Francisella tularensis, l'agent de la tularémie, est une bactérie intracellulaire capable d'infecter un grand nombre de cellules dont les macrophages. Le système immunitaire inné cytosolique est capable de détecter la bactérie à différents stades de son cycle d'infection. Dans un premier temps, le macrophage détecte la bactérie cytosolique et produit de l'interféron de type I. Cet interféron induit l'expression de milliers de gènes. Le macrophage est ensuite capable de détecter l'ADN cytosolique de la bactérie via un récepteur spécifique AIM2. La liaison AIM2-ADN entraine la formation d'un complexe multi-protéique appelé inflammasome et se composant de AIM2-ASC-caspase-1. L'activation de ce complexe conduit à la maturation de la caspase-1. Caspase-1 permet la sécrétion de deux cytokines majeures antimicrobiennes : l'IL-1beta et l'IL-18. De plus, caspase-1 induit une mort programmée des cellules infectées appelée pyroptose. La sécrétion de cytokines et la pyroptose sont deux évènements majeurs pour lutter contre les pathogènes. Ma thèse a consisté à identifier le lien entre l'interféron et l'activation de l inflammasome AIM2 dans des macrophages infectés par la bactérie Francisella. En réalisant un crible a l'aide d'ARNs interférents, j'ai découvert que 2 protéines sont impliquées dans l'activation de cet inflammasome, les guanylate binding proteins 2 et 5 (GBP2 et GBP5). En collaboration avec l'équipe du Dr. Broz en Suisse, nous avons démontré que les GBPs étaient impliquées dans le contrôle de la réplication intracellulaire de Francisella et également dans la lyse de la bactérie permettant le relargage d'ADN et l'activation de l'inflammasome AIM2. Les GBPs sont induites par l'interféron de type I mais très majoritairement par l'interféron de type II (IFN- gamma). Nous avons mis en évidence que le contrôle de la réplication bactérienne est GB dépendant et inflammasome-dépendant en absence d'IFN- gamma mais qu'il devient totalement GB dépendant et inflammasome-indépendant dans des macrophages pré-stimulés avec de l'IFN- gamma. De plus, la mort des macrophages pré-stimulés avec de l'IFN- gamma et infectés par Francisella est également GBP-dépendante et inflammasome-indépendante. En prenant en compte tous ces résultats, nous concluons que les GBPs sont des protéines impliquées dans l'immunité des macrophages infectés par Francisella mais qu'elles ont un double rôle : d'une part celui d'induire l'activation de l'inflammasome (la pyroptose) sous le contrôle de l'interféron de type I et d'autre part, d'induire une mort cellulaire et la lyse des bactéries cytosoliques de manière indépendante de l'inflammasome sous le contrôle d'IFN- gamma. Nos résultats placent donc les GBPs comme les effecteurs majeurs de l'immunité cytosolique antibactérienne suite au traitement par l'IFN-gamma / Francisella tularensis is an intracellular bacterium, and the causative agent of tularemia, capable of infecting a large number of cells including macrophages. The innate cytosolic immune system is capable of detecting the bacterium at different stages of its infection cycle. Macrophages first detect the DNA of the cytosolic bacterium and produce type I interferon. Type I interferon subsequently induces the expression of thousands of genes. The macrophages then detect the cytosolic DNA of the bacterium via a cytosolic DNA sensor called AIM2. The AIM2-DNA binding results in the formation of a multi-protein complex called the AIM2 inflammasome composed of AIM2-ASC-caspase-1. Activation of this complex leads to the maturation of caspase-1. Caspase-1 activation leads to the secretion of two major antimicrobial cytokines, IL-1ß and IL-18. In addition, caspase-1 induces a programmed cell death termed pyroptosis. Cytokine secretion and pyroptosis are two major events in the control of pathogens. My PhD focused in identifying the link between interferon and activation of the AIM2 inflammasome in macrophages infected with the pathogenic