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1

Navet, Benjamin. "Homéogènes Dlx, signalisation RANK/RANKL et ostéosarcomes". Thesis, Nantes, 2016. http://www.theses.fr/2016NANT1018/document.

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L’ostéosarcome (OS), plus fréquente des tumeurs osseuses primitives malignes, se caractérise par une croissance ostéoïde parfois associée à une ostéolyse. Malgré les avancées thérapeutiques, le taux de survie reste faible (30 % à 5 ans si métastases ou chimiorésis-tances). De nouvelles approches thérapeutiques ciblant la cellule tumorale et son environnement sont néces-saires. Les travaux présentés se sont intéressés aux poten-tiels facteurs pro-tumoraux que sont les gènes Dlx et à une signalisation clé de l’environnement osseux (RANKL/RANK) susceptible d’influer l’agressivité tumo-rale. L’OS étant une tumeur ostéoblastique, la famille Dlx a été choisie, car impliquée dans l’ostéoblasto-genèse, et la signalisation RANKL/RANK, car voie car-dinale du couplage entre ostéoblastes et ostéoclastes. De plus un lien entre Dlx et signalisation RANK était suspecté. Les gènes Dlx1, Dlx4 et Rank non-exprimés dans l’ostéoblaste sain le sont dans les lignées d’OS. Des modulations d’expression des Dlx et de Rank ont été réalisées afin d’en évaluer l’impact sur les cellules tumo-rales. L’implication de la signalisation RANK/RANKL dans le microenvironnement tumoral a été analysée. La perturbation du remodelage est en faveur de la tumeur en participant à l’établissement d’un cercle vicieux entre la tumeur et l’environnement. Les travaux ont établi l’implication des Dlx, surtout Dlx4 pour lequel un nouveau transcrit codant a été ca-ractérisé. Cependant des études supplémentaires sont nécessaires. Concernant la signalisation RANK/RANKL, il s’avère qu’au-delà du cercle vicieux, important au stade d’initiation tumorale, l’expression de RANK par la tumeur s’avère être un facteur pro-métastatique
Osteosarcoma (OS), the most common malignant primary bone tumor, is characterized by an osteoid formation occasionally associated with osteolysis. De-spite therapeutic advances, the 5-years survival rate remains low (30% in case of metastasis or drug-resistance). New therapeutic approaches targeting the tumor cell and its environment are needed. Presented studies focused on potential pro-tumor factors namely Dlx genes and a key signaling pathway of the bone environment (RANKL / RANK) that may influence tumor aggressiveness. The OS is an osteo-blastic tumor and Dlx family was chosen due to its in-volvement in osteoblastogenesis. RANKL / RANK path-way was selected as it constitutes a main element in the coupling between osteoblasts and osteoclasts. A link between Dlx genes and RANK signaling was suspected. Dlx1, Dlx4 and Rank genes are not normally ex-pressed in osteoblasts but are present in the OS cell lines. Dlx and Rank expression modulations were real-ized to assess the impact on tumor cells. RANK / RANKL signaling involvement in the tumor microenvi-ronment was analyzed. Disruption of remodeling is in favor of the tumor taking part in the establishment of a vicious circle between tumor and environment. This work established the involvement of Dlx, espe-cially DLX4 to which a new coding transcript has been characterized. However, additional studies are needed. Regarding the RANK / RANKL signaling, it turns out that beyond the vicious circle, leading to tumor initiation stage, the RANK expression by the tumor proves to be pro-metastatic elements

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2

Yoskovitz, Guy. "Genetic Polymorphisms of RANK, RANKL and their relation to osteoporosis (Polimorfismos genéticos de RANK y RANKL y su relación con la osteoporosis)". Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/98294.

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Osteoporosis is a systemic skeletal disorder and the most common metabolic bone disease. It is recognized as one of the most prevalent problems facing postmenopausal women in western society. The World Health Organization definition of osteoporosis uses bone mineral density (BMD) measurements as the gold standard. The genetic complexity of BMD is incompletely defined. Bone turnover, also called bone remodelling, is a lifelong process that refers to the entire cycle of bone resorption and formation, which determines BMD. In general, the cell biology of an adult bone includes 3 cell types, among others, that have opposite functions: osteoblasts produce the extracellular matrix that becomes mineralized; osteoclasts are responsible for the resorptive actions; and osteocytes are involved in the regulation of both resorption and formation (and are even claimed to dominate the process). A complex signal system between these 3 cell types balances their activities to avoid any over-creation or loss of bone tissue. The bone remodelling equilibrium is in part dominated by a set of protein reactions known as the RANK/RANKL/OPG system. The special importance of the Receptor Activator of NF-kappa-B (RANK) and its interaction with its ligand (RANKL) is that the RANK/RANKL complex is one of the main triggers of osteoclast differentiation and survival. Hence, in-depth analysis of variants in these 2 genes may contribute to the understanding of the genetics of BMD. This study had 4 main objectives: (1) association analysis of putative functional SNPs in evolutionary conserved regions of the RANK and RANKL genes with BMD and the occurrence of fractures in our cohort (BARCOS). (2) Replication of previously associated SNPs, in the BARCOS cohort (3) In-silico study followed by in-vitro functional experiments of the BMD-associated SNP(s) in order to reveal its (their) role(s) in the pathological process of osteoporosis and (4) Characterization of the human RANKL promoter and regulatory regions in-silico and in-vitro. We replicated the association of SNP rs9594738, a genetic variant at 184 kb upstream to RANKL gene, with BMD. Statistical analysis for other SNPs in the RANKL gene failed to be associated with osteoporotic phenotypes. The functional experiments’ results demonstrate that this region surrounding rs9594738, between AKAP11 and RANKL, has the capacity to regulate RANKL, with different effects on its expression in the presence or absence of vitamin D. These results suggest that it may play a role in the RANK/RANKL/OPG equilibrium, and might explain the association between the SNPs in this region and BMD. A transcript of minimum 300 bp (with rs9594738 in a central position) has been detected in this region. The existence of this RNA segment suggests its involvement in alternative functions of the region. We also identified 2 SNPs in the RANK 3’UTR (rs78326403 and rs884205) that are associated with low trauma fractures in our cohort. SNP rs78326403 is associated with wrist/forearm fractures, while SNP rs884205 is associated with spine fractures. In addition, we observed a significant interaction between rs78326403 and the RANKL BMD-associated SNP (rs9594738), highlighting the relevance of both microarchitecture and low BMD as genetically determined predictors of fracture risk that should be assessed using multiple techniques. To conclude, our results highlight the importance of the region between AKAP11 and RANKL on RANKL transcription regulation and suggest that it may play an important role in the RANK/RANKL/OPG equilibrium. Furthermore, the site-dependent associations in the RANK gene might be clinically relevant in the future to better profile a more specific approach to the different types of fractures, both to better understand their underlying mechanisms and to search for site-specific therapeutic strategies.
La osteoporosis es la enfermedad metabólica ósea más frecuente. Está definida como un trastorno esquelético sistémico caracterizado por una disminución de la densidad mineral ósea (DMO) y alteraciones en la microarquitectura del tejido óseo, con un consecuente incremento de la fragilidad ósea y del riesgo de fractura. Su complejidad genética permanece incompletamente definida. La DMO viene marcada por un remodelado óseo basado en ciclos de resorción y formación que suceden a lo largo de toda la vida del organismo. Este proceso, está regulado en parte por un conjunto de reacciones proteicas pertenecientes al sistema conocido como RANK/RANKL/OPG. La especial importancia de RANK, así como la interacción de éste con su ligando RANKL, recae en el hecho que, son factores clave tanto en el desencadenamiento de la diferenciación como de la supervivencia osteoclástica. El estudio se centra en el análisis detallado de variantes pertenecientes a ambos genes, seguido del estudio funcional correspondiente. Se ha replicado la asociación del SNP rs9594738 con la DMO, una variante genética localizada a 184 kb 5' del gen RANKL. Los resultados del estudio funcional muestran que la región que engloba dicha variante actúa como un regulador distal de RANKL, ejerciendo efectos en su expresión que varían en presencia ó ausencia de vitamina D. Además, se identificaron dos SNPs (rs78326403 y rs884205) en el 3’UTR de RANK, asociados con fracturas por bajo traumatismo en nuestra cohorte. Por último, una interacción significativa entre rs78326403 y rs9594738 en la determinación del riesgo de fractura, pone de relieve la importancia de la DMO baja y de la microarquitectura como predictores genéticamente determinados del riesgo de fractura que se deben evaluar con el uso de diversas técnicas.

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3

Hamoudi, Dounia. "Implication de la voie RANK/RANKL/OPG dans la physiopathologie musculaire et potentiel thérapeutique de l’anti-RANKL pour la dystrophie musculaire de Duchenne". Doctoral thesis, Université Laval, 2021. http://hdl.handle.net/20.500.11794/69507.

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La dystrophie musculaire de Duchenne (DMD) est une maladie génétique neuromusculaire provoquée par des mutations du gène codant pour la dystrophine situé sur le chromosome Xp21. L'absence de cette protéine membranaire engendre une dégénérescence progressive des cellules, une augmentation de la concentration du calcium intracellulaire, des dommages oxydatifs, inflammatoires et ultimement une fibrose musculaire. Les patients souffrent également de plusieurs autres anomalies dont les plus importantes sont la cardiomyopathie et l'ostéoporose. Il n'y a actuellement aucune stratégie curative pour la DMD. Les corticostéroïdes sont prescrits pour prolonger la mobilité et l'espérance de vie, mais sont associés à une ostéotoxicité élevée. Bien qu'il existe une association entre l'ostéoporose et la dégénérescence musculaire, nous avons été les premiers à étudier le rôle du récepteur-activateur du facteur nucléaire kB (RANK), son ligand RANKL et du récepteur soluble ostéoprotégérine (OPG), principaux régulateurs du remodelage osseux, dans le contexte des maladies musculaires. Nos travaux antérieurs montrent que les myotubes différenciés secrètent l'OPG, expriment le récepteur RANK à leurs surfaces et dans le contexte de DMD l'expression de l'ARNm de RANK est 4 fois plus élevée dans les muscles de souris dystrophiques comparativement aux muscles sains. L'objectif de la présente thèse vise à exploiter cette voie afin de comprendre le mécanisme d'action de ces cytokines sur la physiopathologie musculaire et d'établir une stratégie thérapeutique pour la DMD en traitement unique ou combinée aux glucocorticoïdes. Dans un premier temps, nous avons investigué l'impact de la neutralisation systémique à long terme de RANKL sur l'intégrité et la fonction musculaire et osseuse dans un modèle sévère de dystrophie déficient en dystrophine/haploinsuffisant en utrophine. Ensuite, nous avons étudié les rôles physiopathologiques de l'OPG sur les tissus musculaires en caractérisant la fonction musculaire de souris déficientes en OPG. Finalement, nous avons débuté une étude sur l'effet de neutralisation systémique de RANKL sur l'ostéoporose associée à un traitement au deflazacort, un glucocorticoïde prescrit pour la DMD. Ainsi nous avons démontré que le traitement à long terme à l'anti-RANKL améliore la fonction et l'intégrité musculaire et osseuse chez les souris dystrophiques et protège contre l'ostéoporose induite par les glucocorticoïdes. À l'opposé, l'absence d'OPG induit, possiblement via RANKL, une faiblesse osseuse et musculaire et une atrophie sélective des fibres musculaires les plus puissantes. Ces avancées repoussent les connaissances au sujet de la voie RANK/RANKL/OPG au sein de la communication muscle-os et appuient l'anti-RANKL comme perspective thérapeutique chez les patients atteints de la DMD.

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4

Manfrin, Thais Mara [UNESP]. "Imunomarcação das proteínas OPG, RANK e RANKL em dentes reimplantados de rato". Universidade Estadual Paulista (UNESP), 2007. http://hdl.handle.net/11449/101206.

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O sistema OPG, RANK e RANKL é uma das mais importantes descobertas da biologia óssea. Essas proteínas regulam as atividades celulares na remodelação do tecido ósseo e na literatura há diversas investigações nos tecidos dentários. No entanto, no reimplante dentário, ainda não foram encontrados relatos. Foi objetivo deste trabalho, avaliar a imunomarcação das proteínas OPG, RANK e RANKL em dentes reimplantados de rato. Um grupo controle foi formado com quatro ratos no qual o reimplante dentário não foi realizado. Vinte e quatro ratos (Rattus norvegicus, albinus) tiveram seu incisivo superior extraído e depois reimplantado formando os seguintes grupos: grupo I – reimplante imediato; grupo II - reimplante tardio sem tratamento e grupo III - reimplante tardio com tratamento endodôntico (ressecção da papila dentária e preenchimento do canal com pasta de hidróxido de cálcio) e tratamento da superfície radicular (raspagem mecânica do ligamento periodontal necrosado e imersão em solução de flúor fosfato acidulado de sódio a 2,5%). Ao final dos períodos experimentais (10 e 60 dias) os ratos foram eutanasiados. Foram obtidos cortes longitudinais parafinados com 6μm de espessura. Os cortes foram submetidos à reação imunoístoquímica mediante a utilização de anticorpos primários para OPG, RANK e RANKL. Os resultados mostraram que a imunomarcação de OPG e RANKL ocorreu em todos os grupos e períodos estudados, muito embora RANKL não tenha sido observada no grupo reimplante imediato aos 60 dias. RANK foi observada somente aos 10 dias de todos os grupos no qual o reimplante foi realizado. A análise qualitativa dos resultados demonstrou que o sistema OPG, RANK e RANKL apresentou marcação evidente no reimplante tardio, sugerindo a efetiva participação no início do processo de reabsorção radicular, uma vez que aos 60 dias a imunomarcação foi discreta.
The OPG, RANK and RANKL system is one of the most important discoveries in bone biology. These proteins are key regulators of bone remodeling and in the literature there are several studies of tooth resorption. However, in tooth replantation, reports have not been found. The purpose of this study was to determine the immunolabeling of OPG, RANK and RANKL in replanted teeth in rats, using immunohistochemistry methodology. A control group (no replanted teeth) was formed by four rats. Twenty-four rats (Rattus norvegicus, albinus) were submitted to the extraction of their upper right incisors. The replantation was performed according to the groups below: group I – immediate replantation; group II – delay replantation without treatment and group III – delay replantation with endodontic treatment (extirpation of papilla and the root canal filled with calcium hydroxide) and root surface treatment (periodontal ligament was removed with scalpel and teeth were immersed in 2% acidulated phosphate sodium fluoride). The animals were euthanized at the end of the experimental periods (10 and 60 days). Longitudinal 6μm slices embedded in paraffin were obtained. The slices were submitted to immunohistochemistry reaction by means of the primary antibodies for OPG, RANK and RANKL proteins. The results showed the expression of OPG in all groups and periods. RANKL expression was observed in all groups, except in the immediate replanted teeth group (60 days). RANK expression was observed only at 10 days in all groups which replantation was performed. The qualitative analysis of our findings indicated that the system OPG, RANK and RANKL presented strong expression in delayed replantation, suggesting effective participation at the beginning of root resorption, since at 60 days the immunostained cells were discreet.

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Manfrin, Thais Mara. "Imunomarcação das proteínas OPG, RANK e RANKL em dentes reimplantados de rato /". Araçatuba : [s.n.], 2007. http://hdl.handle.net/11449/101206.

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Resumo: O sistema OPG, RANK e RANKL é uma das mais importantes descobertas da biologia óssea. Essas proteínas regulam as atividades celulares na remodelação do tecido ósseo e na literatura há diversas investigações nos tecidos dentários. No entanto, no reimplante dentário, ainda não foram encontrados relatos. Foi objetivo deste trabalho, avaliar a imunomarcação das proteínas OPG, RANK e RANKL em dentes reimplantados de rato. Um grupo controle foi formado com quatro ratos no qual o reimplante dentário não foi realizado. Vinte e quatro ratos (Rattus norvegicus, albinus) tiveram seu incisivo superior extraído e depois reimplantado formando os seguintes grupos: grupo I - reimplante imediato; grupo II - reimplante tardio sem tratamento e grupo III - reimplante tardio com tratamento endodôntico (ressecção da papila dentária e preenchimento do canal com pasta de hidróxido de cálcio) e tratamento da superfície radicular (raspagem mecânica do ligamento periodontal necrosado e imersão em solução de flúor fosfato acidulado de sódio a 2,5%). Ao final dos períodos experimentais (10 e 60 dias) os ratos foram eutanasiados. Foram obtidos cortes longitudinais parafinados com 6μm de espessura. Os cortes foram submetidos à reação imunoístoquímica mediante a utilização de anticorpos primários para OPG, RANK e RANKL. Os resultados mostraram que a imunomarcação de OPG e RANKL ocorreu em todos os grupos e períodos estudados, muito embora RANKL não tenha sido observada no grupo reimplante imediato aos 60 dias. RANK foi observada somente aos 10 dias de todos os grupos no qual o reimplante foi realizado. A análise qualitativa dos resultados demonstrou que o sistema OPG, RANK e RANKL apresentou marcação evidente no reimplante tardio, sugerindo a efetiva participação no início do processo de reabsorção radicular, uma vez que aos 60 dias a imunomarcação foi discreta.
Abstract: The OPG, RANK and RANKL system is one of the most important discoveries in bone biology. These proteins are key regulators of bone remodeling and in the literature there are several studies of tooth resorption. However, in tooth replantation, reports have not been found. The purpose of this study was to determine the immunolabeling of OPG, RANK and RANKL in replanted teeth in rats, using immunohistochemistry methodology. A control group (no replanted teeth) was formed by four rats. Twenty-four rats (Rattus norvegicus, albinus) were submitted to the extraction of their upper right incisors. The replantation was performed according to the groups below: group I - immediate replantation; group II - delay replantation without treatment and group III - delay replantation with endodontic treatment (extirpation of papilla and the root canal filled with calcium hydroxide) and root surface treatment (periodontal ligament was removed with scalpel and teeth were immersed in 2% acidulated phosphate sodium fluoride). The animals were euthanized at the end of the experimental periods (10 and 60 days). Longitudinal 6μm slices embedded in paraffin were obtained. The slices were submitted to immunohistochemistry reaction by means of the primary antibodies for OPG, RANK and RANKL proteins. The results showed the expression of OPG in all groups and periods. RANKL expression was observed in all groups, except in the immediate replanted teeth group (60 days). RANK expression was observed only at 10 days in all groups which replantation was performed. The qualitative analysis of our findings indicated that the system OPG, RANK and RANKL presented strong expression in delayed replantation, suggesting effective participation at the beginning of root resorption, since at 60 days the immunostained cells were discreet.
Orientador: Wilson Roberto Poi
Coorientador: Roberta Okamoto
Banca: Sônia Regina Panzarini Barioni
Banca: Celso Koogi Sonoda
Banca: Luiz Guilherme Brentegani
Banca: Mirian Marubayashi Hidalgo
Doutor

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Cordeiro, Olga. "From lymph node embryogenesis to homeostasis : new insights into the functions of stromal RANKL (TNFSF11)". Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ075/document.

