Integrating receptor interactions and dynamics and Interference with endocrine disruptors in the mechanisms of action of PPAR nuclear receptors

Abstract Peroxisome Proliferator-Activated Receptors (PPARs) form a family of three nuclear receptors regulating important cellular and metabolic functions. PPARs control gene expression by directly binding to target promoters as heterodimers with the Retinoid X Receptor (RXR), and their transcriptional activity is enhanced upon activation by natural or pharmacological ligands. The binding of PPAR/RXR heterodimers on target promoters allows the anchoring of a series of coactivators and corepressors involved in promoter remodeling and the recruitment of the transcription machinery. The transcriptional output finally depends on a complex interplay between (i) the respective expression levels of PPARs, RXRs and of other nuclear receptors competing for DNA binding and RXR recruitment, (ii) the availability and the nature of PPAR and RXR ligands, (iii) the expression levels and the nature of the different coactivators and corepressors and (iv) the sequence and the epigenetic status of the promoter. Understanding how all these factors and signals integrate and fine-tune transcription remains a challenge but is necessary to understand the specificity of the physiological functions regulated by PPARs. The work presented herein focuses on the molecular mechanisms of PPAR action and aims at understanding how the interactions and mobility of the receptor modulate transcription in the physiological context of a living cell: Such observations in vivo rely on the use of engineered fluorescent protein chimeras and require the development and the application of complementary imaging techniques such as Fluorescence Recovery After Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS). Using such techniques, PPARs are shown to reside solely in the nucleus where they are constitutively associated with RXR but transcriptional activation by ligand binding -does not promote the formation of sub-nuclear structures as observed with other nuclear receptors. In addition, the engagement of unliganded PPARs in large complexes of cofactors in living cells provides a molecular basis for their ligand-independent activity. Ligand binding reduces receptor diffusion by promoting the recruitment of coactivators which further enlarge the size of PPAR complexes to acquire full transcriptional competence. Using these molecular approaches, we deciphered the molecular mechanisms through which phthalates, a class of pollutants from the plastic industry, interfere with PPARγ signaling. Mono-ethyl-hexyl-phthalate (MEHP) binding induces the recruitment of a specific subset of cofactors and translates into the expression of a specific subset of target genes, the transcriptional output being strongly conditioned by the differentiation status of the cell. This selective PPARγ modulation induces limited adipogenic effects in cellular models while exposure to phthalates in animal models leads to protective effects on glucose tolerance and diet-induced obesity. These results demonstrate that phthalates influence lipid and carbohydrate metabolism through complex mechanisms which most likely involve PPARγ but also probably PPARα and PPARß, Altogether, the molecular and physiological demonstration of the interference of pollutants with PPAR action outlines an important role of chemical exposure in metabolic regulations. Résumé Les PPARs (Peroxisome Proliferator-Activated Receptors) forment une famille de récepteurs nucléaires qui régulent des fonctions cellulaires et métaboliques importantes. Les PPARs contrôlent l'expression des gènes en se liant directement à leurs promoteurs sous forme d'hétérodimères avec les récepteurs RXR (Retinoid X Receptor), et leur activité transcriptionnelle est stimulée par la liaison de ligands naturels ou pharmacologiques. L'association des hétérodimères PPAR/RXR avec les promoteurs des gènes cibles permet le recrutement de coactivateurs et de corépresseurs qui vont permettre le remodelage de la chromatine et le recrutement de la machinerie transcriptionnelle. Les actions transcriptionnelles du récepteur dépendent toutefois d'interactions complexes qui sont régulées par (i) le niveau d'expression des PPARs, des RXRs et d'autres récepteurs nucléaires entrant en compétition pour la liaison à l'ADN et l'association avec RXR, (ii) la disponibilité et la nature de ligands de PPAR et de RXR, (iii) les niveaux d'expression et la nature des différents coactivateurs et corépresseurs et (iv) la séquence et le marquage épigénétique des promoteurs. La compréhension des mécanismes qui permettent d'intégrer ces aspects pour assurer une régulation fine de l'activité transcriptionnelle est un défi qu'il est nécessaire de relever pour comprendre la spécificité des fonctions physiologiques régulées par les PPARs. Ce travail concerne l'étude des mécanismes d'action moléculaire des PPARs et vise à mieux comprendre comment les interactions du récepteur avec d'autres protéines ainsi que la mobilité de ce dernier régulent son activité transcriptionnelle dans le contexte physiologique des cellules vivantes. De telles observations reposent sur l'emploi de protéines fusionnées à des protéines fluorescentes ainsi que sur le développement et l'utilisation de techniques d'imagerie complémentaires telles que le FRAP (Fluorescence Recovery After Photobleaching), le FRET (Fluorescence Resonance Energy Transfer) ou la FCS (Fluorescence Corrélation Spectroscopy). En appliquant ces méthodes, nous avons pu montrer que les PPARs résident toujours dans le noyau où ils sont associés de manière constitutive à RXR, mais que l'ajout de ligand n'induit pas la formation de structures sub-nucléaires comme cela a pu être décrit pour d'autres récepteurs nucléaires. De plus, les PPARs sont engagés dans de larges complexes protéiques de cofacteurs en absence de ligand, ce qui procure une explication moléculaire à leur activité ligand-indépendante. La liaison du ligand réduit la vitesse de diffusion du récepteur en induisant le recrutement de coactivateurs qui augmente encore plus la taille des complexes afin d'acquérir un potentiel d'activation maximal. En utilisant ces approches moléculaires, nous avons pu caractériser les mécanismes permettant aux phtalates, une classe de polluants provenant de l'industrie plastique, d'interférer avec PPARγ. La liaison du mono-ethyl-hexyl-phtalate (NERF) à PPARγ induit un recrutement sélectif de cofacteurs, se traduisant par l'induction spécifique d'un sous-ensemble de gènes qui varie en fonction du niveau de différentiation cellulaire. La modulation sélective de PPARγ par le MEHP provoque une adipogenèse modérée dans des modèles cellulaires alors que l'exposition de modèles animaux aux phtalates induit des effets bénéfiques sur la tolérance au glucose et sur le développement de l'obésité. Toutefois, les phtalates ont une action complexe sur le métabolisme glucido-lipidique en faisant intervenir PPARγ mais aussi probablement PPARα et PPARß. Cette démonstration moléculaire et physiologique de l'interférence des polluants avec les récepteurs nucléaires PPAR souligne un rôle important de l'exposition à de tels composés dans les régulations métaboliques.