bacterium Francisella. I performed a RNA interference screening and identified two proteins involved in the activation of the AIM2 inflammasome: guanylate binding proteins 2 and 5 (GBP2 and GBP5). In collaboration with Dr. Broz’s team in Switzerland, we demonstrated that GBPs are involved in the control of intracellular replication of Francisella and also in the lysis of the bacterium allowing the release of bacterial DNA and the activation of inflammasome AIM2. GBPs are induced by type I interferon but to a much greater extent by type II interferon (IFN-gamma). In the second part of my work, we demonstrate that the control of bacterial replication is GBP-dependent and inflammasome-dependent in the absence of IFN-gamma but that it becomes fully GBP-dependent and inflammasome-independent in macrophages primed with IFN-gamma. Cell-death of macrophages primed with IFN-? and infected with Francisella is also GBP-dependent and inflammasome-independent. Taken together, these results demonstrate that GBPs are innate immunity proteins involved in the death of macrophages and the bacterial growth restriction through two differents pathways : one induces the activation of inflammasome (induction of Pyroptosis) controlled with type I interferon signaling and, another induces cell-death and bacterial killing in an inflammasome-independent manner under the control of IFN-gamma. Our results thus discriminates the antimicrobial action of the inflammasome and of GBPs and position GBPs as the master antibacterial effectors of IFN-gamma, a key cytokine to fight cytosolic bacteria

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-06-29T16:18:24Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE - Evellyne - com ficha catalográfica.pdf: 3540470 bytes, checksum: 42d4abc2752fe4f60bc667d7c9829075 (MD5) / Made available in DSpace on 2016-06-29T16:18:24Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE - Evellyne - com ficha catalográfica.pdf: 3540470 bytes, checksum: 42d4abc2752fe4f60bc667d7c9829075 (MD5) Previous issue date: 2015-09-18 / CAPES / Lectinas são proteínas ou glicoproteínas de origem não-imunológica que possuem a capacidade de aglutinar eritrócitos ou outras células e precipitar polissacarídeos ou outras glicoproteínas. Cramoll 1, Cramoll 2, Cramoll 3 e Cramoll 4 são lectinas extraídas das sementes de Cratylia mollis. Cramoll 1,4 é uma mistura de Cramoll 1 e sua isoforma Cramoll 4 e rCramoll foi sintetizada pela técnica de expressão heteróloga, a partir da sequência de aminoácidos de Cramoll 1. Este trabalho objetivou avaliar a atividade indutora de morte celular das lectinas Cramoll 1,4 e rCramoll em células de adenocarcinoma de próstata humano (PC-3), além de verificar as propriedades das lectinas Cramoll 1, Cramoll 2, Cramoll 3 e Cramoll 4 em aglutinar plaquetas lavadas de diferentes tipos sanguíneos humanos e de sangue de coelho. Cramoll 1,4 e rCramoll diminuíram a viabilidade das células PC-3 com o aumento das concentrações das lectinas ou do tempo de exposição. Os valores de concentrações capazes de matar 50% das células PC-3 expostas às lectinas foram 39.69 μg/mL e 29.91 μg/mL para Cramoll 1,4 e rCramoll, respectivamente. Elas apresentaram maior citotoxicidade para células PC-3 quando comparadas à lectina ConA. Tratamento com Cramoll 1,4 e com rCramoll induziu a morte celular por necrose, com um aumento de cerca de 3 vezes nos níveis de superóxido mitocondrial, bem como aumento dos níveis de cálcio citosólico. Quando utilizados inibidores da abertura do poro de transição de permeabilidade mitocondrial não foi observada proteção das células PC-3 no processo de morte celular. Provavelmente, a morte de células PC-3 ocorreu devido aos aumentos nas concentrações de cálcio citosólico e de espécies reativas de oxigênio (EROs) mitocondrial, culminando com uma diminuição na fosforilação oxidativa, deficiência de ATP e privação de energia. Com relação à propriedade em aglutinar plaquetas, a aglutinação