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Aims: This study uses radiological and clinical evaluations of the healing of endosseous titanium implants presented with different surface characteristics in the clinical situation (paper I-III) and experimentally to describe the early bone healing in maxillary sinus membrane elevation with and without the use of grafting material (paper IV). Material and methods: In paper I, 136 patients were treated with 394 dental implants – 199 were oxidized titanium implants (Nobel Biocare TiUnite) and 195 were turned titanium surface implants (Nobel Biocare Mark III). Implant survival rates were retrospectively investigated after a minimum of five months after functional loading of the implants. At the five-year follow-up (paper II), eight patients were deceased and 128 were invited. Twenty-five patients refrained from participating in the study. The remaining 103 patients (287 implants – 133 with a turned surface and 154 with an oxidized surface) were examined after at least five years of functional loading. Clinical examinations of bleeding on probing (BoP) and pocket depth (PD) were performed. Intraoral radiographs were used to assess marginal bone levels (MBLs). In paper III, 28 patients were subjected to autologous bone graft and delayed implant placement, with a total of 92 dental implants. Thirteen patients received 47 implants with a turned surface and 15 patients received 45 implants with an oxidized surface. After a minimum of five years of functional loading, all patients were clinically examined regarding PD and BoP. The MBL was measured in intraoral radiographs. Cone beam computed tomography (CBCT) was used to evaluate the apical bone level (ABL) of the implants and intra-sinus conditions. The experimental study (paper IV) used nine adult male tufted capuchin primates (Cebus apella). Eight animals were subjected to bilateral maxillary sinus membrane elevation using a lateral replaceable bone window technique. One oxidized dental implant was placed in the residual bone of the sinus floor, protruding into the maxillary sinus cavity on both sides. In four animals, one sinus was left without any additional treatment, while the contralateral sinus was filled with autologous bone grafts from the tibia. In two animals, the implants were inserted under the elevated sinus membrane on both sides. In two animals, the sinus membrane was totally removed bilaterally before placement of implants. The animals were euthanized after 10 (n=4) or 45 (n=4) days. One non-operated animal representing pristine tissue conditions served as the control. The maxillary sinuses with implants were retrieved and further processed to prepare light microscopic ground sections or decalcified sections for immunohistochemical analyses. Results: In paper I seven implants were lost in five patients – six in the maxilla and one in the mandible. All failed implants were Mark III turned implants. The overall implant survival rate was 98.2% with a survival rate of 96.4% for implants with turned surface after a minimum of five months after functional loading. In paper II, one additional oxidized implant failed, giving an overall cumulative survival rate of 94.7 and 99.4%, respectively, after at least five years of functional loading. There was no difference for BoP, PD, or MBL between turned and oxidized implants. A total of two implants, three oxidized and one turned, showed a PD > 3 mm, MBL > 4 mm, and BoP. However, none of these were associated with suppurative infection on examination. In paper III no difference was found between the two implants surfaces used in terms of PD, BoP, MBL, or ABL. Pathological reactions to the sinus membrane were seen in four of the patients (14%). Radiographic signs of sinus pathology were not correlated to either survival rate of the implants or any of the investigated parameters. In the experimental paper IV, bone formation started from the bottom of the sinus floor, sprouting into the granulation tissue along the implant surface under the elevated membrane irrespective of time and surgical technique. Bone formation was not seen in direct conjunction with the sinus membrane. A distinct expression of osteopontin was observed in the serous glands of deeper portion of the lamina propria in direct connection with the elevated sinus membrane and close to the implant within all groups. Conclusion: After more than five years of function in non-grafted patients, oxidized implants had a survival rate higher than turned implants, although this was not statistically significant. No difference was found in MBL, PD, or BoP. Grafting of the maxillary sinus floor with intra- orally harvested bone and delayed placement of either turned or oxidized implants resulted in equally high long-term survival rates, MBL, ABL, and BoP. Pathological findings in the maxillary sinus cavity, in terms of sinus membrane health, are few and not correlated to any of the other investigated parameters. In the experimental study bone formation after sinus membrane elevation with or without additional bone grafts started at the sinus floor and sprouted into the elevated space along the implant surface. Removal of the membrane resulted in less bone formation. The sinus membrane did not seem to present osteoinductive potential in sinus membrane elevation procedures.

