No description available.

No description available.

No description available.

No description available.

Yes / The key metabolic regulator, AMP-activated protein kinase (AMPK) is reported to be downregulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491 respectively as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.

Le nombre de gènes codant pour des protéines chez l’'homme, le vers rond et la mouche des fruits est du même ordre de grandeur. Cette absence de correspondance entre le nombre de gènes d’un eucaryote et sa complexité phénotypique s’explique en partie par le caractère alternatif de l’épissage.L'épissage alternatif augmente considérablement le répertoire fonctionnel de protéines codées par un nombre limité de gènes. Ce mécanisme, très actif lors du développement embryonnaire, participe au devenir cellulaire. De nombreux troubles génétiques, hérités ou acquis (en particulier certains cancers), se caractérisent par une altération de son fonctionnement.Les technologies de séquençage à haut débit de l'ARN donnent accès a une information plus riche sur le mécanisme de l’épissage. Cependant, si la lecture à haut débit des séquences d’ARN est plus rapide et moins coûteuse, les données qui en sont issues sont complexes et nécessitent le développement d’outils algorithmiques pour leur interprétation. En particulier, la reconstruction des transcrits alternatifs requiert une étape de déconvolution non triviale.Dans ce contexte, cette thèse participe à l'étude des événements d'épissage et des transcrits alternatifs sur des données de séquençage à haut débit de l'ARN.Nous proposons de nouvelles méthodes pour reconstruire et quantifier les transcrits alternatifs de façon plus efficace et précise. Nos contributions méthodologiques impliquent des techniques de régression parcimonieuse, basées sur l'optimisation convexe et sur des algorithmes de flots. Nous étudions également une procédure pour détecter des anomalies d'épissage dans un contexte de diagnostic clinique. Nous suggérons un protocole expérimental facilement opérant et développons de nouveaux modèles statistiques et algorithmes pour quantifier des événements d’épissage et mesurer leur degré d'anormalité chez le patient. / The number of protein-coding genes in a human, a nematodeand a fruit fly are roughly equal.The paradoxical miscorrelation between the number of genesin an organism's genome and its phenotypic complexityfinds an explanation in the alternative natureof splicing in higher organisms.Alternative splicing largely increases the functionaldiversity of proteins encoded by a limitednumber of genes.It is known to be involved incell fate decisionand embryonic development,but also appears to be dysregulatedin inherited and acquired human genetic disorders,in particular in cancers.High-throughput RNA sequencing technologiesallow us to measure and question splicingat an unprecedented resolution.However, while the cost of sequencing RNA decreasesand throughput increases,many computational challenges arise from the discrete and local nature of the data.In particular, the task of inferring alternative transcripts requires a non-trivial deconvolution procedure.In this thesis, we contribute to deciphering alternative transcript expressions andalternative splicing events fromhigh-throughput RNA sequencing data.We propose new methods to accurately and efficientlydetect and quantify alternative transcripts.Our methodological contributionslargely rely on sparse regression techniquesand takes advantage ofnetwork flow optimization techniques.Besides, we investigate means to query splicing abnormalitiesfor clinical diagnosis purposes.We suggest an experimental protocolthat can be easily implemented in routine clinical practice,and present new statistical models and algorithmsto quantify splicing events and measure how abnormal these eventsmight be in patient data compared to wild-type situations.

Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system. My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for Down Syndrome Cell Adhesion Molecule, which is a Drosophila homolog of human DSCAM. According to genomic analysis, DrosophilaDscam gene can generate more than 38,000 isoforms through alternative splicing in its exons 4, 6, 9 and 17. All Dscam isoforms share similar domain structures, with 10 immunoglobulin domains and 6 fibronectin type III repeats in the ectodomain, a single transmembrane domain and a cytoplasmic endodomain. There are two alternative exons in exon 17 (17.1 and 17.2), which encodes Dscam’s transmembrane domain. Interestingly, in ectopic expression, Dscam isoforms carrying exon 17.1 (Dscam[TM1]) can be preferentially localized to dendrites and cell bodies, while Dscam isoforms carrying exon 17.2 (Dscam[TM2]) are distributed throughout the entire neuron including axons and dendrites. To unravel the mechanisms involved in the differential distribution of Dscam[TM1] versus Dscam[TM2], I conducted a mosaic genetic screening to identify the possible factors affecting dendritic distribution of Dscam[TM1], established an in vivoTARGET system to better distinguish the differential distribution of Dscam, identified the axonal and dendritic targeting motifs of Dscam molecules and further showed that Dscam’s differential roles in dendrites versus axons are correlated with its localization. Several mutants affecting dendritic distribution of Dscam[TM1] have been identified using a MARCM genetic screen. Three of these mutants (Dlis1, Dmn and p24) are components of the dynein/dynactin complex. Silencing of other dynein/dynactin subunits and blocking dynein function with a dominant-negative Glued mutant also resulted in mislocalization of Dscam[TM1] from dendrites to axons. However, microtubule polarity in the mutant axons was maintained. Taken together, this was the first demonstration that the dynein/dynactin complex is involved in the polarized distribution of membrane proteins in neurons. To further examine how dynein/dynactin is involved in the dendritic distribution of Dscam[TM1], I compromised dynenin/dynactin function with dominant-negative Glued and transiently induced Dscam[TM1] expression. The results suggested that dynein/dynactin may not be directly involved in the targeting of newly synthesized Dscam[TM1] to dendrites. Instead, it plays a role in maintaining dendritic restriction of Dscam[TM1]. Notably, dynein/dynactin dysfunction did not alter distribution of another dendritic transmembrane protein Rdl (Resistant to Dieldrin), supporting involvement of diverse mechanisms in distributing distinct molecules to the dendritic membrane. To identify the targeting motifs of Dscam, I incorporated the TARGET (Temporal and regional gene expression targeting) system into mushroom body (MB) neurons, and this allowed the demonstration of the differential distribution of Dscam[TM1] and Dscam[TM2] with more clarity than conventional overexpression techniques. Using the TARGET system, I identified an axonal targeting motif located in the cytoplasmic juxtamemebrane domain of Dscam[TM2]. This axonal targeting motif is dominant over the dendritic targeting motif located in Dscam’s ectodomain. Scanning alanine mutagenesis demonstrated that two amino acids in the axonal targeting motif were essential for Dscam’s axonal distribution. Interestingly, swapping the cytoplasmic juxtamembrane portions between TM1 and TM2 not only reversed TM1’s and TM2’s differential distribution patterns but also their functional properties in dendrites versus axons. My thesis research also involved studying endodomain diversity of Dscam isoforms. Besides the diversity originally found in the ectodomain and transmembrane domain of Dscam, my colleagues and I further demonstrated the existence of four additional endodomain variants. These four variants are generated by skipping or retaining exon 19 or exon 23 through independent alternative splicing. Interestingly, different Dscam endodomain isoforms are expressed at different developmental stages and in different areas of the nervous system. Through isoform-specific RNA interference, we showed the differential involvement of distinct Dscam endodomains in specific neuronal morphogenetic processes. Analysis of the primary sequence of the Dscam endodomain indicated that endodomain variants may confer activation of different signaling pathways and functional roles in neuronal morphogenesis. In Summary, my thesis work identified and characterized several previously unknown mechanisms related to the differential distribution of membrane proteins in neurons. I showed that there may be a dynein/dynactin-independent mechanism for selective transport of dendritic membrane proteins to dendrites. Second, dynein/dynactin plays a maintenance role in dendritic restriction of Dscam[TM1]. Third, different membrane proteins may require distinct combinations of mechanisms to be properly targeted and maintained in certain neuronal compartments. Further analysis of the mutants indentified from my genetic screen will definitely help to resolve the missing pieces of the puzzle. These findings provide novel mechanistic insight into the differential distribution of membrane proteins in polarized neurons.