ocorreu em maior percentual nas plaquetas provenientes de sangue tipo A e em menor intensidade em plaquetas provenientes de sangue tipo AB e apesar da especificidade a carboidratos diferente entre Cramoll 3 e Cramoll 4, estas isoformas apresentaram semelhança quanto à promoção de aglutinação de plaquetas humanas. Quanto aos ensaios de inibição da aglutinação de plaquetas, foi observado que, na maior parte das vezes, os carboidratos específicos não inibiram completamente a aglutinação de plaquetas. / Lectins are proteins or glycoproteins of non-immune origin that have the ability to agglutinate erythrocytes or other cells and precipitating polysaccharides and glycoproteins other. Cramoll 1, Cramoll 2, Cramoll 3 and Cramoll 4 are lectin extracted from seeds Cratylia mollis. Cramoll 1,4 is a mixture of Cramoll 1 and isoform Cramoll 4. rCramoll was synthesized by heterologous expression technique, from the Cramoll amino acid sequence 1. This study evaluated the activity of inducing cell death of lectins Cramoll 1,4 and rCramoll in human prostate adenocarcinoma cells (PC-3), and evaluate the properties of lectins Cramoll 1, Cramoll 2, Cramoll 3 and Cramoll 4 washed platelets agglutinate in different human blood types and rabbit blood. Cramoll 1,4 and rCramoll decreased viability of PC-3 cells with increasing concentrations of lectins or exposure time. The values of effective concentrations to kill 50% of PC-3 cells treated were 39.69 μg/ml and 29.91 μg/mL for the lectins Cramoll 1,4 and rCramoll, respectively. They showed greater toxicity for PC-3 cells compared to the lectin ConA. Treatment with Cramoll 1,4 and rCramoll induced cell death by necrosis, with an increase about 3 times higher the levels mitochondrial superoxide as well as in cytosolic calcium levels. When used inhibitors opening of the mitochondrial permeability transition pore protection was not observed in PC-3 cells in cell death process. Probably, PC-3 cell death occurred due to these previous increases in cytosolic calcium concentrations and reactive oxygen species (ROS) mitochondrial, culminating in a decrease in oxidative phosphorylation, ATP deficiency and deprivation of energy. Platelet agglutination occurred in a higher percentage in platelets from blood type A and to a lesser intensity in platelets from blood type AB. Despite the specificity of different carbohydrates between Cramoll 3 and Cramoll 4, these isoforms were similar as to promoting platelet agglutination human. As for inhibition assays of platelet agglutination it was observed that, in most of times, the specific carbohydrate didn’t completely inhibit platelet agglutination

Recently, they are examined various means for activating the endogenous signalling pathways leading to increased resistance of the myocardium from ischemic/reperfusion (I/R) injury. One of them is the adaptation to chronic hypoxia, which has been shown to reduce the incidence and severity of ventricular arrhythmias, improves the recovery of postischemic contractile function of the heart and particularly reduces the extent of myocardial infarction. Since the function of the heart depends on the maintenance of membrane integrity of cardiomyocytes there are very important phospholipase A2 (PLA2) which are involved in the repair of cellular membranes. Also they are an important component of the protective signalling pathways because they cleave membrane phospholipids to produce lipid signalling molecules. Elucidate the role of PLA2 and the precise mechanism of action of signalling pathways leading to cardioprotection could be important for the prevention and treatment of cardiovascular diseases. Therefore, in this thesis we examined the influence of continuous normobaric hypoxia (CNH) to the relative representation of cardiac PLA2 (secretory - sPLA2IIA, calcium-independent - iPLA2, cytosolic - cPLA2α and its phosphorylated form - p-cPLA2α), and proteins involved in the activation and phosphorylation of...