Made available in DSpace on 2014-06-11T19:30:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-06Bitstream added on 2014-06-13T18:40:39Z : No. of bitstreams: 1 marchesano_lh_dr_arafcf.pdf: 336354 bytes, checksum: 54de5ce8ee681faefedf0567be191430 (MD5) / Universidade Estadual Paulista (UNESP) / O tratamento cirúrgico da fissura congênita do processo alveolar superior compreende o enxerto ósseo, um procedimento bem aceito e de grande importância na restauração da forma e da função perdidas. Associado ao enxerto ósseo tem-se utilizado um produto atóxico, não imunoreativo e de fácil obtenção, denominado plasma rico em plaquetas (PRP). Neste estudo foi analisado o comportamento dos marcadores fosfatase alcalina, fosfatase alcalina isoforma óssea, osteocalcina e fosfatase ácida tartarato resistente em 50 pacientes, com idade entre 10 e 20 anos e que foram submetidos à cirurgia de enxerto ósseo autógeno alveolar pelo serviço de Cirurgia Buco-maxilofacial do Hospital de Reabilitação de Anomalias Craniofaciais da Universidade de São Paulo. O objetivo foi acompanhar de forma sistêmica e em curto período a formação ou reabsorção óssea após a realização do enxerto ósseo alveolar, bem como avaliar a eficácia do uso do plasma rico em plaquetas no processo de formação óssea. O estudo concluiu que as propriedades restauradoras do PRP não puderam ser demonstradas por nenhum dos marcadores bioquímicos do metabolismo ósseo nos primeiros 70 dias do ato cirúrgico; a análise temporal dos marcadores de formação óssea testados demonstrou uma tendência de queda com 35 dias e retorno próximo aos níveis basais com 70 dias do ato cirúrgico nos dois grupos estudados; não houve uma correlação significativa dos marcadores com o número de plaquetas e nem com a área da fissura e o resultado do exame ao raio X foi considerado inconclusivo para a presença ou não de trabeculado ósseo organizado em fase inicial de formação. / The surgical treatment of the congenital cleft of the upper alveolar process understands the bone graft, a well accepted procedure of great importance in the restoration of the lost form and function. Together with the bone graft it is being used a non-toxic, non imunoreactive and easily obtained product, denominated platelet-rich plasma (PRP). In this study it was analysed the behavior of the alkaline phosphatase, bone alkaline phosphatase, osteocalcin and tartrate-resistant acid phosphatase markers in 50 patients, with age between 10 and 20 years and that were undergone to alveolar autogenous bone graft performed by the Bucomaxillofacial Service of the Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo. The aim was follow in a sistemic and early way the bone formation or reabsorption after the accomplishment of the alveolar bone graft, as well as to evaluate the effectiveness of the use of the platelet-rich plasma in the process of bone formation. The study concluded that the restorative properties of the PRP could not be demonstrated by of the biochemistry markers of the bone metabolism in the first 70 days of the surgery; the temporal analisys of the bone formation markers tested demonstrated a fall tendency in 35 days with return near to basal levels in 70 days in the two studied groups; there was not a significant correlation between markers and the number of platelets and neither with the area of the cleft and the result of the x-ray examination was not considered conclusive for the presence or not of organized bone trabeculae in the initial phase of formation.

Activation of tissue-specific genes is a tightly controlled process that normally involves the combined action of several transcription factors and transcriptional co-regulators. The bone-specific osteoca1cin gene (OC) has been used as a prototype to study both tissue-specific and hormonal responsiveness. In this study we have examined the role of Runx2, VDR and C/EBP factors in the regulation of OC gene transcription. Contributions of the Runx and VDRE motifs to OC promoter activity were addressed by introducing point mutations within the context of the rat (-1.1 kb) osteocalcin promoter fused to a CAT-reporter gene. The functional significance of these mutations was assayed following transient transfection and after genomic integration in ROS 17/2.8 osteoblastic cell lines. Furthermore, we tested the effect of these mutations on the chromatin organization of the OC promoter. Our data show that all three Runx sites are required for maximal activation of the OC promoter and that the distal sites contribute significantly to the basal activity. Strikingly, mutation of the three Runx sites abrogates responsiveness of the OC promoter to vitamin D; this loss is also observed when only the Runx sites flanking the VDRE are mutated. Chromatin changes that result in the appearance of DNase I hypersensitive sites during activation of the OC gene are well documented. Mutation of the three Runx sites results in altered chromatin structure as reflected by absence of DNase I hypersensitive sites at the vitamin D response element and over the proximal, tissue-specific basal promoter. These data are consistent with the critical role of Runx2 in osteoblast maturation and bone development. Mutation of the VDRE resulted in a complete loss of vitamin D responsiveness; however, this mutant promoter exhibited increased basal activity. The two DNase I hypersensitive sites characteristic of the transcriptionally active OC gene in osteoblastics cells were not altered upon mutation of the VDRE element, although restriction enzyme accessibility in the proximal promoter region was decreased. We also found an increased level of histone H3 acetylation at the VDRE mutant promoter in comparison to the endogenous gene. Thus binding of VDR to OC promoter is required to achieve a normal transcriptional regulation and chromatin structure of the OC gene. Although Runx2 is considered a master gene for bone development and osteoblast differentiation, it is noteworthy that osteoblast-specific transcription of the rat OC promoter occurs even in the absence of Runx sites. Therefore, other transcription factor(s) should be able to drive OC expression. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteoca1cin gene that resides in close proximity to a Runx element, essential for tissue-specific activation. We find that C/EBPβ or δ and Runx2 factors interact together in a synergistic manner to enhance OC transcription in cell culture systems. Mutational analysis demonstrated that this synergism is mediated through the C/EBP responsive element in the OC promoter and requires a direct interaction between Runx2 and C/EBPβ or δ. Taken together, our findings strongly support a mechanism in which combinatorial interaction of Runx2, VDR, C/EBPβ or δ and probably other transcription factors are needed for regulating OC expression. In this process Runx factors not only act as simple transcriptional trans activators but also by facilitating modifications in promoter architecture and maintaining an active conformation of the target gene promoter.

