Populations of federally-listed gopher tortoises (Gopherus polyphemus) are in decline in Mississippi. Soil and forage quality may be linked to their health and recruitment. To gain a better understanding of existing soil and forage quality conditions on areas inhabited by gopher tortoises, I investigated soil chemistry parameters, forage nutrients, and plant community characteristics from 2012 to 2013. These parameters were collected on 7 soil and habitat management treatment types in uplands on public forest lands in south Mississippi. Soil sample analyses indicated that most pH levels in soils were acidic (pH < 5.0) to strongly acidic (pH < 4.5). Greatest soil calcium levels were detected on growing season burn, moderately suitable soil areas, and soil phosphorus levels were greatest on mowed, less suitable soil areas. Greatest levels of nutrients were detected at 0 – 10 cm soil depths. Weak, positive associations were detected between soil pH and soil calcium and magnesium levels, while weak, negative associations were detected between soil pH and soil phosphorus levels. Greater levels of calcium, magnesium, and phosphorus were detected in plants collected in mowed, less suitable soil areas. Cacti, forbs and, legumes were found to have greatest nutrient levels of all the plant growth forms. Moderate, positive associations were detected between soil pH and calcium levels in legumes and vines. Weak, positive associations were detected between soil pH and forage calcium levels in forbs and native grasses. Very weak, positive associations were detected between soil pH and forage phosphorus levels in vines. I found greatest species richness and percent coverage of legumes and forbs on moderately suitable soils that received growing season fire; whereas, greater species richness and percent coverage of native grasses were detected on moderately suitable soil regardless of season of burn. Greatest percent coverage of cacti (puntia sp.) and greatest quantities of above-ground plant biomass were detected on mowed, less suitable soil areas. This information can be valuable to habitat evaluation and management for gopher tortoises.

Base teórica: A Mucopolissacaridose II (MPS II) é uma doença genética recessiva ligada ao X causada por mutações no gene IDS. Como consequência, há acúmulo dos glicosaminoglicanos (GAGs) no lisossomo, fato que é responsável pelo fenótipo de MPS II. Anormalidades articulares e ósseas são conhecidas nos pacientes com MPS II e os tratamentos existentes não são eficientes para sanar tais anormalidades, portanto, realizamos este estudo de caracterização da doença articular e óssea, buscando evidenciar possíveis mecanismos responsáveis pela progressão da doença. Objetivo: Avaliar a progressão das alterações osteoarticulares em animais com MPS II dos dois aos oito meses de idade. Métodos: Foram utilizados camundongos nocaute B6N.Cg-Idstm1Muen/J, adquiridos do Jackson’s Lab. Os machos foram genotipados para compor o grupo controle (normal) ou o grupo de animais com MPS II. Ambos foram avaliados aos 2, 4, 6 e 8 meses de idade. Foi realizada análise histológica da articulação tíbio-femural, avaliando presença de infiltrado inflamatório, reabsorção óssea, reabsorção cartilaginosa e proliferação fibrocartilaginosa. Também foi realizada a mensuração do tamanho total da placa de crescimento e suas zonas e avaliação de anormalidades ósseas mediante exame de imagem por Raio-X dos ossos fêmur e zigomático. Resultados: Nos animais MPS II foi observado que o focinho era menos afilado (mais arredondado) e, em comparação com os animais controle, os animais MPS II apresentaram peso significativamente maior a partir dos 4 meses de idade. O escore histológico teve como principal característica a presença de reabsorção cartilaginosa, presente em 80% (4/5 animais) dos animais aos 8 meses, outras anormalidades encontradas neste tempo foram presença de infiltrado inflamatório (2/5 animais aos 8 meses) e proliferação fibrocartilaginosa (1/5 animais) Não houve diferença significativa entre animais normais e MPS II no tamanho das zonas de cartilagem da placa de crescimento ósseo. As medidas em diâmetro do osso zigomático apresentaram-se significativamente superiores nos animais MPS II aos 4, 6 e 8 meses. Quanto ao comprimento do fêmur não houve diferença significativa entre os grupos. Já, na medida da espessura do fêmur, os animais MPS II do grupo de 6 meses de idade mostraram diferença significativa. Conclusões: Anormalidades na articulação tíbio-femural foram detectadas nos animais aos 8 meses de idade. Não foram encontradas anormalidades óbvias na estrutura da placa de crescimento. Foi observado aumento na espessura do fêmur e do zigomático nos animais MPS II, sem alterações do tamanho do fêmur. / Background: Mucopolysaccharidosis II (MPS II) is a recessive X-linked genetic disease caused by mutations in the IDS gene. Consequently, there is an accumulation of glycosaminoglycans (GAGs) in the lysosome, fact that is responsible by the MPS II phenotype. Joints and bone abnormalities are known in MPS II patients and existing treatments are not efficient in correcting these abnormalities. Therefore, this study was performed to evidence bone and joint disease description in the animal model, searching for mechanisms responsible for disease progression. Objective: To evaluate the progression of osteoarticular changes in animals with MPS II from two to eight months of age. Methods: Male animals from the MPS II colony (B6N.Cg-Idstm1Muen/J) were genotyped to form the control (normal) group or MPS II group. Both groups were evaluated at the 2, 4, 6 or 8 months. Histological analysis of knee joint (presence of inflammatory infiltrate, bone resorption, cartilaginous reabsorption and fibrocartilaginous proliferation), measurement of total growth plate size and its zones, and evaluation of bone abnormalities by X-ray imaging of the femur and zygomatic bones were performed. Results: MPS II mice presented progressive abnormal features, such as a more rounded snout and a significative increased weight from 4 months of age. The main histological alteration was the presence of cartilage reabsorption, present in 80% (4/5 animals) of the eighth-month old animals. Other abnormalities found at this period were the presence of inflammatory infiltrate (2/5 animals at the eighth months old) and fibrocartilaginous proliferation (1/5 animals). There was no significant difference in the growth plate between normal and MPS II animals. The zygomatic bone diameter was increased in MPS II at fourth, sixth and eighth months There were no significant differences in femur length between groups. Thickness of the femur was increased in MPS II at six months. Conclusions: Abnormalities in the joint were detected in the animals at 8 months of age. No obvious abnormalities were found in the growth plate structure. An increase in femur and zygomatic thickness was observed in MPS II animals, with no changes in femur size.

