Study of interindividual variability in transcriptome data after stimulation

Ce travail de thèse concerne l’étude de la variabilité inter-individuelle du transcriptome soumis à un stimulus. Cette variabilité n’est pas homogène au sein même du transcriptome et varie suite aux perturbations extérieures.Nous avons mis en place une stratégie d’analyse de la variabilité inter-individuelle sur la base des indices de diversité. Nous avons sélectionné trois jeux de données ayant des caractéristiques particulières (stimulus fort, faible, cinétique, données appariées, …). Ces indices nous permettent de mettre en évidence les différences entre les individus; certains ayant des transcriptomes plus divers que d’autre. Ces différences s’attenues après l’application d’un stimulus au système : La variabilité de la diversité diminue. Nous mesurons cette diminution à l’aide d’une mesure de la similarité. Les résultats indiquent un phénomène global. Nous avons par la suite analysé les données sous le regard d’un indice de spécialisation. Les différences entre groupes témoins et groupes sous l’action d’un stimulus indiquent que la spécialisation diminue de manière significative après l’action du stimulus.Les informations obtenues sont différentes de celles obtenues par les stratégies d’analyses statistiques classiques. Nos travaux montrent enfin que ces outils permettent d’établir de nouvelles hypothèses. Ces indices, utilisés pour la première fois pour ce type d’analyse, ouvrent ainsi la voie à un nouvel arsenal d’outils d’analyse pour la compréhension des modifications transcriptomiques globales mais aussi individuelles et s’inscrit donc parfaitement dans le mouvement de la médecine personnalisée.This thesis concern the study of the interindividual variability of transcriptome after stimulation. This variability is not homogenous within the same transcriptome and varies with external stimulus.We designed an analysis strategy of the interindividual variability based on diversity indices. We selected three transcriptome datasets based on particular characteristics (strong stimulus, kinetic, paired data…). The indices highlight differences across the samples, some being more diverse than the others. The differences decrease after applying a stimulation to the samples. We measure this modification with a measure of similarity. The results depict a global effect of the stimulation.We analysed the data using specialisation measure. The samples’ specialisation significantly decrease after stimulation, meaning the interindividual variability decreases when the system is on the pressure of a stimulus.Noteworthy, these information are different from those obtained with classical analyses. Our work describe also that this strategy leads to new hyptotheses. These indices are used for these analyses for the first time and can be added to the statistical and mathematical tools already available for transcriptome analysis. They show their capability for highlighting global but also individual modifications and therefore fits perfectly into the field of personalized medicine

Étude de la variabilité inter-individuelle du transcriptome soumis à un stimulus

This thesis concern the study of the interindividual variability of transcriptome after stimulation. This variability is not homogenous within the same transcriptome and varies with external stimulus.We designed an analysis strategy of the interindividual variability based on diversity indices. We selected three transcriptome datasets based on particular characteristics (strong stimulus, kinetic, paired data…). The indices highlight differences across the samples, some being more diverse than the others. The differences decrease after applying a stimulation to the samples. We measure this modification with a measure of similarity. The results depict a global effect of the stimulation.We analysed the data using specialisation measure. The samples’ specialisation significantly decrease after stimulation, meaning the interindividual variability decreases when the system is on the pressure of a stimulus.Noteworthy, these information are different from those obtained with classical analyses. Our work describe also that this strategy leads to new hyptotheses. These indices are used for these analyses for the first time and can be added to the statistical and mathematical tools already available for transcriptome analysis. They show their capability for highlighting global but also individual modifications and therefore fits perfectly into the field of personalized medicine.Ce travail de thèse concerne l’étude de la variabilité inter-individuelle du transcriptome soumis à un stimulus. Cette variabilité n’est pas homogène au sein même du transcriptome et varie suite aux perturbations extérieures.Nous avons mis en place une stratégie d’analyse de la variabilité inter-individuelle sur la base des indices de diversité. Nous avons sélectionné trois jeux de données ayant des caractéristiques particulières (stimulus fort, faible, cinétique, données appariées, …). Ces indices nous permettent de mettre en évidence les différences entre les individus; certains ayant des transcriptomes plus divers que d’autre. Ces différences s’attenues après l’application d’un stimulus au système : La variabilité de la diversité diminue. Nous mesurons cette diminution à l’aide d’une mesure de la similarité. Les résultats indiquent un phénomène global. Nous avons par la suite analysé les données sous le regard d’un indice de spécialisation. Les différences entre groupes témoins et groupes sous l’action d’un stimulus indiquent que la spécialisation diminue de manière significative après l’action du stimulus.Les informations obtenues sont différentes de celles obtenues par les stratégies d’analyses statistiques classiques. Nos travaux montrent enfin que ces outils permettent d’établir de nouvelles hypothèses. Ces indices, utilisés pour la première fois pour ce type d’analyse, ouvrent ainsi la voie à un nouvel arsenal d’outils d’analyse pour la compréhension des modifications transcriptomiques globales mais aussi individuelles et s’inscrit donc parfaitement dans le mouvement de la médecine personnalisée