Orientador: Tamara Beres Lederer Goldberg / Resumo: O excesso de peso e a osteoporose são grandes problemas de saúde pública, resultando em agravos à saúde da população. Objetivo: Investigar o impacto do excesso de gordura corporal sobre a remodelação óssea de adolescentes sobrepesos, obesos e superobesos, através da realização de densitometria óssea e dosagem de biomarcadores de formação e reabsorção óssea. Casuística e Métodos: Obteve-se peso, estatura e determinou-se o Índice de Massa Corpórea (IMC) de 391 adolescentes de 10 a 20 anos. Foram classificados em: eutróficos, sobrepesos, obesos e superobesos. Obtida a idade óssea, avaliação do conteúdo mineral ósseo (CMO) e densidade mineral óssea (DMO) através do DXA em coluna lombar, fêmur proximal e corpo total e subtotal, além da coleta de sangue para avaliação dos biomarcadores ósseos: osteocalcina (OC), fosfatase alcalina óssea (FAO) e telopeptídeo carboxiterminal (S-CTx). Realizou-se Análise de Variância, seguida do Teste de Tukey para comparação entre grupos. Utilizou-se modelo linear generalizado com distribuição gama para comparação entre grupos estratificados por sexo e, Correlação de Pearson para verificar as associações entre os três biomarcadores ósseos e massa magra (MM), massa de gordura (MG) e percentual de gordura corporal (% GC), IMC, CMO e DMO, com p<0,05. Resultados: No sexo feminino com excesso de peso os biomarcadores apresentaram maiores concentrações na faixa etária de 10 a 13 anos, e não houve diferenças estatísticas entre os grupos segundo estado nutri... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Excess weight and osteoporosis are serious public health problems. Objective: To investigate the impact of excess body fat on bone remodeling in overweight, obese and extremely obese adolescents by bone densitometry and by measuring biomarkers of bone formation and resorption. Materials and Methods: Body weight, height and body mass index (BMI) were determined in 391 adolescents aged 10 to 20 years. The participants were classified as eutrophic, overweight, obese, and extremely obese. Bone age was obtained and bone mineral content (BMC) and bone mineral density (BMD) by DXA were evaluated in the lumbar spine, proximal femur, and total and subtotal body. Blood samples were collected for evaluation of the following bone biomarkers: osteocalcin, bone alkaline phosphatase (BAP), and serum carboxy-terminal telopeptide (S-CTx). Analysis of variance followed by the Tukey test was used for comparison between groups. A generalized linear model with gamma distribution was applied to compare the groups stratified by sex and Pearson‟s correlation test to evaluate associations between the three bone biomarkers and lean mass (LM), fat mass (FM), body fat percentage (BF%), BMI, BMC and BMD (p<0.05). Results: In girls with excess weight, the concentrations of the biomarkers were higher in the 10 to 13-year age group and no significant differences were observed between groups according to nutritional status. In boys with excess weight, the mean levels of the markers were higher in those aged ... (Complete abstract click electronic access below) / Doutor

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T09:47:46Z No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T10:03:57Z (GMT) No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2015-12-10T10:03:57Z (GMT). No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Central Giant Cell Lesion (CGCL) is an intraosseous lesion that can be classified into non aggressive and aggressive. Due to the aesthetic and functional defects of surgical treatment of CGCL, therapies with drugs have been reported, such as glucocorticoid injections and calcitonin. The studies reported in the literature support the use of these drugs through the investigation of the presence of glucocorticoid receptors (RGC) and calcitonin (RCT) in CGCL; however there is no consensus if all lesions express these receptors and if there is any difference between non aggressive and aggressive lesion. In addition, there are no studies that evaluated the bone formation potential through the investigation of Osteocalcin (OC) in aggressive and non-aggressive lesions. The aim of this study was to compare, using immunohistochemistry, the GR and CTR and osteocalcin protein (OC) expression in non aggressive (n = 20) and aggressive (n = 11) CGCL, and the correlation between the OC expression and these receptors determined in both groups of lesions. The number of mononuclear cells in mitosis (MOC), and the number of multinucleated giant cells (MGC) were also investigated using immunohistochemical techniques (hematoxylin and eosin). Our results show that all the cases express the GR and CTR and that there is no difference in the expression of these receptors or the number of mitosis between non aggressive and aggressive lesions. The OC expression was rare and higher in non aggressive lesions, however, not statistically significant (p> 0.05). There was a correlation between the CTR expression in MOC and MGC (r = 0.45; p <0.01). Considering the different variants of