Bone is a highly specialised living tissue and has both mechanical and metabolical functions. Remodelling of the bone ensures a healthy bone mass and is regulated by a trio of secreted proteins, namely receptor-activator of NFKB (RANK), receptor-activator of NFKB ligand (RANKL) and osteoprotegerin (OPG). OPG, a major regulator of osteoclastogenesis, bone resorption and vascular calcification, is produced by various cell types including mesenchymally derived cells, particularly osteoblastic cells. Wnt signalling also plays a role in maintaining healthy bone mass. Dickkopf- 1 (DKK-1) is a soluble inhibitor of Wnt signalling and its excessive expression contributes to bone loss in rheumatoid arthritis and multiple myeloma. Recently, NDKK-1 has been demonstrated to be over-produced in osteoblasts of patients with Paget's disease of bone (PDB). The osteoblastic cell lines MG63 and Saos-2 were subjected to a series of different growth factors, hormones and cytokines to investigate the production of OPG, DKK-1 and the expression of various Wnt proteins. These results demonstrate that during standard culture conditions, both OPG and DKK-1 production in osteoblastic cells depend on a factor present in serum. Serum deprivation resulted in the up-regulation of Wnt4 and Wnt11, while down-regulating the expression of Wnt7b. Serum-induced OPG and DKK-1 production and Wnt expression was found to be regulated via a number of different signalling pathways. OPG production and expression was stimulated by platelet-derived growth factor-AB (PDGF-AB) not only in MG63 and Saos-2 osteosarcoma cells, but also a mouse pre-osteoblastic cell line (MC3T3-E1) and human bone marrow stromal cells (BMSC). PDGF-AB was shown to act through the PDGF receptor, PKC, PI3K, ERK and P38 and not via NFKB or JNK. PDGF isoforms AA, BB and AB demonstrated a similar stimulation of OPG production. The importance of PDGF in fracture healing suggests a role for OPG production in countering bone resorption during the early phase of this process. BIO, an inhibitor of canonical Wnt signalling resulted in the down-regulation of DKK-1 and the up-regulation of WntSa. Phorbol ester (PE), a known stimulator of PKC resulted in the up-regualtion of DKK-1, Wnt4, WntTa and Wnt16. The effects of PE were inhibited by bisindolymaleamide but not staurosporine. DKK-1 production, but not expression, was observed to be stimulated by calcium along with an up-regulation of WntTb and a down-regulation of WntWa and Wnt11. Incubation of pre-stimulated cells with Triton-X demonstrated the ability of calcium to increase DKK-1 secretion. DKK-1 was shown to be significantly elevated in the serum of PDB patients compared to healthy controls and did not correlate with ALP levels. Immunohistochemistry demonstrated that DKK-1 production is increased in both osteoblasts and fibrotic cells within the marrow cavity in PDB patients compared to fracture callus. B-catenin was found to be localised to intercellular membranes of plump osteoblasts, demonstrating its alternate role as a cell adhesion protein. DKK-1 therefore may be a useful biomarker of PDB and that Dkk-1 may play a central role in the aetiology of PDB. In summary, the results presented in this thesis have investigated the ways in which OPG and DKK-1 production in osteoblastic cells can be modulated with various effectors and the effect of Wnt signalling. These results may therefore be beneficial to increase the understanding of bone biology, improve fracture repair and generate further research into the role DKK-1 and the osteoblast in the aetiology of PDB to enable improved treatments to be developed.

[Truncated abstract] Bone is a living tissue and is maintained by the coordinate action of osteoblasts and osteoclasts. The intercellular communication between these two cells is the quintessential mechanism in bone remodelling. Unfortunately, the importance of this interaction is often neglected and its significance is only realised when disruption of this “cross-talk” results in debilitating bone diseases. Additionally, the number of known proteins that are involved in this “cross-talk”, especially those that are osteoclast-derived, and act specifically on osteoblasts, is limited. This discrepancy leads to the question: Can osteoclasts directly control the growth and function of osteoblastic cells by expressing specific proteins that bind directly to osteoblasts? If so, is it possible to use these proteins to control and, possibly, treat bone disease? The objective of this thesis is to identify and characterise osteoclast-derived factors that can modulate bone homeostasis, as well as contribute to the intercellular communication between osteoblasts and osteoclasts ... Collectively, the data in this thesis culminates in one important conclusion: the identification of a novel paracrine secretory factor that has the potential to directly induce the formation of bone. These findings represent the first ever characterisation of a protein that allows the osteoclasts to directly control the growth and function of osteoblasts. Due to the potential function of ODOF to induce bone formation, this protein may be used therapeutically to treat bone disease.