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RANKL et RANK sont membres de la superfamille des TNF et de la superfamille des TNF-récepteurs, respectivement. Ils sont connus pour jouer un rôle important dans la régulation de la masse osseuse et dans le développement et la fonction du système immunitaire. Cependant des questions restent. Nous avons utilisé des souris génétiquement modifiées pour répondre à certaines de ces questions, en particulier en utilisant une souris dont les cellules stromales réticulaires marginales manquent RANKL dans les ganglions lymphatiques. Les résultats obtenus lors de cette thèse fournissent de nouvelles informations importantes sur l'impact positif de RANKL stromal sur les macrophages des ganglions lymphatiques concomitantes avec une fonction des cellules B amélioré et une pathogénicité virale réduit. Nous avons constaté que RANKL stromal régule l'expression de lymphotoxine et CXCL13, deux molécules clés de l'homéostasie des cellules B et de l'intégrité cellulaire des organes lymphoïdes secondaires. L’activité du RANKL semble suivre une hiérarchie temporelle sur lymphotoxine/TNFα, vu que le phénotype causé par le déficit en RANKL a une pénétrance augmenté avec l'âge. De plus, nous démontrons que RANKL active les cellules endotheliales lymphatiques des ganglions lymphatiques et on a trouvé que l'intégrine ITGA2b est un nouvel indicateur pour les cellules endotheliales lymphatiques activés. Ainsi, avec MAdCAM-1, ITGA2b sert comme un nouveau marqueur pour les cellules endothéliales lymphatiques qui sont constitutivement activés par le RANKL stromal. Au total, les données confirment l'importance de RANKL pour l'homéostasie des ganglions lymphatiques et dévoile les mécanismes ci-inconnus des fonctions de RANKL. À la lumière de cela et le fait que RANKL est sensible aux hormones féminines, nous avons étudié le rôle de RANKL dans le syndrome de Sjögren, une maladie inflammatoire chronique des glandes salivaires et lacrymales avec une forte polarisation de sexe féminin. Nous apportons la preuve que la neutralisation du RANKL réduit la taille des organes lymphoïdes tertiaire. En perspective, une éventuelle diaphonie entre les cellules endothéliales lymphatiques et les macrophages ou les cellules réticulaires marginales reste à clarifier. En outre, d'autres travaux sont nécessaires pour élucider le mécanisme par lequel RANKL stimule les maladies inflammatoires chroniques présentant des structures lymphoïdes tertiaires, afin de faire RANKL une nouvelle cible pour la thérapie
RANKL and RANK are members of the TNF-superfamily and TNF-receptor superfamily, respectively. They are known to play an important role in the regulation of bone mass and in the development and the function of the immune system. However questions still remain. We have used genetically modified mice to address some of these questions, in particular by using a mouse whose lymph node marginal reticular stromal cells lack RANKL. The results obtained during this PhD provide important new insights into the positive impact of stromal RANKL on lymph node macrophages concomitant with enhanced B cell function and reduced viral pathogenicity. We found that stromal RANKL regulates lymphotoxin and CXCL13 expression, two key molecules for B cell homeostasis and secondarylymphoid organ cellular integrity. RANKL activity seems to follow a temporal hierarchy over lymphotoxin/TNFα, as the phenotype caused by stromal RANKL-deficiency has increased penetrance with age. Furthermore, we demonstrate that RANKL activates lymph node lymphatic endothelial cells and found that the integrin ITGA2b is a new indicator for activated lymphatic endothelial cells. Thus, together with MAdCAM-1, ITGA2b serves as a novel marker for those lymphatic endothelial cells that are constitutively activated by stromal RANKL. Altogether, the data reinforce the importance of RANKL for the lymph node homeostasis and uncover here to unknown mechanisms of RANKL functions.In light of this and the fact that RANKL is responsive to female hormones, we studied the role of RANKL in the Sjögrens syndrome, a chronic inflammatory disease of salivary and lacrimal glands with a strong female sex bias. We provide evidence that RANKL neutralization reduces tertiary lymphoid organ size. On the perspective side, a possible cross talk between lymph node lymphatic endothelial cells and macrophages or marginal reticular cells remains to be clarified.Furthermore, further work is required to elucidate the mechanism by which RANKL stimulates chronic inflammatory diseases presenting tertiary lymphoid structures, in order to make RANKL a new target for therapy

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Nascimento, Mariele Andrade do. "Associação entre polimorfismos genéticos em RANK, RANKL e OPG com alterações nas dimensões craniofaciais". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/58/58135/tde-20112017-095844/.

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O objetivo do presente estudo foi avaliar, em humanos, a associação entre polimorfismos genéticos no sistema RANK/RANKL/OPG com as dimensões craniofaciais. Foram incluídos neste estudo um total de 100 indivíduos caucasianos brasileiros não relacionados. O DNA foi extraído da saliva de cada um dos participantes e os polimorfismos rs3826620, rs9594738 e rs2073618 em RANK, RANKL e OPG, respectivamente, foram analisados por PCR em tempo real. Para avaliação das dimensões craniofaciais foram avaliadas três medidas angulares (SNA, SNB e ANB) e quatro medidas lineares (Co-Gn, Go-Pg, Co-Go e PTM-A), obtidas de traçados cefalométricos. Para avaliar a distribuição dos genótipos de acordo com os padrões esqueléticos faciais (Classe I, Classe II e Classe III) foi utilizado o teste do qui-quadrado. Para comparar as médias das dimensões maxilares e mandibulares de acordo com os genótipos utilizou-se o teste de Kruskal-Wallis, com o pós-teste de Dunn para comparações múltiplas, ou ANOVA com pós-teste de Tukey. Foi utilizada a análise de regressão linear multivariada para ajustar a possível influência da idade e do gênero em cada medida. O equilíbrio de Hardy-Weinberg foi avaliado utilizando o teste do qui-quadrado para cada polimorfismo. O nível de significância adotado para todas as análises foi 5%. Os resultados obtidos evidenciaram que não houve associação estatisticamente significante entre a distribuição genotípica de RANK, RANKL e OPG com as médias dos ângulos SNA e SNB, nem com a distribuição fenotípica (padrão esquelético Classe I, II ou III) (p>0,05). Observou-se diferença estatisticamente significante entre a distribuição das medidas do comprimento da base mandibular, de acordo com os genótipos de RANK, onde o genótipo GG apresentou maior medida de Go-Pg (p=0,039). Na análise multivariada, observou-se associação significante para os polimorfismos em RANK e medidas mandibulares (Go-Pg e Co-Gn) (p<0,05). A medida da maxila não foi associada a nenhum polimorfismo. Conclui-se que houve associação entre o polimorfismo genético rs3826620 em RANK com maiores dimensões mandibulares, onde o comprimento da mandíbula (Co-Gn) e o comprimento da base da mandíbula (Go-Pg) estavam aumentados.
The objective of the present study was to evaluate, in humans, the association between genetic polymorphisms in the RANK/ RANKL/OPG system with alterations in craniofacial dimensions. A total of 100 unrelated Brazilian Caucasians were included in this study. DNA was extracted from the saliva of each of the participants and the polymorphisms rs3826620, rs9594738 and rs2073618 in RANK, RANKL and OPG, respectively, were analyzed by real-time PCR. To evaluate the craniofacial dimensions, three angular measurements (SNA, SNB and ANB) and four linear measurements (Co-Gn, Go-Pg, Co-Go and PTM-A) were obtained from cephalometric tracings. To compare the difference between the means of the linear and angular measurements according to the genotype, Kruskal-Wallis followed by Dunn post test or ANOVA followed by the Tukey post test was used. Linear regression analysis was also used to adjust the possible influence of age and gender on each linear maxillary and mandibular measure. The Hardy-Weinberg equilibrium was also evaluated using the chi-square test within each polymorphism. The level of significance was 5%. The results demonstrated that there were no statistically significant association between the genotypic distribution of RANK, RANKL, OPG, and the angules SNA, SNB and according to the phenotypic distribution (Class I, Class II or Class III of skeletal pattern) (p> 0.05). A statistically significant difference was observed between the distribution of mandibular base length measurements according to the RANK genotypes, where the GG genotype showed a higher Go-Pg measurement (p=0.039). In the multivariate analysis, a statistically significant association was found for RANK and mandibular (Go-Pg and Co-Gn) polymorphisms (p<0.05). Measurement of the maxilla was not associated with any polymorphism. It was concluded that there was an association between genetic polymorphism rs3826620 in RANK with a greater mandibular dimension, in which the length of the mandible (Co-Gn) and the length of the base of the mandible (Go-Pg) were increased.

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8

Dumont, Nicolas. "Atrophie, croissance et fonction musculaire : l'impact des leucocytes et de la triade RANK/RANKL/OPG". Thesis, Université Laval, 2013. http://www.theses.ulaval.ca/2013/29749/29749.pdf.

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L’atrophie et les dysfonctions musculaires sont des problèmes courants et d’origines variées. À titre d’exemple l’alitement prolongé, le SIDA, les cancers, l’hypogravité et le vieillissement peuvent tous entraîner une perte de masse et de force musculaire. Malgré les coûts économiques et sociaux élevés, relativement peu d’efforts ont été investis dans la caractérisation des processus qui gouvernent l’atrophie/dysfonction musculaire et dans le développement de traitements plus efficaces. Conséquemment, cette thèse vise à approfondir les connaissances actuelles sur les mécanismes qui régulent l’atrophie/dysfonction et la récupération des muscles atrophiés par sous-utilisation. Tout d’abord, nous avons caractérisé l’impact des leucocytes impliqués lors de la remise en charge du muscle soléaire atrophié par hypogravité. Ainsi, nous avons démontré que les mastocytes étaient activés par la remise en charge et que leur dégranulation orchestrait le recrutement des neutrophiles et des monocytes/macrophages. Ensuite, nos recherches ont indiqué que l’activité des neutrophiles pouvait être régulée en fonction de leur microenvironnement et que leur présence dans les muscles atrophiés et inflammés n’était pas nécessairement associée à l’induction de dommages secondaires. Par la suite, nous avons établi que la présence des macrophages était importante pour permettre la récupération optimale de la force des muscles atrophiés. Nous avons également démontré que l’activité myogénique des macrophages pouvait être optimisée en favorisant leur phénotype anti-inflammatoire avec le facteur de croissance hématopoïétique « macrophage-colony stimulating factor » (M-CSF). Finalement, nous avons caractérisé l’impact du « receptor activator of NF-kB » (RANK) et de son ligand RANKL, une voie signalétique impliquée dans le remodelage osseux et l’ostéoporose, sur l’atrophie/dysfonction musculaire. Nous avons démontré qu’une déficience de RANK ou le blocage de RANKL par l’ostéoprotégérine protègent contre la perte de force des muscles dénervés ou dystrophiques. Ce phénomène est associé à des modifications dans l’expression des protéines favorisant une recaptation plus efficace du calcium. De plus, RANK participe à la reconversion des fibres rapides vers lentes durant la période de remise charge. Globalement, nos travaux permettent de mieux comprendre les mécanismes entourant l’atrophie, la croissance et les dysfonctions musculaires et ouvrent la voie à de nouvelles pistes de traitement pour plusieurs maladies neuro-musculaires.
Muscle atrophy and dysfunction are characterized by a loss of muscle mass and force, which are commonly found in many pathologies or conditions such as AIDS, cancers, chronic obstructive pulmonary diseases, cast immobilization, hypogravity, bed rest or aging, to name a few. Muscle atrophy/dysfunction have also very high social and economic costs, but very few laboratories have investigated the cellular and molecular mechanisms behind these muscular problems. The aim of this thesis is therefore to enhance our understanding of different mechanisms governing muscle atrophy/dysfunction and regrowth by using different models of disuse and dystrophy. Thus, we have initially explored the roles of different leucocytes in atrophied and reloaded soleus muscle. Firstly, we looked at the role of mast cells and showed that the mechanical stress associated with muscle reloading is able to stimulate mast cell degranulation, which orchestrates the recruitment of neutrophils and monocytes/macrophages. Secondly, our experiments revealed that neutrophil activity is adaptable and that neutrophil-induced tissue damage is dependent on the microenvironment. In atrophied and reloaded muscles, the presence of neutrophils is not associated with secondary damage or promotion of muscle recovery. Thirdly, we demonstrated that the presence of macrophages is essential for optimal muscle force recovery from atrophy. Fourthly, we showed that the macrophage-colony stimulating factor (M-CSF) promote the myogenic activity of macrophages by stimulating their anti-inflammatory phenotype. Finally, we investigated the impact of the receptor activator of NF-kB (RANK) and its ligand RANKL, a signalling pathway involved in bone remodelling and osteoporosis, on muscle atrophy and dysfunction. Our results showed that the specific deletion of RANK in the muscle or the blockage of RANKL with osteoprotegerin increased significantly force production in denervated and dystrophic muscles. These results were associated with various modifications in calcium handling protein expression favouring efficient calcium uptake. Moreover, we also demonstrated that RANK activation gives preference to the reconversion from fast-to-slow muscle fibers following hindlimb unloading/reloading. Overall, our results bring a better understanding of different mechanisms related to muscle atrophy, dysfunction and regrowth and potentially open new avenues for the treatment of several debilitating skeletal muscle conditions.

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9

Loureiro, Melina Bezerra. "Estudo da associa??o dos genes RANk, RANKL e OPG com a osteopatia diab?tica". Universidade Federal do Rio Grande do Norte, 2014. http://repositorio.ufrn.br/handle/123456789/19413.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq
O objetivo do presente trabalho foi avaliar a express?o de RNAm e os polimorfismos dos genes RANK, RANKL e OPG de crian?as, adolescentes e adultos jovens com DM1, bem como estudar a associa??o dos mesmos com o desenvolvimento de altera??es no metabolismo ?sseo.No total, foram inclu?dos no estudo 119 crian?as e adolescentes com DM1 e 161 indiv?duos normoglic?micos (NG) da mesma faixa et?ria. Foram pesquisados os polimorfismos dos genes OPG (1181 G>C e 163 A>G), RANK (575 C>T e 3'UTR C>A) e RANKL (?ntron A>G) e determinadas as express?es g?nicas de OPG, RANK e RANKL. Al?m disso, foram avaliados o controle glic?mico e par?metros laboratoriais de fun??o ?ssea, al?m da densitometria ?ssea. Os indiv?duos com DM1 apresentaram um controle glic?mico insatisfat?rio e valores diminuidos de c?lcio total, propept?deo do col?geno tipo 1 (CTX), como tamb?m baixa densidade mineral ?ssea, quando comparados com os NG (p<0,05). O polimorfismo OPG 1181 G>C pode estar associado com susceptibilidade ao DM1 (p=0,054). Estudando apenas os indiv?duos com DM1, foi observado que os carreadores do gen?tipo OPG 1181 GG apresentaram maiores concentra??es de c?lcio ionizado no modelo recessivo (p<0,05). Esses resultados sugerem que o polimorfismo OPG 1181 G>C pode contribuir para o desenvolvimento do DM1 e da osteopatia diab?tica.

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Camara, Abdouramane. "Control of lymphoid organ CD169+ macrophage differentiation by stromal cells through the RANK-RANKL axis". Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ102.

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Au-delà de leurs rôles de sentinelles, de reconnaissance du danger et d’initiation des réponses protectrices, les signaux et le mécanisme qui gouvernent la formation des macrophages CD169 + sinusoïdaux ganglionnaires sont mal connus. Au cours de ma thèse, j’ai montré que la cytokine Receptor Activator of NF-kB Ligand (RANKL) est requise pour la formation de ces macrophages dès l’embryogenèse jusqu’aux quatre semaines après la naissance. Celle-ci est contrôlée par les cellules endothéliales lymphatiques (LECs) activées par RANKL produite par les cellules mésenchymateuses. Chez l’adulte, les LECs activées par RANKL sont encore nécessaires pour la reconstitution des populations de ces macrophages en cas de déplétion transitoire induite par un stimulus inflammatoire. En complément à cela, j’ai aussi démontré l’importance générale du double signal RANKL & lymphotoxine LTα1β2 dans la formation des macrophages non-ostéoclastiques de la rate et de la moelle osseuse
Lymph node CD169 + sinusoidal macrophages are sentinel cells that recognize the danger signals and initiate the protective immune responses. However, the signals and the mechanism underlying their formation are not well known. During my thesis, I have shown that the cytokine Receptor Activator of NF-kB Ligand (RANKL) is required for their differentiation, starting from the embryogenesis up to four weeks after birth. The lymphatic endothelial cells (LECs) activated by RANKL expressed by mesenchymal cells form the niches for the primary differentiation of these macrophages. Yet, in adults, RANKL-activated LECs are required for their niche replenishment after transient depletion induced by an inflammatory stimulus. Beyond lymph node, my research has revealed a general requirement of the double signal RANKL & lymphotoxin LTα1β2 for the differentiation of non-osteoclastic CD169 + macrophages of spleen and bone marrow

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11

Khogeer, Asim Abdulaziz Omar. "Cellular, epigenitic, genetic and signalling alterations associated with RANK expression in bone-tropic breast cancer cells". Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25880.

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Bone metastases are a major cause of morbidity in patients of advanced breast cancer. Development of osteolytic bone metastasis depends on the interaction between malignant tumour cells and bone microenvironment. Receptor Activator of Nuclear Factor Kappa B (RANK) is a member of tumour necrosis factor (TNF) superfamily that is expressed by osteoclasts (the bone resorbing cells) and primary breast tumour cells. Previous studies demonstrated that RANK receptor and its ligand (RANKL) play an important role in bone remodelling, mammary gland development and immune system. RANKL was also found to serve as a chemotactic factor that facilitates breast tumour metastasis to bone. However, the role of the RANK receptor in breast cancer cell metastatic behaviour in bone is not fully understood. Therefore, the aim of this thesis was to explore the role of the RANK receptor in parental and bone-tropic breast cancer cell growth, motility and invasion, and assess these cells influence on breast cancer cell induced osteoclastogenesis. Functional studies in breast cancer cells showed that RANKL (100 - 300 ng/ ml) significantly enhanced parental human MDA-231 (MDA-231P) and mouse 4T1 breast cancer cell spreading within minutes. RANKL induced chemotactic cell migration of MDA-231P cells in vitro. I also found that RANKL significantly stimulated random and directional 2D and 3D cell migration of parental MDA-231P and bone-tropic (MDA-231BT) breast cancer cells in vitro. These effects were observed at concentrations (100 – 300 ng/ml) that were sufficient to induce osteoclast formation in the presence and absence of breast cancer cells in vitro. In contrast, high concentrations of RANKL (1000 ng/ ml) dramatically suppressed human MDA-231P breast cancer cell invasion in vitro. These data indicate that the RANK receptor in the breast cancer cell lines tested influences cancer cell spreading, migration and invasion in vitro. Thus, targeting RANK in tumour cells may be of value in the prevention of tumour burden associated with breast cancer bone metastasis. Mechanistic studies revealed that RANKL stimulated the phosphorylation of p38 kinase in human and mouse breast cancer cells. Interestingly, RANKL had no effect on NFᴋB, JNK and AKT pathways in parental human MDA-231 and mouse 4T1 breast cancer cells at concentrations up to 300 ng/ ml. These data implies that the RANK receptor modulates human and mouse breast cancer cell metastatic behaviour via p38 activation and independently of the NFᴋB and PI3K/AKT pathways. Silencing of the RANK receptor in the bone-tropic human breast cancer cells MDA- 231BT2 reduced directional cell migration without affecting cell viability and growth. Functional studies in osteoclast and breast cancer cell revealed that knockdown of RANK expression in both parental and bone-tropic human breast cancer cells significantly inhibited the ability of these cells to stimulate osteoclast formation. Although, I cannot exclude the possibility of the involvement of other signalling pathways downstream of the RANK receptor, these studies suggest that the RANK/P38 signalling in osteoclast and breast cancer cells contributes significantly to breast cancer cell behaviour in bone. Genetic analysis of the RANK gene in human parental and bone-tropic MDA-231 breast cancer cells showed a number of polymorphisms. One variant detected was found to be deleterious for the RANK protein. This variant changes the amino acid sequence from alanine to threonine (Ala ˃ Thr) and only appeared in the RANK gene in the parental human MDA-231P breast cancer cells. Moreover, of the four known RANK isoforms that were detected in the parental and bone-tropic breast cancer cells tested, two lacked the TRAF6 binding motifs associated with NFκB activation. All RANK isoforms detected on the bone-tropic MDA-231BT breast cancer cells expressed the P38 binding motifs. Altogether, these findings support the role of the RANK/P38 signalling pathway in breast cancer cell behaviour in bone. Epigenetic analysis in parental human MDA-231P breast cancer cells showed that continuous and long-term exposure to RANKL (10 and 100 ng/ ml) for up to 50 passages (approximately 120 days) did not induce epigenetic changes, particularly DNA methylation, in the RANK gene. However, I found DNA methylation changes in a set of genes that are known to be involved in cell development and regulation. The methylation status of the altered CpG loci either hypermethylated or hypomethylated are located at different parts in the CpG islands. Whole genome DNA methylation pattern of the bone-tropic breast cancer cells showed a number of genes that appeared in both bone-tropic variants are correlated with different biological function of the cells. I also found that long-term exposure of human MDA-231P to RANKL (100 ng/ ml) enhanced the ability of these cells to stimulate osteoclastogenesis in vitro. These data together indicate that long-term exposure to RANKL induces “boney” epigenetic changes in a set of genes that enhances breast cancer cell behaviour in bone. Overall, this thesis illustrated that the RANK receptor on human parental and bone-tropic breast cancer cells plays an important role in cell motility and ability of these cells to influence osteoclastogenesis and ultimately osteolysis. Therefore, agents that selectively target the RANK receptor may be of value in the treatment of both tumour burden and osteolytic bone disease associated with breast cancer. However, the role of the RANK receptor in bone metastasis will require further in vivo investigation.

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12

Fuji, Hiroaki. "Necrostatin－7 suppresses RANK－NFATc1 signaling and attenuates macrophage to osteoclast differentiation". Kyoto University, 2019. http://hdl.handle.net/2433/242355.

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Penno, Hendrik. "On the Role of Osteoprotegerin/RANK/RANKL System in the Interaction between Prostate Cancer and Bone". Doctoral thesis, Uppsala universitet, Ortopedi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-160751.

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Metastases to bone are observed in around 80% of prostate cancer patients and represent the most critical complication of advanced prostate cancer. Unlike other solid tumors that are associated with osteolytic bone metastases, prostate cancer bone metastases stimulate osteoblastic activity with sclerosis in the bone lesions as a consequence. Osteoprotegerin (OPG) is part of a system with three proteins that play a key role in bone remodeling; namely OPG, RANK and RANKL. RANKL regulates osteoclast activity by binding to RANK on the osteoclasts surface, and this interaction is interrupted by OPG. OPG also plays a role in the lifecycle of tumor cells by blocking TNF-related apoptosis-inducing ligand (TRAIL) making it possible for them to evade cell death. The aim of this thesis was to investigate the interaction between the OPG/RANK/RANKL system and prostate cancer. Data showed that there was production of OPG from prostate cancer cell lines in vitro. This expression was under the influence of cytokines that are present in the microenvironment of bone. Further, there was documented a previously unnoticed cell surface expression of RANKL. Co-culturing the prostate cancer with human osteoblasts increased the expression of RANKL. To connect these findings with in vivo studies, OPG-gene single nucleotide polymorphisms (SNP) were investigated. To evaluate OPG SNPs association with bone, a cohort of elderly men was used. OPG SNPs was shown to be correlated to bone mineral density at hip and spine. There was also an association to fragility fractures. Then there was examined the association of the same SNPs to the incidence of prostate cancer but after a four-year follow-up there was no association to the genetic variants. To summarize this research, we hereby present data that the OPG/RANK/RANKL system might be relevant for prostate cancer growth in bone, and for the skeletal related morbidity in this disease. Future in vitro and in vivo studies will demonstrate the relative importance of this crosstalk, and whether pharmacological interference with the system might be used as a therapeutic tool aiming to decrease skeletal morbidity and possibly also prolong survival in prostate cancer.