Les interneurones GABAergiques constituent une population mineure de cellules par rapport aux neurones glutamatergiques dans le néocortex. Cependant ils contrôlent fortement l'excitabilité neuronale, la dynamique des réseaux neuronaux et la plasticité synaptique. L'importance des circuits GABAergiques dans le processus fonctionnel et la plasticité des réseaux corticaux est soulignée par des résultats récents qui montrent que des modifications très précises et fiables des circuits GABAergiques sont associées à divers troubles du développement neurologique et à des défauts dans les fonctions cérébrales. De ce fait, la compréhension des mécanismes cellulaires et moléculaires impliquant le développement des circuits GABAergiques est la première étape vers une meilleure compréhension de la façon dont les anomalies de ces processus peuvent se produire. La molécule d’adhésion cellulaire neurale (NCAM) appartient à la super-famille des immunoglobulines de reconnaissance cellulaire et est impliquée dans des interactions homophiliques et hétérophiliques avec d’autres molécules. Même si plusieurs rôles de NCAM ont été démontrés dans la croissance neuronale, la fasciculation axonale, la formation et la maturation de synapses, de même que dans la plasticité cellulaire de plusieurs systèmes, le rôle de NCAM dans la formation des synapses GABAergiques reste inconnu. Ce projet visait donc à déterminer le rôle précis de NCAM dans le processus de maturation des synapses GABAergiques dans le néocortex, en modulant son expression à différentes étapes du développement. L’approche choisie a été de supprimer NCAM dans des cellules GABAergiques à paniers avant la maturation des synapses (EP12-18), pendant la maturation (EP16-24), ou durant le maintien de celles-ci (EP24-32). Les méthodes utilisées ont été le clonage moléculaire, l’imagerie confocale, la culture de coupes organotypiques et des techniques morphométriques de quantification de l’innervation GABAergique. Nos résultats montrent que l’inactivation de NCAM durant la phase de maturation des synapses périsomatiques (EP16-24) cause une réduction du nombre de synapses GABAergiques périsomatiques et du branchement de ces axones. En revanche, durant la phase de maintien (EP26-32), l’inactivation de NCAM n’a pas affecté ces paramètres des synapses GABAergiques. Or, il existe trois isoformes de NCAM (NCAM120, 140 et 180) qui pourraient jouer des rôles différents dans les divers types cellulaires ou à des stades développementaux différents. Nos données montrent que NCAM120 et 140 sont nécessaires à la maturation des synapses périsomatiques GABAergiques. Cependant, NCAM180, qui est l’isoforme la plus étudiée et caractérisée, ne semble pas être impliquée dans ce processus. De plus, l’inactivation de NCAM n’a pas affecté la densité des épines dendritiques ou leur longueur. Elle est donc spécifique aux synapses périsomatiques GABAeriques. Finalement, nos résultats suggèrent que le domaine conservé C-terminal KENESKA est essentiel à la maturation des synapses périsomatiques GABAergiques. Des expériences futures nous aiderons à mieux comprendre la mécanistique et les différentes voies de signalisation impliquées. / GABAergic interneurons, though a minor population in the neocortex, play an important role in cortical function and plasticity. Alterations in GABAergic circuits are implicated in various neurodevelopmental disorders. The GABAergic network comprises diverse interneuron subtypes that have different morphological and physiological characteristics, and localize their synapses onto distinct subcellular locations on the postsynaptic targets. Precisely how activity and molecularly driven mechanisms conspire to achieve the remarkable specificity of GABAergic synapse localization and formation is unknown. Therefore, unravelling the cellular and molecular mechanisms involved in this process is crucial for a better understanding of both cortical function and the basis of various neurological disorders. Here we focus our study on a subtype of GABAergic neurons - the basket interneurons which localize synapses, called perisomatic synapses, onto the soma and proximal dendrites of the postsynaptic targets, and tightly regulate their firing patterns. Although recent studies have shown the activity dependence of basket synapse formation, the molecular mechanisms implicated in the perisomatic synapse formation process are poorly understood. NCAM, the neural cell adhesion molecule, is a prime molecular player implicated both in early synaptogenesis events, and during maturation of glutamatergic synapses in the hippocampus. Recent studies have implicated the polysialylated form of NCAM (PSA-NCAM) in basket synapse formation. However, whether and how NCAM per se plays a role in the formation of GABAergic synapses is unknown. Using single cell genetics to knock down NCAM in individual basket interneurons at specific developmental time periods, we characterized the role of NCAM during perisomatic synapse formation and maintenance. Here we show that loss of NCAM during perisomatic synapse formation from equivalent postnatal day (EP) 16 to EP24, in organotypic slices from mouse visual cortex, significantly retards the process of basket cell axonal branching and bouton formation. However, loss of NCAM at a later stage (EP26 to EP32), when the synapses are already formed, did not affect the number or intricacy of perisomatic synapses. NCAM is therefore implicated in perisomatic synapse formation but not in its maintenance. Further studies also show that isoforms of NCAM, such as NCAM140 and NCAM120 are involved in perisomatic GABAergic synapse maturation. However, NCAM180 is not implicated in this process. Also, NCAM does not affect dendritic spine density and length during maturation and maintenance phases, therefore its action is specific only to GABAergic perisomatic synapses. Finally, the highly conserved C-terminal domain KENESKA is essential for GABAergic perisomatic synapse maturation. Future experiments will help us clarify this mechanism and the involved signalling pathways related to NCAM.

PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...

A macroprolactinemia é condição freqüente na hiperprolactinemia e em geral, sem impacto clínico. Os dados sobre a atividade biológica da macroprolactina (bbPRL) são controversos e baseados em bioensaio heterólogo com células de rato Nb2. A atividade biológica da bbPRL é observada in vitro e não in vivo, provavelmente porque seu alto peso molecular evita sua passagem pelos capilares. A bioatividade da bbPRL talvez varie de acordo com a especificidade do receptor de prolactina (PRLR). Avaliamos a bioatividade da bbPRL de indivíduos macroprolactinêmicos (Grupo I, n = 18) e da PRL monomérica (mPRL) de pacientes hiperprolactinêmicos sem bbPRL (Grupo II, n = 5) em Nb2 e em células Ba/F-LLP, transfectadas com o PRLR humano. Enquanto ambos ensaios apresentam resultados similares para a atividade de mPRL, nossos resultados indicam que a atividade da bbPRL é presente em ensaio heterólogo e não em ensaio homólogo. O ensaio Ba/F-LLP é sensível e apresenta melhor correlação com a atividade in vivo da bbPRL / Macroprolactinemia is a frequent finding in hyperprolactinemic individuals, usually without clinical impact. Data on biological activity of macroprolactin (bbPRL) is mostly based on a heterologous bioassay (Nb2 cell). Biological activity of bbPRL observed in vitro but not in vivo maybe due to its high molecular weight preventing its passage through capillary barrier. Alternatively, bbPRL bioactivity may differ depending on the PRL receptor species specificity. BbPRL from macroprolactinemic individuals and monomeric PRL (mPRL) from hyperprolactinemic patients without macroprolactinemia were tested in two bioassays: Nb2 and in Ba/F-LLP, which expresses human prolactin receptor. While both bioassays achieve similar results considering mPRL activity, our results indicate that bbPRL displays activity in a heterologous but not in a homologous bioassay, consistently with the apparent absence of bbPRL bioactivity in vivo