Paget’s disease of bone (PDB) is a common disease with a strong genetic component. Approaches such as linkage analysis and candidate gene studies have shown that mutations in Sequestosome 1 (SQSTM1) explain up to 40% of familial cases and 10% of sporadic cases, however the majority of PDB patients have no mutations in this gene. Genome-wide association studies (GWAS) have recently identified new susceptibility loci for PDB including variants at CSF1, TNFRSF11A, OPTN, TM7SF4, PML, NUP205 and RIN3 loci. These loci were confirmed to be associated with PDB in various European populations. OPTN encodes optineurin, a widely expressed protein involved in many cellular processes but its role in bone metabolism is yet unknown. The aim of this PhD thesis was to investigate the role of OPTN in bone metabolism and PDB using in vitro and in vivo studies. In chapter 3, the OPTN rs1561570 identified by previous GWAS was examined for its association with the severity and clinical outcome of PDB in patients without SQSTM1 mutations. The results showed that rs1561570 was significantly associated with total disease severity score so that carriers of the risk allele “T” had higher severity score compared to non-carriers (P < 0.05). A trend for reduced quality of life physical scores (SF36) was also associated with the rs1561570 risk allele, but the relationship was not statistically significant. In order to identify functional variants within OPTN, the coding regions as well as the exon-intron boundaries were sequenced in 24 familial PDB cases and 19 controls. No mutation was found that could be predicted as pathogenic suggesting that disease susceptibility could be mediated by regulatory polymorphisms that influence gene expression. In chapter 4, the role of OPTN was investigated in osteoclast development using in vitro knockdown experiments. Optn was expressed in mouse bone marrow derived macrophages (BMDMs) as well as all stages of osteoclast development and it was significantly increased three days post RANKL treatment. Optn expression was knocked down in BMDMs and cells were induced to form osteoclast in the presence of RANKL and M-CSF. Compared to non-targeted cells, Optn depleted cells formed significantly more and larger osteoclasts (P< 0.05). Optn knockdown was also found to enhance osteoclast survival as well as RANKL-induced NFκB activation. In chapter 5, the role of OPTN was investigated in vitro from cells obtained from knock in mice with a loss-of-function mutation in Optn (OptnD477N/D477N). In agreement with the in vitro knockdown experiments, osteoclasts were significantly higher and larger in mutant mice compared to WT and the NF-B activity measured by luciferase reporter assay was significantly higher in cells from OptnD477N/D477N compared to WT during most stages of osteoclast development. OPTN from mutant and WT mice was co-precipitated with its CYLD binding-partner, which acts as a negative regulator to RANK signalling by inhibiting the TRAF6 downstream signalling. The data from this immunoprecipitation (IP) experiment revealed that defective OPTN interacted less with CYLD from mutant mice compared to WT. This study also showed that OPTN was expressed in osteoblasts and the expression rate did not change during osteoblast development. The data obtained from the mineralization assay revealed no significant difference between OptnD477N/D477N and WT. In chapter 6, I investigated the effect of the D477N loss of function mutation in Optn on bone metabolism. Bone Histomorphometrical analysis of OptnD477N/D477N mice showed higher bone resorption parameters (Oc.N/BS and Oc.S/BS) compared to wild type (WT). Osteoid analysis showed evidence of increased bone formation parameters (OS/BS and OV/BV) in mutant mice compared to WT. Calcein labelling showed a significant difference in mineral apposition rate (MAR) from mutant mice compared to WT. Analysis of serum biomarkers of bone turnover showed evidence of enhanced bone turnover in mutant mice compared to WT. Micro computed tomography (μCT) analysis of 4 and 14 months old mice showed no significant differences in bone morphology between WT and OptnD477N/D477N mice of both sexes. In conclusion, this study has shown for the first time that OPTN plays a role in regulating bone turnover by acting as a negative regulator of osteoclast differentiation. The data obtained from this study strongly suggest the crucial role of OPTN in RANK signalling. The effect of OPTN on osteoblast activity may be direct or indirect compensation for increased osteoclast activity. Further detailed studies will be required to explore the underlying mechanism of OPTN including downstream RANK signalling and a complete knockout model to corroborate these findings.