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Coutinho, Carolina Chiantelli Cláudio [UNESP]. "Expressão das proteínas osteoprotegerina, RANK e RANKL durante o processo de reparo alveolar em ratos: estudo imunoistoquímico". Universidade Estadual Paulista (UNESP), 2005. http://hdl.handle.net/11449/88947.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Na dinâmica da reparação óssea os fenômenos de reabsorção e neoformação são dependentes e acoplados. Proteínas efetivamente envolvidas na diferenciação celular determinam ativação ou inibição das atividades que regulam o ganho ou perda de massa óssea. Dentre as proteínas ósseas identificadas e envolvidas na dinâmica óssea podemos destacar a OPG, a RANK e RANKL como marcadores de atividades celulares. O presente trabalho tem como objetivo identificar, nos diferentes períodos da cronologia do processo de reparo alveolar através de técnica imunoistoquímica, a presença das proteínas OPG, RANK e RANKL. Para tanto foram utilizados 60 ratos machos submetidos à exodontia do incisivo superior direito e perfundidos aos 14, 21 e 28 dias pós-operatórios. As hemi-maxilas contendo o alvéolo dental em reparação foram removidas, pósfixadas, descalcificadas em EDTA, crioprotegidas e obtidos cortes longitudinais com 14æm em criostato. Os cortes foram submetidos à reação imunoistoquímica mediante a utilização de anticorpos primários para OPG, RANK e RANKL, como amplificador foi utilizado o sistema avidina-biotina e a diaminobenzidina (DAB) como cromógeno. Os resultados mostram que qualitativamente ocorre um balanço na expressão das proteínas que caracterizam reabsorção e neoformação óssea nos diferentes períodos estudados, onde aos 14 e 21 dias ocorre maior expressão de RANK. Com relação às proteínas OPG e RANKL, observa-se que elas apresentam-se expressas nas células da linhagem osteoblástica de forma similar, sendo que 28 dias é o período de maior expressão destas proteínas.
In the bone healing dynamics, the resorption and neoformation processes are dependent. Proteins involved in the cellular differentiation determinate the activation or inhibition of the activities that regulate the gain or loss of bone mass. From all of the identified bone proteins, it may be distinguished the OPG, RANK and RANKL. The present study has the aim to identify, in the different periods of alveolar bone healing chronology, the expression of OPG, RANK and RANKL proteins using the immunohistochemistry methodology. To perform this study, 60 male rats had the right upper incisive extracted and they were perfused at 14, 21 and 28 pos-operative days. The hemimaxilla with the rat extraction socket was removed, pos fixed and decalcified in EDTA. Then, they were cryoprotected and longitudinal slices with 14 ìm thickness were obtained in cryostat. The slices were submitted to immunohistochemistry reaction and the primary antibodies used were against OPG, RANK and RANKL proteins. It was used the avidinbiotin system to amplify the sign and diaminobenzidine was the cromogen. The results show that there is a balance in the expression of the proteins, showing that there is an increase in the expression of RANK at 14 and 21 pos-operative periods. In relation to OPG and RANKL, these proteins presents a similar expression in all of the pos-extraction periods analysed in this study and at 28 days after the extraction there is the greater expression of both proteins.

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Coutinho, Carolina Chiantelli Cláudio. "Expressão das proteínas osteoprotegerina, RANK e RANKL durante o processo de reparo alveolar em ratos : estudo imunoistoquímico /". Araçatuba, 2005. http://hdl.handle.net/11449/88947.

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Resumo: Na dinâmica da reparação óssea os fenômenos de reabsorção e neoformação são dependentes e acoplados. Proteínas efetivamente envolvidas na diferenciação celular determinam ativação ou inibição das atividades que regulam o ganho ou perda de massa óssea. Dentre as proteínas ósseas identificadas e envolvidas na dinâmica óssea podemos destacar a OPG, a RANK e RANKL como marcadores de atividades celulares. O presente trabalho tem como objetivo identificar, nos diferentes períodos da cronologia do processo de reparo alveolar através de técnica imunoistoquímica, a presença das proteínas OPG, RANK e RANKL. Para tanto foram utilizados 60 ratos machos submetidos à exodontia do incisivo superior direito e perfundidos aos 14, 21 e 28 dias pós-operatórios. As hemi-maxilas contendo o alvéolo dental em reparação foram removidas, pósfixadas, descalcificadas em EDTA, crioprotegidas e obtidos cortes longitudinais com 14æm em criostato. Os cortes foram submetidos à reação imunoistoquímica mediante a utilização de anticorpos primários para OPG, RANK e RANKL, como amplificador foi utilizado o sistema avidina-biotina e a diaminobenzidina (DAB) como cromógeno. Os resultados mostram que qualitativamente ocorre um balanço na expressão das proteínas que caracterizam reabsorção e neoformação óssea nos diferentes períodos estudados, onde aos 14 e 21 dias ocorre maior expressão de RANK. Com relação às proteínas OPG e RANKL, observa-se que elas apresentam-se expressas nas células da linhagem osteoblástica de forma similar, sendo que 28 dias é o período de maior expressão destas proteínas.
Abstract: In the bone healing dynamics, the resorption and neoformation processes are dependent. Proteins involved in the cellular differentiation determinate the activation or inhibition of the activities that regulate the gain or loss of bone mass. From all of the identified bone proteins, it may be distinguished the OPG, RANK and RANKL. The present study has the aim to identify, in the different periods of alveolar bone healing chronology, the expression of OPG, RANK and RANKL proteins using the immunohistochemistry methodology. To perform this study, 60 male rats had the right upper incisive extracted and they were perfused at 14, 21 and 28 pos-operative days. The hemimaxilla with the rat extraction socket was removed, pos fixed and decalcified in EDTA. Then, they were cryoprotected and longitudinal slices with 14 ìm thickness were obtained in cryostat. The slices were submitted to immunohistochemistry reaction and the primary antibodies used were against OPG, RANK and RANKL proteins. It was used the avidinbiotin system to amplify the sign and diaminobenzidine was the cromogen. The results show that there is a balance in the expression of the proteins, showing that there is an increase in the expression of RANK at 14 and 21 pos-operative periods. In relation to OPG and RANKL, these proteins presents a similar expression in all of the pos-extraction periods analysed in this study and at 28 days after the extraction there is the greater expression of both proteins.
Orientador: Roberta Okamoto
Coorientador: Roelf Justino Cruz Rizollo
Banca: Idelmo Rangel Garcia Júnior
Banca: Victor Elias Arana-Chavez
Mestre

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Maia, Concei??o Aparecida Dornelas Monteiro. "An?lise imuno-histoqu?mica das prote?nas RANK, RANKL e OPG em dentes de ratos reimplantados". Universidade Federal do Rio Grande do Norte, 2014. http://repositorio.ufrn.br/handle/123456789/19980.

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O sistema RANK/RANKL/OPG contribui para a compreens?o do processo e regula??o da forma??o e reabsor??o do osso, tendo sido o maior avan?o na biologia ?ssea com rela??o a osteoclastog?nese. RANKL e OPG inibem RANK regulando a forma??o, ativa??o e sobreviv?ncia de osteoclastos na remodela??o ?ssea. O objetivo desse estudo foi investigar a express?o dos marcadores RANKL/RANK/OPG em dentes de ratos reimplantados, bem como observar a rela??o entre a express?o desses marcadores com o processo de reabsor??o dent?ria e ?ssea. Foram utilizados 30 incisivos superiores direitos de 30 ratos machos, adultos, da linhagem Wistar (Rattus norvegicus albinus). Os dentes foram avulsionados e divididos em dois grupos que permaneceram extra-alveolar em ar seco: G1 (n=15) - 5 minutos e G2 (n=15) - 60 minutos, e em seguida foram reimplantados e analisados nos intervalos de 1, 3 e 7 dias. Finalizado os per?odos experimentais, ocorreu a eutan?sia dos animais. Cortes longitudinais com 5?m de espessura foram obtidos e corados pela t?cnica H/E para a an?lise histol?gica e cortes com 3?m de espessura foram submetidos ? rea??o imuno-histoqu?mica mediante a utiliza??o de anticorpos prim?rios para ratos como OPG, RANK e RANKL. Os resultados demonstraram que o sistema RANK/RANKL/OPG participa ativamente, tanto do processo de reparo de dentes de ratos reimplantados, quanto das reabsor??es dent?rias e ?sseas; que RANKL apresentou maior imunomarca??o em ambos os grupos, participando em todas as fases da reabsor??o ?ssea e dent?ria; e o aumento da express?o de RANKL foi observada em ambos os grupos em todos os intervalos de tempo, comprovando que a resposta inflamat?ria no ligamento periodontal ocorre no in?cio do processo de reparo; e que a fraca express?o de RANK e OPG e o aumento da express?o de RANKL sugere uma diminui??o da reabsor??o ativa e aumento da reabsor??o reparada no osso e no cemento, sendo maior no osso.
The RANK / RANKL / OPG sy stem plays an important role in bone formation and resorption . This finding has been regarded as one of the m ost important advances in the understanding of bone biology with respect to osteoclastogenesis. The aim of this study was to investigate the expression of RANKL / RANK / OPG markers in reimplanted t eeth of rats, and to observe the relationship between the expression of these markers and to oth and bone resorption. Thirty male Wistar rats (Rattus norvegicus albinos) had their maxillary right incisors extrac ted , and were divided into 2 groups according to the period that the extracted teeth were kept in dry air before reimplantation : G1 (n = 15) - 5 minutes , and G2 (n = 15) - 60 minutes . After reimplantation, teeth were analyzed at intervals of 1, 3 and 7 da ys. After these experimental periods, the animals were euthanized. Longitudinal sections with 5?m thick were obtained and stained with Hematoxylin and Eosin for histological analysis , while 3?m thick sections were subjected to immunohistochemical analysis of OPG , RANK and RANKL. The results showed that the RANK / RANKL / OPG system actively participates in both the repair process, as well as tooth and bone resorption . Extr a - alveolar time of 60 minutes before replantation caused minor expressions of RANKL a nd OPG, not influencing the expression of RANK; RANKL immunostaining showed higher in both groups when compared to other biomarkers, participating in all phases of bone and tooth resorption; RANKL was associated to both osteoclastogenesis and c ell ular proliferation , and was expressed in both groups.

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Waegner, Rena Hinrika [Verfasser], Lutz [Akademischer Betreuer] Trojan y Carsten [Akademischer Betreuer] Gründker. "Expression von RANK / RANKL im Harnblasenkarzinom / Rena Hinrika Waegner. Betreuer: Lutz Trojan. Gutachter: Lutz Trojan ; Carsten Gründker". Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1080361693/34.

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Wang, Cathy Ting-Peng. "Molecular dissection of RANKL signaling pathways in osteoclasts". University of Western Australia. School of Surgery and Pathology, 2007. http://theses.library.uwa.edu.au/adt-WU2008.0037.

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[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.

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Lesky, Thomas. "In vitro Differenzierung von Monozyten der Zelllinine RAW 264.7 zu Osteoklasten, deren Charakterisierung und Wechselwirkung mit Osteoblasten". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1158674933492-53949.

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Das RANKL/RANK/OPG-System spielt eine entscheidende Rolle in der Steuerung der Osteoklastendifferenzierung und -aktivierung durch Osteoblasten/ Knochenmarkbindegewebszellen im Rahmen des Knochenremodelings. Osteoblasten/Knochenmarkbindegewebszellen exprimieren RANKL. Dieses hat im Körper zwei Rezeptoren: RANK und OPG. RANKL kann durch Bindung an RANK auf Osteoklasten/Osteoklastenvorläuferzellen in Gegenwart von M-CSF seine osteoklastenstimulierende Wirkung entfalten. Der ebenfalls von Osteoblasten gebildete „decoy“-Rezeptor OPG blockiert als freies Protein durch Bindung an RANKL dessen Interaktion mit RANK und verhindert somit die Osteoklastogenese und Osteoklastenaktivierung. Das RANKL/RANK/OPG-System erfüllt im Körper noch weitere Funktionen im Immunsystem, in der Organentwicklung lymphatischer Gewebe und in der Entwicklung der laktierenden Brustdrüse. Viele Zytokine greifen hemmend oder aktivierend in die Osteoklastogenese ein. Sie können dies zum einen durch die Beeinflussung des RANKL/OPG-Verhältnisses, zum anderen durch direkte Interaktion mit Osteoklasten/Osteoklastenvorläuferzellen tun. Zytokine, die die Osteoklastogenese begünstigen, werden vor allem bei inflammatorischen Prozessen ausgeschüttet. Zusammen mit dem, bei diesen Zuständen von aktivierten T-Zellen produzierten RANKL kann dies längerfristig zu einem Knochenverlust führen, welcher sich im klinischen Bild der Osteoporose äußert. Aus den in der vorliegenden Dissertation durchgeführten Untersuchungen ergeben sich folgende Schlussfolgerungen: 1. Monozyten der Zelllinie RAW 264.7 lassen sich, wie bereits in der Literatur beschrieben, durch Zugabe von M-CSF und RANKL zu osteoklastenähnlichen Zellen differenzieren. 2. Die Osteoklastogenese lässt sich anhand der Veränderung verschiedener osteoklastenspezifischer Parameter charakterisieren. Es zeigt sich bei den mit M-CSF und RANKL stimulierten Monozyten eine erhöhte Transkription von CTR (Calcitoninrezeptor)- und TRAP (tartratresistente saure Phosphatase)-mRNA, eine erhöhte Expression des CTR-Proteins, eine erhöhte TRAP-Aktivität und eine Formierung TRAP-positiver mehrkerniger Riesenzellen, die in diesen Eigenschaften Osteoklasten entsprechen. Die zusätzliche Zugabe von TGF-b1 in Kombination mit M-CSF und RANKL resultiert in einer verstärkten Expression von CTR-mRNA und CTR-Protein. TRAP-mRNA-Expression und TRAP-Aktivität bleiben davon unbeeinflusst. 3. Als funktionelles Merkmal der in vitro differenzierten Osteoklasten können ihre Fähigkeit zur Ausbildung von Aktinringen und die Resorption von mineralisiertem Kollagen nachgewiesen werden. 4. Im Verlauf ihrer Differenzierung sekretieren Osteoblasten unterschiedliche Mengen an OPG. Das Maximum der Synthese liegt bei Tag 11. Freies RANKL lässt sich in Überständen von MC3T3-E1-Osteoblasten nicht nachweisen. 5. Das von Osteoblasten in das Medium abgegebene OPG ist in der Lage, die durch RANKL induzierte Osteoklastogenese von RAW-Monozyten zu hemmen. 6. In Kokulturen von MC3T3-E1-Osteoblasten und RAW-Monozyten kann keine Osteoklastogenese beobachtet werden, wahrscheinlich durch Fehlen der RANKLExprimierung oder zu starke OPG-Sekretion durch Osteoblasten. Besonders in der westlichen Welt mit ihrer hohen Lebenserwartung haben Krankheiten mit Knochenverlust sowie bösartige Neubildungen mit Knochenbefall eine große medizinische Bedeutung. Die Beeinflussung des RANKL/RANK/OPG-Systems bietet eine vielversprechende Möglichkeit zur Entwicklung hochwirksamer und nebenwirkungsarmer Medikamente zur Behandlung dieser Zustände.

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Moraes, Maiara de. "Express?o imuno-hitoqu?mica das prote?nas RANK, RANKL e OPG em cistos radiculares e cistos dent?geros". Universidade Federal do Rio Grande do Norte, 2010. http://repositorio.ufrn.br:8080/jspui/handle/123456789/17113.

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Receptor ativador nuclear κappa B (RANK), ligante do receptor ativador nuclear κappa B (RANKL) e osteoprotegerina (OPG) s?o membros da fam?lia do fator de necrose tumoral relacionados com o metabolismo ?sseo. A forma??o, diferencia??o e atividade dos osteoclastos s?o reguladas por estas tr?s prote?nas. RANK ? um receptor transmembrana presente em diversos tipos celulares, principalmente em c?lulas de linhagem macrof?gica, linf?citos, c?lulas dendr?ticas e fibroblastos e quando ativado pelo seu ligante, RANKL, promove a diferencia??o e ativa??o de c?lulas osteocl?sticas respons?veis pelo processo de reabsor??o ?ssea. A OPG impede a liga??o RANK/RANKL atuando como um receptor inibit?rio para a atividade osteol?tica. O objetivo deste estudo foi comparar a express?o imuno-histoqu?mica destes biomarcadores em cistos radiculares (n=20) e cistos dent?geros (n=20). A express?o imuno-histoqu?mica destes marcadores foi avaliada no epit?lio e na c?psula dos cistos por escores e percentuais m?dios de imunomarca??o. Para o epit?lio, a an?lise semi-quantitativa revelou um padr?o similar dos escores de imunomarca??o de RANK, RANKL e OPG nas les?es, n?o havendo diferen?a estat?stica significante (p=0.589, p=0.688, p=0.709, respectivamente). Para a c?psula c?stica a an?lise quantitativa, mostrou diferen?a estat?stica significante entre os percentuais m?dios de imunomarca??o do RANK e RANKL (p=0,001 e p=0,005, respectivamente) nos cistos. A correla??o dos escores de imunomarca??o de RANKL e OPG no epit?lio do CR e do CD revelou diferen?a estat?stica significante (p=0,029, p=0,003, respectivamente). No epit?lio dos CRs e dos CDs observou-se uma maior imunoexpress?o da OPG comparada a do RANKL. Os resultados apontam a presen?a de RANK, RANKL e OPG nos cistos radiculares e cistos dent?geros, sugerindo a atua??o destas prote?nas no desenvolvimento e expans?o das les?es no osso adjacente
Receptor ativador nuclear κappa B (RANK), ligante do receptor ativador nuclear κappa B (RANKL) e osteoprotegerina (OPG) s?o membros da fam?lia do fator de necrose tumoral relacionados com o metabolismo ?sseo. A forma??o, diferencia??o e atividade dos osteoclastos s?o reguladas por estas tr?s prote?nas. RANK ? um receptor transmembrana presente em diversos tipos celulares, principalmente em c?lulas de linhagem macrof?gica, linf?citos, c?lulas dendr?ticas e fibroblastos e quando ativado pelo seu ligante, RANKL, promove a diferencia??o e ativa??o de c?lulas osteocl?sticas respons?veis pelo processo de reabsor??o ?ssea. A OPG impede a liga??o RANK/RANKL atuando como um receptor inibit?rio para a atividade osteol?tica. O objetivo deste estudo foi comparar a express?o imuno-histoqu?mica destes biomarcadores em cistos radiculares (n=20) e cistos dent?geros (n=20). A express?o imuno-histoqu?mica destes marcadores foi avaliada no epit?lio e na c?psula dos cistos por escores e percentuais m?dios de imunomarca??o. Para o epit?lio, a an?lise semi-quantitativa revelou um padr?o similar dos escores de imunomarca??o de RANK, RANKL e OPG nas les?es, n?o havendo diferen?a estat?stica significante (p=0.589, p=0.688, p=0.709, respectivamente). Para a c?psula c?stica a an?lise quantitativa, mostrou diferen?a estat?stica significante entre os percentuais m?dios de imunomarca??o do RANK e RANKL (p=0,001 e p=0,005, respectivamente) nos cistos. A correla??o dos escores de imunomarca??o de RANKL e OPG no epit?lio do CR e do CD revelou diferen?a estat?stica significante (p=0,029, p=0,003, respectivamente). No epit?lio dos CRs e dos CDs observou-se uma maior imunoexpress?o da OPG comparada a do RANKL. Os resultados apontam a presen?a de RANK, RANKL e OPG nos cistos radiculares e cistos dent?geros, sugerindo a atua??o destas prote?nas no desenvolvimento e expans?o das les?es no osso adjacente

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Pellegrini, Pasquale 1983. "RANK signaling, a master regulator of mammary stemness and cancer : characterization of RANK and RANKL pathway stem cell fate, tumorigenesis and metastasis". Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/586316.

