Détection de micro-ARNs in situ : utilisation de sondes LNA

Les micro-ARNs sont de petits ARN non-codants de 21 à 25 nucléotides impliqués dans la régulation de l’expression post-transcriptionnelle de gènes. Ils se fixent par complémentarité de séquence à des ARN messagers (ARNm) cibles afin d’induire leur dégradation ou à l’inhibition de leur traduction en protéine. Récemment, ils ont été montrés comme i) étant dérégulés dans différents contextes de pathologies musculaires et ii) jouant un rôle important dans le processus physiopathologique associé à la myopathie de Duchenne par une implication notamment dans la réponse aux dommages musculaires et à la régénération. Une analyse spatiale de l’expression tissulaire et cellulaire des micro-ARNs s’avère nécessaire pour approfondir le champ des connaissances sur leur degré d’implication dans les mécanismes physiologiques et/ou physiopathologiques. Les techniques conventionnelles d’hybridation in situ − technique de référence en histologie permettant une localisation cellulaire des acides nucléiques − ne sont pas adaptées pour la détection de micro-ARNs en raison de la très petite taille de ces derniers. Par conséquent, les sondes classiques d’ADN ou d’ARN sont remplacées par des sondes spécialement développées pour cet usage, dites sondes LNA (Locked Nucleic Acid). Notre équipe s’est intéressée à l’expression de deux micro-ARNs dans le muscle de chiens myopathes. Par hybridation in situ avec des sondes LNA, nous avons documenté leur expression tissulaire et démontré qu’ils s’expriment plus particulièrement dans les myoblastes et dans les fibres en cours de régénération

Fluorescence Bio-imaging practical courses: Immunohistochemistry : Principle

Immunohistochemistry- what’s good about it?•Uses antibodies to detect and visualize antigens in cells from tissue section•Antibodies bind to antigen in specific manner•Gives you a spatial location•Can be used to locate particular cells and proteins•Can be used to identify cellular events – e.g.apoptosis(...

FoxO3a overexpression prevents both glycogen overload and autophagic buildup in skeletal muscle of Pompe disease

FoxO3a overexpression prevents both glycogen overload and autophagic buildup in skeletal muscle of Pompe disease. 6eme congrès international de Myologi

Cadeia produtiva do gengibre (Zingiber officinale ROSCOE) no estado do Paraná : análise e recomendações para melhoria da qualidade