Submitted by Eunice Novais (enovais@uepg.br) on 2018-08-14T18:52:18Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Gabriela Veloso 28-05-2018.pdf: 2164370 bytes, checksum: 4e7d75f83857ad16625e9fb2ea2f374d (MD5) / Made available in DSpace on 2018-08-14T18:52:18Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Gabriela Veloso 28-05-2018.pdf: 2164370 bytes, checksum: 4e7d75f83857ad16625e9fb2ea2f374d (MD5) Previous issue date: 2018-05-28 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / Estudos epidemiológicos têm apontado a doença periodontal como a desordem óssea mais prevalente em humanos. Sua etiologia é definida de forma mul-tifatorial, em que há influência de doenças sistêmicas. De fato, muitos estudos relatam esta última associação e, neste contexto, a sepse é uma doença sistêmica caracteri-zada por uma disfunção orgânica com risco de vida, causada por uma resposta des-regulada do hospedeiro à uma infecção. O objetivo deste estudo foi avaliar por meio de análise histológica e imunohistoquímica se a sepse é capaz de exercer influência sobre o metabolismo ósseo na região do periodonto. Métodos: Foram utilizados dois grupos de ratos Wistar divididos em controle (n=6) e sepse (n=6). Os animais passa-ram pelo modelo de procedimento de ligação e perfuração do ceco (CLP) e, após 24 horas, por meio de sobredose anestésica, suas hemimandíbulas foram coletadas e submetidas a procedimentos histotécnicos. Em seguida, componentes do periodonto como matriz óssea, fibras colágenas, fibroblastos, osteócitos, células inflamatórias, vasos sanguíneos e espaços em branco do ligamento periodontal, osso alveolar e osso da área da furca foram avaliados e quantificados por meio de análise histomor-fométrica. Posteriormente, foram realizadas análises por imunohistoquímica nestas mesmas regiões, visando avaliar o número de células imunomarcadas para BMP-2/4, osteocalcina e RANKL. Resultados: A análise histomorfométrica não revelou dife-renças significativas entre os grupos avaliados. Entretanto, a análise imunohistoquí-mica revelou diferenças significativas no número de células imunomarcadas entre o grupo controle e sepse para osteocalcina e RANKL. Conclusões: Foram observadas algumas modificações no periodonto de ratos após 24 horas da indução da sepse. Contudo, outros estudos são necessários para possibilitar uma avaliação mais abran-gente sobre os possíveis efeitos da sepse na região periodontal, incluindo avaliações moleculares e com períodos experimentais maiores. / Epidemiological studies have pointed the periodontal disease as the most prevalent bone disorder in humans. Its aetiology is defined in a multifactorial way, in which there is influence of systemic illnesses. In fact, many studies report this asso-ciation and, in such context, sepsis is a systemic disease characterized by an organic malfunction that is life threatening, caused by a deregulated response from the host to certain infection. The objective of this study was to evaluate through histological and immunohistochemical analyses whether sepsis is able to have influence on the bone metabolism in the periodontal regions. Methods: Two groups of Wistar rats were used, which were divided into control (n=6) and sepsis (n=6). The animals were submitted to the model of procedure cecum link and perforation (CLP) and, after 24 hours, through an anaesthetic overdose, their hemimandibles were collected and submitted to histo-technical procedures. Next, periodontal components such as bone matrix, collagenous fibers, fibroblasts, osteocytes, inflammatory cells, blood vessels, periodontal ligament blank spaces, alveolar bone and bone from the furcation area were evaluated and quantified using the histomorphometric analysis. Later on, immunohistochemical anal-ysis was carried out in the same regions, aiming at evaluating the number of BMP-2/4, osteocalcin and RANKL immunolabeled cells. Results: The histomorphometric analy-sis did not reveal significant differences between the groups under analysis. However, immunohistochemical analysis revealed significant differences in the number of immu-nolabelled cells between the control and sepsis groups for osteocalcin and RANKL. Conclusions: Some modifications were observed in the periodontium of rats after 24 hours of induction of sepsis. However, further studies are required to enable a broader evaluation of the possible effects of the sepsis on the periodontal region, including molecular evaluations and with longer experimental periods.

