrial localization as well as enzymatic activity is needed to complement the respiratory-deficient phenotype of the mrf1Δ strain from S. cerevisiae suggests that Etr1p and Mrf1p might act as a part of the mitochondrial fatty acid synthesis machinery, the proper function of which is essential for respiration and the maintenance of mitochondrial morphology in yeast. The mammalian enzyme, denoted Nrbf-1p, showed similar localization, enzymatic activity, and ability to rescue the growth of the mrf1Δ strain suggesting that mammals are also likely to possess the ability and required machinery for mitochondrial fatty acid synthesis. This study further included the characterization of another mitochondrial thioester reductase, 2,4-dienoyl-CoA reductase, which acts as an auxiliary enzyme in the β-oxidation of unsaturated fatty acids. The function of this gene was analyzed by creating a knock-out mouse model. While unstressed mice deficient in 2,4-dienoyl-CoA reductase were asymptomatic, metabolically challenged mice showed symptoms including hypoglycemia, hepatic steatosis, accumulation of acylcarnitines, and severe intolerance to acute cold exposure. Although the oxidation of saturated fatty acids proceeds normally, the phenotype was in many ways similar to mouse models of the disrupted classical β-oxidation pathway, except that an altered ketogenic response was not observed. This mouse model shows that a proper oxidative metabolism for unsaturated fatty acids is important for balanced fatty acid and energy metabolism.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

No description available.

Background Multiple myeloma is an incurable cancer disease that emerges from the bone marrow. Bone morphogenetic proteins (BMPs) are ligands that activates intracellular signaling pathways causing activation of transcription factors. Previous studies show that BMP treatment of myeloma cells induce apoptosis, a mechanism dependent on downregulation of c-MYC. BMPs uses different receptors on myeloma cells, but it is still unclear how the intracellular signaling pathway leading to apoptosis works. A recent whole genome CRISPR/Cas9 knockout screening suggested FOXO1 as a gene involved in the mechanism of apoptosis during BMP treatment. We therefore aimed to investigate further on how FOXO1 has an impact on BMP induced apoptosis. Methods Our hypothesis was that knockout of FOXO1 would protect the cells from apoptosis. To begin to address this issue we tested INA-6 FOXO1 knock-out cell clones that was generated before the start of the project and treated them with BMP-9 to look for effects on cell viability and protein expression. We measured cell viability using CellTiter-Glo® 2.0 Cell viability assay and expression of c-MYC and FOXO1 protein using Western blot. Results and conclusions Treatment with BMP-9 for 72 hours showed a decrease in viability of the cells, up to 98%. Protein expression of c-MYC was inhibited by BMP-9 treatment while a constant expression of FOXO1 was seen in all cells clones regardless of BMP treatment. Expression of FOXO1 in the FOXO1 knock-out cells indicates that the knock-out has not worked. More experiments are needed to clarify the role of FOXO1 in BMP-induced apoptosis.

No description available.

Le syndrome des Néoplasies Endocriniennes Multiples de type 1 (NEM1) est une maladie à transmission autosomique dominante liée à l’inactivation du gène MEN1. Le but de mon travail de thèse était d’étudier le rôle biologique et oncosuppressif du gène Men1 dans le pancréas endocrine. La caractérisation d’un nouveau modèle m’a permis de démontrer que l’invalidation du gène Men1 spécifiquement dans les cellules alpha conduit à la fois au développement de glucagonomes et d’insulinomes par un mécanisme de transdifférenciation de cellules exprimant le glucagon en cellules exprimant l’insuline. Parallèlement, en explorant les modèles murins où le gène Men1 est invalidé respectivement dans les cellules alpha et beta Pancréatiques, j’ai pu identifier l’expression altérée de certains facteurs de transcription ayant des fonctions vitales dans ces cellules, notamment Foxa2 et MafB, dans les lésions précoces des cellules endocrines pancréatiques correspondantes. En conclusion, mon travail de thèse a permis de mieux clarifier la fonction biologique du gène Men1 dans les cellules pancréatiques endocrines et de mieux comprendre les mécanismes impliqués dans la survenue du syndrome MEN1 / Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited syndrome caused by mutations of the MEN1 gene. The aim of my work is to investigate the biological and oncosuppressive roles of the Men1 gene in the pancreatic endocrine cells. The analyses carried out in a new mouse model showed that Men1 ablation in alpha cells trigged the development of both glucagonoma and insulinoma by the transdifferentiation from glucagon-producing cells to insulin-expressing cells. Furthermore, the data obtained from the characterization of both alpha- and beta-cell-specific Men1 mutant mice allowed to identify the altered expression of several important endocrine specific transcriptional factors, including Foxa2 and MafB, found in the early lesions of the corresponding pancreatic endocrine cells. Overall, my thesis work provides interesting clues for better understanding the mechanisms involved in the tumorigenesis of MEN1 syndrome

No description available.

