The mechanisms of centriole biogenesis

Résumé : Le centrosome contient une paire de centrioles entourée par du matériel péricentriolaire (PCM) et cet ensemble constitue le centre organisateur des microtubules de la majorité des cellules animales. Tout comme l'ADN, 1'unique centrosome présent au début du cycle cellulaire est dupliqué une et une seule fois pour former deux centrosomes qui vont orchestrer la mise en place du fuseau mitotique. La duplication du centrosome doit être soumise à une régulation précise car la présence d'un seul ou de plus de deux centrosomes peut entraîner la formation d'un fuseau mitotique aberrant, la mauvaise ségrégation des chromosomes et l'aneuploïdie. Bien que la duplication des centrioles soit un phénomène clé pour la duplication du centrosome lui-même, les mécanismes impliqués dans la formation des centrioles sont peu connus et constituent une importante question de biologie cellulaire. Dans cette thèse, nous nous sommes concentrés sur l'analyse de HsSAS-6. Nous avons trouvé que cette protéine est nécessaire pour la formation d'un centriole et qu'elle est localisée spécifiquement à la base des nouveaux centrioles formés. Les niveaux de HsSAS-6 oscillent pendant le cycle cellulaire : la protéine est absente en G1, commence à s'accumuler au niveau du centriole et dans le cytoplasme dès le début de la phase S de synthèse et disparaît abruptement pendant l'anaphase, où probablement APC/CCdlh1 la dirige vers une dégradation par le protéasome 26S. Il est important de noter que la surexpression de HsSAS-6 entraîne la formation de multiples centrioles au lieu d'un seul, ce qui indique que les niveaux de HsSAS-6 déterminent le nombre de centrioles formés. En plus de HsSAS-6, nous avons aussi étudié la lignée mutante sas-2 de C. elegans qui quelques fois assemble un fuseau multi-polaire dans l'embryon à une cellule. Nous avons montré que ce phénotype est la conséquence de la présence de multiples centrioles dans les cellules du sperme. Enfin, nous avons aussi préparé une palette de vecteurs compatibles avec le système Gateway pour permettre la génération rapide de lignées cellulaires humaines exprimant des protéines de manière inductible. De plus, nous avons commencé à développer une méthode pour évaluer la duplication des centrioles par le biais d'une plateforme de criblage d'une librairie de siRNA humains. Dans l'ensemble, notre travail a pu apporter une nouvelle compréhension du processus de duplication des centrioles et a contribué au développement de nouveaux outils de recherche de ce processus. Summary : Centrosomes contain a pair of centrioles surrounded by pericentriolar material (PCM) and serve as the main microtubule organizing centers (MTOCs) of most animal cells. Just like the DNA, the single centrosome present early in the cell cycle duplicates once and only once to give rise to two centrosomes which will then direct assembly of a bipolar spindle. Centrosome duplication must be precisely regulated because the presence of either one or more than two centrosomes can lead to the assembly of an aberrant spindle, chromosome missegregation and aneuploidy. Although duplication of centrioles is key for that of the entire centrosome, the mechanisms underlying centriole formation are poorly understood and represent an important question in cell biology. In this thesis, we focused on the analysis of HsSAS-6. We found that this protein is required for centriole formation and that it is localized specifically at the base of newly forming centrioles. The levels of HsSAS-6 oscillate across the cell cycle. The protein is absent during G1, starts to accumulate at the centriole and in the cytoplasm at the onset of S phase and disappears abruptly during anaphase when it is targeted for 26S proteasome dependent degradation probably by the APC/CCdh1. Importantly, overexpression of HsSAS-6 leads to the formation of multiple centrioles instead of just one, indicating that levels of HsSAS-6 determine the number of centrioles at each cell cycle. Besides HsSAS-6 that is the main focus of this thesis, we have also investigated the C. elegans mutant strain sas-2, which sometimes assembles a multipolar spindle in the one cell stage embryo. We have shown that this phenotype derives from the presence of multiple centrioles in sperm cells. Moreover, we prepared a set of Gateway compatible vectors for fast generation of human cell lines with inducible protein expression. Finally, we started to develop an assay for centriole duplication that can be used in a high throughput setting for screening of human siRNA libraries. Taken together, our work brought novel insights into the process of centriole duplication and lead to the development of new tools for further investigation of this process.