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RESUME RANK signaling is essential for mammary gland development and progesterone induced tumorigenesis. Here we show that RANK controls mammary stem cell fate; increased RANK signaling results in accumulation of stem and progenitor cells leading to spontaneous tumor formation. RANK signaling interferes with alveolar commitment through depletion of CD61+ alveolar progenitors and regulation of Elf-5/STAT5 pathway. Using pharmacological and genetic approaches we show that RANK signaling promotes tumor initiation, progression and metastasis in spontaneous breast cancer models and expands tumor initiating cells and metastasis initiating cells. RANK pathway mediates the crosstalk between tumor cells and their microenvironment modulating the tumor immune response. RANK expression correlates with aggressiveness and metastasis in human tumors. Altogether our data support that RANK signaling could be a novel therapeutic target in breast cancer.
RESUMEN La vía de RANK es esencial para el desarollo de la glándula mamaria y la tumorigénesis inducida por progesterona. Demostramos que RANK controla la especificación de linajes mamarios, y que un aumento de señalización a través de RANK induce una acumulación de células madre y progenitoras de mama con la consiguiente formación espontánea de tumores. RANK bloquea la diferenciación alveolar a través de la disminución de progenitoras alveolares CD61+ y la regulación de la via Elf-5/STAT5. Utilizando estrategias genéticas y farmacológicas demostramos que RANK promueve la formación, progresión tumoral y la metástasis de modelos de tumorigenesis espontánea de mama, y expande las poblaciones de células iniciadoras de tumores y células iniciadoras de metastasis. La vía de RANK media además la interacción entre las células tumorales y su entorno, modulando la respuesta inmune tumoral. El aumento en la expresión de RANK se correlaciona con la agresividad de tumores humanos y la metástasis. Estos resultados sugieren que la vía de RANK puede ser una nueva diana terapéutica en cáncer de mama.

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Amorim, Fernanda Penna Lima Guedes de. "Expressão do receptor ativador de NF-kBETA (Rank), Rank ligante (RANKL), e Osteoproterina (OPG) em sítios de reparo ósseo de ratos diabéticos". reponame:Repositório Institucional da UnB, 2007. http://repositorio.unb.br/handle/10482/1847.

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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2007.
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Os mecanismos envolvidos na modificação do reparo ósseo em diabéticos ainda permanece pouco elucidado. Assim, esse estudo investigou a expressão de reguladores do metabolismo ósseo receptor ativador de NFkB (RANK), RANK ligante (RANKL) e osteoprotegerina (OPG), através da imunohistoquímica e RT-PCR, em sítios de fratura óssea de ratos diabéticos. Foram realizadas fraturas ósseas fechadas em tíbias esquerdas de ratos controle e com diabetes induzido pelo aloxano. A análise histomorfométrica dos sítios de fratura após 7 dias revelaram que os ratos diabéticos (db) apresentaram menor formação óssea e de cartilagem em comparação com o grupo controle. Paralelamente, o número de células RANK e RANKL positivas foi reduzido no grupo diabético. Além disso, células OPG positivas apresentaram-se significantemente diminuídas no grupo diabético comparado ao grupo controle (p=0,05). Entretanto, a razão RANKL/OPG foi similar no grupo controle (0,074) e diabético (0,099) nesse período. Após 14 dias, o número de células RANKL e OPG positivas e a expressão de RNAm desses marcadores foi maior no grupo controle (p=0.008). Apesar de menores níveis, a razão RANKL/OPG no grupo diabético (1,29) foi maior do que no grupo controle (0,90), o que sugere o favorecimento dos mecanismos de reabsorção óssea. Os resultados obtidos demonstram a expressão de marcadores da atividade de formação/remodelação de tecidos duros em sítios de fratura. Modificações no balanço da expressão de RANKL/OPG pode contribuir para o retardo do reparo de fraturas associado ao estado diabético. ____________________________________________________________________________________ ABSTRACT
To clarify the mechanisms of altered bone reapair in diabetic state, we investigate the RANK, RANKL and OPG expression by immunohistochemistry and RT-PCR in the fracture sites of diabetic rats. A closed fracture was performed on the anatomical left tibia in rats either healthy or made diabetic by alloxan. Histomorphometric analysis of fracture site at 7 days after fracture revealed that diabetic rats (db) have significantly lesser bone and cartilage formation at fracture site in comparison with controls. Parallel with this, the number of RANK and RANKL positive cells were slighly decreased in db group. Furthermore, OPG+ cells were significantly lower in db than control (p=0.05). However, the RANKL/OPG ratio was similar in control (0.074) and db (0.099) at this time. At day 14, the numbers of RANKL and OPG positive cells and the mRNA expression for these markers were increased in control group (P=0.008). Despite lower levels, the RANKL/OPG ratio in db group (1.29) was greater than in controls (0.90) what suggets a favoring of pro-resorptive pathways. Our results demonstrate the expression of consistent markers of hard tissue formation/remodeling activities in sites of fractures. The imbalance of RANKL/OPG expression may contribute to the delay of fracture repair during diabetes course.

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Chypre, Mélanie. "Role of receptor activator of NF-kB ligand (RANKL) in adult lymph node homeostasis and identification of inhibitors". Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ016/document.

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Le récepteur activateur de NF-κB (RANK), membre de la famille des récepteurs au TNF, est connu pour son rôle dans l’homéostasie de l’os, mais joue aussi un rôle important dans le système immunitaire. J’ai tout d’abord étudié des outils permettant de cibler RANK/RANKL. J’ai caractérisé et comparé l’activité biologique de deux anticorps anti-RANK. J’ai également criblé une librairie de petites molécules pour identifier des inhibiteurs de l’interaction RANK/RANKL. Dans une deuxième partie, je me suis intéressée au rôle du ligand de RANK (RANKL) dans l’homéostasie du ganglion lymphatique. RANKL joue un rôle dans la différenciation des ostéoclastes mais son rôle dans la différenciation d’autres macrophages n’a pas été étudié. Nous avons étudié des souris déficientes pour RANKL dans les cellules marginales réticulaires (MRC) qui expriment RANKL de manière constitutive dans le ganglion adulte. Nous avons observé une diminution de la population de macrophages sous-capsulaires (SSM). Nous avons également montré que les cellules endothéliales lymphatiques (LEC) expriment l’intégrine alpha 2b (ITGA2b) et que cette expression est sensible à la présence de RANKL
The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation. Firstly I looked for new molecular tools to target RANK/RANKL axis. I characterized and compared the biological activity of two anti-RANK antibodies. Moreover, I screened the Prestwick Chemical Library® of small molecules in order to identify inhibitors of RANK/RANKL interaction. Secondly, I studied the effect of the RANK/RANKL axis in lymph node homeostasis. RANKL is known to promote osteoclast differentiation but whether it also plays a role in the differentiation of other macrophage subsets is not known. We addressed this question by conditionally deleting RANKL from marginal reticular stromal cells (MRCs) that constitutively express RANKL in the lymph node. We observed impaired differentiation of the subcapsular sinus macrophages (SSMs). We also studied lymph node lymphatic endothelial cells (LECs) and showed that integrin alpha 2b (ITGA2b) is expressed by a lymph node subset of LECs and its expression is sensitive to RANKL

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Piedra, León María. "Estudio de la distribución de determinados polimorfismos de un solo nucleótido de los genes OPG,RANK, RANKL, GNAS1 y CLDN14 y su relación con la densidad mineral ósea y diversos marcadores de remodelación ósea en el hiperparatiroidismo primario". Doctoral thesis, Universidad de Cantabria, 2011. http://hdl.handle.net/10803/80773.

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Introducción: analizamos la relación entre fracturas y densidad mineral ósea (DMO) y los SNP (polimorfismos de un solo nucleótido) rs3102735 (163 A/G), rs3134070 (245 T/G) y rs2073618 (1181 G/C) de OPG, el SNP rs2277438 SNP de RANKL, el SNP rs7121 (393 T/C) de GNAS1 y del SNP rs219780 del gen CLDN14 en pacientes con HPP (hiperparatiroidismo primario) esporádico. Métodos: reclutamos 298 pacientes con HPP y 328 voluntarios sanos en un estudio transversal. Analizamos historia de fracturas o litiasis renal, parámetros bioquímicos, DMO en columna lumbar, cadera total, cuello femoral y radio distal y genotipado de los SNP mencionados. Resultados: no encontramos diferencias entre los genotipos de ninguno de los SNP estudiados en relación con la frecuencia de fracturas en HPP o en sujetos control. La DMO fue menor en el radio en los HPP homocigotos para el alelo menor en comparación con el resto de grupos en los SNP de OPG (163 A/G) y (245 T/G) pero no en sujetos control. En el resto de los SNP estudiados no encontramos diferencias entre genotipos y DMO en los sujetos con HPP o control excepto en el SNP de OPG (1181 G/C) en sujetos control con mayor DMO lumbar en el grupo CC respecto del GG. Conclusiones: los sujetos con HPP y homocigotos para el alelo menor (GG) en los SNP rs3102735 (163 A/G) y rs3134070 (245 T/G) de OPG tienen menor DMO en el radio distal. El resto de SNP estudiados no parecen influir en la diferente expresión de las manifestaciones óseas del HPP.
Background: we analyze the relationship between fractures and BMD (bone mineral density) and the rs3102735 (163 A/G), rs3134070 (245 T/G) and rs2073618 (1181 G/C) SNPs of the OPG, the rs2277438 SNP of the RANKL, the rs7121 SNP (393 T/C) of GNAS1 and the rs219780 of CLDN14 in patients with sporadic PHPT (primary hyperparathyroidism). Methods: We enrolled 298 Caucasian patients with PHPT and 328 healthy volunteers in a cross-sectional study. We analyzed history of fractures or renal lithiasis, biochemical determinants, BMD measurements in the lumbar spine, total hip, femoral neck and distal radius, and genotyping for the SNPs to be studied. Results: Regarding the frequency of fractures we found no differences between genotypes in any of the SNPs studied in the PHPT or in the control subjects groups. Significant lower BMD in the distal radius was found in the minor allele homozygotes (GG) compared to heterozygotes and major allele homozygotes in both OPG rs3102735 (163 A/G) and OPG rs3134070 (245 T/G) SNPs in those with PHPT but not in control subjects. We found no difference between genotypes of the rest of the SNPs studied in PHPT or control subjects with the exception of SNP OPG rs2073618 (1181 G/C) in control CC subjects which showed higher lumbar BMD than GG ones. Conclusions: Subjects with PHPT and minor homocygote genotype (GG) for the OPG rs3102735 (163 A/G) and OPG rs3134070 (245 T/G) SNPs have lower BMD in the distal radius. All the other SNPs studied do not appear to influence the different expression of HPP in bone.

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25

Sutton, Kate Maurice. "Functions of receptor activator of NF-κB ligand (RANKL) and its receptors, RANK and OPG, are evolutionarily conserved". Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10041.

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The tumour necrosis factor (TNF) superfamily is a group of cytokines that orchestrate a variety of functions, both in the development of the architecture of immune organs and of the immune response. The mammalian TNF superfamily consists of 19 ligands and 29 receptors, whereas in the chicken only 10 ligands and 15 receptors are present. Chickens do not develop lymph nodes, possibly due to the absence of the lymphotoxin genes (TNF superfamily members) in their genome. New members of the chicken TNF superfamily have recently been identified in the genome, namely chicken receptor activator of NF-κB ligand (chRANKL), its signalling receptor, chRANK, and its decoy receptor, osteoprotegerin (chOPG). In mammals, RANKL and RANK are transmembrane proteins expressed on the surface of Th1 cells and mature dendritic cells (DC), respectively. OPG is expressed as a soluble protein from osteoblasts and DC, regulating the interaction between RANKL and RANK. To investigate the bioactivity of this triad of molecules, the extracellular soluble domains of chRANKL and chRANK and full-length chOPG were identified and cDNAs cloned. ChRANKL, chRANK and chOPG mRNA are ubiquitously expressed across non-lymphoid and lymphoid tissues and immune cells in the chicken. Similar to mammals, chRANK and chOPG mRNA expression levels are upregulated in mature bone marrow-derived DC (BMDC). ChRANKL transcription is regulated by Ca2+-mobilisation and is further enhanced by the activation of the protein kinase C pathway, as seen in mammals. The biological activities of chRANKL, chRANK and chOPG were investigated by the production of recombinant soluble fusion proteins. The extracellular, TNF-homology, domain of chRANKL (schRANKL) was sub-cloned into a modified pCI-neo vector expressing an in-frame isoleucine zipper to encourage trimer formation. FLAG-tagged schRANKL produced in COS-7 cells predominantly forms homotrimers and chOPG is expressed as homodimers, both signatures of their mammalian TNF superfamily orthologues. SchRANKL enhances the mRNA expression levels of pro-inflammatory cytokines in mature BMDC and BM-derived macrophages (BMDM). Pre-incubation with soluble chRANK-Fc or chOPG-Fc blocked the schRANKL-mediated increase in pro-inflammatory cytokine mRNA expression levels in BMDC. Expression of surface markers on BMDC and BMDM were not affected by schRANKL treatment. SchRANKL enhances the survival rates of BMDC and BMDM and can drive osteoclast differentiation from monocyte/macrophage progenitor cells. The chRANKL signalling receptor, chRANK, does not contain an intracellular catalytic domain but requires the binding of intracellular TNF receptor-associated factors (TRAF) to initiate signalling. TRAFs are a family of seven proteins (TRAF1-7) grouped due to their highly conserved RING domains, zinc finger domains, TRAF-N and TRAF-C domains. ChRANK possesses four of the five TRAF peptide-binding motifs found in mammalian RANK. The "missing" chRANK TRAF peptide-binding motif is TRAF6-specific, a vital protein for RANKL-mediated osteoclastogenesis. All seven members of the mammalian TRAF family are present in the chicken genome. To investigate the conservation of RANK-specific TRAF signalling proteins, chicken TRAF2 (chTRAF2), chTRAF5, chTRAF6 and a newly found member, chTRAF7, were identified and their cDNAs cloned. ChTRAF5, chTRAF6 and chTRAF7 had mRNA expression patterns, in non-lymphoid and lymphoid tissues and in a number of immune cells, similar to their orthologues in mammals. Interestingly, chTRAF2 has two variants, the full-length chTRAF2 and a novel isoform (chTRAF2S) lacking exon 4. ChTRAF2S lacks a portion of zinger finger one, all of zinc finger two and a portion of zinc finger three, producing a protein with a hybrid of zinc fingers 1 and 3 and intact zinc fingers 4 and 5. RT-PCR analyses indicated differential expression of both of the chTRAF2 isoforms in a number of non-lymphoid and lymphoid tissues, splenocyte subsets and in a kinetic study of ConA-stimulated splenocytes. ChTRAF2S is biologically active compared to chTRAF2, inducing higher levels of NF-κB activation. Co-transfections indicate that chTRAF2 may regulate chTRAF2S bioactivity as no synergistic effect was identified when cells were transfected with both isoforms. Knowledge gained from this study will help work to further dissect the interactions between chRANKL-expressing T cells and chRANK-expressing DC to drive Th1 immune responses and to understand how the chicken mounts an effective immune response while expressing a minimal essential repertoire of the TNF superfamily.

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26

Dufresne, Sébastien S. "Rôles de rank/rankl/opg dans le muscle squelettique : intérêt thérapeutique potentiel pour la dystrophie musculaire de Duchenne". Doctoral thesis, Université Laval, 2017. http://hdl.handle.net/20.500.11794/30305.

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Une synchronicité existe entre l’apparition de l’atrophie musculaire et osseuse (ostéoporose) mais, très peu de groupes de recherche se sont intéressés à la possibilité qu’une voie de signalisation commune puisse contrôler simultanément ces tissus dans un contexte pathologique. Le but de cette thèse est de caractériser les rôles du sentier signalétique principal du remodelage osseux soit la voie RANK/RANKL/OPG, sur le muscle squelettique sain ou pathologique. Premièrement, nous avons démontré que RANK est exprimé dans le muscle squelettique et que son absence dans ce tissu induit un effet inotropique sur le muscle rapide extensor digitorum longus (EDL), limitant ainsi la perte de force maximale spécifique, tout en augmentant l'atrophie musculaire, la fatigabilité et la proportion de fibres rapides. Ensuite, nous avons montré qu’un blocage pharmacologique de la voie RANKL/RANK par l’OPG atténue la perte de la force musculaire de manière dose-dépendante et préserve l'intégrité musculaire, en particulier des muscles rapides EDL de souris dystrophiques. Cette étude nous a également permis de démontrer que l’OPG-Fc a un effet intéressant mais plus limité sur la préservation de la force du muscle lent soleus (Sol). Par contre, nous avons découvert que l’OPG-Fc potentialise les effets positifs d'une faible dose de formotérol, un membre de la famille des β2-agonistes, et leur combinaison restaure complètement la fonction du Sol des souris dystrophiques. Finalement, nous avons débuté une étude mécanistique sur l’effet protecteur de l’OPG-Fc sur le muscle squelettique dystrophique. Structurellement, l'OPG-Fc pleine longueur contient quatre domaines TNFR (RANKL), deux domaines de la mort cellulaire par apoptose (TRAIL) et un domaine lié à l'héparine. Nos résultats indiquent que les injections d'anti-RANKL, d’anti-TRAIL et d’OPG-Fc tronquée (possédant seulement les domaines TNFR) ou la suppression génétique de RANK dans le muscle sont nettement moins efficaces sur la préservation de la force des muscles dystrophiques que celles d’OPG-Fc pleine longueur. Étonnamment, l'absence de Ca2+ extracellulaire réduit considérablement les effets de l’OPG-Fc pleine longueur sur la force des muscles dystrophiques dans un modèle de contractilité in vitro. Nos analyses en microscopie confocale ont démontré que l’OPG-Fc pleine longueur pourrait se lier à un récepteur présentement non identifié localisé sur les myotubes et que cette liaison entraîne possiblement une activation d’une kinase liée aux intégrines (ILK) et la surexpression d’une pompe calcique ATPase du réticulum sarcoplasmique appelée SERCA-2a, un déterminant clé de la performance musculaire. Les myotubes traités à l'héparinase, une enzyme connue pour cliver les domaines de l'héparine ou encore l’inhibition de l’ILK réduit significativement la surexpression de SERCA-2a induite par l’OPG-Fc. Cette thèse apporte globalement, une meilleure compréhension des fonctions de RANK/RANKL/OPG dans le muscle squelettique dénervé ou dystrophique et s’inscrit dans la liste des travaux pré-cliniques qui pourrait éventuellement contribuer à l’élaboration de nouveaux traitements pour les maladies musculaires et osseuses.
Although there is an obvious dynamic cross-talk between muscle and bone, a common signalling pathway that efficiently and synchronously controls these tissues has barely been investigated in all forms of muscle diseases. The aim of this thesis is to characterize the roles of RANK/RANKL/OPG, key regulators of bone remodeling, on skeletal muscle atrophy, phenotype and dysfunction. Firstly, we show that RANK is expressed in skeletal muscle and that muscle RANK deletion has inotropic effects in denervated fast-twitch extensor digitorum longus (EDL) muscles, preventing on one side the loss of maximum specific force while promoting muscle atrophy and fatigability, and increasing the proportion of fast-twitch fibers. We next demonstrate that a pharmacological treatment of dystrophic mdx mice with recombinant full-length OPG-Fc mitigates the loss of muscle force in a dose-dependent manner and preserves muscle integrity, particularly in EDL muscles. We also found that the full-length OPG-Fc has limited effects on slow-twitch soleus (Sol) muscles. However OPG-Fc potentiates the positive effects of a low dose of formoterol, a member of β2-agonists, and completely restores the function of the Sol dystrophic muscles. Finally, we investigated the mechanism by which the full-length OPGFc protects the dystrophic muscles. Structurally, the OPG protein contains four TNFR domains (RANKL), two death domains ( TRAIL) and a heparin-binding region. Our results indicate that anti-RANKL or anti-TRAIL or truncated OPG treatments (only TNFR domains) or RANK deletion are much less effective in preserving the strength of dystrophic muscles than full-length OPG-Fc. Surprisingly, the absence of extracellular Ca2+ significantly reduces the effects of full-length OPG-Fc on the force production of dystrophic muscles when incubated in a physiological bath in vitro. Confocal microscopy images showed that the full-length OPG-Fc binds directly to myotubes through a receptor that is currently unidentified activating possibly integrin-linked kinase (ILK) which upregulates sarco/endoplasmic calcium ATPase pump (SERCA-2a) expression in C2C12 myotubes. Heparinase, which cleaves heparin and heparin sulphate proteoglycan, or an inhibitor of ILK activity abrogates OPG-induced SERCA-2a expression, suggesting that OPG through ILK upregulates SERCA-2a expression, a key determinant of muscle performance. Overall, this thesis shed some light on RANK/RANKL/OPG functions in skeletal muscle which will potentially contribute to the development of new treatments for several forms of muscle and bone diseases.

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Himelfarb, Silvia Tchernin. "Efeito da pioglitazona sobre o remodelamento ósseo em diabetes tipo 2". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-26042013-153329/.