Cardiovascular diseases, particularly acute myocardial infarction, are the leading causes of death in developed countries including the Czech Republic. One of the ways to increase cardiac resistance against acute ischemia/reperfusion (I/R) injury is adaptation to chronic hypoxia. However, changes at the molecular level associated with this adaptation have still not been fully explored. It is obvious that the myocardial function depends on maintaining membrane integrity and cellular homeostasis of cardiomyocytes. From this perspective, phospholipases A2 (PLA2) are the key enzymes that take part in the remodeling and repairing of the cell membranes. Moreover, PLA2 are also involved in generation of lipid signaling molecules - free long chain fatty acids (FA) and 2-lysophopholipids. In myocardium, members of three major PLA2 classes are present: cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2) and secretoric PLA2 (sPLA2). This thesis aimed to determine the following in the left ventricular myocardium of adult male Wistar rats: 1) The effect of intermittent hypobaric hypoxia (IHH; 8 hours/day, 5 days/week, 5 weeks, ~ 7000 m) on the expression of total cPLA2α and its phosphorylated form (p-cPLA2α, Ser505 ), and further iPLA2 and sPLA2IIA, as well as signaling proteins activating cPLA2α enzyme...

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by the common deletion of 26-28 contiguous genes in the 7q11.23 region, which poses difficulties to the establishment of genotype-phenotype correlations. The use of mouse models would broader the knowledge of the syndrome, the role of deleted genes, affected pathways and possible treatments. In this thesis project, several mouse models, tissues and cells have been used to define the phenotypes at different levels, the deregulated genes and pathways and to discover modifying elements and novel treatments for the cardiovascular phenotype. In addition, a new binding motif has been described for Gtf2i, a deleted gene encoding a transcription factor with a major role in WB, providing new target genes from deregulated pathways. The obtained results reveal the essential role of mouse models for the study of Williams-Beuren syndrome and provide new treatments options and affected pathways and genes which could be future treatment targets. / La síndrome de Williams-Beuren és una malaltia del neurodesenvolupament causada per una deleció comú d’entre 26 i 28 gens contigus a la regió 7q11.23, dificultant l’establiment de relacions genotip-fenotip. L’ús de models de ratolí pot augmentar el coneixement sobre la malaltia, el paper dels gens delecionats, les vies moleculars afectades i els futurs tractaments. En aquesta tesi s’han usat diversos models de ratolí, les seves cèl·lules i teixits per tal de descriure i definir fenotips, gens i vies moleculars desregulades i per descobrir elements modificadors i nous tractaments. Per últim, s’ha definit un nou motiu d’unió per Gtf2i, uns dels gens delecionats que codifica per un factor de transcripció amb un rol central en la síndrome, proporcionats possible nous gens diana de vies moleculars desregulades. Els resultats obtinguts revelen el paper essencial dels models de ratolí per a l’estudi de la síndrome de Williams-Beuren, proporcionen noves opcions terapèutiques i defineixen nous gens i vies moleculars afectades que podrien suposar noves dianes terapèutiques.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 29, 2010). Vita. Thesis advisor: Grace Y. Sun. "May 2008" Includes bibliographical references.