Biogeochemical weathering in sedimentary chronosequences of the Rhône and Oberaar Glaciers (Swiss Alps): rates and mechanisms of biotite weathering

We analysed the composition of phyllosilicate minerals in sediments deposited by the Rhone and Oberaar glaciers (Swiss Alps), in order to identify processes and rates of biogeochemical weathering in relation to glacial erosion. The investigated sediments are part of chronosequences consisting of (A) suspended, "fresh" sediment in melt water; (B) terminal moraines from the Little Ice Age (LIA; approximately 1560-1850); and (C) tilts of the Younger Dryas interval (YD; approximately 11'500y BP). Secondary weathering products associated with the suspended sediment have not been observed: we therefore exclude intermittent subglacial storage and weathering of this material and assume that the suspended sediment is directly derived from mechanically abraded bedrock. This implies that biogeochemical weathering processes started once the glacially-derived sediment was deposited in the proglacial area. The combination of a developing vegetation cover, the generally high permeability allowing the percolation of precipitation, and the chemical reactivity related to the dominance of fine-grained material (<63 pm) drives the weathering process and the initial Umbrepts present in LIA profiles undergo podzolisation and lead to the formation of Humods observed in YD profiles. Systematic XRD analyses of these chronosequences show a progressive decrease in biotite contents and a concomitant increase in pedogenically formed vermiculite with increasing sediment age. Biotite contents decrease by 25-50% in the upper 30 cm of the moraines after 145-275 yr in the proglacial environment. Biotite weathering rates are calculated using the difference in the biotite content between unweathered and weathered glacial sediments within the investigated profiles. The reactive mineral surface area is estimated geometrically, both with regards to the total relative surface (WRT) as well as to the relative edge surface (WRE). WRT Biotite weathering rates are estimated as 10(-13)-10-(15) mol(biotite) m(biotite)(-2) s(-1). WRE Biotite weathering rates are on the order of 10(-13)-10(-14) mol(biotite) m(biotite)(-2) s(-1). Biotite weathering rates obtained by this study are in the order of one magnitude higher in comparison to other published field-based weathering rates. Using biotite as an indicator, we therefore suggest that glacially-derived material in the area of the Oberaar and Rhone glaciers is generally subjected to enhanced biogeochemical weathering, starting immediately after deposition in the proglacial zone and subsequently continuing for thousands of years after glacier retreat.