Base teórica: A Mucopolissacaridose II (MPS II) é uma doença genética recessiva ligada ao X causada por mutações no gene IDS. Como consequência, há acúmulo dos glicosaminoglicanos (GAGs) no lisossomo, fato que é responsável pelo fenótipo de MPS II. Anormalidades articulares e ósseas são conhecidas nos pacientes com MPS II e os tratamentos existentes não são eficientes para sanar tais anormalidades, portanto, realizamos este estudo de caracterização da doença articular e óssea, buscando evidenciar possíveis mecanismos responsáveis pela progressão da doença. Objetivo: Avaliar a progressão das alterações osteoarticulares em animais com MPS II dos dois aos oito meses de idade. Métodos: Foram utilizados camundongos nocaute B6N.Cg-Idstm1Muen/J, adquiridos do Jackson’s Lab. Os machos foram genotipados para compor o grupo controle (normal) ou o grupo de animais com MPS II. Ambos foram avaliados aos 2, 4, 6 e 8 meses de idade. Foi realizada análise histológica da articulação tíbio-femural, avaliando presença de infiltrado inflamatório, reabsorção óssea, reabsorção cartilaginosa e proliferação fibrocartilaginosa. Também foi realizada a mensuração do tamanho total da placa de crescimento e suas zonas e avaliação de anormalidades ósseas mediante exame de imagem por Raio-X dos ossos fêmur e zigomático. Resultados: Nos animais MPS II foi observado que o focinho era menos afilado (mais arredondado) e, em comparação com os animais controle, os animais MPS II apresentaram peso significativamente maior a partir dos 4 meses de idade. O escore histológico teve como principal característica a presença de reabsorção cartilaginosa, presente em 80% (4/5 animais) dos animais aos 8 meses, outras anormalidades encontradas neste tempo foram presença de infiltrado inflamatório (2/5 animais aos 8 meses) e proliferação fibrocartilaginosa (1/5 animais) Não houve diferença significativa entre animais normais e MPS II no tamanho das zonas de cartilagem da placa de crescimento ósseo. As medidas em diâmetro do osso zigomático apresentaram-se significativamente superiores nos animais MPS II aos 4, 6 e 8 meses. Quanto ao comprimento do fêmur não houve diferença significativa entre os grupos. Já, na medida da espessura do fêmur, os animais MPS II do grupo de 6 meses de idade mostraram diferença significativa. Conclusões: Anormalidades na articulação tíbio-femural foram detectadas nos animais aos 8 meses de idade. Não foram encontradas anormalidades óbvias na estrutura da placa de crescimento. Foi observado aumento na espessura do fêmur e do zigomático nos animais MPS II, sem alterações do tamanho do fêmur. / Background: Mucopolysaccharidosis II (MPS II) is a recessive X-linked genetic disease caused by mutations in the IDS gene. Consequently, there is an accumulation of glycosaminoglycans (GAGs) in the lysosome, fact that is responsible by the MPS II phenotype. Joints and bone abnormalities are known in MPS II patients and existing treatments are not efficient in correcting these abnormalities. Therefore, this study was performed to evidence bone and joint disease description in the animal model, searching for mechanisms responsible for disease progression. Objective: To evaluate the progression of osteoarticular changes in animals with MPS II from two to eight months of age. Methods: Male animals from the MPS II colony (B6N.Cg-Idstm1Muen/J) were genotyped to form the control (normal) group or MPS II group. Both groups were evaluated at the 2, 4, 6 or 8 months. Histological analysis of knee joint (presence of inflammatory infiltrate, bone resorption, cartilaginous reabsorption and fibrocartilaginous proliferation), measurement of total growth plate size and its zones, and evaluation of bone abnormalities by X-ray imaging of the femur and zygomatic bones were performed. Results: MPS II mice presented progressive abnormal features, such as a more rounded snout and a significative increased weight from 4 months of age. The main histological alteration was the presence of cartilage reabsorption, present in 80% (4/5 animals) of the eighth-month old animals. Other abnormalities found at this period were the presence of inflammatory infiltrate (2/5 animals at the eighth months old) and fibrocartilaginous proliferation (1/5 animals). There was no significant difference in the growth plate between normal and MPS II animals. The zygomatic bone diameter was increased in MPS II at fourth, sixth and eighth months There were no significant differences in femur length between groups. Thickness of the femur was increased in MPS II at six months. Conclusions: Abnormalities in the joint were detected in the animals at 8 months of age. No obvious abnormalities were found in the growth plate structure. An increase in femur and zygomatic thickness was observed in MPS II animals, with no changes in femur size.