Categorization of bibsonomy tags to apply privacy-preserving mechanisms.

In this technical report, we approach one of the practical aspects when it comes to represent users' interests from their tagging activity, namely the categorization of tags into high-level categories of interest. The reason is that the representation of user profiles on the basis of the myriad of tags available on the Web is certainly unfeasible from various practical perspectives; mainly concerningthe unavailability of data to reliably, accurately measure interests across such fine-grained categorization, and, should the data be available, its overwhelming computational intractability. Motivated by this, our study presents the results of a categorization process whereby a collection of tags posted at BibSonomy #http://www.bibsonomy.org# are classified into 5 categories of interest. The methodology used to conduct such categorization is in line with other works in the field.

Mechanisms of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight

Pseudomonas fluorescens EPS62e was selected during a screening procedure for its high efficacy in controlling infections by Erwinia amylovora, the causal agent of fire blight disease, on different plant materials. In field trials carried out in pear trees during bloom, EPS62e colonized flowers until the carrying capacity, providing a moderate efficacy of fire-blight control. The putative mechanisms of EPS62e antagonism against E. amylovora were studied. EPS62e did not produce antimicrobial compounds described in P. fluorescens species and only developed antagonism in King’s B medium, where it produced siderophores. Interaction experiments in culture plate wells including a membrane filter, which physically separated the cultures, confirmed that inhibition of E. amylovora requires cell-to-cell contact. The spectrum of nutrient assimilation indicated that EPS62e used significantly more or different carbon sources than the pathogen. The maximum growth rate and affinity for nutrients in immature fruit extract were higher in EPS62e than in E. amylovora, but the cell yield was similar. The fitness of EPS62e and E. amylovora was studied upon inoculation in immature pear fruit wounds and hypanthia of intact flowers under controlled-environment conditions. When inoculated separately, EPS62e grew faster in flowers, whereas E. amylovora grew faster in fruit wounds because of its rapid spread to adjacent tissues. However, in preventive inoculations of EPS62e, subsequent growth of EPS101 was significantly inhibited. It is concluded that cell-to-cell interference as well as differences in growth potential and the spectrum and efficiency of nutrient use are mechanisms of antagonism of EPS62e against E. amylovora

Vascular dysfunction in children conceived by assisted reproductive technologies: underlying mechanisms and future implications.

Epidemiological studies in humans have demonstrated a relationship between pathological events during fetal development and increased cardiovascular risk later in life and have led to the so called "Fetal programming of cardiovascular disease hypothesis". The recent observation of generalised vascular dysfunction in young apparently healthy children conceived by assisted reproductive technologies (ART) provides a novel and potentially very important example of this hypothesis. This review summarises recent data in ART children demonstrating premature subclinical atherosclerosis in the systemic circulation and pulmonary vascular dysfunction predisposing to exaggerated hypoxia-induced pulmonary hypertension. These problems appear to be related to the ART procedure per se. Studies in ART mice demonstrating premature vascular aging and arterial hypertension further demonstrate the potential of ART to increase cardiovascular risk and have allowed to unravel epigenetic alterations of the eNOS gene as an underpinning mechanism. The roughly 25% shortening of the life span in ART mice challenged with a western style high-fat-diet demonstrates the potential importance of these alterations for the long-term outcome. Given the young age of the ART population, data on cardiovascular endpoints will not be available before 20 to 30 years from now. However, already now cohort studies of the ART population are needed to early detect cardiovascular alterations with the aim to prevent or at least optimally treat cardiovascular complications. Finally, a debate needs to be engaged on the future of ART and the consequences of its exponential growth for public health.

