There are two classes of transposable elements: DNA transposons and retrotransposons. DNA transposons spread in the genome by “cut and paste” mechanism. In contrast, retrotransposons use copy and paste strategy involving RNA and retrotranscriptase mediated mechanism; these include long interspersed nuclear elements-1 (LINE-1, L1) and short interspersed nuclear elements (SINE). In mammals, in order to maintain genome integrity both types of transposons are tightly repressed. However, some copies of retrotransposons are still active in germ cells contributing to natural variation. Surprisingly, recent reports indicate that also somatic cells support L1 reactivation in early development, in particular in the brain leading to mosaicism. However, whether L1 retrotransposition is a part of other cell lineage developmental programs and its functional significance in the context of cell differentiation remain to be elucidated. To address this question, I investigated whether L1 retrotransposition was occurring during in vitro osteogenic and adipogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). Interestingly, clinical observations have revealed loss of bone density in HIV-infected individuals treated with nucleoside analogs that inhibit HIV retrotranscriptase, as well as the endogenous one encoded by L1s. This observation made us to hypothesize that transposable elements played a positive role in post-natal bone homeostasis. I found that while adipogenesis is “retrotransposition free”, osteogenic differentiation is a “retrotransposition-prone” process and its inhibition blocks its genetic program. Indeed, L1 DNA content does not change during adipogenic differentiation and that of retrotranscriptase does not have any effect on the acquisition of a terminally differentiated phenotype. In contrast, soon after MSCs commitment into pre-osteoblasts, L1 retrotransposable elements increase their expression and actively transpose. Inhibition of retrotransposition and knock down of L1 mRNA strongly impairs matrix deposition. Moreover, I forced L1 expression in in vitro adipogenesis, by directly delivering L1 mRNA to the cells. Interestingly, overexpression of L1 elements was detrimental for in vitro adipogenesis. Then, I performed loss of function experiments in osteogenesis by directly targeting and degrading the L1 endogenous transcript. This experiment confirmed the positive role of L1 reactivation in the osteogenic context, suggesting also a possible role for L1 RNA, distinct from retrotransposition.

Le muscle strié squelettique est un tissu fascinant qui permet d’assurer les fonctions essentielles à notre existence : se mouvoir, maintenir sa posture, se nourrir, communiquer ou tout simplement respirer. De nombreuses situations, engendrant principalement une hypoactivité, peuvent provoquer un déconditionnement musculaire caractérisé par une perte de masse et de force ainsi qu’un développement d’infiltrations graisseuses (IMAT), altérant ainsi la fonction musculaire. Le développement d’IMAT est également observé lorsque les processus de régénération musculaire sont altérés. Les fibro-adipogenic progenitors (FAPs) représentent la population de cellules souches principalement impliquée dans le développement d’IMAT. L’interaction entre FAPs et cellules satellites/immunitaires semble être un trio indispensable pour une régénération optimale, sans développement d’IMAT. Au regard de la littérature scientifique, une modulation du niveau d’activité contractile permet de faire varier le niveau d’expression de nombreuses cytokines impliquées dans la modulation des FAPs et donc dans l’apparition d’IMAT. Nos travaux ont contribué à l’accroissement des connaissances scientifiques relatives à la thématique des infiltrations graisseuses et à leurs exacerbations dans des situations d’hypoactivité ou de régénération musculaire. Nous avons montré que 3 jours d’hypoactivité chez l’homme, induite par le modèle novateur de dry immersion, suffisent à augmenter le contenu musculaire en IMAT. Dans un contexte de régénération musculaire, induite par le modèle glycérol chez la souris, nous avons démontré une inhibition de l’apparition des IMAT en diminuant les contraintes mécaniques appliquées au muscle lésé. Nous avons également précisé le rôle de l’axe TNFα/TGF-β1, et donc celui des processus inflammatoires nécessaires dans l’apoptose des FAPs afin de limiter le développement des IMAT dans ce modèle. Ces trois études ouvrent de nombreuses perspectives, afin i) de préciser le rôle des IMAT dans la dysfonction musculaire, ii) de définir les mécanismes de régulation qui contrôlent le développement et l’accumulation d’IMAT. / Skeletal muscle is a fascinating tissue that ensures core functions: moving, maintaining postures, feeding, communicating or just breathing. Many situations, associated