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Alterações morfológicas no tecido ósseo têm sido descritas nos usuários de hipoglicemiantes orais da classe das tiazolidinedionas (TZDs). Hipotetiza-se que alguns genes relacionados com a osteogênese e osteoclastogênese podem ser influenciados pelo tratamento farmacológico, entretanto, o exato mecanismo ainda não está bem esclarecido. O objetivo do estudo foi avaliar o efeito da pioglitazona no remodelamento ósseo através de genes envolvidos na osteoclastogênese em indivíduos recentemente diagnosticados com DM2 e modelos animais, com a finalidade de identificar marcadores genéticos sensíveis de alterações ósseas. Foram convidados para participar do estudo 199 indivíduos (100 diabéticos e 99 normoglicêmicos), no ambulatório de dislipidemias do Instituto Dante Pazzanese de Cardiologia. Os indivíduos diabéticos foram tratados com pioglitazona (15, 30, 45, 45 mg/ dia/ via oral) por 16 semanas. Foram colhidas amostras de sangue, antes e após o tratamento para avaliações laboratoriais, extração de DNA genômico e de RNA total. Os polimorfismos e a expressão do mRNA nas células sanguíneas foram determinados pela PCR em tempo real através do sistema TaqMan®. Para o estudo em modelo animal após a indução da dieta hiperlipídica por 32 semanas, foram utilizados 12 camundongos machos da linhagem C57BL/J6, os quais foram divididos em três grupos: controle (n=4); diabéticos induzidos pela dieta hiperlipídica (DH, n=4) e diabéticos induzidos pela dieta hiperlipídica e tratados com pioglitazona 35mg/Kg/dia por 16 semanas (DHP, n=4). Para os grupos experimentais foram colhidos: amostras de sangue, para exames laboratoriais; fêmures, para a extração do RNA total; e tíbias, para determinação dos parâmetros histomorfométricos. Os pacientes DM2 apresentaram diminuição nas concentrações séricas de osteocalcina e na expressão de OPG e aumento na expressão de VDR em comparação ao grupo NG (p<0,05). A expressão de RANKL e IL6 foi maior entre as mulheres, enquanto que a expressão de PPARG foi maior entre os homens com DM2 em comparação ao grupo NG (p=0,032). Pacientes DM2 antes do tratamento apresentaram glicemia e expressão do mRNA de IL6 negativamente associados ao cálcio ionizado, enquanto que as transcrições de TNFA e VDR foram associadas positivamente e negativamente com bALP respectivamente (p<0,05). O tratamento com pioglitazona reduziu a glicemia de jejum, glicemia pós-prandial, insulina, HOMA-IR, triglicerídeos, VLDL-C, tALP e bALP e aumentou a HDL, tACP, TNF-α e a transcrição de OPG (p<0,05). A glicemia basal associou-se positivamente com o cálcio ionizado. A expressão basal de OPG foi associado negativamente com tALP, enquanto que a expressão basal de TNFA foi associada positivamente com tALP e negativamente com tACP. A expressão basal IL6 foi associada positivamente com tALP, enquanto que a expressão basal de VDR foi associada negativamente com osteocalcina e positivamente com bALP em resposta ao tratamento (p<0,05). O polimorfismo RANK rs1805034 foi associado com redução na transcrição do gene RANK nos indivíduos DM2 e com o remodelamento ósseo após o tratamento com pioglitazona (p<0,05). O polimorfismo RANKL rs9525641 foi associado com aumento da transcrição gênica de RANKL nos indivíduos NG e DM2 e melhora da resposta farmacológica nos indivíduos DM2 tratados com pioglitazona (p<0,05). O polimorfismo rs3102735 do gene OPG foi associado com aumento da formação óssea nos indivíduos DM2 antes e após o tratamento (p<0,05). O genótipo CG do polimorfismo OPG rs2073618 foi associado com alteração da transcrição de OPG no grupo DM2 pré e pós-tratamento (p<0,05). O polimorfismo PPARG rs1801282 foi associado com menor risco para o desenvolvimento de diabetes (p<0,05). O polimorfismo PPARG rs2972162 foi associado com melhora da resistência insulínica nos indivíduos DM2 tratados com pioglitazona (p=0,017). O polimorfismo ESRI rs9340799 foi associado com redução da formação óssea nos indivíduos DM2 (p=0,038). Nos camundongos, após a indução da dieta hiperlipídica por 32 semanas, observou-se aumento do peso, da glicemia, do colesterol total, da expressão do mRNA de RANK, RANKL, IL6 e TNFA em fêmures e aumento de Tb.Sp e diminuição de BV/TV em comparação ao grupo controle (p<0,05). O tratamento com pioglitazona diminuiu a expressão de TNFA (p=0,028). As medidas histomorfométricas não alteraram-se após o tratamento (p>0,05). Os resultados sugerem que o estado hiperglicêmico e o tratamento influenciam os marcadores bioquímicos e moleculares. Os polimorfismos dos genes RANK, RANKL, OPG e ESRI parecem estar envolvidos no remodelamento ósseo independentemente da hiperglicemia e do tratamento e os polimorfismos do gene PPARG parecem estar envolvidos com menor risco para desenvolver diabetes e com a melhora da resistência insulínica em resposta ao tratamento com pioglitazona.
Morphological changes in bone tissue have been reported in users of oral hypoglycemic class of thiazolidinediones (TZDs). It is hypothesized that some genes related to osteogenesis and osteoclastogenesis may be influenced by pharmacological treatment, however, was not aware exact mechanism. The study aims was to evaluate pioglitazone effect on bone remodeling through genes involved in osteoclastogenesis in individuals newly diagnosed with DM2 and animal models, in order to identify sensibles genetics markers of bone alterations. Were invited to participate in study 199 patients (100 diabetics and 99 normoglycemic), in dyslipidemia ambulatory of Institute Dante Pazzanese of Cardiology. Diabetic subjects were treated with pioglitazone (15, 30, 45 or 45 mg /day/oral) for 16 weeks. Blood samples were collected before and after treatment for laboratory evaluations, extraction of genomic DNA and total RNA. Polymorphisms and mRNA expression in blood cells was determined by real time PCR using TaqMan® system. For study in animal model after 32 weeks of fat diet induction, was used 12 male mice C57BL/J6, which were divided into three groups: control (n=4); induced diabetic fat diet (DH, n=4) and induced diabetic fat diet and treated with pioglitazone 35mg/Kg/day for 16 weeks (DHP, n=4). For experimental groups were collected: blood samples for laboratory tests; femurs, for extraction of total RNA; and tibias, to determine histomorphometric parameters. DM2 patients showed decrease in serum osteocalcin and OPG expression and increased VDR expression compared to NG group (p<0.05). RANKL and IL6 expression were higher among women, whereas PPARG expression was higher among men with DM2 compared to NG group (p=0,032). DM2 patients before treatment showed blood glucose and IL6 mRNA expression negatively associated with ionized calcium, whereas TNFA and VDR transcription are positively and negatively associated with bALP respectively (p<0.05). Pioglitazone treatment reduced fasting glucose, postprandial glucose, insulin, HOMA-IR, triglycerides, VLDL-C, tALP and bALP and increased HDL, tACP, TNF-α and OPG transcription (p<0.05). Basal blood glucose was positively associated with ionized calcium. Basal OPG expression was negatively associated with tALP, whereas basal TNFA expression was positively associated with tALP and negatively with tACP. Basal IL6 expression was positively associated with tALP, whereas basal VDR expression was negatively associated with osteocalcin and positively with bALP in response to treatment (p<0.05). RANK rs1805034 polymorphism was associated with RANK gene transcription reduction in subjects with DM2 and bone remodeling after treatment with pioglitazone (p<0.05). RANKL rs9525641 polymorphism was associated with increased RANKL gene transcription in NG and DM2 subjects and pharmacological response improvement in DM2 subjects treated with pioglitazone (p<0.05). OPG rs3102735polymorphism was associated with increased bone formation in DM2 subjects before and after treatment (p<0.05). CG genotype of OPG rs2073618 polymorphism was associated with OPG transcription change in DM2 group before and after treatment (p<0.05). PPARG rs1801282 polymorphism was associated with lower risk for diabetes development (p<0.05). PPARG rs2972162 polymorphism was associated with insulin resistance improvement in DM2 subjects treated with pioglitazone (p=0,017). ESRI rs9340799 polymorphism was associated with reduced bone formation in DM2 subjects (p=0,038). In mice, after 32 weeks of fat diet induction, was observed increase weight, blood glucose, total cholesterol and RANK, RANKL, IL6 and TNFA mRNA expression in femurs and Tb.Sp increase and BV/TV decrease compared to control group (p<0.05). Treatment with pioglitazone decrease TNFA (p=0,028). Histomorphometrics measurements not change after treatment (p>0.05). Results suggest that hyperglycemic state and treatment influence biochemical and molecular markers. RANK, RANKL, OPG and ESRI polymorphisms seens to be involved in bone remodeling regardless of hyperglycemia and treatment and PPARG gene polymorphisms seens to be associated with lower risk for diabetes development and with insulin resistance improvement in response to treatment with pioglitazone.

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Santos, Fernanda Regina Ribeiro. "Ciclooxigenase-2 modula in vivo a expressão de marcadores da osteoclastogênese e genes envolvidos no metabolismo ósseo em resposta ao lipopolissacarídeo bacteriano". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/58/58135/tde-04072012-135640/.

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Durante a resposta inflamatória, diversos mediadores são liberados localmente com o objetivo de estimular a resposta imune celular e humoral. Por meio da ação das enzimas ciclooxigenases e lipoxigenases ocorrerão modificações estruturais na cadeia do ácido araquidônico levando a síntese de prostaglandinas ou leucotrienos e lipoxinas, respectivamente. Tais mediadores são responsáveis pela regulação da expressão dos genes RANK, RANKL e OPG, moduladores da osteoclastogênese. Dessa maneira, o objetivo deste estudo foi avaliar a expressão do RNA mensageiro (RNAm) para as enzimas envolvidas no metabolismo do ácido araquidônico, ciclooxigenase-2 (COX-2) e 5- lipoxigenase (5-LO), e para os mediadores da osteoclastogênese (RANK, RANKL e OPG) no tecido ósseo, após inoculação de lipopolissacarídeo bacteriano (LPS) nos canais radiculares de molares de camundongos. Posteriormente foi investigado o efeito do bloqueio farmacológico da via COX-2 induzida pelo LPS, na expressão de mediadores da osteoclastogênese e de genes envolvidos no metabolismo ósseo. Foram utilizados 144 camundongos C57BL/6, com 6 semanas de idade, pesando de 18 a 20 gramas, nos quais os canais radiculares dos primeiros molares foram inoculados com uma solução contendo lipopolissacarídeo bacteriano de E. coli (0,1, 1,0 e 10mg/ml). Decorridos os períodos experimentais de 7, 14, 21 e 28 dias, os animais foram submetidos à eutanásia e os blocos contendo dente e osso foram removidos para extração do RNA total. Em seguida, foi realizada a avaliação da expressão gênica por meio de transcrição reversa e reação da polimerase em cadeia em tempo real (qRT-PCR). A análise global da expressão de RNAm para proteínas envolvidas no metabolismo ósseo foi realizada por meio de um ensaio de PCR Array (Osteogenesis RT² Profiler PCR Array). Os valores de expressão relativa de cada RNAm, para cada grupo, foram comparados por meio da análise de variância (ANOVA) de duas vias seguido pelo pós-teste de Bonferroni ou por ANOVA de uma via seguido pelo pós-teste de Dunnett (&alpha = 0,05). A inoculação de LPS nos canais radiculares de molares de camundongos foi capaz de induzir a expressão dos genes PTGS2 e ALOX5, responsáveis pela codificação das enzimas COX-2 e 5-LO, envolvidas no metabolismo do ácido araquidônico, concomitantemente à modulação da expressão dos genes TNFRSF11A, TNFSF11 e TNFRSF11B, responsáveis pela codificação dos moduladores da osteoclastogênese RANK, RANKL e OPG, respectivamente. A administração de Indometacina, um inibidor não seletivo de COX-2, inibiu a expressão de RNAm para RANK e RANKL e estimulou a expressão de OPG durante os períodos iniciais de resposta à inoculação de LPS nos canais radiculares. A inibição da via COX-2 de metabolismo do ácido araquidônico nos períodos iniciais de resposta à inoculação de LPS nos canais radiculares modulou diferencialmente a expressão de genes envolvidos no catabolismo e anabolismo ósseo, indicando possíveis papéis para os mediadores derivados no ácido araquidônico na regulação do metabolismo ósseo. Estes resultados sugerem alvos terapêuticos importantes para intervenção precoce em doenças inflamatórias, como lesões periapicais para evitar a reabsorção do tecido ósseo.
During an inflammatory response, several mediators are locally released in order to stimulate cellular and humoral immune response. Through the action of cyclooxygenase and lipoxygenase enzymes structural changes occur in the arachidonic acid chain leading to synthesis of prostaglandins or leukotrienes and lipoxins, respectively. Such mediators are responsible for the regulation of RANK, RANKL and OPG gene expression, osteoclastogenesis modulators. Thus, the objective of this study was to evaluate the expression of messenger RNA (mRNA) for the enzymes involved in arachidonic acid metabolism, cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), and the osteoclastogenesis mediators (RANK, RANKL and OPG) in bone tissue after injection of bacterial lipopolysaccharide (LPS) in murine dental root canals. Then, COX-2 pathway was pharmacologically blocked for investigation of expression of osteoclastogenesis mediators and genes involved in bone metabolism. We used 144 C57BL/6 mice, 6 weeks-old, weighing 18-20 grams, which had the first molars root canals inoculated with a solution containing LPS from E. coli (0.1, 1.0 and 10 mg/ml). After 7, 14, 21 and 28 days the animals were euthanized and the tooth-and-bone blocks were removed for total RNA extraction. Subsequently, the evaluation of gene expression was performed by reverse transcription and polymerase chain reaction in real time (qRT-PCR). Global analysis of mRNA expression for proteins involved in bone metabolism was performed using PCR arrays (Osteogenesis RT² Profiler PCR Array). The values for relative expression of each mRNA for each group were compared using two-way analysis of variance (ANOVA) followed by Bonferroni post-test or one-way ANOVA followed by Dunnett\'s test (α=0.05). The injection of LPS into the root canals was induced expression of genes PTGS2 and ALOX5, responsible for encoding COX-2 and 5-LO enzymes, involved in the metabolism of arachidonic acid, simultaneously to the modulation of gene expression of TNFRSF11A, TNFSF11 and TNFRSF11B, responsible for encoding the osteoclastogenesis modulators RANK, RANKL and OPG, respectively. Administration of Indomethacin, a non-selective inhibitor of COX-2, inhibited the expression of mRNA for RANK and RANKL and stimulated the expression of OPG during the initial response to the root canals contamination with LPS. Inhibition of the COX-2 pathway from arachidonic acid metabolism in the initial periods of response to LPS injection into the root canals differentially modulated the expression of genes involved in bone catabolism and anabolism, indicating possible roles for mediators derived from arachidonic acid in the regulation of bone metabolism. These results suggest important therapeutic targets for early intervention in inflammatory diseases such as apical periodontitis to avoid resorption of bone tissue.

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Lesky, Thomas. "In vitro Differenzierung von Monozyten der Zelllinine RAW 264.7 zu Osteoklasten, deren Charakterisierung und Wechselwirkung mit Osteoblasten". Doctoral thesis, Technische Universität Dresden, 2005. https://tud.qucosa.de/id/qucosa%3A24881.

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Das RANKL/RANK/OPG-System spielt eine entscheidende Rolle in der Steuerung der Osteoklastendifferenzierung und -aktivierung durch Osteoblasten/ Knochenmarkbindegewebszellen im Rahmen des Knochenremodelings. Osteoblasten/Knochenmarkbindegewebszellen exprimieren RANKL. Dieses hat im Körper zwei Rezeptoren: RANK und OPG. RANKL kann durch Bindung an RANK auf Osteoklasten/Osteoklastenvorläuferzellen in Gegenwart von M-CSF seine osteoklastenstimulierende Wirkung entfalten. Der ebenfalls von Osteoblasten gebildete „decoy“-Rezeptor OPG blockiert als freies Protein durch Bindung an RANKL dessen Interaktion mit RANK und verhindert somit die Osteoklastogenese und Osteoklastenaktivierung. Das RANKL/RANK/OPG-System erfüllt im Körper noch weitere Funktionen im Immunsystem, in der Organentwicklung lymphatischer Gewebe und in der Entwicklung der laktierenden Brustdrüse. Viele Zytokine greifen hemmend oder aktivierend in die Osteoklastogenese ein. Sie können dies zum einen durch die Beeinflussung des RANKL/OPG-Verhältnisses, zum anderen durch direkte Interaktion mit Osteoklasten/Osteoklastenvorläuferzellen tun. Zytokine, die die Osteoklastogenese begünstigen, werden vor allem bei inflammatorischen Prozessen ausgeschüttet. Zusammen mit dem, bei diesen Zuständen von aktivierten T-Zellen produzierten RANKL kann dies längerfristig zu einem Knochenverlust führen, welcher sich im klinischen Bild der Osteoporose äußert. Aus den in der vorliegenden Dissertation durchgeführten Untersuchungen ergeben sich folgende Schlussfolgerungen: 1. Monozyten der Zelllinie RAW 264.7 lassen sich, wie bereits in der Literatur beschrieben, durch Zugabe von M-CSF und RANKL zu osteoklastenähnlichen Zellen differenzieren. 2. Die Osteoklastogenese lässt sich anhand der Veränderung verschiedener osteoklastenspezifischer Parameter charakterisieren. Es zeigt sich bei den mit M-CSF und RANKL stimulierten Monozyten eine erhöhte Transkription von CTR (Calcitoninrezeptor)- und TRAP (tartratresistente saure Phosphatase)-mRNA, eine erhöhte Expression des CTR-Proteins, eine erhöhte TRAP-Aktivität und eine Formierung TRAP-positiver mehrkerniger Riesenzellen, die in diesen Eigenschaften Osteoklasten entsprechen. Die zusätzliche Zugabe von TGF-b1 in Kombination mit M-CSF und RANKL resultiert in einer verstärkten Expression von CTR-mRNA und CTR-Protein. TRAP-mRNA-Expression und TRAP-Aktivität bleiben davon unbeeinflusst. 3. Als funktionelles Merkmal der in vitro differenzierten Osteoklasten können ihre Fähigkeit zur Ausbildung von Aktinringen und die Resorption von mineralisiertem Kollagen nachgewiesen werden. 4. Im Verlauf ihrer Differenzierung sekretieren Osteoblasten unterschiedliche Mengen an OPG. Das Maximum der Synthese liegt bei Tag 11. Freies RANKL lässt sich in Überständen von MC3T3-E1-Osteoblasten nicht nachweisen. 5. Das von Osteoblasten in das Medium abgegebene OPG ist in der Lage, die durch RANKL induzierte Osteoklastogenese von RAW-Monozyten zu hemmen. 6. In Kokulturen von MC3T3-E1-Osteoblasten und RAW-Monozyten kann keine Osteoklastogenese beobachtet werden, wahrscheinlich durch Fehlen der RANKLExprimierung oder zu starke OPG-Sekretion durch Osteoblasten. Besonders in der westlichen Welt mit ihrer hohen Lebenserwartung haben Krankheiten mit Knochenverlust sowie bösartige Neubildungen mit Knochenbefall eine große medizinische Bedeutung. Die Beeinflussung des RANKL/RANK/OPG-Systems bietet eine vielversprechende Möglichkeit zur Entwicklung hochwirksamer und nebenwirkungsarmer Medikamente zur Behandlung dieser Zustände.

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30

ELIAS, Larissa Santana Arantes. "Expressão de reguladores da reabsorção óssea (RANK/RANKL/OPG) e formação óssea (osteocalcina) em lesões realcionadas ao osso e osteossarcoma". Universidade Federal de Goiás, 2010. http://repositorio.bc.ufg.br/tede/handle/tde/1374.