CGCL, there was a correlation between CTR expression in MOC and MGC in non aggressive lesions (r = 0.66; p <0.01) and between the CTR and OC expression in MGC (r = 0.718; p = 0.01). There was a higher number of MGC in aggressive lesions (p = 0.01). The results indicate that all cases express GR and CTR and that there are no differences between non aggressive and aggressive CGCL lesions of these receptors expression, these results strengthens CGCL treatment with glucocorticoids and calcitonin. Aggressive lesions have a higher number of MGC. The CGCL express glucocorticoid and calcitonin receptors and this finding give biological basis to the CGCL treatment with intralesional glucocorticoid and calcitonin either in non aggressive and aggressive cases. It was also identified osteocalcin positive cells, that may be related to bone repair, it is believed that these cells may also serve as a therapeutic target. / A Lesão Central de Células Gigantes (LCCG) é uma lesão intraóssea que pode ser classificada em não agressiva e agressiva. Devido aos defeitos estéticos e funcionais do tratamento cirúrgico da LCCG, terapias medicamentosas tem sido relatadas, como injeções de glicocorticoide e calcitonina. Há na literatura estudos que suportam o uso desses medicamentos através da investigação da presença de receptores de glicocorticoides (RGC) e de calcitonina (RCT) em LCCG. No entanto não existe consenso se todas as LCCG expressam esses receptores e se existe alguma diferença entre lesões agressivas e não agressivas. Além disso, não existem estudos sobre a avaliação do potencial de formação óssea através da Osteocalcina (OC) em lesões agressivas e não agressivas. O propósito deste estudo foi avaliar comparativamente, por meio de imunohistoquímica, a expressão de RGC e RCT e da OC em LCCG não agressivas (n= 20) e agressivas (n= 11) e a correlação entre a expressão da OC e desses receptores nos dois grupos de lesões estudados. O número de mitoses nas células mononucleares e o número de células gigantes multinucleadas também foram investigados, utilizando técnica histoquímica (hematoxilina e eosina). Nossos resultados mostram que todos os casos analisados expressam o RGC e RCT e que não existe diferença na expressão do RGC, RCT ou do número de mitoses entre lesões não agressivas e agressivas. A expressão de OC em células mononucleares foi rara e maior em lesões não agressivas, no entanto, sem diferenças estatisticamente significantes (p>0,05). Houve correlação entre a expressão do RCT em células mononucleares e células gigantes multinucleadas (r=0,45; p<0,01). Considerando as diferentes variantes foi verificada correlação do RCT entre o componente mononuclear e as células gigantes multinucleadas nas lesões não agressivas (r=0,66; p<0,01) e entre a expressão de OC e RCT em células gigantes multinucleadas (r= 0,718; p=0,01). Houve maior número de células gigantes em lesões agressivas (p= 0,01). Os resultados indicam que todos os casos expressam RGC e RCT e que não há diferenças entre lesões agressivas e não agressivas de LCCG quanto à expressão desses receptores, fortalecendo a recomendação o tratamento da LCCG com o uso de glicocorticoide e calcitonina. Lesões agressivas apresentam maior número de CGM. As células da LCCG expressam o RGC e RCT e esse achado pode fornecer bases biológicas para o tratamento com injeções intralesionais de glicocorticoides e o uso de calcitonina, seja em lesões não agressivas ou agressivas. Adicionalmente, foram identificadas células expressando OC, que podem estar relacionadas ao reparo ósseo, acredita-se que essa linhagem celular também pode se tornar um alvo terapêutico.

Distúrbios do metabolismo mineral e ósseo são altamente prevalentes e considerados como causa relevante da morbidade e mortalidade dos pacientes com doença renal crônica. Diversas estratégias diagnósticas e terapêuticas têm sido estudadas nesses doentes; entretanto, pouco valor é dado ao cálcio do dialisato, apesar do impacto que possa exercer sobre o balanço de cálcio durante a hemodiálise. Os fatores determinantes da transferência de cálcio durante o procedimento são ainda controversos. Nesse estudo prospectivo, avaliamos a influência da remodelação óssea sobre o balanço de cálcio em dez pacientes dialíticos em três situações consecutivas: hiperparatireoidismo grave (Pré paratireoidectomia), durante a \"síndrome de fome óssea\" (Fome óssea) imediatamente após a paratireoidectomia e após estabilização clínica (Paratireoidectomia tardia). Durante cada fase os participantes foram submetidos a três sessões randômicas de hemodiálise com diferentes concentrações de cálcio no dialisato: 2,5; 3,0 e 3,5 mEq/L. Todos os pacientes foram submetidos à biópsia óssea para análise histomorfométrica e quantificação de proteínas ósseas no início do estudo. A transferência de cálcio variou grandemente entre os pacientes em cada fase do estudo mesmo usando o mesmo cálcio no dialisato, com valores negativos de medianas no Pré paratireoidectomia e Fome óssea (-161mg e -218mg, respectivamente) e discretamente positivo no Paratireoidectomia tardio (39mg; p < 0,05 versus