Introduced ant species and mechanisms of competition on Floreana Island (Galapagos, Ecuador) (Hymenoptera : Formicidae)

Simultaneous presence of several tramp ant species of relatively recent introduction on a remote island is an excellent opportunity to study competition mechanisms that lead to the establishment of invasive species. Using attractive food baits we collected 14 ant species among which 10 are well-known tramp species. The most important change between 1996-97 and 2003 is the spread of the tropical fire ant Solenopsis geminata at the detriment of Tetramorium simillimum, suggesting that the colonization process on Floreana is still very dynamic. The follow-up of 400 food baits for 21 hours permitted us to calculate indices of competition abilities for 11 species, revealing distinct strategies. The two small tramp species Monomorium floricola and Tapinoma melanocephalum are typically opportunists when large-sized Odontomachus bauri (possibly native species) and Camponotus macilentus (endemic species) are good interference competitors, out-competing other species at food baits. Dominant species S. geminata and Monomorium destructor reach high scores for all indices due to their high abundance.

Allergen-specific antibody and cytokine responses, mast cell reactivity and intestinal permeability upon oral challenge of sensitized and tolerized mice.

BACKGROUND: Food allergy has reached an epidemic level in westernized countries and although central mechanisms have been described, the variability associated with genetic diversity underscores the still unresolved complexity of these disorders. OBJECTIVE: To develop models of food allergy and oral tolerance, both strictly induced by the intestinal route, and to compare antigen-specific responses. METHODS: BALB/c mice were mucosally sensitized to ovalbumin (OVA) in the presence of the mucosal adjuvant cholera toxin, or tolerized by intra-gastric administrations of OVA alone. Antibody titres and cytokines were determined by ELISA, and allergic status was determined through several physiologic parameters including decline in temperature, diarrhoea, mast cell degranulation and intestinal permeability. RESULTS: OVA-specific antibodies (IgE, IgGs and IgA in serum and feces) were produced in sensitized mice exclusively. Upon intra-gastric challenge with OVA, sensitized mice developed anaphylactic reactions associated with a decline of temperature, diarrhoea, degranulation of mast cells, which were only moderately recruited in the small intestine, and increased intestinal permeability. Cytokines produced by immune cells from sensitized mice included T-helper type 2 cytokines (IL-5, IL-13), but also IL-10, IFN-gamma and IL-17. In contrast, all markers of allergy were totally absent in tolerized animals, and yet the latter were protected from subsequent sensitization, demonstrating that oral tolerance took place efficiently. CONCLUSION: This work allows for the first time an appropriate comparison between sensitized and tolerized BALB/c mice towards OVA. It highlights important differences from other models of allergy, and thus questions some of the generally accepted notions of allergic reactions, such as the protective role of IFN-gamma, the importance of antigen-specific secretory IgA and the role of mucosal mast cells in intestinal anaphylaxis. In addition, it suggests that IL-17 might be an effector cytokine in food allergy. Finally, it demonstrates that intestinal permeability towards the allergen is increased during challenge.

Molecular chaperones and associated cellular clearance mechanisms against toxic protein conformers in Parkinson's disease.

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder marked by the loss of dopaminergic neurons (in particular in the substantia nigra) causing severe impairment of movement coordination and locomotion, associated with the accumulation of aggregated α-synuclein (α-Syn) into proteinaceous inclusions named Lewy bodies. Various early forms of misfolded α-Syn oligomers are cytotoxic. Their formation is favored by mutations and external factors, such as heavy metals, pesticides, trauma-related oxidative stress and heat shock. Here, we discuss the role of several complementing cellular defense mechanisms that may counteract PD pathogenesis, especially in youth, and whose effectiveness decreases with age. Particular emphasis is given to the 'holdase' and 'unfoldase' molecular chaperones that provide cells with potent means to neutralize and scavenge toxic protein conformers. Because chaperones can specifically recognize misfolded proteins, they are key specificity factors for other cellular defenses, such as proteolysis by the proteasome and autophagy. The efficiency of the cellular defenses decreases in stressed or aging neurons, leading to neuroinflammation, apoptosis and tissue loss. Thus, drugs that can upregulate the molecular chaperones, the ubiquitin-proteasome system and autophagy in brain tissues are promising avenues for therapies against PD and other mutation-, stress- or age-dependent protein-misfolding diseases.