Regulatory T Cells Orchestrate Similar Immune Evasion of Fetuses and Tumors in Mice

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Statistical and computer modelling of T lymphocyte population's dynamics in mice through aging

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Distinct hepatitis C virus core and F protein quasispecies in tumoral and nontumoral hepatocytes isolated via microdissection

International audienceHepatitis C virus (HCV) genetic variability may be involved in liver carcinogenesis. We investigated HCV core and corresponding putative F protein genetic variability in hepatocellular carcinoma (HCC) and cirrhotic nodules. Hepatocyte clusters from 7 patients with HCC and HCV1b-related cirrhosis were isolated via microdissection of HCC tissues and 2 nontumoral cirrhotic nodules. The HCV core complementary DNA was cloned and sequenced from each liver compartment and from the serum of 2 patients. Nucleotide diversity and synonymous and nonsynonymous substitutions were analyzed within and between compartments via phylogenetic analysis and Mantel's test. Liver HCV RNA accumulation was lower in HCC. Increased quasispecies diversity and complexity was observed with HCC in 6 of 7 patients. Mantel's test demonstrated marked compartmentalization of quasispecies between HCC and cirrhotic nodules in all 7 patients and also between the 2 nontumoral nodules in 5 of them. Synonymous-nonsynonymous substitution analysis indicated low selection against tumoral core quasispecies in all patients and a more selective pressure against F protein quasispecies in all compartments. In the 2 subjects analyzed, HCC and nontumoral hepatocyte quasispecies were only minor or undetected in serum. Conclusion: In tumoral hepatocytes, low-replicating hepatitis C quasispecies are compartmentalized and more diversified and are subjected to low selective pressure. Our study supports the importance of core genetic variability in hepatocellular carcinogenesis. (HEPATOLOGY 2007.

Contribution of laser microdissection-based technology to proteomic analysis in hepatocellular carcinoma developing on cirrhosis.

International audienceHepatocellular carcinoma (HCC) is a major cause of cancer worldwide. Proteomic studies provide opportunities to uncover targets for the diagnosis and treatment of this disease. However, in HCC developing in a setting of cirrhosis, the detection of proteome alterations may be hampered by the increased cellular heterogeneity of tissue when analysing global liver homogenates. The aim of this study was to evaluate whether the identification of proteome alterations in these HCC cases was improved when the differential protein profile between tumour and non-tumour areas of liver was determined using hepatocytes isolated by laser microdissection (LM). Differential profiles established with LM-hepatocytes and liver section homogenates using 2-DE and MS exhibited noticeable differences: 30% of the protein spots with deregulated expression in tumorous LM-samples did not display any modification in homogenates; conversely 15% of proteins altered in tumorous homogenates were not impaired in LM-hepatocytes. These alterations resulted from the presence in cirrhotic liver of fibrotic stroma which displayed a protein pattern different from that determined in LM-hepatocytes. In conclusion, our data demonstrate the interest of LM in distinguishing between fibrotic and hepatocyte proteome alterations and thus the benefit of LM to proteome studies of HCC developing in a context of cirrhosis