All cells maintain essential metal nutrients at optimal levels by metal homeostasis. P-type ATPases, a crucial superfamily of integral membrane proteins, are involved in the active transport of metal ions across biological membranes driven by the motive force of ATP- hydrolysis. The PIB-type ATPase subfamily, also called CPx-ATPases, fulfills a key role in heavy metal homoeostasis among the most widespread species from bacteria to human. In humans, the defect in copper transporters is the direct cause of severe neurological and hepatic disorders such as Wilson and Menkes diseases, therefore, understanding the molecular function of these pumps is of paramount importance in human health. Cu+-ATPases have two transmembrane metal binding sites (TM-MBS) and three cytosolic domains, namely the actuator (A-domain) and phosphorylation and nucleotide-binding domain (PN), and regulatory N-terminal heavy metal binding domain (HMBD). Here, we have studied the Legionella pneumophila CopA (LpCopA) and its isolated cytosolic domains to improve our understanding of the functional interaction of the protein domains during metal transport relate this to the known structure of this ATPase. To elucidate how cytosolic ligands (Cu+ and nucleotide) stimulate the interactions among the cytosolic domains and may transmit conformational changes to the TM-MBS, the interactions among recombinant isolated cytosolic domains were first examined biochemically by co-purification and spectroscopically by circular dichroism, time-resolved fluorescence and site-directed fluorescent labeling assays. The Cu+-dependent interaction between the A-domain and HMBD has been postulated as a mechanism for activating the ATPase cycle. This question was addressed here by studying copper-dependent interactions between the isolated expressed domains. Spectroscopic evidence is provided that an HMBD-A complex is formed in the presence of Cu+ which binds with 100-200 nM affinity to the recombinant HMBD. In contrast, the A-domain interacts with the PN domain in a nucleotide-dependent fashion. This molecular recognition is required for the dephosphorylation step in the catalytic cycle. The interaction was investigated in more detail by the use of a decameric peptide derived from the PN-binding interface of the A-domain and carrying the conserved TGE-motif involved in dephosphorylation. Its binding to the isolated PN domain in a weakly nucleotide-dependent manner, is demonstrated here by stopped-flow fluorescence spectroscopy. Several ATPase assays were modified to assess the functionality of the PN-domain and full length LpCopA. The peptide was found to reduce the catalytic turnover of full length LpCopA. This agrees with the expected slowing down of the reformation of the PN-A-domain interaction since the peptide occupies their binding interface. Thus, the synthetic peptide provides a means to study specifically the influence of PN-A-domain interactions on the structure and function of LpCopA. This was done by time-correlated single photon counting (TCSPC) method. The time-dependent Stokes shift of the environmentally sensitive fluorophore BADAN which was covalently attached to the conserved CPC-motif in the TM-MBS was measured. The data indicate that the interior of the ATPase is hydrated and the mobility of the intra-protein water varies from high to low at C382 at the “luminal side” and C384 at the “cytosolic side” of the TM-MBS, respectively. This finding is consistent with the recent MD simulation of LpCopA, bringing the first experimental evidence on a luminal-open conformation of E2~P state. The A-domain-derived decapeptide, although binding to the cytosolic head piece, induces structural changes also at the TM-MBS. The peptide-stabilized state (with a disrupted PN-A interface) renders the C384 environment more hydrophobic as evidenced by TCSPC. Taken together, the data from cytosolic domain interactions, ATPase assays and of time-dependent Stoke shift analyses of BADAN-labeled LpCopA reveal the presence of hydrated intramembraneous sites whose degree of hydration is regulated by the rearrangement of cytosolic domains, particularly during the association and dissociation of the PN-A domains. Copper affects this arrangement by inducing the linkage of the A-domain to the HMBD. The latter appears to play not only an autoinhibitory but also a chaperone-like role in transferring Cu+ to the TM-MBS during catalytic turnover.