Weißbüschelaffen sind häufig verwendete Tiermodelle in der Forschung. Jedoch gibt es zu wenige Untersuchungen zu ihrem Knochenaufbau und Knochenstoffwechsel, um sie auch als Modell auf dem Gebiet der Knochenkrankheiten einsetzen zu können. Somit war es das Ziel dieser Studie, Richtwerte der Knochendichte von Weißbüschelaffen aufzustellen, diese mit denen des Menschen und anderer nichtmenschlicher Primaten zu vergleichen, sowie bestimmte Einflussfaktoren, wie Gewicht, Alter oder hormonelle Umstellungen auf die Knochendichte abzuklären. Desweiteren sollte mittels einer histologischen Darstellung der Knochen, sowie mittels immunhistologischen Nachweisen verschiedener knochenspezifischer Antigene, Gemeinsamkeiten oder Unterschiede zum Menschen im Hinblick auf Veränderungen im Knochen aufgedeckt werden. Abschließend stellte sich dann noch die Frage, ob ein therapeutisches Eingreifen bei niedriger messbarer Knochendichte möglich ist, um die Lebenssituation wieder zu verbessern. Hierzu wurde von 58 C.j. mit Hilfe des Aloka®-CT knochenspezifische Parameter im Bereich des vierten Lendenwirbels (L4) gemessen. Zusätzlich wurde Ca, Pi, AP und Östrogen im Blut bestimmt. Knochenschnitte von L4, Femurkopf/Femurhals und Femurschaft von fünf euthanasierten C.j. (4 männliche, 1 weibliches) wurden histologisch, sowie immunhistologisch (Bestimmung von OPN, OC, RUNX 2, Kollagen Typ 1, Kollagen Typ 5) untersucht. Abschließend wurde bei 2 Tieren eine Therapie mittels Zufütterung von Ca und Vitamin D durchgeführt. Trotz Unterschieden in der Höhe des BMD zum Menschen, konnten Gemeinsamkeiten bei der Reaktion auf Gewichtsveränderungen und Alter festgestellt werden. Eine Zunahme des Gewichts führte zu einer signifikanten Zunahme des BMD. Männliche Weißbüschelaffen zeigten einen Anstieg des BMD bis zu einem Alter von 96 Monaten, was bei Menschen Knochenmassepeak genannt wird, und danach einen signifikanten Abfall. Das bedeutet, dass die Knochen im Alter, genau wie beim Menschen, eine größere Frakturneigung zeigen, als bei jungen Tieren. Desweiteren zeigten Tiere mit einem hohen BMD eine signifikant niedrigere AP, als Tiere mit einem niedrigen BMD. Somit konnte dargestellt werden, dass die AP auch beim C.j. einen Marker für die Osteopenie darstellt. Dies ist ebenfalls eine Gemeinsamkeit mit dem Menschen. Immunhistologisch konnten die Knochenformationsmarker OPN, OC und RUNX 2 nur in den stabilen, bruchfesten Knochen nachgewiesen werden. Kollagen Typ I und V wurden in allen Knochen detektiert. Eine Therapie mittels Ca und Vitamin D bei Tieren mit einem pathologisch niedrigem BMD und klinischen Symptomen, zeigte einen deutlichen Anstieg der Knochendichte nach einem halben Jahr, sowie ein Verschwinden der klinischen Symptome. Schlussfolgernd lässt sich sagen, dass der Weißbüschelaffe trotz unterschiedlicher Knochendichtewerte, ein sehr gutes Tiermodell darstellt zur Untersuchung von Erkrankungen, die sich im Knochen manifestieren (z.B. Osteoporose, Ostemalazie).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Indivíduos que permanecem longo tempo em cadeira de rodas apresentam importante perda de massa óssea, principalmente nos membros inferiores, possivelmente agravada pela baixa ingestão de cálcio dietético e pelo inadequado estado nutricional de vitamina D. O exercício físico pode contribuir para a manutenção ou aumento da massa óssea em diferentes populações e nos indivíduos com lesão medular pode contribuir para atenuar a perda de massa óssea. O objetivo do presente estudo foi avaliar a influência da prática regular de exercício físico sobre a adequação da massa óssea, indicadores bioquímicos do metabolismo ósseo e estado nutricional de vitamina D em indivíduos com lesão medular cervical há pelo menos um ano. Em vinte e cinco homens de 19 a 56 anos sendo 15 fisicamente ativos e 10 sedentários, foi realizada análise sérica de cálcio, PTH, 25(OH)D, IGF-1, osteocalcina e NTx. As medidas do conteúdo mineral ósseo, densidade mineral óssea (DMO), massa magra e massa gorda foram realizadas por DXA. A pigmentação da pele (constitutiva e por bronzeamento) foi determinada por colorimetria com o objetivo de investigar sua influência sobre o estado de vitamina D. A ingestão habitual de cálcio foi registrada em um questionário de frequência alimentar direcionado para alimentos fonte. As comparações entre os dois grupos foram realizadas pela aplicação do Teste t de Student exceto para as variáveis ósseas que foram realizadas após ajustes pela massa corporal total, tempo de lesão e ingestão de cálcio utilizando-se análise de co-variância. Associações entre as variáveis estudadas foram avaliadas através de análise de correlação de Pearson. Valores de p<0.05 foram considerados significativos. Não foram observadas diferenças estatisticamente significativas entre os grupos para nenhuma variável óssea com exceção do z-score da DMO da coluna lombar, que foi significativamente maior no grupo de indivíduos sedentários (0,9 1,7 vs -0,7 0,8; p<0,05). No entanto, entre os indivíduos ativos, aqueles que iniciaram a prática de exercício físico com menos tempo decorrido após a lesão apresentaram