with hypoactivity, are able to involve muscle deconditioning defined by a loss of mass and strength, as well as fat infiltration development (IMAT), altogether impairing muscle function. IMAT development occurs also with disrupted regeneration processes. Fibro-adipogenic progenitors (FAPs) appear as the main stem cell population involved in IMAT development. The interaction between FAPs and satellite/immune cells seems to be a crucial trio for an efficient regeneration, without IMAT development. According to the literature, the degree of contractile activity is able to affect the expression levels of different cytokines involved in FAPs fate, and therefore in IMAT accumulation. Our work contributed to increase scientific knowledge on muscle fatty infiltrations and their exacerbations in hypoactivity or regeneration situations. We showed that 3 days of hypoactivity in human, induced by the innovative model of dry immersion, are sufficient to promote an increase in IMAT content. In a context of muscle regeneration, induced by the mouse glycerol model, we highlighted an almost complete inhibition of IMAT accumulation by decreasing mechanical constraints applied to the injured muscle. We also investigated the role of the TNFα/TGF-β1 axis, and therefore the potential role of the inflammatory stage in FAPs apoptosis and inhibition of IMAT development. Our work open up new prospects 1°) clarifying the role of IMAT in muscle dysfunction, and 2°) defining the underlying mechanisms controlling IMAT development and accumulation.

Background: Rhabdomyosarcoma (RMS) is a soft tissue malignancy of the skeletal muscle that occurs primarily in pediatric populations. The prevailing treatment for RMS is a combination of chemoradiation therapy and surgery which has contributed to its 5-year survival rate of 75%. However, the combination of RMS and chemoradiation therapy can lead to impaired muscle growth and development which results in life-long skeletal muscle atrophy and weakness for RMS survivors. Skeletal muscle is a highly plastic tissue due, in part, to dynamic interactions between muscle-resident stem and progenitor cells (i.e., satellite cells (SCs) and fibro/adipogenic progenitors (FAPs)), which are necessary for muscle maintenance, growth, and adaptation to anabolic stimuli such as resistance exercise training. There is a clear gap in research investigating whether resistance endurance training (RET) stimulates muscle growth and preserves muscle function after juvenile chemoradiation therapy. Purpose: To determine the extent to which RET ameliorates the skeletal muscle defects in a preclinical model of RMS survivorship. Hypothesis: RET will improve physical performance, muscle cross-sectional area (CSA), and stem/progenitor cell populations compared to sedentary mice following RMS and chemoradiation therapy. Methods: RMS (M3-9-M cells) was injected into a single hindlimb of juvenile (4 week) C57Bl/6 mice that underwent systemic chemotherapy followed by targeted, fractionated radiation therapy to the RMS-injected limb. Following therapy, mice underwent RET (RET; n=10) or remained sedentary (SED; n=10) for 8 weeks. Body composition and performance tests were completed pre- and post-therapy and throughout the exercise intervention. Fibre typing, cross-sectional area, myonuclear characteristics and trichrome staining were evaluated following muscle harvest and progenitor cell populations were assessed using flow cytometry. Results: RET led to increased endurance performance (p<0.0001) as well as a reduction in body fat percentage (p=0.0004). RET rescued atrophy induced by RMS+therapy as evidenced by a significant increase in gastrocnemius/soleus to body weight ratio for the RET group compared to the SED group (p=0.0303), despite the decrease in muscle weight observed in the treated limb compared to the control limb (p=0.015). A distinct increase in intramuscular fibrosis was noted in the treated limb compared to the control limb in both groups (p=0.0283). Furthermore, RET resulted in larger myofibre cross-sectional area (p<0.05) and a shift from Type IIX to IIA fibres (p<0.05). We also noted a greater Type IIA myonuclear domain in the RET group compared to the SED group (p=0.0015) and an overall decrease in myonuclear domain (the cytoplasmic volume controlled by each myonucleus) for the treated limb compared to the control limb (p<0.05). Interestingly, we noticed overall cell death and an increase in immune cells in the RMS treated limb, while exercise resulted in increased endothelial and mesenchymal stromal cells. Significance: These preclinical findings will provide the rationale for further investigation of the mechanisms responsible for the beneficial effects of RET as well as optimizing the RET protocol in this juvenile cancer survivorship model.