Pré paratireoidectomia e Fome óssea). Análise de regressão multivariada mostrou que o gradiente de cálcio entre o cálcio iônico sérico inicial e o cálcio do dialisato, a diferença entre o cálcio iônico sérico final e o inicial e a forma não carboxilada da osteocalcina foram preditores independentes da transferencia de cálcio (R2=0.48; p < 0.05). Pelo fato da remodelação óssea também influenciar os níveis séricos de cálcio iônico e suas variações durante a diálise, nesse estudo demonstramos que o esqueleto tem papel fundamental no balanço de cálcio e essas variáveis devem ser consideradas na individualização do cálcio do dialisato de nossos pacientes / Disturbances in mineral and bone metabolism are highly prevalent and are a major cause of morbidity and mortality in chronic kidney disease patients. Different diagnostic and therapeutic strategies have been studied in these patients. However, little attention is paid to the calcium concentration in the dialysate, despite the impact it could exert over calcium balance during dialysis. The variables that determine calcium transfer during hemodialysis are still controversial. In this study, we have prospectively investigated the influence of bone remodeling on calcium balance in ten dialysis patients in three consecutive situations: severe hyperparathyroidism (Pre parathyroidectomy), during \"hungry bone syndrome\" (Hungry bone) right after surgery and after stabilization of clinical status (Late parathyroidectomy). During each phase participants were submitted to 3 random hemodialysis sessions, with different dialysate calcium: 2.5, 3.0 and 3.5 mEq/L. Bone biopsy for hystomorphometric analysis and bone proteins quantification were performed in all patients at baseline. Calcium mass transfer varied widely among patients in each study phase even using the same dialysis calcium with negative median values in Pre parathyroidectomy and Hungry Bone (-161 and -218mg, respectively) and slightly positive in Late parathyroidectomy (39mg; p<0.05 versus Pre parathyroidectomy and Hungry Bone). Multiple regression analysis showed that calcium gradient between initial serum ionic calcium and the dialysate calcium, the difference between final and initial serum ionic calcium and serum undercarboxylated form of osteocalcin were independent predictors of calcium mass transfer (R2=0.48; p<0.05). As bone remodeling also influences the serum levels of ionic calcium and its variance during dialysis, in this study we have added new data by demonstrating that the skeleton plays a key role on calcium balance and these variables must be considered when individualizing calcium dialysate for our patients

Les fonctions endocriniennes des os sont médiées par au moins deux hormones, l’ostéocalcine et le facteur de croissance fibroblastique 23 « Fibroblast growth factor 23 » (FGF23), ces derniers sont secrétés par les cellules osseuses, les ostéoblastes et les ostéocytes. L’ostéocalcine est produite par les ostéoblastes et régule le métabolisme du glucose et énergétique. Elle améliore ainsi la tolérance au glucose et la sensibilité à l’insuline. Également, elle favorise la sécrétion d’insuline et la prolifération des cellules β, elle augmente la dépense énergétique et réduit l’accumulation de graisse. L'ostéocalcine est gamma-carboxylée au niveau de trois résidus d'acide glutamique (Glu), un processus qui inhibe sa fonction endocrinienne chez la souris et l'humain. Le pH acide de la lacune de résorption décarboxyle l'ostéocalcine et libère sa forme non carboxylée (ucOCN), la forme active de cette hormone. Nos connaissances sur la régulation des fonctions endocriniennes d’ostéocalcine sont encore limitées à sa gamma-carboxylation. Puisque cette hormone est secrétée par les ostéoblastes et les ostéocytes, des cellules endocriniennes non classique, nous avons émis l’hypothèse que l'ostéocalcine pourrait être soumise à d'autres modifications post-traductionnelles (PTMs) au niveau de la voie de sécrétion contrôlant ses fonctions endocriniennes. Dans la première partie de cette thèse, nous avons montré que le propeptide de l'ostéocalcine pouvait être clivé dans son extrémité C-terminale au niveau du motif de base « RLRR » par la pro-protéine convertase furine, un processus qui se produit indépendamment de la gamma-carboxylation de l'ostéocalcine. L’inactivation du gène codant pour la furine, spécifiquement dans les ostéoblastes et les ostéocytes chez la souris, abolit totalement le clivage de la pro-ostéocalcine et altère son activation et sa libération lors de la résorption osseuse. Par conséquent, ces souris sont caractérisées par un niveau bas d'ucOCN dans le sérum, ce qui entraîne une altération de la tolérance au glucose, une diminution de la sécrétion d'insuline et de la dépense énergétique ainsi qu’une augmentation de l'accumulation de graisses. De plus, ces souris ont une perte d'appétit indépendamment de l'ostéocalcine. La restriction de la nourriture pour les souris contrôles ou « pair feeding » rend le phénotype des souris déficientes en furine plus apparent. Il apparait à un plus jeune âge avec une résistance à