maior DMO do fêmur (r=-0,60; p<0,05). Nos indivíduos ativos, a freqüência do exercício apresentou associação negativa com a concentração sérica de i-PTH (r = -0,50; p =0,05) e positiva com a concentração de 25(OH)D (r= 0,58; p <0,05). Após ajustes pela massa corporal total e tempo de lesão foram observadas associações positivas entre a ingestão diária de cálcio e z-score da DMO da coluna lombar (r = 0,73 e p <0,01) e DMO do rádio (r = 0,56 e p <0,05). Os resultados do presente estudo apontam para um efeito benéfico do exercício físico sobre a massa óssea e o perfil hormonal relacionado ao metabolismo ósseo. O início da prática regular de exercício físico o quanto antes após a lesão parece contribuir para atenuar a perda de massa óssea nos membros inferiores. Além disso, os resultados deste estudo sugerem uma possível potencialização do efeito osteogênico do exercício físico quando combinado a uma adequada ingestão de cálcio. / Individuals who stay long time in a wheelchair have significant bone loss, especially in lower limbs that may be aggravated by low calcium intake and inadequate vitamin D status. Physical exercise contributes to maintain or increase bone mass in different populations and may be useful in reducing bone loss in spinal cord injured individuals. The aim of this study was to evaluate the influence of regular physical exercise on bone mass adequacy, biochemical markers of bone metabolism and vitamin D status in individuals with cervical spinal cord injury. Twenty five male adults (19-56 years) with cervical spinal cord injury for at least one year, were recruited and divided into physically active (n=15) and sedentary (n=10) groups. Fasting blood samples were collected and serum samples were stored at -20oC until analysis of calcium, PTH, 25(OH)D, IGF-1, osteocalcin and NTx. Bone mineral content and density (BMD), lean mass and fat mass were evaluated by DXA. Skin pigmentation (constitutive and facultative) was evaluated by reflectance colorimetry in order to investigate its influence on vitamin D status. Habitual calcium intake was recorded using a food frequency questionnaire directed to calcium food sources. Comparisons between groups were performed using Students t test except for bone variables that were performed after adjustments for total body mass, duration of injury and calcium intake by analysis of covariance. Associations between variables were evaluated using Pearson's correlation analysis. P values <0.05 were considered significant. There were no significant differences between groups for bone measurements except for lumbar spine Z-score, that was significantly higher in the sedentary group (0.9 1.7 vs -0.7 0.8; p< 0.05). However, in the active group, it was observed that subjects who initiated the practice of physical exercise with less time after injury had higher BMD at the femur (r=-0.60; p<0.05). In active subjects, exercise frequency was negatively associated with serum i-PTH (r = -0.50, p = 0.05) and positively associated with serum 25(OH)D (r= 0.58; p <0.05). After adjustments for total body mass and duration of injury daily calcium intake was positively associated with lumbar spine Z-score (r = 0.73 and p < 0.01) and with radius BMD (r = 0.56 and p <0.05). The results of this study suggest a beneficial effect of regular exercise practice on bone mass and bone-related hormonal profile. The earlier initiation of regular physical exercise after the injury appears to contribute to attenuate the loss of bone mass in lower limbs. Moreover, our results suggest that the osteogenic effect of exercise may be potentiated when combined with an adequate calcium intake.

abstract: Applications of non-traditional stable isotope variations are moving beyond geosciences to biomedicine, made possible by advances in multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) technology. Mass-dependent isotope variation can provide information about the sources of elements and the chemical reactions that they undergo. Iron and calcium isotope systematics in biomedicine are relatively unexplored but have great potential scientific interest due to their essential nature in metabolism. Iron, a crucial element in biology, fractionates during biochemically relevant reactions. To test the extent of this fractionation in an important reaction process, equilibrium iron isotope fractionation during organic ligand exchange was determined. The results show that iron fractionates during organic ligand exchange, and that isotope enrichment increases as a function of the difference in binding constants between ligands. Additionally, to create a mass balance model for iron in a whole organism, iron isotope compositions in a whole mouse and in individual mouse organs were measured. The results indicate that fractionation occurs during transfer between individual organs, and that the whole organism was isotopically light compared with food. These two experiments advance our ability to interpret stable iron isotopes in biomedicine. Previous research demonstrated that calcium isotope variations in urine can be used as an indicator of