Molecular signatures of neural connectivity in the olfactory cortex

International audienceThe ability to target subclasses of neurons with defined connectivity is crucial for uncovering neural circuit functions. The olfactory (piriform) cortex is thought to generate odour percepts and memories, and odour information encoded in piriform is routed to target brain areas involved in multimodal sensory integration, cognition and motor control. However, it remains unknown if piriform outputs are spatially organized, and if distinct output channels are delineated by different gene expression patterns. Here we identify genes selectively expressed in different layers of the piriform cortex. Neural tracing experiments reveal that these layer-specific piriform genes mark different subclasses of neurons, which project to distinct target areas. Interestingly, these molecular signatures of connectivity are maintained in reeler mutant mice, in which neural positioning is scrambled. These results reveal that a predictive link between a neuron's molecular identity and connectivity in this cortical circuit is determined independent of its spatial position

Molecular signatures of neural connectivity in the olfactory cortex

The ability to target subclasses of neurons with defined connectivity is crucial for uncovering neural circuit functions. The olfactory (piriform) cortex is thought to generate odour percepts and memories, and odour information encoded in piriform is routed to target brain areas involved in multimodal sensory integration, cognition and motor control. However, it remains unknown if piriform outputs are spatially organized, and if distinct output channels are delineated by different gene expression patterns. Here we identify genes selectively expressed in different layers of the piriform cortex. Neural tracing experiments reveal that these layer-specific piriform genes mark different subclasses of neurons, which project to distinct target areas. Interestingly, these molecular signatures of connectivity are maintained in reeler mutant mice, in which neural positioning is scrambled. These results reveal that a predictive link between a neuron’s molecular identity and connectivity in this cortical circuit is determined independent of its spatial position

Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes by boosting Tregs

CD4+CD25+Foxp3+ Tregs play a major role in prevention of autoimmune diseases. The suppressive effect of Tregs on effector T cells (Teffs), the cells that can mediate autoimmunity, has been extensively studied. However, the in vivo impact of Teff activation on Tregs during autoimmunity has not been explored. In this study, we have shown that CD4+ Teff activation strongly boosts the expansion and suppressive activity of Tregs. This helper function of CD4+ T cells, which we believe to be novel, was observed in the pancreas and draining lymph nodes in mouse recipients of islet-specific Teffs and Tregs. Its physiological impact was assessed in autoimmune diabetes. When islet-specific Teffs were transferred alone, they induced diabetes. Paradoxically, when the same Teffs were cotransferred with islet-specific Tregs, they induced disease protection by boosting Treg expansion and suppressive function. RNA microarray analyses suggested that TNF family members were involved in the Teff-mediated Treg boost. In vivo experiments showed that this Treg boost was partially dependent on TNF but not on IL-2. This feedback regulatory loop between Teffs and Tregs may be critical to preventing or limiting the development of autoimmune diseases

Restoration of regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythematosus patients through vitamin D supplementation.

International audienceABSTRACT: INTRODUCTION: Systemic lupus erythematosus (SLE) is a T and B cell-dependent autoimmune disease characterized by the appearance of autoantibodies, a global regulatory T cells (Tregs) depletion and an increase in Th17 cells. Recent studies have shown the multifaceted immunomodulatory effects of vitamin D, notably the expansion of Tregs and the decrease of Th1 and Th17 cells. A significant correlation between higher disease activity and lower serum 25-hydroxyvitamin D levels [25(OH)D] was also shown. METHODS: In this prospective study, we evaluated the safety and the immunological effects of vitamin D supplementation (100 000 IU of cholecalciferol per week for 4 weeks, followed by 100 000 IU of cholecalciferol per month for 6 months.) in 20 SLE patients with hypovitaminosis D. RESULTS: Serum 25(OH)D levels dramatically increased under vitamin D supplementation from 18.7±6.7 at day 0 to 51.4±14.1 (p<0.001) at 2 months and 41.5±10.1 ng/mL (p<0.001) at 6 months. Vitamin D was well tolerated and induced a preferential increase of naïve CD4+ T cells, an increase of regulatory T cells and a decrease of effector Th1 and Th17 cells. Vitamin D also induced a decrease of memory B cells and anti-DNA antibodies. No modification of the prednisone dosage or initiation of new immunosuppressant agents was needed in all patients. We did not observe SLE flare during the 6 months follow-up period. CONCLUSIONS: This preliminary study suggests the beneficial role of vitamin D in SLE patients and needs to be confirmed in randomized controlled trials