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The RANK (receptor activator of nuclear factor Kappa-Beta)-RANKL (receptor activator of nuclear factor-kappa beta ligand)-OPG (osteoprotegerin) system is the principal means of differentiating and activating osteoclasts. Changes along this path have been associated with various bone related lesions (BRL), whether benign or malignant, such as osteosarcoma (OS). This system induces resorption when it is deregulated, and in the case of LROs, by replacing the bone tissue for fibrous tissue with the presence of various forms of ossification. And in this same context another protein, osteocalcin (OC), a marker of late ossification, plays a key role in the diagnosis of these lesions. This being so, the objective of this study was to identify, quantify and compare cell RANK+, RANKL+, OPG+ and OC+ in lesions of the jaw with bone involvement: ossifying fibroma (OF), fibrous dysplasia (FD), simple bone cysts (SBC), central giant cell lesions (CGCL) and osteosarcoma (OS) so as to contribute to understanding the pathogenesis and establishing the diagnosis of these lesions. RANK+, RANKL+, OPG+ and OC+ cells were identified by the technique of immunohistochemistry, a method of immunoperoxidase and polymer, in 10 samples of OF, FD, SBC, CGCL and 5 samples of OS. Our results showed that all samples were positive for RANK, RANKL, OPG and OC. In the stromal fibroblast-like cells, the OF (P<0.001), CGCL (P=0.007) and OS (P=0,058) presented a greater expression of RANKL than OPG, in contrast with both the SBC (P=0.003) and the FD (P<0.001). As for bone-matrix (cells around bone/osteoid-osteoblast and osteoclast), the OS (P=0.24) and OF (P=0.001) samples demonstrated a higher RANKL immunoreactivity and and a lower in FD (P=0.001) and SBC (P=0.4) samples. In terms of OC, a higher expression was shown in FD, SBC, and OS (P=0.008). Our results suggest that OF, CGCL and OS express bone metabolism regulators, which may be related to increased bone resorption in these lesions. In addition, osteoblastic involvement was seen in FD and OS. Note: The superscript + is where it appears. Programs to copy some formatting errors.
O sistema RANK (receptor activator of nuclear factor Kappa-Beta)-RANKL (receptor activator of nuclear factor-kappa beta ligand)-OPG (osteoprotegerin) constitui uma das principais vias de diferenciação e ativação dos osteoclastos e alterações nessa via tem sido associadas a diversas lesões relacionadas ao osso (LRO), benignas e maligna como no osteossarcoma (OS). Esse sistema quando desregulado induz reabsorção, e no caso das LROs, através da substituição do tecido ósseo por um tecido fibroso com a presença de várias formas de ossificação. E nesse contexto outra proteína, a osteocalcina (OC), que é um marcador tardio de ossificação, desempenha um papel fundamental no diagnóstico destas lesões. Portanto, o objetivo do presente estudo foi identificar, quantificar e comparar células RANK+, RANKL+, OPG+ e OC+ em lesões dos maxilares com envolvimento ósseo: fibroma ossificante (FO), displasia fibrosa (DF), cisto ósseo simples (COS), lesão central de células gigantes (LCCG) e osteossarcoma (OS). As células RANK+, RANKL+, OPG+ e OC+ foram identificadas pela técnica da imunoistoquímica, método da imunoperoxidase e do polímero, em 10 amostras de FO, DF, COS, LCCG e 5 amostras de OS. Quando comparado as lesões entre si, tanto nas células fibroblásticas estromais quanto da matriz óssea, nossos resultados demonstraram que os ativadores da reabsorção óssea (RANK/RANKL) apresentam uma maior expressão no FO e LCCG e, o inibidor da reabsorção (OPG) e a OC apresentaram maior na DF e COS. Em adição, nossos achados revelam que o OS apresenta alta expressão de todas as proteínas avaliadas, quando comparadas àquelas das LROs. Todavia, uma maior expressão de RANKL em relação à OPG e OC foi evidenciada nesta neoplasia. Nossos resultados sugerem que o FO, a LCCG e o OS expressam reguladores do metabolismo ósseo que podem estar relacionados com a reabsorção óssea aumentada nessas lesões, sendo que na DF e no OS foi observado envolvimento osteoblástico. OBS: A + está sobrescrita onde aparece. Programas copiam com erros certas formatações.

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31

Lima, Mariana dos Reis. "Effect of calendula officinalis in rats submitted to experimental periodontitis: participation of RANK-RANKL-OPG and WNT / Β-CATENIN PATHWAYS". Universidade Federal do CearÃ, 2016. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=18394.

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FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
Periodontitis is an infecto-inflammatory disease that leads to connective tissue and alveolar bone loss. Calendula officinalis (CLO) has been used due to its anti-inflammatory effects. Therefore the aim of this study was to evaluate the effect of CLO on alveolar bone loss (ABL) in rats focusing on RANK-RANKL-OPG and WNT signaling pathways. Experimental periodontitis (EP) was induced through placement of a nylon ligature around the upper left 2nd molar, and the hemimaxilla used as control. The animals were divided in groups: Normal, subjected to no treatment; Saline (SAL), that received 2 ml/kg of 0,9% saline solution orally; or CLO at 90 mg/kg orally, 30 minutes before EP and daily for 11 days until euthanasia. In order to evaluate the periodontal tissue, it macroscopic, micro-tomographic, electron scanning microscopy (SEM), confocal microscopy and polarized light microscopy analyses were performed, as well as immunohistochemistry for WNT 10b, β-catenin, DKK-1, RANK, RANKL, and OPG. During euthanasia the gingival tissue was removed for malonaldehyde (MDA) assay. Treatment with CLO significantly prevented ABL, preserved bone internal microstructure (p<0.05) and topography, and also preserved collagen fibers from the periodontal ligament, when compared to SAL. CLO significantly increased the number of immunopositive cells for WNT 10b, β-catenin and OPG and reduced DKK-1, RANK (p>0.05) and RANKL. CLO reduced the gingival levels of MDA compared to SAL (p<0.05). In this way, we can conclude that CLO prevented ABL via RANK-RANKL-OPG and WNT signaling pathway.
A periodontite Ã uma doenÃa infecto-inflamatÃria que causa perda de tecido conjuntivo e osso alveolar. A Calendula officinalis (CLO) tem sido utilizada pelos seus efeitos anti-inflamatÃrios. Nesse contexto, o objetivo deste trabalho foi avaliar efeito da CLO na perda Ãssea alveolar (POA) em ratos com foco na participaÃÃo do eixo RANK-RANKL-OPG e da via WNT/β-catenina. A periodontite experimental (PE) foi induzida atravÃs da inserÃÃo do fio (nailon 3.0) em torno do 2Â molar superior esquerdo, e hemiarcada contralateral usada como controle. Os animais foram divididos em grupos: Normal, nÃo submetido a nenhum procedimento; Salina (SAL), que receberam 2 ml/kg de soluÃÃo salina 0,9% - v.o.; ou CLO na dose de 90 mg/kg - v.o. 30 min antes da PE e diariamente durante por 11 dias atÃ eutanÃsia. Para avaliaÃÃo do tecido periodontal realizaram-se anÃlises macroscÃpica, por microtomografia computadorizada, por microscopia eletrÃnica de varredura (MEV), microscopia confocal e microscopia por luz polarizada, imunohistoquÃmica para WNT 10b, β-catenina, DKK-1, RANK, RANKL e OPG. Por ocasiÃo da eutanÃsia foi removido tecido gengival para avaliaÃÃo dos nÃveis de malondialdeÃdo (MDA). O tratamento com CLO preveniu de forma significante a POA, preservou a microestrutura interna (p<0,05) e topografia do tecido Ãsseo, e preservou tambÃm as fibras colÃgenas do ligamento periodontal, quando comparado a SAL. A CLO provocou aumento significante de cÃlulas imunopositivas para WNT 10b, β-catenina e OPG e reduÃÃo na imunomarcaÃÃo de DKK-1, RANK (p>0,05) e RANKL. CLO reduziu os nÃveis de MDA gengivais comparados a SAL (p<0,05). Desta forma, podemos concluir que a CLO previne a POA com participaÃÃo do eixo RANK-RANKL-OPG e da via WNT/β-catenina.

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Lima, Mariana dos Reis. "Efeito da calendula officinalis em ratos submetidos a periodontite experimental: participação das vias RANK-RANKL-OPG e WNT B-CATENINA". reponame:Repositório Institucional da UFC, 2016. http://www.repositorio.ufc.br/handle/riufc/21662.

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LIMA, M. R. Efeito da calendula officinalis em ratos submetidos a periodontite experimental: participação das vias RANK-RANKL-OPG e WNT B-CATENINA. 2016. 66 f. Dissertação (Mestrado em Ciências Morfofuncionais) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2016.
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Periodontitis is an infecto-inflammatory disease that leads to connective tissue and alveolar bone loss. Calendula officinalis (CLO) has been used due to its anti-inflammatory effects. Therefore the aim of this study was to evaluate the effect of CLO on alveolar bone loss (ABL) in rats focusing on RANK-RANKL-OPG and WNT signaling pathways. Experimental periodontitis (EP) was induced through placement of a nylon ligature around the upper left 2nd molar, and the hemimaxilla used as control. The animals were divided in groups: Normal, subjected to no treatment; Saline (SAL), that received 2 ml/kg of 0,9% saline solution orally; or CLO at 90 mg/kg orally, 30 minutes before EP and daily for 11 days until euthanasia. In order to evaluate the periodontal tissue, it macroscopic, micro-tomographic, electron scanning microscopy (SEM), confocal microscopy and polarized light microscopy analyses were performed, as well as immunohistochemistry for WNT 10b, β-catenin, DKK-1, RANK, RANKL, and OPG. During euthanasia the gingival tissue was removed for malonaldehyde (MDA) assay. Treatment with CLO significantly prevented ABL, preserved bone internal microstructure (p<0.05) and topography, and also preserved collagen fibers from the periodontal ligament, when compared to SAL. CLO significantly increased the number of immunopositive cells for WNT 10b, β-catenin and OPG and reduced DKK-1, RANK (p>0.05) and RANKL. CLO reduced the gingival levels of MDA compared to SAL (p<0.05). In this way, we can conclude that CLO prevented ABL via RANK-RANKL-OPG and WNT signaling pathway.
A periodontite é uma doença infecto-inflamatória que causa perda de tecido conjuntivo e osso alveolar. A Calendula officinalis (CLO) tem sido utilizada pelos seus efeitos anti-inflamatórios. Nesse contexto, o objetivo deste trabalho foi avaliar efeito da CLO na perda óssea alveolar (POA) em ratos com foco na participação do eixo RANK-RANKL-OPG e da via WNT/β-catenina. A periodontite experimental (PE) foi induzida através da inserção do fio (nailon 3.0) em torno do 2º molar superior esquerdo, e hemiarcada contralateral usada como controle. Os animais foram divididos em grupos: Normal, não submetido a nenhum procedimento; Salina (SAL), que receberam 2 ml/kg de solução salina 0,9% - v.o.; ou CLO na dose de 90 mg/kg - v.o. 30 min antes da PE e diariamente durante por 11 dias até eutanásia. Para avaliação do tecido periodontal realizaram-se análises macroscópica, por microtomografia computadorizada, por microscopia eletrônica de varredura (MEV), microscopia confocal e microscopia por luz polarizada, imunohistoquímica para WNT 10b, β-catenina, DKK-1, RANK, RANKL e OPG. Por ocasião da eutanásia foi removido tecido gengival para avaliação dos níveis de malondialdeído (MDA). O tratamento com CLO preveniu de forma significante a POA, preservou a microestrutura interna (p<0,05) e topografia do tecido ósseo, e preservou também as fibras colágenas do ligamento periodontal, quando comparado a SAL. A CLO provocou aumento significante de células imunopositivas para WNT 10b, β-catenina e OPG e redução na imunomarcação de DKK-1, RANK (p>0,05) e RANKL. CLO reduziu os níveis de MDA gengivais comparados a SAL (p<0,05). Desta forma, podemos concluir que a CLO previne a POA com participação do eixo RANK-RANKL-OPG e da via WNT/β-catenina.

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Chu, Chia-Yi. "The Role of Rankl in Prostate Cancer Progression and Bone Metastasis". Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/118.

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This study focused on the role of RANKL in prostate cancer EMT progression and metastasis. Activation of RANK, a receptor activator of NF-kB, by its ligand RANKL, in a paracrine manner is responsible for osteoclast differentiation and bone remodeling. RANK activation in cancer cells, however, is thought to be promoted by both autocrine and paracrine mechanisms because RANKL has been shown to be derived from either tumor or its microenvironment, such as osteoblasts, infiltrating inflammatory cells and stromal fibroblasts. In the present study, we demonstrated that autocrine and paracrine RANKL-RANK signaling could be responsible for driving prostate cancer bone metastasis by promoting epithelial to mesenchymal transition (EMT). We further characterized a novel converging RANKL-c-Met signaling network in which the activation of RANKL was found to promote the expression of both RANKL and c-Met in an autocrine manner in prostate cancer cells. The induced RANKL and c-Met in prostate cancer cells is biologically functional and contributes to increased osteoclastogenesis, epithelial to mesenchymal transition (EMT), cell motility, migration and invasion and conferred bone and soft tissue metastases. Remarkably, RANKL expression by 1,000 prostate cancer cells can provoke bone and soft tissue metastases of a “dormant” population of prostate cancer cells which by themselves failed to form tumors and colonize mouse skeleton, suggesting RANKL can serve as a factor in “reawakening” cancer dormancy to initiate the re-growth and metastasis of cancer cells. We also showed that RANKL-induced RANKL feed-forward autocrine regulation is mediated through cMyc transactivation, allowing the establishment of a “vicious cycle” further promoting prostate cancer growth and metastasis. The converging RANKL-c-Met signaling network is therefore a novel target that could be further manipulated for delaying the lethal progression of castration-resistant human prostate cancer bone metastasis.

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34

Fiallos, Ana Cristina de Mello. "Estudo do ranelato de estrÃncio no reparo Ãsseo de defeitos crÃticos em calvÃria de ratos: participaÃÃo da via RANK/RANKL/OPG". Universidade Federal do CearÃ, 2013. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=9577.

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O reparo Ãsseo Ã um processo multifuncional com a participaÃÃo de vÃrios mediadores. Dentre os fÃrmacos que interferem nesse processo, destaca-se o Ranelato de EstrÃncio (RSr), o qual apresenta um mecanismo de aÃÃo dual, estimulando a neoformaÃÃo ao mesmo tempo que inibe a reabsorÃÃo Ãssea. Para avaliar a capacidade osteoindutiva, modelos que favorecem o estudo do potencial de reparo Ãsseo local tÃm sido utilizados, tais como o de induÃÃo de defeitos de tamanhos crÃticos (CSD) em calvÃrias de ratos. O objetivo deste estudo foi avaliar o RSr no reparo Ãsseo de defeitos crÃticos de 8 mm de diÃmetro induzidos em calvÃria de ratos. Para tanto, imediatamente apÃs a cirurgia, os CSD receberam uma Ãnica aplicaÃÃo de RSr (2,1 e 6,3 mg) ou nenhum tratamento (Controle). Grupos de animais foram sacrificados a 0 h e aos 15, 45, 90 e 120 dias apÃs a induÃÃo do CSD e calvÃrias foram processadas para anÃlise macroscÃpica, por meio de Tomografia Computadorizada tipo Cone Beam (TCCB), histolÃgica (HE) e imunohistoquÃmica para RANKL e OPG. Na anÃlise por TCCB, verificou-se que, no grupo RSr 6,3 mg, o RSr causou reduÃÃo significativa da Ãrea do CSD aos 90 dias (67,79 Â 2,32 mmÂ) e 120 dias (62,28 Â 4,17 mmÂ), quando comparadas Ãs calvÃrias recÃm-induzidas (0 h) (78,61 Â 0,96 mmÂ) (p<0,05), mas nÃo em relaÃÃo ao grupo Controle apÃs 90 dias (74,2 Â 2,73 mmÂ) e 120 dias (72,04 Â 1,74 mmÂ) (p>0,05). Na anÃlise histolÃgica das calvÃrias dos animais do grupo Controle foram observadas alteraÃÃes histolÃgicas significantes relacionadas ao reparo Ãsseo como neoformaÃÃo Ãssea restrita Ãs bordas do CSD quando comparados aos animais do grupo normal em todos os perÃodos experimentais (p<0,05). Os animais do grupo RSr (2,1 mg) nÃo apresentaram alteraÃÃes histolÃgicas significantes quando comparados ao grupo Controle em todos os perÃodos experimentais (p>0,05) enquanto que, nos animais do grupo RSr (6,3 mg), foram observados aspectos histolÃgicos compatÃveis com reparo Ãsseo aos 90 dias e aos 120 dias como neoformaÃÃo Ãssea em borda e no centro do CSD com diferenÃas significativas quando comparados aos grupos Controle ou RSr 2,1 mg (p<0,05). Complementando esses resultados, as calvÃrias dos animais apÃs 120 dias da aplicaÃÃo local de RSr (6,3 mg) apresentaram intensa imunoexpressÃo para OPG e negativa para RANKL, enquanto que as calvÃrias do grupo Controle apresentaram imunoexpressÃo moderada apenas para RANKL. Assim, pode-se concluir que o tratamento local com RSr evidenciou seu papel osteoindutor favorecendo a reparaÃÃo Ãssea do CSD pela modulaÃÃo da via RANKL/RANK/OPG.
The bone repair is a multifunctional process involving various mediators. Among the many drugs that interfere with this process, we highlight the Strontium Ranelate (SrR), which has a dual mechanism of action, stimulating neoformation at the same time, which inhibits bone resorption. To evaluate the osteoinductive capacity, models of study that investigate the potential for bone repair site have been used, such as induction of critical size defects (CSD) in rat calvaria. The aim of this study was to evaluate bone healing induced by SrR in critical defects of 8 mm in diameter in rat calvaria. For this purpose, immediately after surgery, the CSD received a single application of SrR (2.1 and 6.3 mg) or no treatment (Control). Groups of animals were sacrificed at 0 h and at 15, 45, 90 and 120 days after induction of CSD and calvarial samples were removed and processed for analysis by macroscopic type Cone Beam Computed Tomography (CBCT), histological (HE) and immunohistochemical for RANKL and OPG. In CBCT analysis, it was found that induction of CSD group SrR 6.3 mg caused a significant reduction of the areas of CSD at 90 days (67.79 Â 2.32 mmÂ) and at 120 days (62.28 Â 4.17 mmÂ) compared to calvariae newly induced (0 h) (78.61 mmÂ Â 0.96) (p<0.05) but not compared to Control groups at 90 days (74.2 Â 2.73 mmÂ) and at 120 days (72.04 Â1.74 mmÂ) (p>0.05). We observed in the histological analysis of calvariae of Control groups significant changes related to bone repair when compared to normal group (p<0.05). The animals that received SrR (2.1 mg) showed no significant histological changes, compared to the Control groups in all experimental periods (p>0.05), while animals of SrR 6.3 mg group showed significantly histological features consistent with bone repair at 90 days and at 120 days as neoformation in edge and center of the CSD when compared to Control or SrR 2.1 mg groups (p<0.05). To complement these results, the calvariae of animals after 120 days of topical application of SrR (6.3 mg) showed intense immunostaining for OPG and RANKL negative, whereas the calvariae of Control groups showed moderate immunoreactivity only for RANKL. Thus, it can be concluded that the local treatment with SrR (6.3 mg) revealed its role favoring osteoinductive bone repair by modulating the CSD RANK/RANKL/OPG.

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35

Heymann, Marie-Françoise. "Implication de la triade OPG/RANK/RANKL en physiopathologie vasculaire : à propos d'une étude comparée entre plaques athéromateuses carotidiennes et fémorales". Nantes, 2011. http://archive.bu.univ-nantes.fr/pollux/show.action?id=0df9b7bd-ab6b-49f2-aecf-5c9f8e8a0102.

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La triade moléculaire Osteoprotegerin (OPG)/Receptor Activator of NFkB (RANK)/RANK Ligand (RANKL) exerce plus spécifiquement son activité sur les systèmes ostéoarticulaire, immunitaire et vasculaire. Ce travail a permis l'étude de son implication dans la genèse de la plaque athéromateuse, en comparant deux sites d'artériopathie périphérique : carotide et fémorale. Actuellement, les résultats du traitement endovasculaire sont limités par la survenue de resténose et diffère selon le territoire artériel. A partir d'une biocollection de pièces d'endartériectomie, une analyse histologique comparative a été réalisée : les plaques carotidiennes sont fibrolipidiques avec un abondant infiltrat macrophagique alors que les fémorales sont plus calcifiées avec souvent métaplasie ostéoïde. En immunohistochimie, les plaques carotidiennes expriment plus fréquemment l'OPG par rapport aux fémorales, cette expression étant corrélée avec l'infiltrat macrophagique. L'expression de RANK et RANKL est équivalente dans les deux lits artériels. L'OPG semble donc jouer un rôle dans la différence de calcification des plaques carotidiennes et fémorales. L'analyse des acteurs cellulaires tels les macrophages, les cellules musculaires lisses et péricytes, éléments clés du processus, permettra d'identifier de nouvelles cibles thérapeutiques
The molecular triad osteoprotegerin (OPG)/Receptor Activator of NFkB (RANK)/RANK Ligand (RANKL) exerts its activity on the osteoarticular, immunologic and vascular systems. The present work demonstrates its implication in the atheromatous plaque genesis, comparing two beds of peripheral arterial disease: carotid and femoral. Currently, the results of endovascular treatment are limited by stenosis and differ according to the arterial territory. A comparative histologic study has been carried out from a biocollection of endarteriectomies : carotid plaques are fibrolipidic with abundant macrophagic population whereas femoral plaques reveal more calcifications associated with osteoid metaplasia. Carotid plaques exhibit more frequently OPG compared to femoral arteries as shown by immunohistochemistry, correlating with the macrophagic population. RANK and RANKL are similarly expressed in the two arterial beds. OPG appears to play a role in the differential calcification process of carotid and femoral plaques. The study of cellular actors like macrophages, vascular smooth muscle cells and pericytes will allow to identify novel therapeutic targets

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36

Fiallos, Ana Cristina de Mello. "Estudo do ranelato de estrôncio no reparo ósseo de defeitos críticos em calvária de ratos : participação da via RANK/RANKL/OPG". reponame:Repositório Institucional da UFC, 2013. http://www.repositorio.ufc.br/handle/riufc/6873.