changes in net bone mineral balance. In order to measure calcium isotopes by MC-ICP-MS, a chemical purification method was developed to quantitatively separate calcium from other elements in a biological matrix. Subsequently, this method was used to evaluate if calcium isotopes respond when organisms are subjected to conditions known to induce bone loss: 1) Rhesus monkeys were given an estrogen-suppressing drug; 2) Human patients underwent extended bed rest. In both studies, there were rapid, detectable changes in calcium isotope compositions from baseline - verifying that calcium isotopes can be used to rapidly detect changes in bone mineral balance. By characterizing iron isotope fractionation in biologically relevant processes and by demonstrating that calcium isotopes vary rapidly in response to bone loss, this thesis represents an important step in utilizing these isotope systems as a diagnostic and mechanistic tool to study the metabolism of these elements in vivo. / Dissertation/Thesis / Ph.D. Chemistry 2011

O corpo de evidências científicas a respeito da inter-relação entre câncer oral e doenças periodontais vem aumentando consideravelmente. Entretanto, estudos avaliando os mecanismos biológicos que permeiam esta associação ainda são escassos. O objetivo do estudo foi avaliar o efeito da exposição ao carcinógeno 4-nitroquinolina 1-óxido (4NQO), sobre a perda óssea alveolar induzida por ligadura e espontânea em ratos Wistar. Foram utilizados 84 ratos Wistar machos, divididos em quatro grupos experimentais: Grupo 1 (Controle total); Grupo 2 (Exposição ao 4NQO); Grupo 3 (Perda óssea alveolar); Grupo 4 (Exposição ao 4NQO e perda óssea alveolar). Os grupos 2 e 4 foram expostos ao carcinógeno, diluído em água (50ppm), por vinte semanas. A perda óssea alveolar foi induzida, por meio de ligadura com fio de seda no segundo molar da hemiarcada direita dos animais, nos grupos 3 e 4, sendo no grupo 4, após a exposição ao 4NQO. O peso corporal dos animais foi monitorado ao longo do estudo. Após decorridas vinte semanas do período experimental, os animais foram mortos e as peças maxilares e línguas foram analisadas em relação à perda óssea alveolar e lesões macroscópicas linguais. 71 animais completaram o estudo. Todos os animais expostos ao 4NQO apresentaram lesões macroscópicas na língua, demonstrando que o modelo experimental de exposição ao carcinógeno foi efetivo. A análise da perda óssea alveolar demonstrou diferenças significativas entre grupos expostos em relação a não expostos ao 4NQO na ocorrência de perda óssea alveolar espontânea. Essas diferenças não foram observadas nos lados com ligadura. Pode-se concluir que a exposição ao carcinógeno 4NQO potencialmente influencia a perda óssea alveolar espontânea em ratos. / Scientific evidence about the inter-relationship between periodontal disease and oral cancer has increased considerably. However, studies evaluating the biological mechanisms related to this association are still scarce. The aim of this study was to evaluate the effect of exposure to 4-nitroquinoline 1-oxide (4NQO) on spontaneous and ligature-induced alveolar bone loss. For this, 84 male Wistar rats were divided into four groups: Group 1 (Full Control), Group 2 (4NQO exposure), Group 3 (Alveolar bone loss), Group 4 (4NQO exposure and alveolar bone loss). Groups 2 and 4 were exposed to the carcinogen, diluted in water (50ppm) for twenty weeks. Alveolar bone loss was induced by silk ligatures at the upper right maxillae, around the second molar, for animals in groups 3 and 4; in group 4, ligatures were placed after exposure to 4NQO. Body weight of the animals was monitored throughout the study. After twenty weeks of the experimental period, the animals were killed and the maxillae and tongues were analyzed. Morphometric analysis of alveolar bone loss and macroscopic lesions in tongue were analyzed in 71 animals which completed the study. All animals exposed to 4NQO presented macroscopic lesions in dorsal part of tongue, demonstrating that the experimental model of carcinogen exposure was effective. The analysis of alveolar bone loss, was performed by medians and interquartile ranges and showed significant differences between animals exposed or not to 4NQO in terms of spontaneous alveolar bone loss. This was not observed in sites with ligatures. It can be concluded that exposure to carcinogenic 4NQO potentially influences spontaneous alveolar bone loss in rats.