Selective neurodegeneration induced in rotation-mediated aggregate cell cultures by a transient switch to stationary culture conditions: a potential model to study ischemia-related pathogenic mechanisms.

Aggregating brain cell cultures at an advanced maturational stage (20-21 days in vitro) were subjected for 1-3 h to anaerobic (hypoxic) and/or stationary (ischemic) conditions. After restoration of the normal culture conditions, cell loss was estimated by measuring the release of lactate dehydrogenase as well as the irreversible decrease of cell type-specific enzyme activities, total protein and DNA content. Ischemia for 2 h induced significant neuronal cell death. Hypoxia combined with ischemia affected both neuronal and glial cells to different degrees (GABAergic neurons>cholinergic neurons>astrocytes). Hypoxic and ischemic conditions greatly stimulated the uptake of 2-deoxy-D-glucose, indicating increased glucose consumption. Furthermore, glucose restriction (5.5 mM instead of 25 mM) dramatically increased the susceptibility of neuronal and glial cells to hypoxic and ischemic conditions. Glucose media concentrations below 2 mM caused selective neuronal cell death in otherwise normal culture conditions. GABAergic neurons showed a particularly high sensitivity to glucose restriction, hypoxia, and ischemia. The pattern of ischemia-induced changes in vitro showed many similarities to in vivo findings, suggesting that aggregating brain cell cultures provide a useful in vitro model to study pathogenic mechanisms related to brain ischemia.

PPARs in diseases: control mechanisms of inflammation.

The three isotypes of peroxisome proliferator-activated receptors (PPARs), PPARalpha, beta/delta and gamma, are ligand-inducible transcription factors that belong to the nuclear hormone receptor family. PPARs are implicated in the control of inflammatory responses and in energy homeostasis and thus, can be defined as metabolic and anti-inflammatory transcription factors. They exert their anti-inflammatory effects by inhibiting the induction of pro-inflammatory cytokines, adhesion molecules and extracellular matrix proteins or by stimulating the production of anti-inflammatory molecules. Furthermore, PPARs modulate the proliferation, differentiation and survival of immune cells including macrophages, B cells and T cells. This review discusses the molecular mechanisms by which PPARs and their ligands modulate the inflammatory response. In addition, it presents recent developments implicating PPAR specific ligands in potential treatments of inflammation-related diseases, such as atherosclerosis, inflammatory bowel diseases, Parkinson's and Alzheimer's diseases.

Cellular and molecular mechanisms underlying the effects of sleep loss on hippocampal synaptic plasticity