No description available.

ABSTRAKT:Isolering och karaktärisering av mesenkymala stamceller från periodontalligamentet hos friskatänderSYFTE: Att isolera och odla celler från periodontalligamentet samt karaktärisera dem sommesenkymala stamceller.MATERIAL OCH METOD: Friska premolarer gjordes tillgängliga vid ortodontiskaextraktioner. Den mellersta 1/3 av periodontalligamentet skrapades varpå en enzymatiskmetod användes för isolering av individuella celler. Resulterande celler odlades understandardiserade metoder. Karaktärisering av celler skedde genom flödescymetri med 2 olikapaneler av cellyta markörer; en för etablerat positiva uttryck och en för kända negativauttryck hos mesenkymala stamceller. Möjlighet av celler att differentieras in vitro tilladipocyter och osteocyter testades genom tillförsel av specifika substanser till odlingsmediet.RESULTAT: Celler från 11 av 13 tänder isolerades och odlades framgångsrikt adherenta tillodlingsytan i upp till 8 generationer. Celluttryck av de positiva markörerna CD73, CD90 samtCD44 bekräftades genom flödescymetri. Inget uttryck observerades för den negativa panelenCD45, CD34, CD11b, CD19 eller HLA class II. Uttrycket av CD105 kunde inte fastställas pgaofullständigt data. Försök till differentiering av celler till adipocyter och osteocyter visade påfenotypiska förändringar efter 21 dagar.SLUTSATS: Den här studien har bidragit till framgångsrik isolering och delvis karaktäriseringav mesenkymala stamceller från periodontalligamentet hos friska tänder. En icke-invasivmetod av detta slag, resulterande i tillgång till denna cellpopulation utgör ett lovande verktygför framtida studier med goda möjligheter till ytterligare kunskap applicerbart till kliniskasituationer inom tandvården. / ABSTRACT:Isolation and Characterization of Mesenchymal Stem Cells from the Periodontal Ligament ofHealthy TeethAIM: To isolate and culture viable cells from the periodontal ligament and confirming theiridentity as mesenchymal stem cells.METHODS AND MATERIALS: Healthy premolars were collected at the time oforthodontic extractions. The middle 1/3 of the periodontal ligament was scraped andsubsequent cell isolation was performed using an enzymatic method; yielding single cellisolates. Cells were cultured and maintained under standard culture conditions. Cellcharacterization was performed by flow cytometry using two sets of cell surface markers; oneknown to be present and one known to be absent in mesenchymal stem cells. Ability of thecells for in vitro differentiation into adipogenic and osteogenic lineages was tested usingspecifically formulated media supplements.RESULTS: Cells were successfully isolated from 11 of 13 teeth and were maintained asadherent cultures for up to 8 generations. Cellular expression of positive markers; CD73, CD90and CD44 were confirmed by flow cytometry. For the negative marker panel, expression ofCD45, CD34, CD11b, CD19 and HLA class II were not detectable. The expression of CD105was inconclusive. As determined by phenotypic changes, cells appeared to have undergoneadipogenic and osteocytic differentiation at 21 days.CONCLUSION: This study has resulted in successful isolation and partial characterization ofmesenchymal stem cells from the periodontal ligament of healthy teeth. Non-invasive accessto these cells, provides an excellent tool for future studies, potentially leading to beneficialknowledge transferable to the dental clinical situation.