Les oligomères solubles de peptide Bêta-amyloïde (A-bêta) apparaissent comme les acteurs majeurs de la perte synaptique précoce observée au cours de la maladie d'Alzheimer. Notre équipe a précédemment montré que ces oligomères de peptide A-bêta activent la phospholipase A2 cytosolique (cPLA2), qui entraîne la libération d'acide arachidonique à partir des phospholipides membranaires. En utilisant un modèle d'injection intra cérébro ventriculaire unique d'une faible quantité de peptide A-bêta, nous avons pu observer que l'inactivation constitutive du gène de la cPLA2 protége les souris KO contre les perturbations mnésiques et empêche la réduction de l'expression de protéines synaptiques au sein de l'hippocampe, ces deux effets délétères étant constatés chez les animaux wild-type. Par la suite, nous avons montré que l'activation des sphingomyélinases, consécutive à l'exposition aux oligomères A-bêta, est indétectable dans des neurones en culture issus de souris KO. Dans ces mêmes neurones KO, nous avons constaté que la phosphorylation de Akt/PKB n'est pas altérée suite à l'exposition des cellules aux oligomères A-bêta. Enfin, nous avons pu mettre en évidence une diminution de l'expression de la protéine précurseur du peptide A-bêta (protéine APP), tant au niveau d'homogénats hippocampiques que de neurones en cultures, issus de souris KO. Néanmoins, des travaux supplémentaires sont requis pour établir le lien exact entre cette réduction de l'expression d'APP et la résistance aux oligomères A-bêta, tant in vitro qu'in vivo. Toutefois, ces résultats soulignent l'implication de la cPLA2 dans la neuro dégénérescence entrainée par les oligomères A-bêta, et font apparaitre cette enzyme comme une cible thérapeutique potentielle pour le traitement de la maladie d'Alzheimer / Soluble beta-amyloid (A-beta) oligomers putatively play a critical role in the early synapse loss and cognitive impairment observed in Alzheimer's disease. We previously demonstrated that A-beta oligomers activate cytosolic phospholipase A2 (cPLA2) which specifically releases arachidonic acid from membrane phospholipids. By using a single A-beta oligomers intra cerebro ventricular injection, we observed that cPLA2 gene suppression prevents both the alterations of cognitive abilities and the reduction of hippocampal synaptic markers levels which are observed in wild type mice. We further demonstrated that the A-beta oligomers-induced sphingomyelinase activation is suppressed and that the phosphorylation of Akt/PKB is preserved in neuronal cells isolated from KO mice. Interestingly, expression of the A-beta precursor protein (APP) is reduced in hippocampus homogenates and neuronal cells from KO mice, but the relationship with the resistance of these mice to the A-beta oligomers toxicity requires further investigation. These results therefore show that cPLA2 plays a key role in the A-beta oligomers-associated neurodegenerative effects, and as such represents a potential therapeutic target for the treatment of Alzheimer's disease

L’objectif de cette thèse porte sur l’étude de la compartimentalisation cellulaire et de la prise en charge de l’uranium (U) par les protéines cytosoliques des cellules branchiales du poisson zèbre (Danio rerio, espèce modèle en toxicologie aquatique) après exposition contrastées (chronique vs. aiguë, 20 et 250 µg.L-1) par voie directe. Cette étude a nécessité le développement, l’utilisation et le couplage d’outils analytiques de pointe (SEC, IEF hors-gel, RP-UHPLC pour la séparation, ICP-SFMS, ESI-FTMS/MS pour la détection) avec comme défis majeurs la conservation des interactions non-covalentes U biomolécule et une sensibilité maximale pour travailler à des niveaux d’exposition proches de ceux rencontrés dans l’environnement. Après extraction, 24 à 32% de la charge branchiale totale en U est contenue dans le cytosol dans lequel la distribution de l’U sur les biomolécules (en fonction de leur PM mais aussi de leur pI) diffère selon le niveau d’exposition. Enfin, une cartographie des biomolécules cibles de l’U a permis (i) de mettre en évidence une affinité particulière de l’U pour les protéines à caractère acide et/ou contenant du phosphore et (ii) d’identifier 24 protéines candidates pour lier U. / The objective of this thesis is to study the cellular compartmentalization and the chelation of uranium (U) by cytosolic proteins of gill cells of the zebrafish (Danio rerio, model species in aquatic toxicology) under different direct exposure conditions (chronic vs. acute, 20 and 250 µg.L 1). This study required the development of hyphenated techniques (SEC, IEF off-gel, RP-UHPLC for the separation, ICP-SFMS, ESI-FTMS/MS for the detection) with the main challenges of maintaining the non-covalent U-biomolecule interactions and enhancing sensitivity for the analysis of environmentally relevant samples. After extraction, 24% to 32% of the total U detected in the gills were present in the cytosolic fraction, in which the U distribution on the biomolecules (as a function of their MW and pI) varied depending on the exposure level. Finally, U target biomolecules mapping allowed us (i) to highlight a particular affinity of U for acidic and/or P-containing proteins and (ii) to identify 24 protein candidates for U binding.