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.

Gut-associated lymphoid tissues such as Peyer’s patches (PP) are inductive sites for immune response in the intestine. Unlike other peripheral lymphoid tissues, gut-associated lymphoid tissues lack afferent lymphatics and can directly sample mucosal antigens by specialized epithelial cells in the follicular associated epithelia (FAE), known as M cells. M cells derive from Lgr5+ intestinal stem cells in intestinal crypts, where the daughter cells of Lgr5+ cells differentiate into M cells after stimulation from the cytokine receptor activator of nuclear factor-κB ligand (RANKL). RANKL is produced by stromal cells within the sub-epithelial dome (SED) residing below the FAE. The transcytosis of antigens across the FAE by M cells is an important initial step in the induction of efficient mucosal immune responses against certain pathogenic bacteria as well as the commensal bacterial flora. However some pathogens, for example orally-acquired prions, may also exploit M cells to infect the host. M cells have been implicated in the uptake of orally acquired prions from the gut lumen. After oral exposure, the accumulation of prions in PP is important for their efficient spread to the nervous system. Previous studies have also shown that pathogen-induced inflammation increases M cell density and this effect can be mimicked by exogenous administration of RANKL. This has led to the hypothesis tested in this thesis that inflammation-related enhancement of M cell differentiation aids the delivery of prions into the lamina propria of villi. The administration of RANKL resulted in increased M cell density in the gut epithelium of mice. Consequently, RANKL treatment enhanced the accumulation of orally-administered prions in PP, decreased disease incubation time and increased prion disease susceptibility. These data indicate the importance of M cells in prion disease pathogenesis and highlight the potential of M cells as vaccine targets against prion disease. The fate and terminal differentiation of distinct intestinal epithelial cell lineages from their uncommitted precursors is dependent on their intrinsic expression of one or more specific transcription factors during their development. Alongside inducing M cell differentiation, RANKL stimulation can also induce the nuclear translocation of the NF-κB transcription factor subunit c-Rel. A comparison of the genes encoding the individual NF-κB subunits c-Rel, Rel-A and Rel-B revealed that they were expressed at the mRNA level in the FAE and by M cells. A c-Rel-deficiency in mice did not influence the expression of RANKL or RANK in PP. The subsequent induction of M cell maturation in the FAE was also unaffected in, indicating that c-Rel is dispensable for the RANKL-mediated differentiation and functional maturation of M cells. The factors implicated in Lgr5+ intestinal stem cell proliferation and their differentiation into M cells are poorly understood. Some reports have indicated that crypt-associated macrophages may provide extrinsic factors that assist Lgr5+ intestinal stem cell proliferation. In this thesis, the ablation of macrophages in the gut resulted in dysregulation of crypt microarchitecture, depleting Paneth cells and the Lgr5+ stem cells. This adversely affected the subsequent differentiation of intestinal epithelial cell lineages and impeded the functional development of M cells. These data reveal a previously unknown role for macrophages in the maintenance of intestinal crypts and intestinal stem cell proliferation and differentiation.

Made available in DSpace on 2014-06-11T19:23:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-19Bitstream added on 2014-06-13T18:19:49Z : No. of bitstreams: 1 coutinho_ccc_me_araca.pdf: 114919 bytes, checksum: 49f558e8cba145ffdabb2e45dc62ada3 (MD5) / 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.

5 ABSTRACT Background: Thromboembolic disease is one of the most common causes of pregnant women morbidity and mortality. The pregnancy period is often the first time, when the apparent congenital or acquired thrombophilia is identi- fied. Patients with thrombophilia have an increased risk of pregnancy compli- cations. The optimal anticoagulant prophylaxis helps to prevent these compli- cations. Methods: The presented work is focused on monitoring of coagulation param- eters, blood counts and acute phase proteins in pregnant women (N = 68) with thrombophilia treated with enoxaparin during pregnancy and it is also con- cerned with evaluation of effectiveness of anticoagulant therapy during preg- nancy using anti FXa activity determination based on inhibition of FXa weight, coagulation parameters and acute phase proteins. In the first and the second part of the study, there is no control group of pregnant patients with severe thrombophilia, but no anticoagulation - this is justified by ethical rea- sons. In the third part, we examined by questionnaire our patients for enoxap- arin adverse reactions at the injection site. Finally, the last part is focused on evaluation of enoxaparin effects on bone remodelling markers, compared with a group of pregnant women without anticoagulation. Results: During the...