Mechanisms of angiogenesis impairment by radiation therapy

Summary : Clinical evidence indicates that tumors recurring within previously irradiated fields are highly invasive and metastatic, suggesting a role of the tumor stroma in this effect. Angiogenesis plays a critical role in tumor progression. Ionizing radiation is known to induce apoptosis of angiogenic endothelial cells, while the effect on quiescent endothelial cells and de novo angiogenesis is not well characterized. We recently observed that irradiation of normal tissue prevents tumor- and growth factor-induced angiogenesis. The main aim of my thesis work was to characterize the mechanisms of radiation-mediated inhibition of angiogenesis. To this purpose we used a combination of in vivo and ex vivo studies on irradiated healthy tissue, and in vitro irradiation experiments using angiogenesis models and isolated endothelial cells. We found that irradiation did not induce endothelial cell apoptosis and did not disrupt quiescent vessels within irradiated skin. Radiation reduced the recruitment of leukocytes to angiogenic Matrigel plugs, but this effect was rather secondary to decreased angiogenesis, as exogenous addition of leucocytes to Matrigel plugs did not rescue the angiogenesis defects. To ascertain the direct effect of radiation on endothelial cells, we used the mouse aortic ring assay to test the sprouting capacity of irradiated endothelial cells ex vivo and in vitro, and found that irradiation completely suppressed endothelial cell sprouting. Using HUVEC cells, we showed that irradiation of quiescent confluent endothelial cells did not induce cell death but suppressed subsequent migration and cell proliferation and induced senescence. By Western blotting, we observed a rapid and sustained increase in p21 levels, previously shown to be activated by p53 in response to double strand break, and mediating senescence in human cells. Current experiments focus on the mechanism of sustained p21 upregulation and its role in reduced migration. Inhibition of endothelial cell migration and proliferation by radiation may explain reduced angiogenesis in tumors growing in previously irradiated fields.

Interregional compensatory mechanisms of motor functioning in progressing preclinical neurodegeneration.

Understanding brain reserve in preclinical stages of neurodegenerative disorders allows determination of which brain regions contribute to normal functioning despite accelerated neuronal loss. Besides the recruitment of additional regions, a reorganisation and shift of relevance between normally engaged regions are a suggested key mechanism. Thus, network analysis methods seem critical for investigation of changes in directed causal interactions between such candidate brain regions. To identify core compensatory regions, fifteen preclinical patients carrying the genetic mutation leading to Huntington's disease and twelve controls underwent fMRI scanning. They accomplished an auditory paced finger sequence tapping task, which challenged cognitive as well as executive aspects of motor functioning by varying speed and complexity of movements. To investigate causal interactions among brain regions a single Dynamic Causal Model (DCM) was constructed and fitted to the data from each subject. The DCM parameters were analysed using statistical methods to assess group differences in connectivity, and the relationship between connectivity patterns and predicted years to clinical onset was assessed in gene carriers. In preclinical patients, we found indications for neural reserve mechanisms predominantly driven by bilateral dorsal premotor cortex, which increasingly activated superior parietal cortices the closer individuals were to estimated clinical onset. This compensatory mechanism was restricted to complex movements characterised by high cognitive demand. Additionally, we identified task-induced connectivity changes in both groups of subjects towards pre- and caudal supplementary motor areas, which were linked to either faster or more complex task conditions. Interestingly, coupling of dorsal premotor cortex and supplementary motor area was more negative in controls compared to gene mutation carriers. Furthermore, changes in the connectivity pattern of gene carriers allowed prediction of the years to estimated disease onset in individuals. Our study characterises the connectivity pattern of core cortical regions maintaining motor function in relation to varying task demand. We identified connections of bilateral dorsal premotor cortex as critical for compensation as well as task-dependent recruitment of pre- and caudal supplementary motor area. The latter finding nicely mirrors a previously published general linear model-based analysis of the same data. Such knowledge about disease specific inter-regional effective connectivity may help identify foci for interventions based on transcranial magnetic stimulation designed to stimulate functioning and also to predict their impact on other regions in motor-associated networks.

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.

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.

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.

Identification of novel elements of the Drosophila blisterome sheds light on potential pathological mechanisms of several human diseases.

Main developmental programs are highly conserved among species of the animal kingdom. Improper execution of these programs often leads to progression of various diseases and disorders. Here we focused on Drosophila wing tissue morphogenesis, a fairly complex developmental program, one of the steps of which - apposition of the dorsal and ventral wing sheets during metamorphosis - is mediated by integrins. Disruption of this apposition leads to wing blistering which serves as an easily screenable phenotype for components regulating this process. By means of RNAi-silencing technique and the blister phenotype as readout, we identify numerous novel proteins potentially involved in wing sheet adhesion. Remarkably, our results reveal not only participants of the integrin-mediated machinery, but also components of other cellular processes, e.g. cell cycle, RNA splicing, and vesicular trafficking. With the use of bioinformatics tools, these data are assembled into a large blisterome network. Analysis of human orthologues of the Drosophila blisterome components shows that many disease-related genes may contribute to cell adhesion implementation, providing hints on possible mechanisms of these human pathologies.