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Indivíduos que permanecem longo tempo em cadeira de rodas apresentam importante perda de massa óssea, principalmente nos membros inferiores, possivelmente agravada pela baixa ingestão de cálcio dietético e pelo inadequado estado nutricional de vitamina D. O exercício físico pode contribuir para a manutenção ou aumento da massa óssea em diferentes populações e nos indivíduos com lesão medular pode contribuir para atenuar a perda de massa óssea. O objetivo do presente estudo foi avaliar a influência da prática regular de exercício físico sobre a adequação da massa óssea, indicadores bioquímicos do metabolismo ósseo e estado nutricional de vitamina D em indivíduos com lesão medular cervical há pelo menos um ano. Em vinte e cinco homens de 19 a 56 anos sendo 15 fisicamente ativos e 10 sedentários, foi realizada análise sérica de cálcio, PTH, 25(OH)D, IGF-1, osteocalcina e NTx. As medidas do conteúdo mineral ósseo, densidade mineral óssea (DMO), massa magra e massa gorda foram realizadas por DXA. A pigmentação da pele (constitutiva e por bronzeamento) foi determinada por colorimetria com o objetivo de investigar sua influência sobre o estado de vitamina D. A ingestão habitual de cálcio foi registrada em um questionário de frequência alimentar direcionado para alimentos fonte. As comparações entre os dois grupos foram realizadas pela aplicação do Teste t de Student exceto para as variáveis ósseas que foram realizadas após ajustes pela massa corporal total, tempo de lesão e ingestão de cálcio utilizando-se análise de co-variância. Associações entre as variáveis estudadas foram avaliadas através de análise de correlação de Pearson. Valores de p<0.05 foram considerados significativos. Não foram observadas diferenças estatisticamente significativas entre os grupos para nenhuma variável óssea com exceção do z-score da DMO da coluna lombar, que foi significativamente maior no grupo de indivíduos sedentários (0,9 1,7 vs -0,7 0,8; p<0,05). No entanto, entre os indivíduos ativos, aqueles que iniciaram a prática de exercício físico com menos tempo decorrido após a lesão apresentaram maior DMO do fêmur (r=-0,60; p<0,05). Nos indivíduos ativos, a freqüência do exercício apresentou associação negativa com a concentração sérica de i-PTH (r = -0,50; p =0,05) e positiva com a concentração de 25(OH)D (r= 0,58; p <0,05). Após ajustes pela massa corporal total e tempo de lesão foram observadas associações positivas entre a ingestão diária de cálcio e z-score da DMO da coluna lombar (r = 0,73 e p <0,01) e DMO do rádio (r = 0,56 e p <0,05). Os resultados do presente estudo apontam para um efeito benéfico do exercício físico sobre a massa óssea e o perfil hormonal relacionado ao metabolismo ósseo. O início da prática regular de exercício físico o quanto antes após a lesão parece contribuir para atenuar a perda de massa óssea nos membros inferiores. Além disso, os resultados deste estudo sugerem uma possível potencialização do efeito osteogênico do exercício físico quando combinado a uma adequada ingestão de cálcio. / Individuals who stay long time in a wheelchair have significant bone loss, especially in lower limbs that may be aggravated by low calcium intake and inadequate vitamin D status. Physical exercise contributes to maintain or increase bone mass in different populations and may be useful in reducing bone loss in spinal cord injured individuals. The aim of this study was to evaluate the influence of regular physical exercise on bone mass adequacy, biochemical markers of bone metabolism and vitamin D status in individuals with cervical spinal cord injury. Twenty five male adults (19-56 years) with cervical spinal cord injury for at least one year, were recruited and divided into physically active (n=15) and sedentary (n=10) groups. Fasting blood samples were collected and serum samples were stored at -20oC until analysis of calcium, PTH, 25(OH)D, IGF-1, osteocalcin and NTx. Bone mineral content and density (BMD), lean mass and fat mass were evaluated by DXA. Skin pigmentation (constitutive and facultative) was evaluated by reflectance colorimetry in order to investigate its influence on vitamin D status. Habitual calcium intake was recorded using a food frequency questionnaire directed to calcium food sources. Comparisons between groups were performed using Students t test except for bone variables that were performed after adjustments for total body mass, duration of injury and calcium intake by analysis of covariance. Associations between variables were evaluated using Pearson's correlation analysis. P values <0.05 were considered significant. There were no significant differences between groups for bone measurements except for lumbar spine Z-score, that was significantly higher in the sedentary group (0.9 1.7 vs -0.7 0.8; p< 0.05). However, in the active group, it was observed that subjects who initiated the practice of physical exercise with less time after injury had higher BMD at the femur (r=-0.60; p<0.05). In active subjects, exercise frequency was negatively associated with serum i-PTH (r = -0.50, p = 0.05) and positively associated with serum 25(OH)D (r= 0.58; p <0.05). After adjustments for total body mass and duration of injury daily calcium intake was positively associated with lumbar spine Z-score (r = 0.73 and p < 0.01) and with radius BMD (r = 0.56 and p <0.05). The results of this study suggest a beneficial effect of regular exercise practice on bone mass and bone-related hormonal profile. The earlier initiation of regular physical exercise after the injury appears to contribute to attenuate the loss of bone mass in lower limbs. Moreover, our results suggest that the osteogenic effect of exercise may be potentiated when combined with an adequate calcium intake.