Submitted by Etelvina Domingos (etelvina.domingos@ufpe.br) on 2015-04-08T19:41:57Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE Rodrigo Alves Ribeiro.pdf: 1374775 bytes, checksum: d36ffe53b7c0883541e67e218c484dee (MD5) / Made available in DSpace on 2015-04-08T19:41:57Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE Rodrigo Alves Ribeiro.pdf: 1374775 bytes, checksum: d36ffe53b7c0883541e67e218c484dee (MD5) Previous issue date: 2014 / INTRODUÇÃO: Os implantes dentais constituem uma opção de tratamento confiável e previsível para reabilitação oral. Apesar dos benefícios reconhecidos, complicações biológicas e mecânicas podem ocorrer, comprometendendo a estabilidade da reabilitação. OBJETIVO: estimar o índice de sucesso através dos critérios propostos na literatura e verificar a associação do polimorfismo do gene RANKL [região -438 (A/G)] e do gene IL-10 [região -1082(A/G)] ao insucesso de implantes dentários. MATERIAIS E MÉTODOS: A amostra aleatória foi composta por noventa pacientes, de ambos os gêneros, parcialmente edêntulos reabilitados com 245 implantes dentários Straumann® (Waldenburg, Switzerland), os quais foram avaliados clínica e radiograficamente para os seguintes parâmetros: mobilidade, queixas subjetivas persistentes, infecção peri-implantar recorrente com supuração, radiolucência contínua ao redor do implante, profundidade de sondagem ≥ 5mm e sangramento à sondagem. Estes parâmetros foram agrupados pelos seguintes autores: Schnitman & Schulman (1979), Albrektsson et al. (1986), Buser et al. (1990), Mombelli & Lang (1994) e Ong et al. (2008). Na presença de algum destes critérios, o implante foi considerado como insucesso. O tempo de carga dos implantes foi categorizado em 3 grupos: Grupo 1: < 1 ano de carga; Grupo 2: ≥ 1 e < 5 anos de carga e Grupo 3: ≥ 5 anos de carga. Também foram coletadas células da mucosa jugal para a análise do polimorfismo dos genes RANKL e IL-10 através da reação em cadeia da polimerase. RESULTADOS: Quando as diferentes classificações para insucesso de implante foram consideradas, houve diferença significativa na ocorrência de insucessos. A prevalência de insucesso variou de 3,3%, segundo os critérios de Buser et al. 1990 a 36,7%, de acordo com os critérios de Ong et al. (2008). Não houve associação entre o insucesso dos implantes e os genótipos dos genes RANKL e IL-10 (p>0,05). CONCLUSÃO: Pode-se concluir que de acordo com o critério adotado há grande variação na ocorrência de insucesso nos implantes e que não houve associação entre o polimorfismo genético da RANKL 438 e IL-10 e o insucesso do implante dentário na população avaliada.

5 ABSTRACT Background: Thromboembolic disease is one of the most common causes of pregnant women morbidity and mortality. The pregnancy period is often the first time, when the apparent congenital or acquired thrombophilia is identi- fied. Patients with thrombophilia have an increased risk of pregnancy compli- cations. The optimal anticoagulant prophylaxis helps to prevent these compli- cations. Methods: The presented work is focused on monitoring of coagulation param- eters, blood counts and acute phase proteins in pregnant women (N = 68) with thrombophilia treated with enoxaparin during pregnancy and it is also con- cerned with evaluation of effectiveness of anticoagulant therapy during preg- nancy using anti FXa activity determination based on inhibition of FXa weight, coagulation parameters and acute phase proteins. In the first and the second part of the study, there is no control group of pregnant patients with severe thrombophilia, but no anticoagulation - this is justified by ethical rea- sons. In the third part, we examined by questionnaire our patients for enoxap- arin adverse reactions at the injection site. Finally, the last part is focused on evaluation of enoxaparin effects on bone remodelling markers, compared with a group of pregnant women without anticoagulation. Results: During the...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21622号 / 医博第4428号 / 新制||医||1033(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 松田 秀一, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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.

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.