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-23T22:33:49Z No. of bitstreams: 1 Arquivototal.pdf: 8097104 bytes, checksum: 8976cc960e57607b6bc0686e00fc41c3 (MD5) / Made available in DSpace on 2018-04-23T22:33:49Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 8097104 bytes, checksum: 8976cc960e57607b6bc0686e00fc41c3 (MD5) Previous issue date: 2016-04-01 / Skeletal fluorosis is a chronic metabolic disorder caused by chronic ingestion or inhalation of high concentrations of fluoride. The main consequences are bone alterations and deformities leading to disability. It is a disease of difficult diagnosis because of its pre-clinical signs resembling other bone diseases. In this sense, this epidemiological, observational, transversal and descriptive study aimed at mapping clinical, radiological and molecular biomarkers of exposure. Initially the sample consisted of 103 individuals of both sexes and varied ages. From the total, 45 individuals were analyzed for the identification of the radiological biomarker and 25 individuals were monitored for the biochemical and proteomic markers. For the analysis of fluoride intake in the region, drinking water was collected and the biomarkers were evaluated. As for identification of the samples regarding fluoride exposure: 25 hours urine analysis was performed through the direct method using TISAB II and III on an specific electrode and for the fluoride dosage on fingernail and hair the analysis was done though the indirect method (HMDS). The radiographs were carried out on a Kodak K 9000 C 3d apparatus (by the same operator) with exposure of 16 S, 10 Ma, 60-70 kvp and the maximum skin intake (DEP) of 5 mgy. The volunteers underwent digital panoramic radiographs under same circumstances. For biochemical parameters it was performed a complete blood count, hormonal dosage, calcium and enzymes dosage (creatinine and alkaline phosphatase). For the proteomic identification it was performed a mass spectrophotometry (MALDI-TOF). Data were analyzed on the statistical package for social sciences (SPSS), version 18, using p<0.05. The results pointed out that 46% of the drinking water from São João do Peixe River presented [F] above the ideal value for human consumption of 0.7mg/L and 84% showed values above 1.5mg/L, being indicative of fluorosis in the region. The average age of the volunteers was 50 years, with the prevalence of women, and they showed a time of exposure around 40 years. Regarding the symptomatology 85 individuals reported pain, of which 77% (chi-squared) presented symptoms on more than 3 regions. However, the lumbar region was the indicative variable of presence of bone fluorosis (linear regression, p < 0.005). As for the radiological biomarker, it was verified that the most discriminatory bone conditions were osteopenia, ossification of soft tissues, bone deformit ies, jaw density, and thin cortical of jaw and jaw. There was evaluation divergence between orthodontists and radiologist. Panoramic radiographs proved to be an auxiliaar method of diagnosis and can only be an indicative of bone changes related to skeletal fluorosis, it cannot be discriminatory in cases of grades I and II skeletal fluorosis. The biochemical variables showed that the average volume of urine excreted was 890 ml in 24 hours, whose concentration contained an average of 3.9 mgF/liter (0.2-3.2 VR), presenting a high fluoride excretion. The average fluoride concentration on fingernails was 14.2 being the highest level ever recorded, probably due to the diffusion capture of F by the HMDS. Serum calcium had an average of 9.0 mg/dl (8.9 -10, 3VR); phosphorus, 3.8 mg/dl (2.4-4.7 VR); alkaline phosphatase, 288 IU/litre (32-91 VR); Alkaline phosphatase bone specific, 96 ug/litre (4.5-16.9 VR); Osteocalcin, 309 ng/ml (11-50 VR); and PTH, 203 pg/ml (10-65 VR). Electrolytes, glucose, albumin, urea and creatinine were with 70% normal, as well as platelet count. Among the sick and non-patient studied groups, there were no differences between the groups, according to the Mann-Whitney test, p < 0.05. For proteomics analysis, there was a large quantity of proteins in the serum, being the affected volunteers more altered when compared to the other group. Few proteins were found on the saliva. Data indicate that mass spectrophotometry is capable of detecting differential proteins so it can be useful for fluoride biomarkers identification as well as it can help understand the skeletal fluorosis mechanism. / A fluorose ó ssea é uma doença metabólica crônica, causada pela alta concentração do flúor ingerido ou inalado. Tem como principal consequência alterações e deformidades ó sseas levando a incapacidade. Mordidade de difíc il diagnó stico, devido aos sinais pré- clínicos assemelhar-se a de outras doenç as ó sseas, gerando problemas para a realizaç ão do diagnó stico. Neste sentido, este estudo epidemiológico, observacional, transversal e descritivo teve como objetivo mapear biomarcadores de exposiç ão, clínicos, radiológicos e moleculares para fluorose ó ssea . A amostra constitui inicialmente de 103 indivíduos de ambos os sexos e variadas faixas etárias. Sendo 45 analisados quanto a identificaç ão do biomarcador radiológico e 25 monitorados quanto ao marcador bioquímico e proteô mico. Para investigaç ão das concentraç ões de ingestão de flúor na região foi realizada a coleta de água para consumo e para identificaç ão das amostras quanto a exposiç ão ao fluoreto foram avaliados os biomarcadores: urina (25 horas) ambos realizado através do método direto com TISAB II e III respectivamente e a dosagem da unha e cabelo, pelo meio indireto por difusão com hexametildisiloxano (HMDS). As radiografias foram realizadas em aparelho Kodak K 9000 C 3D (pelo mesmo operador) com exposiç ão de 16 s, 10 mA, 60- 70 Kvp e dose de entrada de pele (DEP) máxima de 5 mGy. Os indivíduos foram submetidos a