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FIALLOS, Ana Cristina de Mello. Estudo do ranelato de estrôncio no reparo ósseo de defeitos críticos em calvária de ratos : participação da via RANK/RANKL/OPG. 2013. 106 f. Tese (Doutorado em Odontologia) - Universidade Federal do Ceará. Faculdade de Farmácia, Odontologia e Enfermagem, Fortaleza, 2013.
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The bone repair is a multifunctional process involving various mediators. Among the many drugs that interfere with this process, we highlight the Strontium Ranelate (SrR), which has a dual mechanism of action, stimulating neoformation at the same time, which inhibits bone resorption. To evaluate the osteoinductive capacity, models of study that investigate the potential for bone repair site have been used, such as induction of critical size defects (CSD) in rat calvaria. The aim of this study was to evaluate bone healing induced by SrR in critical defects of 8 mm in diameter in rat calvaria. For this purpose, immediately after surgery, the CSD received a single application of SrR (2.1 and 6.3 mg) or no treatment (Control). Groups of animals were sacrificed at 0 h and at 15, 45, 90 and 120 days after induction of CSD and calvarial samples were removed and processed for analysis by macroscopic type Cone Beam Computed Tomography (CBCT), histological (HE) and immunohistochemical for RANKL and OPG. In CBCT analysis, it was found that induction of CSD group SrR 6.3 mg caused a significant reduction of the areas of CSD at 90 days (67.79 ± 2.32 mm²) and at 120 days (62.28 ± 4.17 mm²) compared to calvariae newly induced (0 h) (78.61 mm² ± 0.96) (p<0.05) but not compared to Control groups at 90 days (74.2 ± 2.73 mm²) and at 120 days (72.04 ±1.74 mm²) (p>0.05). We observed in the histological analysis of calvariae of Control groups significant changes related to bone repair when compared to normal group (p<0.05). The animals that received SrR (2.1 mg) showed no significant histological changes, compared to the Control groups in all experimental periods (p>0.05), while animals of SrR 6.3 mg group showed significantly histological features consistent with bone repair at 90 days and at 120 days as neoformation in edge and center of the CSD when compared to Control or SrR 2.1 mg groups (p<0.05). To complement these results, the calvariae of animals after 120 days of topical application of SrR (6.3 mg) showed intense immunostaining for OPG and RANKL negative, whereas the calvariae of Control groups showed moderate immunoreactivity only for RANKL. Thus, it can be concluded that the local treatment with SrR (6.3 mg) revealed its role favoring osteoinductive bone repair by modulating the CSD RANK/RANKL/OPG.
O reparo ósseo é um processo multifuncional com a participação de vários mediadores. Dentre os fármacos que interferem nesse processo, destaca-se o Ranelato de Estrôncio (RSr), o qual apresenta um mecanismo de ação dual, estimulando a neoformação ao mesmo tempo que inibe a reabsorção óssea. Para avaliar a capacidade osteoindutiva, modelos que favorecem o estudo do potencial de reparo ósseo local têm sido utilizados, tais como o de indução de defeitos de tamanhos críticos (CSD) em calvárias de ratos. O objetivo deste estudo foi avaliar o RSr no reparo ósseo de defeitos críticos de 8 mm de diâmetro induzidos em calvária de ratos. Para tanto, imediatamente após a cirurgia, os CSD receberam uma única aplicação de RSr (2,1 e 6,3 mg) ou nenhum tratamento (Controle). Grupos de animais foram sacrificados a 0 h e aos 15, 45, 90 e 120 dias após a indução do CSD e calvárias foram processadas para análise macroscópica, por meio de Tomografia Computadorizada tipo Cone Beam (TCCB), histológica (HE) e imunohistoquímica para RANKL e OPG. Na análise por TCCB, verificou-se que, no grupo RSr 6,3 mg, o RSr causou redução significativa da área do CSD aos 90 dias (67,79 ± 2,32 mm²) e 120 dias (62,28 ± 4,17 mm²), quando comparadas às calvárias recém-induzidas (0 h) (78,61 ± 0,96 mm²) (p<0,05), mas não em relação ao grupo Controle após 90 dias (74,2 ± 2,73 mm²) e 120 dias (72,04 ± 1,74 mm²) (p>0,05). Na análise histológica das calvárias dos animais do grupo Controle foram observadas alterações histológicas significantes relacionadas ao reparo ósseo como neoformação óssea restrita às bordas do CSD quando comparados aos animais do grupo normal em todos os períodos experimentais (p<0,05). Os animais do grupo RSr (2,1 mg) não apresentaram alterações histológicas significantes quando comparados ao grupo Controle em todos os períodos experimentais (p>0,05) enquanto que, nos animais do grupo RSr (6,3 mg), foram observados aspectos histológicos compatíveis com reparo ósseo aos 90 dias e aos 120 dias como neoformação óssea em borda e no centro do CSD com diferenças significativas quando comparados aos grupos Controle ou RSr 2,1 mg (p<0,05). Complementando esses resultados, as calvárias dos animais após 120 dias da aplicação local de RSr (6,3 mg) apresentaram intensa imunoexpressão para OPG e negativa para RANKL, enquanto que as calvárias do grupo Controle apresentaram imunoexpressão moderada apenas para RANKL. Assim, pode-se concluir que o tratamento local com RSr evidenciou seu papel osteoindutor favorecendo a reparação óssea do CSD pela modulação da via RANKL/RANK/OPG.

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37

Tan, Jamie We-Yin. "The investigation of RANKL TNF-like core domain by truncation mutation". University of Western Australia. School of Surgery and Pathology, 2003. http://theses.library.uwa.edu.au/adt-WU2004.0032.

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Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.

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38

Barreiros, Driely. "Aspectos moleculares da gênese e progressão de lesões periapicais induzidas experimentalmente em camundongos". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/58/58135/tde-01092017-093300/.

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O conhecimento dos eventos biológicos que ocorrem no periápice dos dentes com necrose pulpar se torna importante para compreender o desenvolvimento das lesões periapicais. Muitas são as moléculas e mediadores que participam na instalação da lesão periapical, a partir da infecção bacteriana que ocorre no interior dos canais radiculares. Assim, o objetivo do presente trabalho foi avaliar moléculas do sistema imune inato, da osteoclastogênese e metaloproteinases em lesões periapicais (LP) induzidas experimentalmente em camundongos knockout e wild type. Para esse objetivo, o presente estudo foi dividido em dois trabalhos distintos. O primeiro teve como objetivo avaliar a expressão de metaloproteinase 2 (MMP2) e metaloproteinase 9 (MMP9) durante a progressão da LP em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO), em comparação com camundongos wild type (WT). O segundo estudo avaliou a correlação da expressão gênica e imunomarcação de RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da LP em camundongos WT. No primeiro estudo lesões periapicais foram induzidas em molares inferiores de 54 camundongos TLR2 KO, MyD88 KO e WT (n=18/grupo). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. Os cortes histológicos foram submetidos a imunohistoquímica e posteriormente foi avaliada presença ou ausência de MMP2 e MMP9 nos diferentes grupos. No segundo estudo, 35 camundongos WT foram utilizados. As lesões periapicais foram induzidas nos primeiros molares inferiores de ambos os lados. Após 0 (G0), 7 (G7), 21 (G21) e 42 (G42) dias, os animais foram anestesiados e eutanasiados para que as mandíbulas fossem dissecadas e divididas ao meio.O lado direito das mandíbulas foi para o processamento histotécnico, para posterior marcação de RANK, RANKL, OPG, TLR2 e MyD88, por meio da imuno-histoquímica do lado esquerdo da mandíbula foi utilizado para a extração de RNA, para a determinação da expressão gênica de RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) e MyD88 (Myd88) utilizando quantificação em Tempo Real da Reação da Polimerase em Cadeia (qRT-PCR). Para ambos os estudos, testes paramétricos e não paramétricos foram realizados com nível de significância de 5%. Foi possível observar, no primeiro estudo, que nos períodos iniciais da progressão da lesão periapical, houve um aumento na imunomarcação de MMP9 nos camundongos TLR2 KO e MyD88 KO, quando comparados aos WT, diferente da MMP2 que não se observou nenhum aumento na imunomarcação. No entanto, aos 42 dias observou-se uma redução da imunomarcação de MMP2 e um aumento da MMP9 nos camundongos TLR2 KO. Adicionalmente, no segundo estudo, foi possível observar um aumento da imunomarcação para RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da lesão periapical (p<0,05). O aumento da expressão de Tnfrsf11 foi diferente entre os grupos G0 e G42, e G21 e G42 (p=0,006). No entanto, a expressão de Tnfrsf11b foi diferente entre os grupos G0 e G7, G7, G21 e G42, sendo possível observar uma diminuição dessa expressão ao longo do tempo (p<0,001). Tlr2 foi mais expresso entre os grupos G0 e G42 (p=0,03). E a expressão da molécula Myd88 foi estatisticamente significante entre os grupos G0 e G7, G21 e G42 (p=0,01). A razão Tnfrsf11/Tnfrsf11b aumentou durante a progressão da lesão periapical (p=0,002). Também foi possível observar uma correlação moderada entre Myd88 e Rankl (r=0,42; p=0,03) e entre Myd88 e Tlr2 (r=0,48; p<0,0001). Após as metodologias empregadas e os dados analisados, concluímos que a produção de MMP2 e MMP9 foi modulada por TLR2 e Myd88 durante a progressão da lesão periapical. Alem disso, podemos sugerir que existe uma correlação positiva entre o sistema RANK/RANKL/OPG e as proteínas do sistema imune inato, TLR2 e MyD88, durante a perda óssea decorrente da infecção bacteriana dos canais radiculares e posterior progressão da lesão periapical.
Knowledge of the biological events occurring inteeth apex with pulp necrosis becomes important to understand the development of periapical lesions. There are manymolecules and mediators that participate in the installation of the periapical lesion, from the bacterial infection that occurs inside the root canals. Thus, the aim of the present study was to evaluate molecules of the innate immune system, osteoclastogenesis and metalloproteinases in experimentally apical periodontitis (AP) induced in knockout and wild type mice. For this purpose, the present study was divided into two distinct studies. The first one aimed to evaluate the expression of metalloproteinases 2 (MMP2) and metalloproteinases 9 (MMP9) during the progression of AP in TLR2 knockout mice (TLR2 KO) and MyD88 knockout mice (MyD88 KO), compared to wild type mice (WT). The second study evaluated the correlation of gene expression and immunostaining of RANK, RANKL, OPG, TLR2 and MyD88 during LP progression in WT mice. In the first study AP were induced in lower molars of 54 TLR2 KO, MyD88 KO and WT mice (n = 18 / group). After 7, 21 and 42 days, the animals were euthanized and the jaws were dissected and submitted to histotechnical processing. The histological sections were submitted to immunohistochemistry and subsequently the presence or absence of MMP2 and MMP9 in the different groups was evaluated. In the second study, 35 WT mice were used. Periapical lesions were induced in the lower first molars on both sides. After 0 (G0) to 7 (G7), 21 (G21) and 42 (G42) days, the animals were anesthetized and euthanized so that the jaws were dissected and divided in half. The right side of the jaws was for the histotechnic processing, for subsequent imunostaining of RANK, RANKL, OPG, TLR2 and MyD88, through immunohistochemistry and the left side of the jaws was used for the extraction of RNA, for the determination of expression of RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) and MyD88 (Myd88) using Quantification Real Time of Polymerase Chain Reaction (qRT-PCR). For both studies, parametric and non-parametric tests were performed with significance level of 5%. It was possible to observe in the first study that in the initial periods of AP progression there was an increase in MMP9 immunostaining in TLR2 KO and MyD88 KO mice when compared to WT, different from MMP2 that no increase in immunostaining was observed. However, at 42 days there was a reduction in MMP2 immunostaining and an increase of MMP9 in TLR2 KO mice was observed. Additionally, in the second study, it was possible to observe an increase in the immunostaining for RANK, RANKL, OPG, TLR2 and MyD88 during periapical lesion progression (p <0.05). The increase in Tnfrsf11 expression was different between groups G0 and G42, and G21 and G42 (p = 0.006). However, the expression of Tnfrsf11b was different between the G0 and G7, G7, G21 and G42 groups, and a decrease in expression over time (p <0.001) was observed. Tlr2 was more expressed between the G0 and G42 groups (p = 0.03). And the expression of the Myd88 molecule was statistically significant between the G0 and G7, G21 and G42 groups (p = 0.01). The Tnfrsf11 / Tnfrsf11b ratio increased during the AP progression (p = 0.002). It was also possible to observe a moderate correlation between Myd88 and Rankl (r = 0.42, p = 0.03) and between Myd88 and Tlr2 (r = 0.48, p <0.0001). After the methodologies used and the data analyzed, we conclude that the production of MMP2 and MMP9 was modulated by TLR2 and Myd88 during the AP progression. In addition, we can suggest that there is a positive correlation between the RANK / RANKL / OPG system and the proteins of the innate immune system, TLR2 and MyD88, during bone loss due to bacterial infection of the root canals and subsequent progression of the apical periodontitis.

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39

Palafox, Sánchez Marta. "Estudio de la vía de señalización de RANK y RANKL en células de mama humanas y generación de ortoxenopacientes de càncer de mama". Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/145786.

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Este proyecto de tesis doctoral se ha centrado en tres objetivos principales: el primero es el estudio de la función de la vía de señalización de RANK-RANKL-OPG en el desarrollo y progresión del cáncer de mama en humanos. El segundo es el análisis de la expresión de RANK en muestras de tumores humanos y análisis modificaciones en el gen que afecten en la incidencia de cáncer de mama en humanos. El tercero es la generación de modelos ortotópicos de ratón de cáncer de mama humano que sirvan como herramienta para la caracterización de nuevas dianas terapéuticas así como para testar la eficacia de nuevas drogas dirigidas a pacientes de cáncer de mama resistente a los tratamientos ya existentes. Respecto al primero objetivo, los resultados obtenidos indican que la sobre-expresión de RANK en células epiteliales de mama humanas no transformadas MCF10A induce la adquisición de un fenotipo asociado al proceso de transición epitelio-mesenquina (EMT) así como la adquisición de un fenotipo característico de células madre. El mecanismo por el cual la sobre-expresión de RANK induce EMT está relacionado con el aumento de la producción del factor de crecimiento transformate beta (TGF-beta) mediante la activación de las vías de señalización del factor nuclear potenciador de las cadenas ligeras kappa de las células B activadas (NF-κB) y la proteína quinasa activada por mitógenos p38. La inducción del fenotipo de célula madre está asociado a la activación de vía de señalización de las quinasas reguladas por señales extracelulares 1 y 2 (ERK 1/2). Los resultados referentes al segundo objetivo indican que en muestras de pacientes humanos, los niveles de expresión de RANK son más elevados en tumores con un peor pronóstico clínico (fenotipo triple negativo, alto índice de proliferación y algo grado histológico) y mediante la expresión de los niveles de mARN de RANK y RANKL se puede diferenciar entre tumores metastáticos y no metastáticos a nódulo linfático. Además, se ha descrito la existencia de un polimorfismo de nucleótido simple (SNP) presente en la región 5´del gen de RANK que está asociado con una menor probabilidad de padecer cáncer de mama en pacientes que poseen mutaciones en el gen BRCA2 (“breast cancer 2”). Finalmente, en este trabajo se describe la generación de cinco modelos ortotópicos de cáncer de mama a partir de muestras clínicas de pacientes. Estos modelos mantienen las características histológicas de los tumores humanos de origen así como los patrones de metástasis de los pacientes de los que proceden. De los cinco modelos generados, dos tienen unas características histológicas relativas a tumores triples negativos (TNBC), dos a tumores luminales (positivos para la expresión del receptor de estrógenos y progesterona) y uno a tumor Her2+. Los tumores Luminales y Her2+ manifiestan diferentes comportamientos de crecimiento en respuesta a hormonal, siendo un reflejo de los distintos tipos de comportamientos que existen en clínica. Por último, se ha testado la sensibilidad de estos modelos a taxanos (Docetaxel), una de las drogas quimioterapéuticas mas empleadas en la actualidad. Los dos modelos TNBC muestran más sensibilidad a la droga mientras que los modelos hormono-dependientes son resistentes. A partir de un modelo sensible TNBC se ha desarrollado un segundo modelo de ratones resistente a Docetaxel mediante la administración continuada del fármaco. Lo que se pretende en futuros proyectos es emplear estos modelos generados en el estudio de nuevas dianas terapéuticas en cáncer de mama así como testar nuevos fármacos en su fase pre-clínica de desarrollo.
This study has been focused in three main goals: study of RANK and RANKL pathway in the development and progression of human breast cancer; analysis of RANK expression in clinical human samples and analysis in RANK gene modifications implicated in human breast cancer; generation of orthoxenopatients of human breast cancer. The results of first objective showed RANK over-expression in human mammary cells MCF10A induces epithelial-mesenchymal transition (EMT) and Stemness phenotype. The molecular mechanism though RANK induces these effects implicates the increment of transforming growth factor beta (TGF-beta) ligands expression thorugh activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen activated kinase protein p38. On the other hand, the induction of Stemnes phenotype implicates the activation of Extracellular signal-regulated kinases 1/2 (ERK 1/2). The analysis of RANK expression in clinical human samples showed that tumor with a poor prognosis (such as triple negative tumors, tumors with high proliferative index and high histological grade) has higher levels of RANK expression. Moreover, through analysis of RANK and RANKL expression is able discriminate metastatic and non metastatic tumors. In addition, a lower probability to undergo breast cancer in patients with mutations in BRCA2 gene has been correlated with a SNP in the 5´region of RANK gene. Finally, five models of ortothopic mouse models of human breast cancer have been generated. The models obtained were two triple negative, two luminal and one Her2+. These models resemble de histological characteristics of original tumors and the metastasis patterns of the patients. Also the growth rate, latency and dependence to hormones were evaluated in these models. The sensitivity of these tumors was tested to Docetaxel. The triple negative tumors were sensitive whereas Luminal and Her2+ tumors were resistant to Docetaxel. Moreover, resistant models to Docetaxel were generated from sensitive models by continued Docetaxel administration. Samples were collected during the process in order to investigate the mechanisms implicated in resistance acquisition to Decetaxel in the future.

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40

Keller, Stephanie [Verfasser] y Gunter [Akademischer Betreuer] AßMann. "Assoziation von Genpolymorphismen in den Genen des RANK-RANKL-OPG-System bei Patienten mit Multiplem Myelom und MGUS / Stephanie Keller. Betreuer: Gunter Aßmann". Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2014. http://d-nb.info/1054055440/34.

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Allouche, Farouk. "Role of RANKL in the differentiation of B cell associated stroma in secondary lymphoid organs". Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ002.

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RANKL (ligand du récepteur activateur de NF-KB) est un membre de la famille des TNF dont la signalisation passe par RANK et qui joue un rôle important dans la régulation immunitaire. Chez l'adulte, RANKL est exprimé constitutivement par des cellules réticulaires marginales (MRC) des ganglions lymphatiques. Comme les MRCs sont physiquement proches des lymphocytes B (LB) et ont été proposé d’être des précurseurs de cellules dendritiques folliculaires (FDC), RANKL pourrait jouer un rôle dans la différenciation du stroma associé aux LB et dans la réponse humorale. Afin de mieux comprendre la fonction de RANKL exprimé par les MRC, nous avons généré des souris déficitaires pour RANKL dans les cellules stromales. Nous avons constaté que la formation du follicule B était perturbée ainsi que le réseau FDC. Bien que RANKL ne soit pas requis pour la formation des MRC, il est nécessaire pour l'expression de la chimiokine CXCL13 par ces mêmes cellules. Parmi les TNFRSF dont la signalisation est requise pour l’expression de CXCL13 et la différenciation des FDC, le TNFR1 était significativement réduit dans les cellules stromales des souris dépourvues de RANKL stromal. Ainsi, RANKL pourrait constituer une nouvelle cible thérapeutique contre les immunopathologies des LB en agissant sur son stroma
RANKL (receptor activator of NF-κB ligand), a member of the TNF family that signals via RANK, plays an important role for immune regulation. In the adult, RANKL is constitutively expressed by marginal reticular cells (MRCs) of the lymph nodes. Because MRCs are positioned in close vicinity to B cells and may be precursors of follicular dendritic cells (FDCs), RANKL could play a role in the differentiation of B cell-associated stroma and the humoral immune response. In order to better understand the role of RANKL expressed by the MRCs, we generated mice with conditional RANKL deficiency in the stromal compartment. We found that the B cell follicle structure was disrupted and FDC network formation was reduced. Although RANKL was not required for MRC formation, it was necessary for the expression of B cell attracting chemokine CXCL13. Among the TNFRSF members known to control CXCL13 expression and FDC formation, we found that TNFR1 was significantly reduced in the RANKL cKO mice. Thus, RANKL may present a novel therapeutic strategy against B cell-mediated immunopathologies by acting on its stroma

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Valente, Fabrício Luciani. "Histomorfometria e expressão imunoistoquímica de RANKL em fêmur e vértebra de ratos com osteoporose secundária". Universidade Federal de Viçosa, 2007. http://locus.ufv.br/handle/123456789/4972.