Orientador : Raquel R.B.NegrelleCo-orientadores: Luiz Doni Filho, Neusa G.A. RuckerTese (doutorado) - Universidade Federal do Paraná. Setor de Ciências Agrárias. Curso de Pós-Graduação em Agronomia - área de concentração Produção Vegetal, Departamento de Fitotecnia e FitossanitarismoInclui bibliografia e glossárioResumo: O presente trabalho teve como objetivo o estudo prospectivo da cadeia produtiva do Zingiber officinale Roscoe no Estado do Paraná, identificando os principais agentes envolvidos e paralelamente proceder análise dos pontos de estrangulamentos, especificamente no que concerne à qualidade microbiológica deste produto em todos os níveis, desde a produção até a fase final da comercialização, visando identificar causas e propor soluções no sentido da melhoria do sistema como um todo. Este estudo foi desenvolvido no período de 2000 a 2002 englobando pesquisa de campo, entrevistas abertas com representantes de estabelecimentos de comercialização, produtores e demais atores da cadeia produtiva, além de análises laboratoriais do produto "in natura" disponível no mercado consumidor da Região Metropolitana de Curitiba - PR. Este documento está organizado em 9 capítulos, que apresentam vastas informações englobando aspectos botânicos, ecológicos, fisico-químicos e farmacológicos do gengibre, e que estão incluídas nos capítulos 1 e 2. O capítulo 3 apresenta características gerais da principal região produtora brasileira, Morretes - PR, englobando localização, aspectos sócio-econômicos, geológicos e geomorfológicos e caracterização climática. O capítulo 4 apresenta o estudo prospectivo da cadeia produtiva do gengibre no Estado do Paraná, que engloba panorama mundial, brasileiro e paranaense do volume de produção agrícola de gengibre. Inclui-se também caracterização da comunidade produtora agrícola do litoral paranaense, além da identificação e caracterização dos outros diferentes níveis da cadeia produtiva do gengibre e a detecção dos principais pontos de estrangulamento nos diferentes níveis desta cadeia produtiva. A caracterização do cultivo e beneficiamento do gengibre no litoral paranaense é apresentada no capítulo 5, evidenciando suas particularidades frente ao descrito na literatura especializada. No capítulo 6 avaliou-se as condições higiênico-sanitárias dos estabelecimentos produtores, do processo de beneficiamento pós-colheita (lavagem, limpeza, secagem e embalagem) e das condições de manipulação do gengibre "in natura" no litoral paranaense. De maneira geral, as condições de higiene e limpeza observadas foram consideradas precárias, determinando alta potencialidade de contaminação do gengibre com agentes que poderiam colocar em risco a saúde do consumidor. No capítulo 7 são apresentados os resultados dos estabelecimentos de comercialização da Região Metropolitana de Curitiba - PR, no que tange à qualidade e adequação às normas vigentes. Dos aspectos observados, as condições dos vestuários e dos equipamentos de proteção individual (manipuladores) foram considerados precários, o que pode potencializar a contaminação do produto e colocar em risco a saúde do consumidor. O capítulo 8 teve como objetivo caracterizar o perfil microbiológico do gengibre "in natura" comercializado na Região Metropolitana de Curitiba - PR, tendo como base a Resolução - CNPPA n. 12 - Brasil, 1978 e a Resolução - RDC n. 12 - Brasil, 2001. Para tanto, foram realizadas a determinação do número mais provável (NMP) de coliformes totais, coliformes a 45° C/g e Escherichia coli, e a presença de Salmonella sp em 25 gramas. Sintetizando, um conjunto de propostas e recomendações aos agentes econômicos que atuam e processam a cadeia produtiva do gengibre no Estado do Paraná, em especial o município de Morretes - PR, é apresentado no capítulo 9Abstract: Prospective studies of the productive chain of the Zingiber officinale Roscoe on Parana State identifying the main agents envolved and parallel procedure analysis of the strangulation points, in order to microbiology qualify this product in whole levels from the production until commercialization last phase, aiming identify causes and propose solutions to the improvement of the system in general. This survey developed from 2000 to 2002 involved field research, interviews with representative of the commercialization establishments, producers and others actors of the productive chain, besides made in laboratories of the quality of crude product available to the consumer market in Curitiba. This paper is organized in nine chapters that present information involving botanical, ecological, physique-chemical and pharmacological aspects of ginger that are included in chapters 1 and 2. The chapter 3 presents the characteristics of the main brazilian productive area, embody localization, social and economic aspects, geologycal, geomorphologycal and climate characterization. The chapter 4 the study of the productive chain of this product in Parana State, which includes the paranaense, the brazilian and the worldwide panoramic view of the production volume and commercialization of the ginger; characterization of the agricultural community; identification and characterization of others different levels of the productive chain and also, the main strangulation points on these different levels of the productive chain. In the chapter 5 the agricultural process of the ginger benefit and cultivation in Morretes Town, Paraná, Brazil was caracterized. In the chapter 6 the hygienic-sanitary conditions of the producers establishments, postharvest benefit system and manipulation conditions of crude ginger in the paranaense coastland, Brazil were evaluated with an aim to subsidy the ofert of the best quality product to consumer market. In general, the cleaning and hygienic conditions were precarious, determining a high potential of ginger contamination with agents that can put in risk the consumers' health. The chapter 7 presents the results of the commercialization establishments in the metropolitan area of Curitiba considering adequation to the current norm. Of the observed items, the manipulation conditions was considered precarious due to the inadequate conditions that contributes to contamination, bringing about serious problems to consumers' health. The chapter 8 aimed the microbiological quality of the ginger market in the metropolitan area of Curitiba based on the Brazilian legislation. The microbiological analysis found total coliforms, 45° C/g coliforms, Escherichia coli most probable number (MPN) and Salmonella sp presence. Synthetizing, a group of considerations and recommendations to economic agents that actuate and carry on the productive chain of the ginger in the Parana State, specially in Morretes Town, is presented in the chapter

Human neural crest cells display molecular and phenotypic hallmarks of stem cells

The fields of both developmental and stem cell biology explore how functionally distinct cell types arise from a self-renewing founder population. Multipotent, proliferative human neural crest cells (hNCC) develop toward the end of the first month of pregnancy. It is assumed that most differentiate after migrating throughout the organism, although in animal models neural crest stem cells reportedly persist in postnatal tissues. Molecular pathways leading over time from an invasive mesenchyme to differentiated progeny such as the dorsal root ganglion, the maxillary bone or the adrenal medulla are altered in many congenital diseases. To identify additional components of such pathways, we derived and maintained self-renewing hNCC lines from pharyngulas. We show that, unlike their animal counterparts, hNCC are able to self-renew ex vivo under feeder-free conditions. While cross species comparisons showed extensive overlap between human, mouse and avian NCC transcriptomes, some molecular cascades are only active in the human cells, correlating with phenotypic differences. Furthermore, we found that the global hNCC molecular profile is highly similar to that of pluripotent embryonic stem cells when compared with other stem cell populations or hNCC derivatives. The pluripotency markers NANOG, POU5F1 and SOX2 are also expressed by hNCC, and a small subset of transcripts can unambiguously identify hNCC among other cell types. The hNCC molecular profile is thus both unique and globally characteristic of uncommitted stem cells

ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation

Background: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease that leads to fibre necrosis and progressive paralysis. At present, DMD remains a lethal disease without any effective treatment, requiring a better understanding of the pathophysiological processes and comprehensive assessment of the newly identified therapeutic strategies. MicroRNAs including members of the muscle-specific myomiR family have been identified as being deregulated in muscle of DMD patients and in mdx mice used as a model for DMD. In recent years, the Golden Retriever muscular dystrophy (GRMD) dog has appeared as the crucial animal model for objectively assessing the potential of new innovative approaches. Here, we first aim at establishing the muscle expression pattern of five selected miRNAs in this clinically relevant model to determine if they are similarly affected compared with other DMD contexts. Second, we attempt to show whether these miRNAs could be impacted by the systemic delivery of a promising stem cell candidate (referred to as MuStem cells) to implement our knowledge on its mode of action and/or identify markers associated with cell therapy efficacy. Methods: A comparative study of miRNAs expression levels and cellular localization was performed on 9-monthold healthy dogs, as well as on three sub-sets of GRMD dog (without immunosuppression or cell transplantation, with continuous immunosuppressive regimen and with MuStem cell transplantation under immunosuppression), using RT-qPCR and in situ hybridization. Results: We find that miR-222 expression is markedly up-regulated in GRMD dog muscle compared to healthy dog, while miR-486 tends to be down-expressed. Intriguingly, the expression of miR-1, miR-133a and miR-206 does not change. In situ hybridization exploration reveals, for the first time, that miR-486 and miR-206 are mainly localized in newly regenerated fibres in GRMD dog muscle. In addition, we show that cyclosporine-based immunosuppression, classically used in allogeneic cell transplantation, exclusively impacts the miR-206 expression. Finally, we demonstrate that intra-arterial administration of MuStem cells results in up-regulation of miR-133a and miR-222 concomitantly with a down-expression of two sarcomeric proteins corresponding to miR-222 targets. Conclusion: We point out a differential muscle expression of miR-222 and miR-486 associated with the pathophysiology of the clinically relevant GRMD dog model with a tissue localization focused on regenerated fibres. We also establish a modified expression of miR-133a and miR-222 subsequent to MuStem cell infusion

Differential gene and miRNA profiling in dystrophic dog and impact of MuStem cell-based therapy

Differential gene and miRNA profiling in dystrophic dog and impact of MuStem cell-based therapy. Annual Congress of the European Society of Gene and Cell Therapy (ESGCT

Exploration of muscle from GRMD dogs tranplanted with MuStem cells using “omics” approaches

Duchenne Muscular Dystrophy (DMD), the most common form of inherited neuromuscular disorder, is caused by mutations in the dystrophin gene leading to the protein lack. Membrane disorganization and subsequent alterations in signaling pathways and energy metabolism play important roles in muscle fibre necrosis. Systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generate a remodeling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. To pursue investigation of the consequences on the skeletal muscle tissue 6 months after cell transplantation with undedicated approach, we used here a combined analysis of transcriptomics (gene expression microarrays) and quantitative proteomics (ICPL/LC-MS/MS) (Robriquet et al., 2015). At molecular level, we determined that MuStem cell administration enhances muscle regeneration, promotes ubiquitin-mediated protein degradation in parallel with a decrease expression of genes associated with lipid homeostasis and energy metabolism. Furthermore, the proteomic approach confirmed a main impact of MuStem cell delivery on muscle regeneration, metabolism as well as homeostasis pathways. In addition, we establish that the analysis of a limited set of miRNAs in skeletal muscle clearly discriminates between immunosuppression context and MuStem cell therapyrelated effects on GRMD dogs. Overall, the combination of transcriptomics, proteomics and miRNA approaches allowed to pave the way to the understanding of MuStem cell action modalities as stimulation of muscle fibre formation. This strategy has a great potential to considerably contribute to the identification of therapeutic biomarkers of MuStem cell transplantation and thus represents an interesting tool to monitor therapeutic effects during DMD-dedicated preclinical studies