Submitted by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:24:49Z No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Approved for entry into archive by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-11-26T16:25:15Z (GMT) No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) / Made available in DSpace on 2014-11-26T16:25:15Z (GMT). No. of bitstreams: 1 Dissertação Ana Carolina Origa Alves - 02.pdf: 2465539 bytes, checksum: 9ad1e3c8193f72874f714fe1c073b599 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Células-tronco (CT) são células autorrenováveis e não especializadas, com o potencial de diferenciação multidirecional. Células-tronco de tecido adiposo (CT-TA) são um tipo de células-tronco adultas multipotentes, de fácil isolamento e cultura. Nos últimos anos, CT-TA têm mostrado grande potencial para engenharia de tecidos e terapias baseadas em células. Apesar do interesse em aplicações clínicas deste tipo de célula, os mecanismos moleculares fundamentais a sua autorrenovação e diferenciação ainda não foram completamente elucidados. miRNAs são pequenos RNAs não-codificadores, com 21-25 nucleotídeos de comprimento, que tem se mostrado como importantes reguladores da expressão gênica em nível póstranscricional. miRNAs podem atuar por meio de clivagem direta de mRNAs alvo ou através da repressão da tradução, dependendo da complementaridade entre o mRNA e a sequência do miRNA. Perfis de miRNAs de CT adultas sugerem que estes pequenos reguladores podem contribuir para as propriedades intrínsecas das CT. Para entender melhor os mecanismos de ação dos miRNAs em CT-TA, miRNAs associados ao polissomos de CT-TA foram isolados durante a diferenciação celular. Procurando miRNAs reguladores das etapas iniciais de diferenciação ou envolvidos na autorrenovação de CT, as culturas de células foram induzidas a diferenciação adipogênica durante 72 h. O lisado celular foi submetido à ultracentrifugação em gradiente de sacarose para separar monosomos, polissomos e fração livre de ribossomos. O RNA total associado aos ribossomos foi extraído, os fragmentos de RNA (<200 nt) foram enriquecidos e a seleção de tamanho de fragmentos de RNA apropriados ocorreu durante a preparação das amostras para o sequenciamento em larga escala. As amostras foram sequenciadas utilizando a plataforma SOLiD ™, e as frações polissomais de culturas Não Induzida e 72h de indução foram comparadas e dezesseis miRNAs foram identificados. miRNAs encontrados em um trabalho prévio do grupo foram adicionados a esses dados, e sete miRNAs (hsamiR-29b-1-5p, hsa- miR-29c-5, hsa-miR-30c-5p, hsa-miR-143-5p, hsa-miR-210-3p, hsa-miR-210- 5p e hsa-miR-6775- 5p) foram testados por RT-qPCR para confirmar a expressão diferencial, sendo que um deles (hsa-miR-210-5p) mostrou diferença estatisticamente significativa. / Stem cells (SC) are self-renewing and non-specialized cells with the potential of multi-directional differentiation. Adipose Stem Cell (ADSC) is a type of multipotent adult stem cell, easy to isolate and culture. In the past few years, hADSCs have shown great potential for tissue engineering and cell-based therapies. Despite the interest in clinical applications of this kind of cell, the molecular mechanisms underlying their self-renewal and differentiation have yet to be fully elucidated. miRNAs are small noncoding RNAs, 21-25 nucleotides in length, that have been shown to be important regulators of posttranscriptional gene expression. miRNAs can act through direct cleavage of target mRNAs or through translational repression, depending of complementary pairing between the mRNA and miRNA sequence.miRNA profile of adult SCs suggests that these small regulators can contribute to the intrinsic properties of SCs. To better understand the mechanisms of action of miRNAs in hADSCs, we isolate miRNAs associated to polysomes of hADSC during cellular differentiation. Looking for miRNAs regulators of early steps of differentiation or involved in ADSC self-renewing, cell cultures were induced to adipogenic differentiation for 72 h. The cell lysate was submitted to ultracentrifugation on a sucrose gradient to separate monosomes, polysomes and the fraction free of ribosomes. The total RNA associated to ribosomes was extracted and the RNA fragments (<200 nt) were enriched and the size selection of appropriate RNA fragments occurred during the preparation of samples for deep sequencing. The samples were sequenced using SOLiD™ platform, and polysomal fraction of cell cultures non induced and 72h of induction were compared and sixteen miRNAs were identified. miRNAs found in a previous work of our group were added to these data and seven miRNAs (hsa-miR-29b-1-5p, hsa-miR-29c-5, hsa-miR-30c-5p, hsa-miR- 143-5p, hsa-miR-210-3p, hsa-miR-210-5p e hsa-miR-6775-5p) were tested by RTqPCR to confirm differential expression, and one of them (hsa-miR-210-5p) showed statistical significant difference.