radiografias panorâ micas (digital) em aparelhos de RX do mesmo modelo e em condiç ões similares. Para os parâ metros bioquímicos foram realizados hemograma completo, dosagem hormonal, dosagem de cá lcio e enzimologia (creatina e fosfatase alcalina). Para identificaç ão de proteomas utilizou-se espectometria de massa (MALDI- TOF). Os dados foram avaliados por meio do Programa Statistical Package for Social Sciences (SPSS) versão 18, com p<0,005. Os resultados apontaram que 46% da água presente em poç os artesianos em São João do Rio do Peixe-PB, apresentam [F] acima do valor ideal de 0,7mg/L para ingestão de água para consumo humano e 84% aponta valores acima de1,5mg/L, sendo indicativo de fluorose na região. A média de idade foi de 50 anos, com predomínio de mulheres, a amostra apresentou um tempo de exposiç ão em torno de 40 anos. Quanto a sintomatologia 85 relataram dor, destes 77% (qui-quadrado), apresentam mais de 3 regiões acometidas. Porém a região lombar foi a variável indicativa de presenç a de fluorose ó ssea (regressão linerar, p<0,005). No que concerne o biomarcador radiológico, verifica-se que as condiç ões ó sseas mais discriminantes: osteopenia, ossificaç ão dos tecidos moles, deformidades ó sseas, densidade da maxila, e cortical fina de maxila e mandíbula. Houve divergê ncia na avaliaç ão entre ortodontistas e radiologias. A radiografia panorâ mica se mostrou como um recurso auxiliar e apenas indicativa de alteraç ões ó sseas relacionadas com a fluorose ó ssea, não podendo ser discriminató ria nos casos de fluorose ó ssea de graus I e II. Quanto as biomarcadores de exposiç ão foi evidenciado. As variáveis bioquímicas que o volume médio excretado foi de 890 ml em 24 h, cuja concentraç ão continha uma média de 3,9 mg / litro F (0,2-3,2 VR), constatando uma excreç ão elevada. A concentraç ão média da unha encontrado foi de 14,2 sendo o mais elevado nível de concentraç ão já registrada, provavelmente devido à captura de difusão de F pelo HMDS.Cá lcio sérico teve uma média de 9,0 mg / dl (8,9-10,3VR); fó sforo, 3,8 mg / dl (2,4-4,7 VR); fosfatase alcalina, 288 UI / litro (32-91 VR); específica do osso da fosfatase alcalina, 96 ug / litro (4,5-16,9 VR); osteocalcina, 309 ng /ml (11-50 VR); e PTH, 203 pg/ml (10-65 VR). Eletró litos, glicose, albumina, uré ia e creatina estavam com 70% normais, assim como contagem de plaquetas. Entre os grupos estudados doentes e não doentes, não houve diferenç as entre os grupos, segundo o teste de Mann-Whitney, p<0,05. Para aná lise proteô mica, verificou-se a presenç a de uma grande quantidade de proteínas no soro, estando os doentes mais alterado quando comparado com o outro grupo. A saliva poucas proteínas foram encontradas. Os dados indicam que a espectometria de massa é capaz de detectar proteínas diferecialmente expressa. Podendo ser útil para identificaç ão de biomarcadores para F, além de ajudar no avanç o do mecanismo envolvido da fluorose ó ssea.

Base teórica: A Mucopolissacaridose II (MPS II) é uma doença genética recessiva ligada ao X causada por mutações no gene IDS. Como consequência, há acúmulo dos glicosaminoglicanos (GAGs) no lisossomo, fato que é responsável pelo fenótipo de MPS II. Anormalidades articulares e ósseas são conhecidas nos pacientes com MPS II e os tratamentos existentes não são eficientes para sanar tais anormalidades, portanto, realizamos este estudo de caracterização da doença articular e óssea, buscando evidenciar possíveis mecanismos responsáveis pela progressão da doença. Objetivo: Avaliar a progressão das alterações osteoarticulares em animais com MPS II dos dois aos oito meses de idade. Métodos: Foram utilizados camundongos nocaute B6N.Cg-Idstm1Muen/J, adquiridos do Jackson’s Lab. Os machos foram genotipados para compor o grupo controle (normal) ou o grupo de animais com MPS II. Ambos foram avaliados aos 2, 4, 6 e 8 meses de idade. Foi realizada análise histológica da articulação tíbio-femural, avaliando presença de infiltrado inflamatório, reabsorção óssea, reabsorção cartilaginosa e proliferação fibrocartilaginosa. Também foi realizada a mensuração do tamanho total da placa de crescimento e suas zonas e avaliação de anormalidades ósseas mediante exame de imagem por Raio-X dos ossos fêmur e zigomático. Resultados: Nos animais MPS II foi observado que o focinho era menos afilado (mais arredondado) e, em comparação com os animais controle, os animais MPS II apresentaram peso significativamente maior a partir dos 4 meses de idade. O escore histológico teve como principal característica a presença de reabsorção cartilaginosa, presente em 80% (4/5 animais) dos animais aos 8 meses, outras anormalidades encontradas neste tempo foram presença de infiltrado inflamatório (2/5 animais aos 8 meses) e proliferação fibrocartilaginosa (1/5 animais) Não houve diferença significativa entre animais normais e MPS II no tamanho das zonas de cartilagem da placa de crescimento ósseo. As medidas em diâmetro do osso zigomático apresentaram-se significativamente superiores nos animais MPS II aos 4, 6 e 8 meses. Quanto ao comprimento do fêmur não houve diferença significativa entre os grupos. Já, na medida da espessura do fêmur, os animais MPS II do grupo de 6 meses de idade mostraram diferença significativa. Conclusões: Anormalidades na articulação tíbio-femural foram detectadas nos animais aos 8 meses de idade. Não foram encontradas anormalidades óbvias na estrutura da placa de crescimento. Foi observado aumento na espessura do fêmur e do zigomático nos animais MPS II, sem alterações do tamanho do fêmur. / Background: Mucopolysaccharidosis II (MPS II) is a recessive X-linked genetic disease caused by mutations in the IDS gene. Consequently, there is an accumulation of glycosaminoglycans (GAGs) in the lysosome, fact that is responsible by the MPS II phenotype. Joints and bone abnormalities are known in MPS II patients and existing