l'insuline. Dans la deuxième partie de cette thèse, nous avons découvert que l'ostéocalcine de souris est O-glycosylée au niveau de la sérine 8, un processus qui se produit indépendamment de sa gamma-carboxylation et de son clivage. Cette modification, qui n'est pas présente chez l'ostéocalcine humaine, augmente la demi-vie de l'ostéocalcine de souris dans le plasma ex vivo et in vivo. Il est intéressant de noter que la tyrosine 12 dans l'ostéocalcine humaine correspond à la sérine 8 dans la séquence de la souris, tandis que la mutation Tyr12Ser est suffisante pour générer une ostéocalcine humaine O-glycosylée et lui conférer une demi-vie plus longue dans le plasma de la souris comparativement à la forme native. FGF23 est une hormone secrétée par les ostéoblastes et les ostéocytes. Elle régule la réabsorption de phosphate et la production de vitamine D dans le tubule proximal du rein. Sa fonction endocrine est inhibée par un clivage endoprotéolytique qui libère ses fragments N- et C-terminaux. La mutation du motif « RHTR », un site de clivage consensus pour les proprotéines convertases PC(s), a été identifié chez les patients atteints du rachitisme hypophosphatémique génétiquement déterminés ou « Autosomal dominant hypophosphatemic rickets » (ADHR). Ces patients se caractérisent par une augmentation du taux de FGF23 intact, une hypophosphatémie et une ostéomalacie. Malgré l’importance de FGF23 dans plusieurs maladies, l’identité de l’enzyme responsable du clivage de FGF23 n’est pas encore connue, même si la furine et la proprotéine convertase subtilisine/kexine type 5 (PC5) peuvent cliver FGF23 in vitro. Dans la troisième partie de cette thèse, nous tentons de répondre à cette question en utilisant des souris déficientes en furine et/ou PC5 spécifiquement dans les ostéoblastes et les ostéocytes. Sous des conditions physiologiques, l’inactivation du gène de furine dans les ostéoblastes et les ostéocytes augmente le niveau du FGF23 intact par 25%. Malgré cette augmentation ces souris maintiennent une phosphatémie normale et elles ne montrent pas de signe d’ostéomalacie. On a aussi montré qu’une déficience en fer, une condition qui augmente la production de FGF23 au niveau de l’ARN messager et protéique, le FGF23 est totalement en forme intact dans les souris déficientes en furine, montrant que le clivage de FGF23 est totalement inhibé dans cette condition. En revanche, l’injection d’érythropoïétine ou d’interleukine 1-β, des conditions qui augmentent la production de FGF23, induit une augmentation significative du taux de FGF23 total dans le sérum des souris déficientes en furine et/ou PC5 dans les ostéoblastes et les ostéocytes, tandis que le niveau du FGF23 intact n’a pas augmenté de la même façon, suggérant que la FGF23 est correctement clivée chez ces souris. D’une façon intéressante et malgré les défauts développementaux et le retard dans la minéralisation osseuse observée dans les souris complètement déficientes en PC5, la suppression conditionnelle de PC5 dans les ostéoblastes et les ostéocytes chez la souris n'a entraîné aucun défaut osseux. Cependant, l’inactivation du gène codant pour la furine dans les ostéoblastes et les ostéocytes chez la souris a augmenté les paramètres osseux trabéculaires et a diminué l'épaisseur de l’os cortical. De plus, ces souris ont eu une diminution de la densité minérale et la rigidité des os reflétant une mauvaise qualité osseuse. En résumé, nous avons décrit pour la première fois que la furine est un régulateur multifonctionnel de la fonction des ostéoblastes et des ostéocytes in vivo. Elle régule le métabolisme du glucose en assurant le clivage de la pro-ostéocalcine, qui est nécessaire à la maturation et à la bio-activité de l'ostéocalcine, et en régulant l'appétit indépendamment de l'ostéocalcine. Ces résultats suggèrent la présence d'ostéokines supplémentaires régulant l'appétit et contrôlées par la furine. De plus, dans les ostéoblastes, la furine régule partiellement le clivage de FGF23 en assurant une phosphatémie normale, suggérant que la régulation de l'accumulation de masse osseuse par la furine est indépendante du FGF23. En outre, nous avons découvert que l'ostéocalcine de souris est soumise à l’O-glycosylation, une modification qui n'est pas conservée chez l'humain, ni chez d’autres espèces, et qui augmente la demi-vie de l'ostéocalcine de souris. La glycosylation artificielle confère à l'ostéocalcine humaine une demi-vie plus longue, offrant ainsi une approche permettant d'augmenter potentiellement la bio-activité de l'ostéocalcine humaine dans les futures applications thérapeutiques de l'ostéocalcine dans les maladies humaines. / Bone endocrine functions are mediated by at least two hormones, osteocalcin and fibroblast growth factor 23 (FGF23) which are secreted by the bone cells, osteoblasts and osteocytes. Osteocalcin is an osteoblast-derived hormone regulating glucose and energy metabolism. It improves glucose tolerance and insulin sensitivity, promotes insulin secretion and β-cell proliferation, increases energy expenditure and reduces fat accumulation. Osteocalcin is