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Osteoporosis is a common human disease affecting both men and women, and it can classified as (1) primary, related to sexual hormones deficiency or senility, or (2) secondary, for what the most common example is the chronic therapies with glucocorticoids. Whatever is its cause, the osteoporosis outcomes are bone loss and increased fracture risk. Although osteoporosis is considered a systemic condition, bone loss seen in osteoporosis is not homogeneous throughout the skeleton. RANKL is a cytokine able to activate the osteoclasts function and its expression is inducible in osteoblasts and T cells by a range of stimuli. Function of RANKL has been considered a possible therapeutic target for the treatment of osteoporosis. To evaluate the trabecular bone loss related to the RANKL expression, immunochemistry and histomorphometric assays were used in femur and vertebra of castrated and/or glucocorticoid-treated male and female rats, at the day 56 after induction. RANKL expression was evident only in the castrated group, both male and female, but not in the group of castrated rats that also received glucocorticoid therapy. But histomorphometric data showed that bone loss was similar in both groups. That could happen because glucocorticoid can inhibit osteoblast metabolism. Histomorphometry also reveals that trabecular bone mass in male is similar to female, and bone loss is not homogenous between distal and proximal femur and vertebra body. At the day 56 after induction, bone loss, in femur and vertebra, both male and female, was compatible to osteoporosis.
A osteoporose é uma doença comum em humanos, acometendo tanto mulheres quanto homens. A doença pode ter origem primária, relacionada à deficiência de hormônios sexuais ou a senilidade, ou secundária, cujo exemplo mais comum é o uso crônico de glicocorticóides. Independente da causa, a conseqüência da osteoporose é a diminuição da massa óssea, aumentando o risco de fraturas. Apesar de ser considerada uma doença sistêmica, a redução de massa óssea na osteoporose não é uniforme no esqueleto. RANKL é uma citocina capaz de ativar a função osteoclástica e sua expressão é indutível em osteoblastos e linfócitos T. A função desta citocina tem sido considerada um possível alvo terapêutico no tratamento da osteoporose. Para avaliar a relação da perda óssea trabecular e a expressão de RANKL, foram realizados testes imunoistoquímicos e histomorfométricos em fêmur e vértebras de ratos castrados e/ou tratados com glicocorticóides, 56 dias após a indução. A expressão imunoistoquímica de RANKL pôde ser verificada nos animais castrados, tanto machos quanto fêmeas, mas não no grupo castrado que também recebeu glicocorticóide. Entretanto, a diminuição da massa óssea em ambos os grupos foi similar na avaliação histomorfométrica. Isso pode ocorrer por causa do efeito inibitório que os glicocorticóides têm sobre o metabolismo. As análises histomorfométricas revelaram ainda, que a massa óssea trabecular avaliada por este método é similar em machos e fêmeas, e que a perda óssea não é uniforme entre o colo femoral, o côndilo femoral e o corpo vertebral. Aos 56 dias de indução, o quadro de perda óssea instalado tanto em machos quanto em fêmeas, para todos os fragmentos ósseos analisados, é compatível com o quadro de osteoporose.

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43

Rankl, Tobias [Verfasser]. "Performance and Bounds of Optical Receivers with Electronic Detection and Decoding / Tobias Rankl". Aachen : Shaker, 2010. http://d-nb.info/112254619X/34.

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Breuil, Véronique. "Recrutement et activité des osteoclastes humains : effets des bisphosphonates et rôle de rankl". Nice, 2003. http://www.theses.fr/2003NICE4011.

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Les ostéoclastes présentent des différences importantes entre espèces rendant difficile l’application des données animales à la biologie de l’ostéoclaste humain. Dans une première partie de ce travail, nous avons participé à la caractérisation des cultures primaires d’ostéoclastes humains. Ainsi, après 21 jours de culture, nous disposons d’un nombre suffisant de cellules présentant les caractéristiques classiques de l’ostéoclaste. Dans ce modèle, nous avons montré que l’alendronate agit en inhibant l’activité de résorption ostéoclastique sans modification du recrutement ostéoclastique. Par ailleurs, une simple pré-incubation des lamelles osseuses avec l’alendronate induit une inhibition de résorption osseuse comparable à celle obtenue avec une exposition continue des cellules à l’alendronate, soulignant l’importance de l’interaction entre l’ostéoclaste et la matrice osseuse. Enfin, nous avons mis au point une technique d’évaluation de l’activité de résorption : la mesure des produits de dégradation du collagène dans le milieu de culture, qui présente de nombreux avantages, permet d’évaluer de manière fiable la résorption osseuse in vitro. Dans une deuxième partie, nous avons montré que RANKL est un facteur chimiotactique pour les monocytes. RANKL induit la migration des cellules MonoMac-6 et de cellules normales (cellules mononuclées du sang périphérique et CD14+ purifiées), confirmant la pertinance physiologique de nos observations. L’effet chémoattractant de RANKL est dose-dépendant et comparable à celui de MCP-1, chimiokine de référence des monocytes. La migration induite par RANKL est abolie par l’addition concomitante d’OPG, confirmant la spécificité des propriétés chémoattractantes de RANKL. RANKL ne module pas l’expression de chimiokines connues pour attirer les monocytes et ses effets correspond bien à du chimioactisme et non pas de la chimiokinésie. Enfin, l’effet chémoattractant de RANKL est additif à celui de MCP-1, suggérant des voies de signalisation différentes
In a second part, we demonstrate that RANKL is a chemotactic factor for human monocytes. RANKL induces the migration of MonoMac-6 and normal cells (peripheral blood mononuclear cells and CD14+ purified cells), strengthening the physiological relevance of our observations. Chemotactic effect of RANKL is dose dependant and similar to MCP-1, the reference chémokines for monocytes. Addition of the RANKL decoy receptor osteoprotegerin abrogates the RANKL-induced migration, hallmarking a true specific activity. RANKL does not modulate the expression of chémokines known to attract monocytes and its effects correspond to chemotaxis and not chemokinesis. Finally, chemotactic effect of RANKL is additive to MCP-1, suggesting that RANKL mediates its action through its own signalling pathways

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45

Malinovska, Alexandra [Verfasser] y H. R. [Akademischer Betreuer] Salih. "Die Blockade der RANK-RANKL Interaktion durch Denosumab bei der Immunüberwachung der Akuten Myeloischen Leukämie durch Natürliche Killerzellen / Alexandra Malinovska ; Betreuer: H. R. Salih". Tübingen : Universitätsbibliothek Tübingen, 2017. http://d-nb.info/1199469084/34.

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Gosselin, Rachelle. "Influence de la voie RANK / RANKL / OPG sur la force et le phénotype musculaire dans un modèle murin de myopathie acquise aux soins intensifs". Master's thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/25879.

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Les troubles musculo-squelettiques entraînent une dégradation simultanée des tissus osseux et musculaires. L’objectif de ce mémoire était d’évaluer si la voie RANK/RANKL/OPG, connue dans les os, joue un rôle important dans la régulation du tissu musculaire. Des souris spécifiquement déficientes en RANK musculaire (RANKmKO) et des souris contrôles (RANKf/f) ont subi une dénervation des nerfs sciatiques suivie d’une injection quotidienne de dexaméthasone pendant 7 jours (modèle animal de myopathie acquise aux soins intensifs). Après le sacrifice, les propriétés contractiles étaient mesurées et les propriétés immunohistochimiques étaient obtenues sur les muscles soléaires et longs extenseurs des doigts. Nos résultats démontrent que RANK musculaire est un régulateur de la fonction et du phénotype musculaire et que son absence améliore significativement la force musculaire des muscles rapides. Globalement, ces travaux nous permettent de mieux comprendre les dysfonctions musculaires et ouvrent la voie à de nouvelles pistes de traitement pour plusieurs formes de myopathies.

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47

Suzuki, Selly Sayuri. "Avaliação histomorfométrica, imunoistoquímica e microtomográfica da ação da terapia laser de baixa potência no processo de reabsorção radicular durante movimentação ortodôntica induzida em ratos". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/85/85134/tde-22082016-144925/.

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A movimentação dentária induzida é um processo biológico complexo mediado por estímulos mecânicos, levando a um subsequente processo de remodelação óssea, podendo haver reabsorção indesejada da raiz dentária provocada pelo excesso de força. Uma vez que a movimentação ortodôntica se baseia em um processo inflamatório localizado, o propósito deste estudo foi avaliar os efeitos do laser de baixa potência no processo de remodelação óssea e reabsorção radicular, buscando correlacionar as mudanças metabólicas observadas a nível celular ocorridas nos dias iniciais da movimentação dentária às alterações teciduais observadas microscopicamente e à arquitetura e morfologia do trabeculado e cortical ósseo. Primeiros molares de sessenta e oito ratos machos Wistar foram submetidos à movimentação induzida, divididos em 3 grupos: controle negativo (nenhuma movimentação), não irradiado (movimentação sem irradiação) e laser (movimentação e irradiação com laser de baixa potência de comprimento de onda de 810 nm, potência de 100 mW, área de 0,02cm2 e energia de 1,5 J/ponto) e eutanasiados nos dias 3, 6, 9, 14 e 21. Mensurações da movimentação dentária e análises histomorfométricas foram realizadas em todos os dias estudados. Análise imunoistoquímica dos marcadores RANKL, OPG e TRAP e avaliações por microscopia eletrônica de varredura (MEV) foram feitas nos dias 3, 6 e 9 e o ensaio Western Blotting para proteínas RANKL e SOFAT e imagens de microtomografia computadorizada (MicroCT) nos dias 14 e 21. Os resultados deste estudo mostraram que a movimentação dentária foi significantemente maior no grupo Laser (aumento em média de 40%) em todos os dias avaliados. O lado de compressão mostrou maior expressão de RANKL e osteoclastos TRAP-positivos nos dias 3, 6 e 9 (p<0,05), promovendo significativa redução na área de osso alveolar presente no lado de compressão nos dias 6, 9 e 14 (p<0,05), e alterações microestruturais, como menor fração de volume ósseo/volume total, menor densidade óssea mineral aos 14 dias. A irradiação com laser também aumentou a expressão de RANKL e a citocina SOFAT no dia 14. No lado de tensão, houve maior expressão de OPG especialmente aos 9 dias (p<0,001) e significativo aumento na área de osso alveolar presente nos dias 14 (p<0,01) e 21 (p<0,05) histomorfometricamente e maior densidade óssea mineral e espessura das trabéculas aos 21 dias (p<0,01). Com relação às áreas de hialinização presentes, os resultados mostraram áreas significantemente reduzidas nos dias 3, 6 e 9 nos grupos irradiados, o que explica o menor número de odontoclastos na superfície radicular nestes dias e a redução significante das áreas de reabsorção radicular observadas nas lâminas histológicas nos dias 9, 14 e 21 e nas imagens de MEV nos dias 3 e 9. Os grupos irradiados também mostraram menor volume das lacunas de reabsorção radicular medidas no MicroCT nos dias 14 e 21, especialmente nos lados de compressão. O estudo concluiu que o laser de baixa potência influenciou a remodelação óssea, aumentou a atividade dos osteoclastos no lado da compressão, e estimulou a formação óssea no de tensão, acelerando significativamente o movimento dentário e potencialmente reduzindo as áreas de necrose no ligamento periodontal e, consequentemente, a reabsorção radicular.
Tooth movement is a complex biological process induced by mechanical stimulation, leading to a subsequent process of bone remodeling, concomitantly unwanted root resorption may occur caused by excessive force. Since orthodontic movement is based on a localized inflammatory process, the purpose of this study was to evaluate the effects of low-level laser therapy on the process of bone remodeling and root resorption, searching to correlate metabolic changes observed at cellular level in the initial days of tooth movement to tissue changes observed microscopically and both architecture and morphology of trabecular and cortical bone. Upper first molars of sixtyeight male Wistar rats were submitted to induced movement, divided into 3 groups: negative control (no movement), non-irradiated (movement without irradiation) and Laser (movement and irradiation using low level laser of 810 nm wavelength, 100 mW power, 0.02cm2 area, energy of 1.5J/point) and euthanized on days 3, 6, 9, 14 and 21. Measurements of tooth movement and histomorphometric analysis were performed at all days. Immunohistochemistry analysis of RANKL, OPG and TRAP markers and scanning electron microscopy (SEM) were made on days 3, 6 and 9. Western Blotting method to evaluate RANKL and SOFAT proteins and MicroCT images were performed on days 14 and 21. The results of this study showed that tooth movement was significantly greater in the irradiated side (increased in average of 40%) in all evaluated days. The compression side showed higher expression of RANKL and TRAP-positive osteoclasts on days 3, 6 and 9 (p <0.05), promoting significant reduction in alveolar bone area in the compression side on days 6, 9 and 14 ( p <0.05), and leading to microstructural changes such as decrease of the fraction of bone volume / total volume (BV/TV) and the bone mineral density (BMD) at 14 days. The laser also increased RANKL expression and SOFAT on day 14. On the tension side there was an increased expression of OPG especially after 9 days (p <0.001), a significant increase in alveolar bone area on days 14 (p < 0.01) and 21 (p <0.05) histomorphometrically and increase in bone mineral density and trabecular thickness after 21 days (p <0.01). Regarding hyalinized areas, the results showed significant reduced areas on days 3, 6 and 9 in irradiated groups, which explains the lower number of clastic cells on the root surface in these days, and a significant reduction of areas of root resorption observed in histology on days 9, 14 and 21 and on days 3 and 9 by SEM images. Irradiated groups also showed less volume of root resorption lacunaes measured by MicroCT on days 14 and 21, especially in the compression side. The study concludes that the low-level laser therapy had an effect on bone remodeling, increasing osteoclast activity on the compression side, and stimulating bone formation in tension side, resulting in significant tooth movement acceleration and potentially reducing the areas of necrosis in the periodontal ligament and consequently the root resorption process.

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Amorim, Ingrid Aquino. "Análise de peptídeos de defesa do hospedeiro na osteoclastogênese mediada por RANKL in vitro". reponame:Repositório Institucional da UnB, 2016. http://repositorio.unb.br/handle/10482/21557.

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Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.
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A remodelação óssea representa um processo de suma importância no sistema esquelético humano. Entretanto, um desequilíbrio na função ou ativação excessiva de osteoclastos pode resultar em extensas reabsorções ósseas. Nesse contexto, peptídeos de defesa do hospedeiro (PDHs) podem apresentar um potencial no desenvolvimento de novas terapias. Nessa perspectiva, o presente estudo avaliou o efeito dos peptídeos clavanina A, clavanina MO e LL-37, na supressão da osteoclastogênese in vitro mediada pelo ligante do receptor de ativação do fator nuclear kappa B (RANKL). Estes resultados foram comparados com medicações utilizadas clinicamente, hidróxido de cálcio P.A. e doxiciclina, em terapias endodônticas e periodontais, respectivamente. Os parâmetros analisados em culturas da linhagem celular RAW 264.7, com ou sem recombinante (r) RANKL, PDHs e controles clínicos, foram: (1) viabilidade celular pelo método de MTT; (2) produção de óxido nítrico (NO); e número de osteoclastos diferenciados, após coloração de fosfatase ácida tartarato resistente (TRAP). Os resultados demonstraram que os PDHs e os controles clínicos não foram citotóxicos às células, exceto na presença de 128 μg.mL-1 de doxiciclina após 72 h, na presença e ausência de rRANKL, que apresentou redução de cerca de 50% da viabilidade celular. A produção de NO foi mantida estável ou reduzida na presença de todas as concentrações dos PDHs e controles clínicos, comparados ao grupo controle, na ausência de rRANKL. Como esperado, a presença de rRANKL elevou sutilmente os níveis da produção de NO. No entanto, a presença dos PDHs e controles clínicos permitiram ora estabilidade, ora redução dos níveis de NO, quando comparados ao grupo controle, exceto na presença de 2 μg.mL-1 de doxiciclina após 7 dias, que promoveu aumento significativo na produção de NO. Na osteoclastogênese, todos os PDHs e controles clínicos foram capazes de reduzir a diferenciação de osteoclastos. Em conclusão, os PDHs podem atuar como potenciais supressores da osteoclastogênese in vitro. Dessa forma, o uso dos PDHs se apresenta como uma forma terapêutica promissora para o tratamento de reabsorções ósseas perirradiculares e periodontais. ________________________________________________________________________________________________ ABSTRACT
Bone remodeling is an important process in the human skeletal system. Nevertheless, an imbalance in the osteoclast function or its excessive activation, may result in extensive bone resorption. In this context, host defense peptides (HDPs) may have a potential for novel therapies development. This study evaluated the potential of HDPs clavanin A, clavanin MO and LL-37 in down-regulate in vitro receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis. HDPs results were compared to currently available medications for endodontic and periodontal therapies, calcium hydroxide P.A. and doxycycline, respectively. The parameters analyzed in cell line RAW 264.7 cultures stimulated with or without recombinant (r) RANKL and HDPs and clinical controls were: (1) cell viability by MTT method; (2) nitric oxide production (NO); and (3) number of differentiated osteoclasts, after tartrate-resistant acid phosphatase (TRAP) staining. Results showed that HDPs and clinical controls were not cytotoxic, except in the presence of 128μg.mL-1 of doxycycline after 72 h, in the presence and absence of rRANKL, which decreased about 50% the cell viability. The NO production was kept stable or reduced in the presence of all concentrations of HDPs and clinical controls, compared to the control group, in the absence of rRANKL. Otherwise, the presence of rRANKL subtly up-regulated the NO production levels. However, the presence of HDPs and clinical controls remained stable or reduced the NO production compared to the control group, except in the presence of 2 μg.mL-1 of doxycycline after 7 days, which up-regulated NO production. During the osteoclastogenesis process, all HDPs and clinical controls were capable of reducing the osteoclasts differentiation. In conclusion, host defense peptides can act as potential suppressors of in vitro osteoclastogenesis. Thus, HDPs represent promising drugs for periradicular and periodontal bone resorption treatments.

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49

Vargas, Franco Jorge William. "Impacts on the growing and adult skeleton of different genetically-achieved RANKL activity levels : consequences on the response to zoledronic acid". Thesis, Nantes, 2019. http://www.theses.fr/2019NANT1035.

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Rationnel et hypothèse : Les amino-bisphosphonates sont de puissants inhibiteurs de la résorption osseuse. Ils sont actuellement utilisés en clinique pour traiter les maladies ostéolytiques de l’enfant et de l’adulte. Des variations entre les individus dans l’intensité de leurs effets et effets secondaires ont été signalés, sans explication claire de leurs origines. L’hypothèse selon laquelle de telles variations pourraient éventuellement être associées à différents niveaux d’activité de la signalisation RANKL dans l’os pendant et après le traitement a été posée ici. Objectifs et méthodologie : Une série de souris transgéniques présentant des niveaux graduels de la signalisation RANKL a été générée par l’accouplement de souris invalidées pour l’Opg et surexprimant Rank. Ces souris ont été soumises à des protocoles d’acide zolédronique imitant ceux utilisés en oncopédiatrie ou chez les patients adultes ostéoporotiques, et les phénotypes squelettiques obtenus ont été comparés un mois après la dernière injection à la fin de la croissance pour le protocole pédiatrique et six mois après la dernière injection à l’âge de dix mois pour le protocole adulte. Résultats : Les résultats obtenus ont validé l’hypothèse avec toutefois une surprise majeure : l’absence d’une stricte corrélation entre la sévérité des phénotypes squelettiques et l’augmentation de l’activité de signalisation RANKL. Plus précisément, la réduction allélique graduelle de l’Opg semblait améliorer le phénotype squelettique observé à la fin de la croissance comme chez l’adulte, tandis que la surexpression de Rank l’empirait. Conclusion : En conclusion, il a été démontré que le niveau d’activité de la signalisation RANKL dans le microenvironnement osseux était impliqué dans la modulation de la réponse phénotypique du squelette aux bisphosphonates, avec des impacts différents de l’invalidation de l’Opg et de la surexpression de Rank, dont le décryptage moléculaire constituera le prochain défi
Rational and hypothesis: Amino-bisphosphonates are powerful inhibitors of bone resorption. They are currently used in clinical practice to treat pediatric and adult osteolytic diseases. Variations between individuals in the intensity of their effects and side-effects have been reported with no clear explanation of their origins. The hypothesis that such variations could potentially be associated with different levels of activity in the RANKL signaling in bone during and after treatment was questioned here. Objectives and methodology: A series of transgenic mice with graded levels of RANKL signaling was generated by mating mice invalidated for Opg and overexpressing Rank. These mice were subjected to zoledronic acid protocols mimicking those used in onco-pediatrics or in osteoporotic adult patients, and the skeleton phenotypes were compared one month after the last injection at the end of growth for the pediatric protocol and six months after the last injection at ten months of age for the adult protocol. Results: The results validated the hypothesis with however one main surprise: the absence of a strict reverse correlation between the severity of the skeleton phenotypes and the increase in RANKL signaling activity. More precisely, the graded allelic reduction in Opg appeared to improve the skeleton phenotype observed both at the end of growth as in adult, while Rank overexpression made it worse. Conclusion: In conclusion, the level of activity of RANKL signaling in the bone microenvironment was shown to be implicated in the modulation of the skeleton’s phenotypic response to bisphosphonates, with differing impacts of Opg invalidation and Rank overexpression, whose molecular deciphering will form the next challenge

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Schilling, Eleonore. "Inducible expression of RANKL in transgenic pigs under the control of the Tet-On system". Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-127661.

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