treatments are not efficient in correcting these abnormalities. Therefore, this study was performed to evidence bone and joint disease description in the animal model, searching for mechanisms responsible for disease progression. Objective: To evaluate the progression of osteoarticular changes in animals with MPS II from two to eight months of age. Methods: Male animals from the MPS II colony (B6N.Cg-Idstm1Muen/J) were genotyped to form the control (normal) group or MPS II group. Both groups were evaluated at the 2, 4, 6 or 8 months. Histological analysis of knee joint (presence of inflammatory infiltrate, bone resorption, cartilaginous reabsorption and fibrocartilaginous proliferation), measurement of total growth plate size and its zones, and evaluation of bone abnormalities by X-ray imaging of the femur and zygomatic bones were performed. Results: MPS II mice presented progressive abnormal features, such as a more rounded snout and a significative increased weight from 4 months of age. The main histological alteration was the presence of cartilage reabsorption, present in 80% (4/5 animals) of the eighth-month old animals. Other abnormalities found at this period were the presence of inflammatory infiltrate (2/5 animals at the eighth months old) and fibrocartilaginous proliferation (1/5 animals). There was no significant difference in the growth plate between normal and MPS II animals. The zygomatic bone diameter was increased in MPS II at fourth, sixth and eighth months There were no significant differences in femur length between groups. Thickness of the femur was increased in MPS II at six months. Conclusions: Abnormalities in the joint were detected in the animals at 8 months of age. No obvious abnormalities were found in the growth plate structure. An increase in femur and zygomatic thickness was observed in MPS II animals, with no changes in femur size.

Orientadores: Andréa Trevas Maciel Guerra, Maricilda Palandi de Mello / Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-16T03:18:52Z (GMT). No. of bitstreams: 1 Lima_Renatade_D.pdf: 4015661 bytes, checksum: e48a1ea1afcdb807a4d9186f4bb5777c (MD5) Previous issue date: 2010 / Resumo: A Discondrosteose de Leri-Weill (DLW), caracterizada por baixa estatura, encurtamento mesomélico dos membros e deformidade de Madelung, deve-se a alterações no gene SHOX, localizado na região pseudoautossômica dos cromossomos sexuais e que atua como fator de transcrição. Alterações em um dos alelos são encontradas em casos de baixa estatura com ou sem DLW, e em ambos os alelos na Displasia Mesomélica de Langer (DML), na qual há alterações esqueléticas mais graves. Há variação fenotípica na DLW, atribuída ao envolvimento de outros genes ou a alterações nas regiões reguladoras à jusante ao gene. No presente estudo, realizou-se a investigação do gene SHOX de 10 pacientes com DLW e um com DML por meio de análises de microssatélites, PCR em tempo real, análise de MLPA e sequenciamento direto. Essas análises permitiram confirmar a etiologia genética do quadro apresentado por sete pacientes, dos quais três apresentavam deleção total ou parcial de um dos alelos. Em um destes pacientes a clinica foi associada a mutação de ponto (IVS3+21G>A no íntron 3), outro paciente (DML) a deleção dos dois alelos, outros pacientes (DLW) a deleção na região downstream do gene, outros dois mutações de ponto (c.439C>A no éxon 3 e c.523delC no éxon 4), outro a deleção (c.523delC) associada a mutação na região 5'UTR. Quatro outros pacientes apresentaram alterações de patogenicidade ainda indefinida: mutações não descritas na região 5'UTR, que poderia interferir no processo de tradução do gene (dois casos), e deleção das regiões referentes às sondas 8, 10 e 12 na análise de MLPA (um caso) e outro caso com relação à deleção da sonda SHOX reg. Os resultados mostram a grande heterogeneidade alélica associada à DLW e indicam a necessidade de que a investigação molecular nesses casos seja ampla, permitindo um diagnóstico molecular mais preciso. / Abstract: Leri-Weill Dyschondrosteosis (LWD) is characterized by short stature, mesomelic shortening of members and Madelung deformity. It results from changes in the SHOX gene, located in the pseudoautosomal region of the sex chromosomes. The protein it codes acts as a transcription factor. Changes in one SHOX allele are found in cases of short stature with or without LWD, whereas Langer Mesomelic Dysplasia (LMD), in which more serious skeletal disorders occur, results from alterations in both alleles. There is phenotypic heterogeneity in LWD, attributed to the possible involvement of other genes, or to changes in regulatory regions downstream of the gene. In the present study, the SHOX gene and the chromosomal SHOX gene region were evaluated in 11 patients with LWD and LMD by using microsatellite PCR, real time PCR analysis, MLPA analysis, and direct sequencing. Those tests confirmed the genetic etiology of the clinical characteristics in seven patients. Three of them carried alleles bearing a partial or total deletion of the SHOX gene, one of which bearing a mutation located in intron 3 (IVS3+21G>A) that has not been described previously; the patient with LMD carried SHOX gene deletion in both alleles; and another had a deletion in the dpwnstream region of the gene. Two patients bore mutations that have not been described previously in exons 3 (C.4390A, Arg147Ser) and 4 (c.523delC, Gln175Lisfs44x219). The later was also associated with a mutation in 5'UTR. Four other patients showed nucleotide changes and deletions of undefined pathogenicity: novel mutations in the 5'UTR, which might interfere with the translation process of the gene (two cases), and deletion of regions related to the probes 8, 10 and 12 in the MLPA analysis (one case) and deletion oh probe SHOX reg. Results showed great molecular heterogeneity associated with LWD, and pointed