Thesis (M.Sc.). / Written for the Division of Experimental Medicine. Title from title page of PDF (viewed 2009/06/18). Includes bibliographical references.

Parathyroid hormone (PTH) analogs are the main anabolic pharmacological agent for osteoporosis. PTH is an endogenous hormone, of which amino acids 1-34 bind the parathyroid hormone receptor (PTH1r), a G-coupled protein receptor expressed in kidney, fat, and bone. PTH increases trabecular bone mass by promoting the differentiation of the mesenchymal stem cell (MSC) into the osteogenic lineage. The Zinc Finger Protein 467 (Zfp467) is a potential downstream target of PTH1r and an important mediator of the MSC into the adipogenic lineage. Taken together, we ask whether Zfp467 knockout cells will show greater osteogenic potential and increased sensitivity to PTH treatment. We also seek to investigate a mechanistic signaling pathway of PTH1r involving Zfp467. Calvarial osteoblast (COB) and bone marrow stromal cells (BMSCs) from Zfp467 wild type (WT) and knockout (KO) mice were osteogenically differentiated and treated with either continuous (48h) or intermittent (6h/42h) PTH for 7-14 days. At 7 and 14 days, alkaline phosphatase (ALP) and von Kossa staining were conducted, respectively. At 7 days after differentiation, qPCR was used to analyze genes involved in osteogenesis, adipogenesis, WNT signaling, and mitochondrial respiration. ELISA was used to measure cAMP levels. Seahorse XF96 assays were used to measure oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs). Western blot was used to measure PTH1r. Additionally, adipogenic differentiation and Oil Red O staining were performed on BMSCs. ALP and von Kossa results showed that Zfp467 KO cells exhibited increased osteogenesis and an increased response to PTH treatment (continuous and intermittent) as compared to WT controls. qPCR analysis of Alp, Rankl, and Sp7 further supported an increased osteogenic potential of the KO. Also, Oil Red O staining revealed suppressed adipogenesis in KO BMSCs and qPCR analysis showed suppressed Adiponectin and Ppary in KO COBs. Additionally, Pth1r and PTH1r expressions were significantly higher in KO and short PTH treatments (~10m) induced a remarkable reduction in Zfp467 of WT cells. Furthermore, the KO showed suppressed Pgc1a, similar OCR, and increased ECAR as compared to WT. The KO also exhibited higher cAMP levels and was more responsive to PTH-induced increases of cAMP at 10 minutes of PTH exposure. However, qPCR analysis of Lef1 and Sost showed no difference regarding the WNT pathway. Our data support an anti-osteogenic and pro-adipogenic role for Zfp467. The KO displays less adipogenesis, more osteogenesis, and is consistently more sensitive to the osteogenic effects of PTH. The upregulation of Pth1r and PTH1r in KO cells offers an explanation for this increased sensitivity. We propose a mechanism where the suppression of Zfp467 upregulates Pth1r and PTH1r activation suppresses Zfp467, resulting in a constitutively active positive feedback loop. Further still, the KO shows potentially suppressed mitochondrial biogenesis (through Pgc1a analysis), similar oxidative phosphorylation (through OCRs), increased glycolysis (through ECARs), and increased PKA signaling (through cAMP assays), yet their exact connections to the PTH1r-Zfp467 signaling pathway have yet to be investigated.

No description available.

Bisphosphonates are the most widely prescribed anti-resorptive agents and work by preventing the post-translational modification (prenylation) of small GTPases in osteoclasts, subsequently leading to cell death by apoptosis.  Phosphonocarboxylate analogues of bisphosphonates also have anti-resorptive activity and work by inhibiting the enzyme Rab GGTase, thereby preventing the prenylation of Rab GTPases specifically.  Rab GTPases comprise a large family of related proteins that coordinate vesicular trafficking, which involves the processing, transportation and delivery of cellular cargo in a strict temporal and spatial manner.  In osteoclasts, vesicular trafficking is vital for the formation of the ruffled border (the resorptive organelle of the cell), the delivery of lytic enzymes and acid into the resorption space, and the uptake and disposal of bone degradation products.  However, the role that specific Rabs play in this functionally unique cell type remains poorly defined, and the Rab expression profile in osteoclasts is incomplete.  The work presented here aimed to increase our understanding of the role that Rabs play in osteoclasts.  Results indicate that the 70% reduction of Rab GGTase activity observed in <i>gunmetal </i>mice is detrimental to the activity of osteoclasts and osteoblasts <i>in vitro</i>, therefore highlighting the importance of Rabs for bone resorption and deposition.  Furthermore, this study is the first to determine the Rab expression profile of human osteoclasts, following a proteomic approach, and describes the transfection methods devised to characterise these candidate Rabs in osteoclasts.  Finally, this study details the characterisation of Rab18 in human osteoclasts, following its discovery during proteomic analysis.

No description available.

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pathology. Title from title-page of electronic thesis. Bibliography: leaves 139-149.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 94-106).