gamma-carboxylated on three of its glutamic acid residues (Glu), a process that inhibits its endocrine function in mice and humans. It is the acidic pH in the resorption lacuna which decarboxylates osteocalcin releasing the uncarboxylated osteocalcin (ucOCN), the active form of this hormone. Our knowledge on osteocalcin regulation by post-translational modifications is limited to its gamma-carboxylation. Since osteocalcin is secreted by differentiated osteoblasts, a non-classical endocrine cell, we hypothesized that osteocalcin may be subjected to additional post translational modifications (PTMs) in the secretory pathway that regulates its endocrine functions. In the first part of the thesis we showed that osteocalcin’s putative pro-peptide is cleaved in its C-terminus at the basic motif «RLRR», by the proprotein convertase furin. This process occurs independently of osteocalcin gamma-carboxylation. Furin inactivation specifically in osteoblasts in mice totally abolishes osteocalcin processing and impairs its activation and release during bone resorption. Consequently, these mice have decreased serum level of ucOCN resulting in impaired glucose tolerance, reduced insulin secretion and energy expenditure, and increased fat accumulation. Moreover, these mice have a decrease in the appetite independently of osteocalcin. Pair feeding of control mice resulted in more apparent phenotype in furin deficient mice, as it appears at younger age alongside with insulin resistance. In the second part of this thesis, we discovered that mouse osteocalcin is O-glycosylated on serine 8, a process that occurs independently of its gamma-carboxylation and processing. This modification is not conserved in human or any other species and it increases mouse osteocalcin half-life in plasma ex vivo and in vivo. Interestingly, tyrosine 12 in human osteocalcin corresponds to the serine 8 in the mouse sequence. Tyr12Ser mutation was sufficient to O-glycosylate human osteocalcin and to confer this hormone a longer half-life in mouse plasma compared to the native form. FGF23 is a hormone secreted by osteoblasts and osteocytes which regulates phosphate reabsorption and vitamin D production in the kidney proximal tubule. Its endocrine function is inhibited by endoproteolytic cleavage which releases its N-terminal and C-terminal fragments. Mutations in the «RHTR» motif, a consensus cleavage site for proprotein convertases (PCs), were found in patients with autosomal dominant hypophosphatemic rickets (ADHR). These patients are characterized by an increased intact FGF23 levels, hypophosphatemia and osteomalacia. Despite the importance of FGF23 in the pathology of multiple diseases, the identity of the enzyme(s) involved in FGF23 cleavage is yet unclear, even though furin and the proprotein convertase subtilisin/kexin type 5 (PC5) were shown to cleave FGF23 in vitro. In the third part of the thesis, we addressed this question using mice model deficient in furin and/or PC5 in osteoblasts and osteocytes in mice. Under physiological conditions, furin inactivation resulted in a 25% increase in intact FGF23; however, these mice maintained normal phosphate level and did not shown any sign of osteomalacia. We also showed that under iron restriction, a condition that induce FGF23 expression at the mRNA and protein level, FGF23 processing is totally impaired in furin deficient mice. However, the injection of erythropoietin or interleukin 1-β, two conditions that increase FGF23 production, induce FGF23 serum level while it is still properly processed in mice deficient in furin and/or PC5 in osteoblasts and osteocytes. Interestingly, despite the patterning defects observed in global inactivation of PC5, conditional inactivation of PC5 in osteoblasts and osteocytes in mice did not result in any bone defect. However, furin inactivation in osteoblasts and osteocytes in mice increases trabecular bone parameters and decreases cortical thickness. Moreover, these mice have decreased bone mineral density and bone strength reflecting a poor bone quality. In summary, we described for the first time that furin is a pleotropic regulator of osteoblast and osteocyte function in vivo. It regulates glucose and energy metabolism by mediating pro-osteocalcin processing which is required for osteocalcin maturation and bioactivity, and by regulating appetite independently of osteocalcin. These findings suggest the presence of additional osteokines controlling appetite and which are regulated by furin. Moreover, furin partially regulates FGF23 processing while maintaining normal phosphate homeostasis, suggesting that the regulation of bone mass accrual by furin occurs independently of FGF23. Additionally, we discovered that mouse osteocalcin is subjected to O-glycosylation, a species-specific modification that is not conserved in humans or any other species and increases mouse osteocalcin half-life. Artificial O-glycosylation confer human osteocalcin a longer half-life, thus providing an approach to increase human osteocalcin bioactivity in future therapeutic applications of osteocalcin in human diseases.