out the need of a more detailed research to allow more accurate molecular diagnosis. / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple myeloma (MM) is the most frequent cancer to involve the skeleton, with over 80% of myeloma patients developing lytic bone disease (MMBD). Importantly, MM-associated bone lesions rarely heal even when patients are in complete remission. Bone marrow stromal cells (BMSCs) isolated from MM patients have a distinct genetic profile and an impaired osteoblast (OB) differentiation capacity when compared to BMSCs from healthy donors. Utilizing an in vivo model of MMBD and patient samples, we showed that BMSCs from tumor-bearing bones failed to differentiate into OBs weeks after removal of MM cells. Both Runx2 and Osterix, the master transcription factors for OB differentiation, remained suppressed in these BMSCs. However, the molecular mechanisms for MM-induced long-term OB suppression are poorly understood. We characterized both Runx2 and Osterix promoters in murine pre-osteoblast MC4 cells by chromatin immunoprecipitation (ChIP). The transcriptional start sites (TSSs) of Runx2 and Osterix in untreated MC4 cells were co-occupied by transcriptionally active histone 3 lysine 4 tri-methylation (H3K4me3) and transcriptionally repressive histone 3 lysine 27 tri-methylation (H3K27me3), termed the “bivalent domain”. These bivalent domains became transcriptionally silent with increasing H3K27me3 levels when MC4 cells were co-cultured with MM cells or treated with TNF-α, an inflammatory cytokine increased in MM bone marrow microenvironment. The increasing H3K27me3 levels induced by MM cells or TNF-α were associated with the downregulation of the H3K27 demethylase JMJD3 in MC4 cells and murine BMSCs. Knockdown of JMJD3 in MC4 cells was sufficient to inhibit OB differentiation. Further, ectopic overexpression of JMJD3 in MC4 cells partially rescued the suppression of osteoblast differentiation induced by TNFa. We also found that pre-incubation of MC4 cells with the NF-kB inhibitor quinazoline (QNZ) before TNF-a treatment prevented the downregulation of JMJD3. In agreement with our in vitro findings, BMSCs from MM patients had persistently decreased JMJD3 expression compared to healthy BMSCs. Our findings together demonstrate that decreased JMJD3 expression in BMSCs contributes to the long-term OB suppression in MMBD by remodeling histone landscapes at the Runx2 and Osterix TSSs. Thus, developing strategies to restore JMJD3 expression in BMSCs should increase bone formation and possibly decrease tumor burden in MM.

Bone mineral density (BMD) is a reflection of the action of osteoblasts compared to osteoclasts. An imbalance in the activity of osteoblasts or osteoclasts, results in bone disease such as osteoporosis caused by overactive osteoclasts. BMD is influenced by genetic and environmental factors as demonstrated through twin studies, association studies and linkage analysis (Ralston, 1999). Several polymorphisms involved in the determination of BMD have been identified, with Vitamin D receptor and Collagen Type 1 showing reproducible associations. To identify genes influencing BMD two distinct strategies have been employed: 1) To determine if DNA polymorphism within the runt related transcription factor (RUNX2) gene is a determinant of BMD and fracture in women. 2) The identification of RANKL target genes in osteoclastogenesis. RUNX2 is a runt domain transcription factor (Werner et al., 1999) essential for osteoblast differentiation (Lee et al., 1997). RUNX2 gene knock-out mice have no osteoblasts due to a failure in osteoblast differentiation and consequently unmineralised skeletons, (Komori et al., 1997; Otto et al., 1997). In humans, mutations in RUNX2 cause cleidocranial dysplasia (CCD), a disorder characterised by hypoplasia or aplasia of the clavicles, short stature, supernumerary teeth, patent fontanelles and other changes in skeletal patterning and growth (Mundlos et al., 1997). RUNX2 contains a poly-glutamine poly-alanine (polyQ/polyA) repeat where mutations causing cleidocranial dysplasia have been observed. BMD has not been routinely examined in CCD, two studies have identified CCD patients with lower BMD with one fracture case identified (Quack et al., 1999; Bergwitz et al., 2001). The central role of RUNX2 in determining osteoblast differentiation makes RUNX2 a prime candidate gene for regulating adult bone density. To determine if polymorphism was present in the polyQ/polyA tract the repeat was amplified within the upper and lower deciles of femoral neck (FN) BMD in the Geelong Osteoporosis study (GOS). The upper and lower deciles of FN BMD acted as a surrogate for genotyping the entire cohort. This study identified two common variants within the polyA repeat: an 18 base pair deletion (11Ala) and a synonymous alanine codon polymorphism with alleles, GCA and GCG (noted as A and G alleles, respectively). The 11Ala and SNP polymorphism are found on codon 64 and 66 respectively (RUNX2 MRIPV variant). A allele frequencies were significantly different in a comparison of the upper and lower deciles of FN BMD (p=0.019). In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The association was maximal at the ultra-distal radius (p=0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The 11Ala polymorphism was not related to BMD in GOS. To further decipher the role of the RUNX2 A allele we genotyped 992 women from a Scottish cohort. The alleles of RUNX2 within the glutamine/alanine repeat were determined by MspA1I restriction digest. To examine the possible influence on estrogen related therapies or estrogen status on the potential genetic effect conferred by RUNX2, we divided the cohort by menopausal and hormone replacement therapy status. Within postmenopausal Scottish women the RUNX2 A allele was associated with significantly higher FN BMD (p=0.028, n=312) but not lumbar spine (LS) BMD. The A allele was associated with higher FN BMD (p=0.035) within a postmenopausal subgroup of the population (n=312). To investigate the effect of weight on the RUNX2 alleles the Scottish cohort was segregated into thin/normal (BMI ≥ 25 kg/m2) and overweight /obese (BMI > 25 kg/m2). RUNX2 A allele showed a stronger effect on FN BMD in postmenopausal women above the median BMI. The 11Ala RUNX2 deletion allele was significantly associated with decreased LS BMD (p=0.018) within overweight/obese women (n=546). The 11Ala allele was significantly associated with increased levels of pyridinoline (p=0.014) and deoxypyridinoline (p=0.038) in the HRT treated subgroup of the population (n=492). Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 11Ala and A alleles exert differing affects on BMD showing preference for different skeletal sites in a weight dependent manner. We genotyped 78 individuals from an osteoarthritic population to elucidate the role of the RUNX2 alleles on markers of bone turnover and inflammation. The RUNX2 11Ala allele was significantly associated with decreased osteocalcin (OC) serum levels (p = 0.01). The RUNX2 A allele was significantly related to reduced tumor necrosis factor alpha (TNF-alpha) serum levels (p = 0.004). RUNX2 is known to bind to the OC promoter. An OC promoter polymorphism is found 7bp upstream from a putative RUNX2 binding site. We hypothesized that OC polymorphism may effect the RUNX2 transactivation of the OC gene and thus affect OC serum levels. OC promoter polymorphism was not related to OC serum levels (n=78). These data present a novel link between RUNX2 alleles and OC and TNF serum levels, providing putative mechanisms of action for the RUNX2 alleles. Further studies in larger populations are required to confirm these findings. Ten individuals within the GOS and the Scottish cohort were found to carry rare mutations of the polyQ/polyA repeat. All polyQ variants had a normal polyA repeat (17 amino acids) and were heterozygous for a normal 23Q/17A allele. Variants observed were 15, 16, 24 and 30Q. One individual was observed with an extended polyA repeat (24A). Patient records indicated otherwise unremarkable clinical history except for fracture in 4/10 individuals from GOS (hip and spine). BMD data from the LS and the FN were expressed as T-scores, a measure that relates BMD in terms of standard deviations below the young normal value. In addition, BMD data were also expressed as Z-scores around the age-mean. Under the null hypothesis, where RUNX2 Q repeat variation has no effect on BMD, Z scores would be expected to be distributed around a mean of zero. However, when all variants were pooled the BMD was significantly lower than expected. This effect persisted when deletion variants were considered alone. The effect was stronger on FN BMD (p=0.001) rather than LS BMD (p=0.096), reflecting either difference in precision of BMD measurements at these sites or perhaps a differential genetic effect on different skeletal sites. These data suggest that polyQ and polyA variants are associated with significantly lower BMD, and may be an important determinant for fracture. Glutamine variants exist at high frequency (~0.7%): this rate of mutation could be important when considering large populations at risk of age related osteoporosis. Considering that these subjects are heterozygous for a normal allele, it suggests that a more severe phenotype might be expected in rare subjects homozygous for glutamine repeat variants. In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles' fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD. - 2) The identification of RANKL target genes in osteoclastogenesis. Osteoclastogenesis is regulated in vivo by the action of osteoblast/stromal cells that express membrane bound, receptor activator of NF-kB ligand (RANKL). Monocytes treated in vitro with a soluble form of RANKL and macrophage colony stimulating factor (M-CSF) differentiate to osteoclasts, whereas monocytes treated with M-CSF alone differentiate to macrophage-like cells. The gene expression profile of human osteoclasts has not been extensively explored. Genes highly expressed by rabbit osteoclasts were identified through random sequencing of an osteoclast cDNA library (Sakai et al., 1995). Differential gene expression of mouse osteoclastogenesis was elucidated by array analysis (Cappellen et al., 2002). To identify genes important for human osteoclastogenesis, total RNA was isolated from monocytes treated for three weeks with either M-CSF alone or with RANKL and M-CSF. RANKL treatment for 3 weeks and 12 hours was investigated in this study, to complement previous data. Differential display was performed on RNA (12 hour treatment with RANKL) and differential gene expression profiles examined. The differential display products were pooled to generate a probe for screening a gene array system derived from a human osteoclast cDNA library. cDNA (3 week treatment with RANKL) hybridisation experiments against the array revealed additional regulated genes. Gene clones that showed significant regulation in M-CSF and RANKL treated cells compared M-CSF treated cells represent genes that are targets for RANKL-specific regulation. Osteopontin, creatine kinase and various mitochondrial genes were up regulated by the treatment of RANKL. Changes in gene expression observed in the array data were confirmed with real-time PCR using mRNA derived from in vitro induced osteoclasts. Cathepsin K gene expression was more than 300 fold greater in osteoclasts compared to macrophage-like cells after one week treatment with RANKL and M-CSF. Cystatin C expression showed a six-fold induction at two weeks of RANKL and M-CSF treatment and cystatin B showed a steady increase in expression. Some of these regulated genes may provide useful targets for influencing BMD.