Banca: Antonio Olavo Cardoso Jorge / Banca: Yasmin Rodarte Carvalho / Banca: Eduardo Dias de Andrade / Banca: Ana Maria Duarte Dias Costa / Banca: Maria Cristina Volpato / Resumo: Considerando que o álcool tem sido identificado, na literatura, como um fator de risco evidente para desenvolvimento de osteoporose, induzindo a perda de massa óssea, propusemos-nos neste trabalho a avaliar o efeito do álcool no Fêmur de ratos machos e fêmeas. Para tanto, foram utilizados 35 ratos machos e 35 fêmeas divididos em sete grupos de cinco animais por gênero. Os grupos foram: grupo controle, que receberam água e ração à vontade ; grupo álcool nas concentrações de 10, 20 e 30% e o grupo isocalórico correspondente às concentrações de álcool. Após 8 semanas os animais foram sacrificados e avaliados quanto ao peso corpóreo e condição nutricional. Os fêmures unilaterais removidos e analisados, quanto ao comprimento, largura, espessura da cortical, porcentagem de trabéculas e densidade óptica. Os dados obtidos foram analisados pela ANOVA (Tukey;p<0,05). Quanto ao peso, houve perda de peso dos animais dos grupos álcool 20% nas fêmeas e nos machos 30%. Quanto à condição nutricional as fêmeas com as doses de 10,20 e 30% de álcool ingeriram em média 25, 39 e 53% de Kcal/dia e os machos 22, 36 e 45 Kcal/dia provinientes do álcool. O álcool diminuiu o comprimento do fêmur somente no grupo MG6. A largura mésio-distal foi maior no grupo FG6, no entanto a largura ântero-posterior não se modificou. A espessura da cortical tanto nos machos como nas fêmeas não foi alterado; no entanto a porcentagem do osso trabeculado diminui tanto nas fêmeas álcool 30% como nas três concentrações alcoólicas nos machos. A densidade optica apresentou-se reduzida nas concentrações de 30 % tanto nos machos como nas fêmeas. Dentre as condições experimentais concluiu- se que o efeito do álcool foi evidente nos machos e no osso trabecular, e que a concentração alcoólica de 30 % foi a mais deletéria para o tecido óseeo ósseo provalvelmente, levando a diminuição da massa óssea e à osteopenia / Abstract: Considering that alcohol has been identified, in the literature, as an evident risk factor for osteoporosis development, inducing loss of bone mass, the aim of this study was to evaluate the effect of the alcohol on the femur of male and female rats. For this purpose, 35 male and 35 female rats, divided into seven groups of five animals per genera were included. The groups were: control group that received food and water as much as they wished; alcohol group in the concentrations of 10, 20 and 30% and the isocalorie: groups corresponding to the concentration of alcohol. After 8 weeks, the animals were sacrificed and evaluated regarding to the body weight and nutritional conditions. Unilateral femurs were removed and analyzed in relation to the length, width, and thickness of the bone cortical, trabeculae percentage and optical density. Data obtained were analyzed by ANOVA (Tukey; p<0.05). Regarding to the weight, loss of weight was observed in the animals of the group alcohol 20% among females and 30% among the males. In relation to the nutritional conditions, the females with the dosages of 10, 20 and 30% of alcohol swallowed a mean of 25, 39 and 53% of Kcall and the males 22, 36 and 45 Kcal Jday coming from the alcohol. Alcohol reduced the length of the femur only in the group MG6. Mesio-distal width was higher in the group FG6 although the antero-posterior width was not modified. The cortical thickness among males and females was not altered however the percentage of trabeculae bone was reduced among females alcohol 30% and among males with the three alcoholic concentrations. Optical density was reduced in the concentration of 30% among the males and females. Under the experimental conditions, it could be concluded that the effect of alcohol was more evident among the males and in the trabeculae bone, and that the alcoholic concentration of 30% was the most harmful for the bone tissue, ....

Indiana University-Purdue University Indianapolis (IUPUI) / The effect of weightlessness on bone and osteoblast precursor cells has previously been studied. A marked decrease in bone formation, an increase in less differentiated committed osteogenic cells (A+A'), and a decrease in preosteoblast cells (C+D) was noted. To date, the effect of hypergravity (2g) on osteoblast histogenesis has not been studied in vivo. In vitro studies using nonphysiologic high levels of gravity (20,40g) have shown an increased proliferation of cloned osteoblast-like cells. The purpose of the present study was to evaluate the effect of hypergravity (2g) on osteoblast histogenesis in the rat periodontal ligament (PDL), on the width of the mesial PDL, and on the percentage of forming bone surface on the mesial side of the tooth. Twenty male Wistar rats (SPF: Harlan Sprague Dawley) were randomly assigned to the centrifuge (experimental) or to the stationary (control) group. The experimental group was centrifuged for 14 days at 2g and the stationary group was housed in identical cages in the centrifuge room. The PDL of the mesial and distal surface of the mesial root of the first maxillary molar was analyzed microscopically 100 μm above and below the midroot area. Nuclear volume morphometry was used to classify periodontal ligament cells as: L cells (<40 μm3), A+A' cells (40-79 μm3), B cells (80-119 μm3), C cells (120-169 μm3), and D cells (>170 μm3). The percent of forming bone surface on the mesial side and the width of the PDL were also measured. A 2x2 factorial ANOVA with repeat measures revealed a significant (p < 0.05) decrease in the C cell population and a nearly significant (p < 0.06) increase in the A+A' cell population in the centrifuge group. Comparing bone surfaces, the forming surface had a significant (p < 0.01) increase in the C and D cell populations, a significant (p < 0.01) decrease in the L and A+A' cell populations, and a significant (p < 0.05) decrease in the B cell population. The stationary group weighed significantly (p < 0.01) more than the centrifuge group post-experiment. And an unpaired t-test revealed a nearly significant (p < 0.06) increase in the percent forming bone surface on the mesial side of the maxillary first molar and no significant difference in mesial PDL width. The results showed that the centrifuge group had a trend toward a block in preosteoblast formation. This is similar to that seen with hypogravity. However, it cannot be concluded at this time if this is a direct gravitational effect or related to other factors such as physiological response to stress. Physical stress has been suggested as a potential mechanism for the observed decrease in weight seen in centrifuged animal, while the PDL width does not seem to be affected by gravitational forces, and thus may not be a sensitive marker to osteoblast differentiation inhibition. Finally, the reason for the increase in forming bone surface in the centrifuged group is unclear.

No description available.