INTRODUÇÃO: O hipoestrogenismo pode determinar perda da massa mineral óssea, diminuindo a qualidade do osso. Assim, vários fármacos são ministrados para evitar esta perda, porém, podem determinar efeitos colaterais importantes. Portanto, questiona-se se o emprego do estrogênio associado a estas substâncias poderia minimizar os efeitos adversos e manteria a massa mineral óssea. Contudo, há poucas informações sobre os efeitos destas combinações. Esta pesquisa tem como objetivo avaliar a ação do estrogênio, raloxifeno e do extrato de soja rico em gensteína, isolado ou combinado no osso de ratas ovariectomizadas. MATERIAIS E MÉTODOS: No nono dia de nascimento, todas as ratas receberam propionato de testosterona (0,1 g/g). No sexto mês de idade, os animais do controle fisiológico foram identificados como GI e receberam apenas o veículo (propilenoglicol em 0,5 ml/dia) durante o experimento e os outros que receberam testosterona foram ovariectomizados e divididos aleatoriamente em seis grupos: GII veículo (controle castrado, n=6); GIII - estrogênio conjugados eqüinos (ECE, (50 g/Kg/dia, n=8); GIV raloxifeno (RAL, 0,75 mg/kg/dia, n=8); GV extrato de soja enriquecido com genisteína (ESG, 300 mg/kg/dia, n=7); GVI ECE + ESG (50 g/Kg/dia + 300 mg/kg/dia, n=7); GVII - ECE+RAL (50 g/Kg/dia + 0,75mg/kg/dia, n=6). Após três meses da cirurgia, os fármacos foram ministrados por 120 dias consecutivos. Posteriormente, os animais foram sacrificados sob anestesia, sendo retirada a tíbia esquerda para rotina histológica. Os cortes histológicos foram corados pela hematoxilina-eosina para avaliar a microarquitetura óssea. Foram feitos procedimentos imunoistoquímicos, de imunofluorescência e PCR para quantificar as principais proteínas ósseas estruturais (colágeno tipo I, osteocalcina, osteopontina e osteoprotegerina), bem como de seus respectivos RNA mensageiros. Os dados foram analisados pelos testes de ANOVA e Tukey. RESULTADOS: Todos os tratamentos determinaram aumento da quantidade de osso trabecular (p<0,05). As fibras totais de colágeno apresentaram-se aumentadas em todos os grupos tratados, exceto com o raloxifeno. Já as fibras finas de colágeno diminuíram apenas no grupo tratado com estrogênio. As frações de colágeno tipo I, mostraram-se aumentadas nos grupos tratados com estrogênio e sua asssociação com o raloxifeno. O colágeno tipo III esteve aumentado no grupo tratado com estrogênio em associação com extrato de soja rico em genisteína. Em relação às proteínas não colagenosas, a osteoprotegerina apresentou-se aumentada nos grupos tratados com estrogênio, suas associações e com o extrato de soja rico em genisteína. A osteopontina esteve diminuída em todos os grupos tratados e a osteocalcina mostrou-se aumentada apenas no grupo tratado com ralolxifeno, em comparação ao grupo castrado (p<0,05). Não houve diferença estatística significante do PCR em tempo real na análise dos transcritos entre os grupos estudados. CONCLUSÃO: A combinação de estrogênio com raloxifeno ou extrato de soja rico em genisteína não trouxe benefícios adicionais na qualidade do tecido ósseo, como ocorreu com esses fármacos isoladamente / INTRODUCTION: Hypoestrogenism can determine bone mineral loss, resulting in decreased bone quality. To prevent that process, several drugs are administered, which can lead, however, to important side effects. Therefore, it is questionable whether the use of estrogen associated with those substances could minimize the adverse effects and maintain bone mineral mass. There is little information on the effects of those compounds. This research aims to evaluate the action of unopposed estrogen or combined with raloxifene and genistein-rich soy extract on ovariectomized adult female rats. MATERIALS AND METHODS: On the ninth day of birth, rats received, testosterone propionate (0.1 mg / g). On the sixth month, animals in the physiological control were identified as GI and received only the vehicle (propylene glycol at 0.5 ml / day) during the experiment and the other which was administered testosterone underwent ovariectomy and divided randomly into six groups: GII - vehicle (control castrated, n = 6); GIII - conjugated equine estrogen (CEE, 50 mg / kg / day, n = 8); GIV - raloxifene (RAL, 0.75 mg / kg / day, n = 8) ; GV - soy extract enriched with genistein (ESG, 300 mg / kg / day, n = 7), GVI - ECE + ESG (50 mg / kg / day + 300 mg / kg / day, n = 7); GVII - ECE + RAL (50 mg / kg / day + 0.75mg/kg/day, n = 6).Three months after the surgery, drugs were consecutively administered for 120 days. Subsequently, the animals were sacrificed on anesthesia and their left tibiae were removed for routine histology. The histological sections were stained by hematoxylin-eosin to evaluate bone microarchitecture. Immunohistochemical, immunofluorescence and PCR procedures were performed to quantify the main structural bone proteins (type I collagen, osteocalcin, osteopontin, and osteoprotegerin) as well as their mRNA. The data were analyzed by ANOVA and Tukey test. RESULTS: All treatments led to increased amounts of trabecular bone (p <0.05). The total collagen fibers had to be enlarged in all treated groups, except with raloxifene. Already thin collagen fibers decreased only in the group treated with estrogen. The fractions of type I collagen, were increased in groups treated with estrogen and its asssociação with raloxifene. Type III collagen was increased in the group treated with estrogen in combination with soybean extract rich in genistein. Regarding the non-collagenous proteins, the increased osteoprotegerin presented in groups treated with estrogen, and their associations with soy extract rich in genistein. The osteopontin was decreased in all treated groups and osteocalcin was increased only in the treated group ralolxifeno, compared to the castrated group (p <0.05). There was no statistically significant difference from the real-time PCR analysis of transcribed between the groups. CONCLUSION: The combination of estrogen with raloxifene or genistein-rich soy extract was uncapable of bringing additional benefits to the quality of bone tissue as observed with those drugs alone

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