SUMMARY : The function of sleep for the organism is one of the most persistent and perplexing questions in biology. Current findings lead to the conclusion that sleep is primarily for the brain. In particular, a role for sleep in cognitive aspects of brain function is supported by behavioral evidence both in humans and animals. However, in spite of remarkable advancement in the understanding of the mechanisms underlying sleep generation and regulation, it has been proven difficult to determine the neurobiological mechanisms underlying the beneficial effect of sleep, and the detrimental impact of sleep loss, on learning and memory processes. In my thesis, I present results that lead to several critical steps forward in the link between sleep and cognitive function. My major result is the molecular identification and physiological analysis of a protein, the NR2A subunit of NMDA receptor (NMDAR), that confers sensitivity to sleep loss to the hippocampus, a brain structure classically involved in mnemonic processes. Specifically, I used a novel behavioral approach to achieve sleep deprivation in adult C57BL6/J mice, yet minimizing the impact of secondary factors associated with the procedure,.such as stress. By using in vitro electrophysiological analysis, I show, for the first time, that sleep loss dramatically affects bidirectional plasticity at CA3 to CA1 synapses in the hippocampus, a well established cellular model of learning and memory. 4-6 hours of sleep loss elevate the modification threshold for bidirectional synaptic plasticity (MT), thereby promoting long-term depression of CA3 to CA 1 synaptic strength after stimulation in the theta frequency range (5 Hz), and rendering long-term potentiation induction.more difficult. Remarkably, 3 hours of recovery sleep, after the deprivation, reset the MT at control values, thus re-establishing the normal proneness of synapses to undergo long-term plastic changes. At the molecular level, these functional changes are paralleled by a change in the NMDAR subunit composition. In particular, the expression of the NR2A subunit protein of NMDAR at CA3 to CA1 synapses is selectively and rapidly increased by sleep deprivation, whereas recovery sleep reset NR2A synaptic content to control levels. By using an array of genetic, pharmacological and computational approaches, I demonstrate here an obligatory role for NR2A-containing NMDARs in conveying the effect of sleep loss on CA3 to CAl MT. Moreover, I show that a genetic deletion of the NR2A subunit fully preserves hippocampal plasticity from the impact of sleep loss, whereas it does not alter sleepwake behavior and homeostatic response to sleep deprivation. As to the mechanism underlying the effects of the NR2A subunit on hippocampal synaptic plasticity, I show that the increased NR2A expression after sleep loss distinctly affects the contribution of synaptic and more slowly recruited NMDAR pools activated during plasticity-induction protocols. This study represents a major step forward in understanding the mechanistic basis underlying sleep's role for the brain. By showing that sleep and sleep loss affect neuronal plasticity by regulating the expression and function of a synaptic neurotransmitter receptor, I propose that an important aspect of sleep function could consist in maintaining and regulating protein redistribution and ion channel trafficking at central synapses. These findings provide a novel starting point for investigations into the connections between sleep and learning, and they may open novel ways for pharmacological control over hippocampal .function during periods of sleep restriction. RÉSUMÉ DU PROJET La fonction du sommeil pour l'organisme est une des questions les plus persistantes et difficiles dans la biologie. Les découvertes actuelles mènent à la conclusion que le sommeil est essentiel pour le cerveau. En particulier, le rôle du sommeil dans les aspects cognitifs est soutenu par des études comportementales tant chez les humains que chez les animaux. Cependant, malgré l'avancement remarquable dans la compréhension des mécanismes sous-tendant la génération et la régulation du sommeil, les mécanismes neurobiologiques qui pourraient expliquer l'effet favorable du sommeil sur l'apprentissage et la mémoire ne sont pas encore clairs. Dans ma thèse, je présente des résultats qui aident à clarifier le lien entre le sommeil et la fonction cognitive. Mon résultat le plus significatif est l'identification moléculaire et l'analyse physiologique d'une protéine, la sous-unité NR2A du récepteur NMDA, qui rend l'hippocampe sensible à la perte de sommeil. Dans cette étude, nous avons utilisé une nouvelle approche expérimentale qui nous a permis d'induire une privation de sommeil chez les souris C57BL6/J adultes, en minimisant l'impact de facteurs confondants comme, par exemple, le stress. En utilisant les techniques de l'électrophysiologie in vitro, j'ai démontré, pour la première fois, que la perte de sommeil est responsable d'affecter radicalement la plasticité bidirectionnelle au niveau des synapses CA3-CA1 de l'hippocampe. Cela correspond à un mécanisme cellulaire de l'apprentissage et de la mémoire bien établi. En particulier, 4-6 heures de privation de sommeil élèvent le seuil de modification pour la plasticité synaptique bidirectionnelle (SM). Comme conséquence, la dépression à long terme de la transmission synaptique est induite par la stimulation des fibres afférentes dans la bande de fréquences thêta (5 Hz), alors que la potentialisation à long terme devient plus difficile. D'autre part, 3 heures de sommeil de récupération sont suffisant pour rétablir le SM aux valeurs contrôles. Au niveau moléculaire, les changements de la plasticité synaptiques sont associés à une altération de la composition du récepteur NMDA. En particulier, l'expression synaptique de la protéine NR2A du récepteur NMDA est rapidement augmentée de manière sélective par la privation de sommeil, alors que le sommeil de récupération rétablit l'expression de la protéine au niveau contrôle. En utilisant des approches génétiques, pharmacologiques et computationnelles, j'ai démontré que les récepteurs NMDA qui expriment la sous-unité NR2A sont responsables de l'effet de la privation de sommeil sur le SM. De plus, nous avons prouvé qu'une délétion génétique de la sous-unité NR2A préserve complètement la plasticité synaptique hippocampale de l'impact de la perte de sommeil, alors que cette manipulation ne change pas les mécanismes de régulation homéostatique du sommeil. En ce qui concerne les mécanismes, j'ai .découvert que l'augmentation de l'expression de la sous-unité NR2A au niveau synaptique modifie les propriétés de la réponse du récepteur NMDA aux protocoles de stimulations utilisés pour induire la plasticité. Cette étude représente un pas en avant important dans la compréhension de la base mécaniste sous-tendant le rôle du sommeil pour le cerveau. En montrant que le sommeil et la perte de sommeil affectent la plasticité neuronale en régulant l'expression et la fonction d'un récepteur de la neurotransmission, je propose qu'un aspect important de la fonction du sommeil puisse être finalisé au règlement de la redistribution des protéines et du tracking des récepteurs aux synapses centraux. Ces découvertes fournissent un point de départ pour mieux comprendre les liens entre le sommeil et l'apprentissage, et d'ailleurs, ils peuvent ouvrir des voies pour des traitements pharmacologiques dans le .but de préserver la fonction hippocampale pendant les périodes de restriction de sommeil.