La maturation lymphocytaire B est sous le contrôle des éléments cis-régulateurs des gènes d’Ig. Les quatre activateurs transcriptionnels (hs3a, hs1,2, hs3b et hs4) de la région régulatrice en 3’ du locus IgH (3’RR) murin sont les éléments clés pour les hypermutations somatiques (SHM) et les recombinaisons isotypiques (CSR). La 3’RR stimule aussi la transcription d’Ig au stade mature du développement B. La 3’RR, en association avec un oncogène, est un dérégulateur puissant conduisant au développement de lymphomes B. Actuellement rien n’est connu sur le rôle de la 3’RR lors de réactions immunes et/ou inflammatoires normales. Durant ma thèse, nous avons étudié l’impact de la délétion de la 3’RR sur le développement d’une réaction inflammatoire en réponse au pristane chez les souris BALB/c. Ces études nous ont permis de montrer que l’absence de la 3’RR ne perturbe pas l’apparition d’une réaction inflammatoire (cinétique de l’ascite, volume, cellularité, recrutement en cellules inflammatoires et leur capacité à produire des cytokines inflammatoires et anti-inflammatoires). Ceci suggère la présence de lymphocytes B physiologiquement aptes à induire/propager/maintenir une réaction inflammatoire et immune robuste et efficiente en absence d’une 3’RR fonctionnelle. Après avoir étudié le rôle de la délétion de la 3’RR dans un contexte inflammatoire, nous avons analysé son rôle dans un contexte de lymphomagenèse. Pour cela nous avons mis l’absence de la 3’RR dans un fond génétique murin apte à développer des lymphomes B matures (souris Igλ-Myc). On observe un changement important dans le phénotype des lymphomes B en absence de la 3’RR avec un plus grand nombre de lymphomes B immatures, une baisse du nombre de lymphomes B matures CD43+ et une augmentation de lymphomes B CD5+. Le rôle de la 3’RR dans la survenue de mutations aux stades B matures durant la SHM et la CSR est suggéré pour expliquer ces résultats. En conclusion, le ciblage pharmacologique de la 3’RR pour bloquer son effet pro-oncogènique transcriptionnel sur un oncogène transloqué au locus IgH pourrait se révéler une approche prometteuse pour le traitement de certains lymphomes B matures humains. / B-cell maturation is under the control of the cis-regulatory elements of Ig genes. The four transcripional enhancers (hs3a, hs1,2, hs3b and hs4) of the mouse IgH 3’ regulatory region (3’RR) are the key elements for somatic hypermutation (SHM) and class-switch recombination (CSR). The 3'RR also stimulates Ig transcription during the mature B-cell stage. When associated with an oncogene, the 3’RR is a potent deregulator leading to B-cell lymphomas. Currently nothing is known about its role in the development of immune and inflammatory reactions. During my thesis, we have studied the impact of the 3’RR deletion on the development of inflammatory reactions in response to pristane in BALB/c mice. These studies have allowed us to show that the lack of the 3'RR does not disturb the development of an efficient inflammatory reaction in response to pristane (kinetics of appearance of ascites, volume, cellularity, recruitment of inflammatory cells and their capacity to produce inflammatory and anti-inflammatory cytokines). This suggests the presence of B-cells physiologically capable to induce/spread/maintain a robust and efficient inflammatory immune response. After studying the role of the deletion of the 3'RR in an inflammatory context we have investigated the role of the 3’RR in a lymphomagenesis. For this we brought the 3’RR deficiency in a genetic background able to develop mature B-cell lymphomas (Igλ-Myc mice). While wt mice develop mature and immature B-cell lymphomas, 3’RR-deficient mice exhibit a strong preference for immature lymphomas. Furthermore 3’RR-deficiency leads to a lowered frequency of CD43+ activated B-cell lymphomas and to an increased frequency of CD5+ B-cell lymphomas. CSR and SHM are abrogated in 3’RR-deficient mice lowering the probability of oncogenic mutations during these stages. Pharmacological targeting of 3'RR to block its transcriptional pro-oncogenic effect on translocated oncogene into IgH locus might be a promising approach for the treatment of some mature human B-cell lymphomas.