Orientador: Lucy Marie Ribeiro Muniz / Resumo: Os efeitos do hipertireoidismo experimental (150 g/kg/dia/42 dias) na homeostase do cálcio e nos marcadores do metabolismo ósseo foram estudados em 14 gatos sem raça definida, com idade entre um e três anos. Houve uma clara tendência de aumento das concentrações séricas de PTH intacto a partir do momento inicial com diferença significativa entre este e os demais momentos. O cálcio ionizado demonstrou uma diminuição significativa aos 14 dias em relação ao momento inicial e aos 42 dias em relação aos 14 dias. Os hormônios tireoidianos apresentaram correlação positiva com o PTH e negativa com o cálcio ionizado. Já a densidade mineral óssea (DMO) apresentou tendência de correlação negativa com o PTH a partir dos 28 dias. Observou-se correlação negativa do PTH com o cálcio ionizado aos 14, 28 e 42 dias. Conclui-se que o hipertireoidismo em gatos adultos jovens sem doenças concomitantes apresenta hiperparatireoidismo secundário. As concentrações séricas da OC apresentaram diferença significativa (p<0,05) entre si, nos quatro momentos. O ICTP, um marcador específico da reabsorção óssea, não apresentou diferença significativa entre os momentos. Provavelmente o remodelamento ósseo foi provocado pelo estado hipertireóideo, visto que tanto a OC como o ICTP apresentou forte correlação positiva com a TT4 e um pouco inferior com a FT4. A FT4 não apresentou correlação positiva com o ICTP, excetuando-se aos 28 dias. Observou-se baixa correlação, em todos os momentos, entre os marcadores do metabolismo ósseo e a densidade mineral óssea. Conclui-se que o excesso dos hormônios tireoidianos em gatos provocou aumento do remodelamento ósseo visto que ocorreu alta correlação entre estes hormônios e os marcadores do metabolismo ósseo. O hipertireoidismo provocou diminuição da DMO óssea, porém a OC e o ICTP apresentaram baixa correlação com esta variável. / Abstract: The effect of experimental hyperthyroidism (150 g/kg/day/42 days) on calcium homeostasis and markers of bone metabolism was studied in fourteen shorthair cats from one to three years of age. Serum concentrations of unbroken PTH had a clear tendency to increase from beginning with significant differences from the initial to other moments. The ionized calcium significantly decreased at the 14 days in comparison to the initial moment and at the 42 days in comparison to the 14 days. The thyroid hormones showed positive correlation with PTH and negative with ionized calcium. In contrast, bone mineral density had a tendency of negative correlation with the PTH from the 28 days. Negative correlation of the PTH and calcium ionized was observed at 14, 28 and 42 days. In the present study, hyperthyroidism in young adult cats without concomitant illnesses did not present secondary hyperparathyroidism. However, increase of PTH and reduction of ionized calcium were observed. Serum concentrations of osteocalcin (OC) were significantly different among all four moments. The carboxi-terminal telopeptides of collagen type I (ICTP), a specific marker of the bone reabsorption, did not significantly differ (p<0.05) between moments. Bone turnover was probably caused by the hyperthyroid state, since OC and ICTP presented strong positive correlation with TT4 and a little less with free T4 (FT4). The FT4 did not present positive correlation with the ICTP, excepting at the 28 days. Positive correlation in all the moments between markers of bone metabolism and bone mineral density was very low. In conclusion, the high correlation between thyroid hormones and markers of bone metabolism indicates that the excess of thyroid hormones in cats may cause an increase of the bone turnover. Moreover, hyperthyroidism may cause reduction of the bone DMO, although OC and the ICTP had low correlation with DMO. / Doutor

O corpo de evidências científicas a respeito da inter-relação entre câncer oral e doenças periodontais vem aumentando consideravelmente. Entretanto, estudos avaliando os mecanismos biológicos que permeiam esta associação ainda são escassos. O objetivo do estudo foi avaliar o efeito da exposição ao carcinógeno 4-nitroquinolina 1-óxido (4NQO), sobre a perda óssea alveolar induzida por ligadura e espontânea em ratos Wistar. Foram utilizados 84 ratos Wistar machos, divididos em quatro grupos experimentais: Grupo 1 (Controle total); Grupo 2 (Exposição ao 4NQO); Grupo 3 (Perda óssea alveolar); Grupo 4 (Exposição ao 4NQO e perda óssea alveolar). Os grupos 2 e 4 foram expostos ao carcinógeno, diluído em água (50ppm), por vinte semanas. A perda óssea alveolar foi induzida, por meio de ligadura com fio de seda no segundo molar da hemiarcada direita dos animais, nos grupos 3 e 4, sendo no grupo 4, após a exposição ao 4NQO. O peso corporal dos animais foi monitorado ao longo do estudo. Após decorridas vinte semanas do período experimental, os animais foram mortos e as peças maxilares e línguas foram analisadas em relação à perda óssea alveolar e lesões macroscópicas linguais. 71 animais completaram o estudo. Todos os animais expostos ao 4NQO apresentaram lesões macroscópicas na língua, demonstrando que o modelo experimental de exposição ao carcinógeno foi efetivo. A análise da perda óssea alveolar demonstrou diferenças significativas entre grupos expostos em relação a não expostos ao 4NQO na ocorrência de perda óssea alveolar espontânea. Essas diferenças não foram observadas nos lados com ligadura. Pode-se concluir que a exposição ao carcinógeno 4NQO potencialmente influencia a perda óssea alveolar espontânea em ratos. / Scientific evidence about the inter-relationship between periodontal disease and oral cancer has increased considerably. However, studies evaluating the biological mechanisms related to this association are still scarce. The aim of this study was to evaluate the effect of exposure to 4-nitroquinoline 1-oxide (4NQO) on spontaneous and ligature-induced alveolar bone loss. For this, 84 male Wistar rats were divided into four groups: Group 1 (Full Control), Group 2 (4NQO exposure), Group 3 (Alveolar bone loss), Group 4 (4NQO exposure and alveolar bone loss). Groups 2 and 4 were exposed to the carcinogen, diluted in water (50ppm) for twenty weeks. Alveolar bone loss was induced by silk ligatures at the upper right maxillae, around the second molar, for animals in groups 3 and 4; in group 4, ligatures were placed after exposure to 4NQO. Body weight of the animals was monitored throughout the study. After twenty weeks of the experimental period, the animals were killed and the maxillae and tongues were analyzed. Morphometric analysis of alveolar bone loss and macroscopic lesions in tongue were analyzed in 71 animals which completed the study. All animals exposed to 4NQO presented macroscopic lesions in dorsal part of tongue, demonstrating that the experimental model of carcinogen exposure was effective. The analysis of alveolar bone loss, was performed by medians and interquartile ranges and showed significant differences between animals exposed or not to 4NQO in terms of spontaneous alveolar bone loss. This was not observed in sites with ligatures. It can be concluded that exposure to carcinogenic 4NQO potentially influences spontaneous alveolar bone loss in rats.