Making sense of AMPA receptor trafficking by modeling molecular mechanisms of synaptic plasticity.

Synaptic plasticity involves a complex molecular machinery with various protein interactions but it is not yet clear how its components give rise to the different aspects of synaptic plasticity. Here we ask whether it is possible to mathematically model synaptic plasticity by making use of known substances only. We present a model of a multistable biochemical reaction system and use it to simulate the plasticity of synaptic transmission in long-term potentiation (LTP) or long-term depression (LTD) after repeated excitation of the synapse. According to our model, we can distinguish between two phases: first, a "viscosity" phase after the first excitation, the effects of which like the activation of NMDA receptors and CaMKII fade out in the absence of further excitations. Second, a "plasticity" phase actuated by an identical subsequent excitation that follows after a short time interval and causes the temporarily altered concentrations of AMPA subunits in the postsynaptic membrane to be stabilized. We show that positive feedback is the crucial element in the core chemical reaction, i.e. the activation of the short-tail AMPA subunit by NEM-sensitive factor, which allows generating multiple stable equilibria. Three stable equilibria are related to LTP, LTD and a third unfixed state called ACTIVE. Our mathematical approach shows that modeling synaptic multistability is possible by making use of known substances like NMDA and AMPA receptors, NEM-sensitive factor, glutamate, CaMKII and brain-derived neurotrophic factor. Furthermore, we could show that the heteromeric combination of short- and long-tail AMPA receptor subunits fulfills the function of a memory tag.

Mechanisms of proteasome inhibitor-induced cytotoxicity in malignant glioma.

The 26S proteasome constitutes an essential degradation apparatus involved in the consistent recycling of misfolded and damaged proteins inside cells. The aberrant activation of the proteasome has been widely observed in various types of cancers and implicated in the development and progression of carcinogenesis. In the era of targeted therapies, the clinical use of proteasome inhibitors necessitates a better understanding of the molecular mechanisms of cell death responsible for their cytotoxic action, which are reviewed here in the context of sensitization of malignant gliomas, a tumor type particularly refractory to conventional treatments.

Mechanisms of Symptomatic Spinal Cord Ischemia After TEVAR: Insights From the European Registry of Endovascular Aortic Repair Complications (EuREC).

Abstract Purpose: To test the hypothesis that simultaneous closure of at least 2 independent vascular territories supplying the spinal cord and/or prolonged hypotension may be associated with symptomatic spinal cord ischemia (SCI) after thoracic endovascular aortic repair (TEVAR). Methods: A pattern matching algorithm was used to develop a risk model for symptomatic SCI using a prospective 63-patient single-center cohort to test the positive predictive value (PPV) of prolonged intraoperative hypotension and/or simultaneous closure of at least 2 of 4 the vascular territories supplying the spinal cord (left subclavian, intercostal, lumbar, and hypogastric arteries). This risk model was then applied to data extracted from the multicenter European Registry on Endovascular Aortic Repair Complications (EuREC). Between 2002 and 2010, the 19 centers participating in EuREC reported 38 (1.7%) cases of symptomatic spinal cord ischemia among the 2235 patients in the database. Results: In the single-center cohort, direct correlations were seen between the occurrence of symptomatic SCI and both prolonged intraoperative hypotension (PPV 1.00, 95% CI 0.22 to 1.00, p = 0.04) and simultaneous closure of at least 2 independent spinal cord vascular territories (PPV 0.67, 95% CI 0.24 to 0.91, p = 0.005). Previous closure of a single vascular territory was not associated with an increased risk of symptomatic spinal cord ischemia (PPV 0.07, 95% CI 0.01 to 0.16, p = 0.56). The combination of prolonged hypotension and simultaneous closure of at least 2 territories exhibited the strongest association (PPV 0.75, 95% CI 0.38 to 0.75, p<0.0001). Applying the model to the entire EuREC cohort found an almost perfect agreement between the predicted and observed risk factors (kappa 0.77, 95% CI 0.65 to 0.90). Conclusion: Extensive coverage of intercostal arteries alone by a thoracic stent-graft is not associated with symptomatic SCI; however, simultaneous closure of at least 2 vascular territories supplying the spinal cord is highly relevant, especially in combination with prolonged intraoperative hypotension. As such, these results further emphasize the need to preserve the left subclavian artery during TEVAR.