Genome-wide screen of genes required for caffeine tolerance in fission yeast

Background: An excess of caffeine is cytotoxic to all eukaryotic cell types. We aim to study how cells become tolerant to atoxic dose of this drug, and the relationship between caffeine and oxidative stress pathways.Methodology/Principal Findings: We searched for Schizosaccharomyces pombe mutants with inhibited growth on caffeinecontainingplates. We screened a collection of 2,700 haploid mutant cells, of which 98 were sensitive to caffeine. The genes mutated in these sensitive clones were involved in a number of cellular roles including the H2O2-induced Pap1 and Sty1 stress pathways, the integrity and calcineurin pathways, cell morphology and chromatin remodeling. We have investigated the role of the oxidative stress pathways in sensing and promoting survival to caffeine. The Pap1 and the Sty1 pathways are both required for normal tolerance to caffeine, but only the Sty1 pathway is activated by the drug. Cells lacking Pap1 aresensitive to caffeine due to the decreased expression of the efflux pump Hba2. Indeed, ?hba2 cells are sensitive to caffeine, and constitutive activation of the Pap1 pathway enhances resistance to caffeine in an Hba2-dependent manner. Conclusions/Significance: With our caffeine-sensitive, genome-wide screen of an S. pombe deletion collection, we havedemonstrated the importance of some oxidative stress pathway components on wild-type tolerance to the drug.

Student modeling based on fuzzy inference mechanisms

The paper presents a competence-based instructional design system and a way to provide a personalization of navigation in the course content. The navigation aid tool builds on the competence graph and the student model, which includes the elements of uncertainty in the assessment of students. An individualized navigation graph is constructed for each student, suggesting the competences the student is more prepared to study. We use fuzzy set theory for dealing with uncertainty. The marks of the assessment tests are transformed into linguistic terms and used for assigning values to linguistic variables. For each competence, the level of difficulty and the level of knowing its prerequisites are calculated based on the assessment marks. Using these linguistic variables and approximate reasoning (fuzzy IF-THEN rules), a crisp category is assigned to each competence regarding its level of recommendation.