O corpo de evidências científicas a respeito da inter-relação entre câncer oral e doenças periodontais vem aumentando consideravelmente. Entretanto, estudos avaliando os mecanismos biológicos que permeiam esta associação ainda são escassos. O objetivo do estudo foi avaliar o efeito da exposição ao carcinógeno 4-nitroquinolina 1-óxido (4NQO), sobre a perda óssea alveolar induzida por ligadura e espontânea em ratos Wistar. Foram utilizados 84 ratos Wistar machos, divididos em quatro grupos experimentais: Grupo 1 (Controle total); Grupo 2 (Exposição ao 4NQO); Grupo 3 (Perda óssea alveolar); Grupo 4 (Exposição ao 4NQO e perda óssea alveolar). Os grupos 2 e 4 foram expostos ao carcinógeno, diluído em água (50ppm), por vinte semanas. A perda óssea alveolar foi induzida, por meio de ligadura com fio de seda no segundo molar da hemiarcada direita dos animais, nos grupos 3 e 4, sendo no grupo 4, após a exposição ao 4NQO. O peso corporal dos animais foi monitorado ao longo do estudo. Após decorridas vinte semanas do período experimental, os animais foram mortos e as peças maxilares e línguas foram analisadas em relação à perda óssea alveolar e lesões macroscópicas linguais. 71 animais completaram o estudo. Todos os animais expostos ao 4NQO apresentaram lesões macroscópicas na língua, demonstrando que o modelo experimental de exposição ao carcinógeno foi efetivo. A análise da perda óssea alveolar demonstrou diferenças significativas entre grupos expostos em relação a não expostos ao 4NQO na ocorrência de perda óssea alveolar espontânea. Essas diferenças não foram observadas nos lados com ligadura. Pode-se concluir que a exposição ao carcinógeno 4NQO potencialmente influencia a perda óssea alveolar espontânea em ratos. / Scientific evidence about the inter-relationship between periodontal disease and oral cancer has increased considerably. However, studies evaluating the biological mechanisms related to this association are still scarce. The aim of this study was to evaluate the effect of exposure to 4-nitroquinoline 1-oxide (4NQO) on spontaneous and ligature-induced alveolar bone loss. For this, 84 male Wistar rats were divided into four groups: Group 1 (Full Control), Group 2 (4NQO exposure), Group 3 (Alveolar bone loss), Group 4 (4NQO exposure and alveolar bone loss). Groups 2 and 4 were exposed to the carcinogen, diluted in water (50ppm) for twenty weeks. Alveolar bone loss was induced by silk ligatures at the upper right maxillae, around the second molar, for animals in groups 3 and 4; in group 4, ligatures were placed after exposure to 4NQO. Body weight of the animals was monitored throughout the study. After twenty weeks of the experimental period, the animals were killed and the maxillae and tongues were analyzed. Morphometric analysis of alveolar bone loss and macroscopic lesions in tongue were analyzed in 71 animals which completed the study. All animals exposed to 4NQO presented macroscopic lesions in dorsal part of tongue, demonstrating that the experimental model of carcinogen exposure was effective. The analysis of alveolar bone loss, was performed by medians and interquartile ranges and showed significant differences between animals exposed or not to 4NQO in terms of spontaneous alveolar bone loss. This was not observed in sites with ligatures. It can be concluded that exposure to carcinogenic 4NQO potentially influences spontaneous alveolar bone loss in rats.