La régénération musculaire implique des interactions fonctionnelles entre différents types de cellules mononucléées. Parmi elles, citons les progéniteurs myogéniques (MPs), qui fusionnent pour générer de nouvelles myofibres en réponse à une blessure, et les cellules immunitaires qui envahissent les muscles endommagés. Des dépôts transitoires fibrotiques et d’adipocytes sont observés dans les muscles en régénération qui cependant persistent dans la dystrophie musculaire de Duchenne (DMD) et au cours du vieillissement. Nous avons démontré que les progéniteurs fibro-adipogéniques (FAPs) exprimant le marqueur de surface PDGFRα, contribueraient au développement des dépôts non myogéniques dans les muscles sains. En effet, ces progéniteurs se différencient en adipocytes blancs fonctionnels, bien qu’étant insensibles à l’insuline, et génèrent des myofibroblastes. Quant à la fibrose des muscles DMD, elle se formerait à partir de la différenciation à la fois des MPs et des FAPs. Dans les muscles sains, les FAPs stimulent la myogenèse des MPs au cours de la régénération, alors que les myotubes et les macrophages pro-inflammatoires inhibent l’adipogenèse et la fibrogenèse des FAPs. Pour les progéniteurs âgés ou dystrophiques, les interactions cellulaires entre les FAPs et les MPs sont perturbées. De manière intéressante, la régulation des FAPs DMD ou âgés peut être restaurée en remplaçant les MPs DMD ou âgés par des MPs jeunes et sains. Nos résultats montrent que les muscles humains contiennent des progéniteurs fibro-adipogéniques qui jouent un rôle central dans la régulation de l’homéostasie musculaire en interagissant avec les progéniteurs myogéniques et les macrophages / Muscle regeneration involves functional interactions between different types of mononuclear cells including myogenic progenitors (MPs) and macrophages. Following injury, damaged muscles are invaded by immune cells and MPs fuse to generate new myofibres. Transient fibrotic and adipocyte deposits are observed in regenerating muscles, which however persist in Duchenne muscular dystrophy (DMD) and during aging. We demonstrated that fibro-adipogenic progenitors (FAPs) expressing the PDGFRα surface marker would contribute to the development of non-myogenic deposits in healthy muscles. Indeed, these progenitors differentiate into functional white adipocytes that have the feature to be insulino-resistant, and give rise to myofibroblastes. Intramuscular fibrosis in DMD patients could be formed from both FAPs and MPs differentiation. In healthy muscles, FAPs stimulate myogenesis of MPs during regeneration, while myotubes and pro-inflammatory macrophages inhibit the adipogenesis and fibrogenesis of FAPs. Cellular interactions between FAPs and MPs are disrupted for DMD or aged progenitors. Interestingly, they are restored if aged or DMD FAPs are replaced by healthy and young MPs. Our results show that the human muscles contain fibro-adipogenic progenitors that play a crucial role in the control of muscle homeostasis by interacting with myogenic progenitors and macrophages

No description available.