Although there are now many theories describing empirical relationships between strength properties of bone and various explanatory variables, the need for improved non-invasive diagnostic techniques to assess bone fragility is of core importance in clinical problems such as osteoporosis. The aim of this thesis was to develop non-invasive radiological methods to assess trabecular bone architecture. Measures of structural anisotropy and bone structure from X-ray or radiological projections have been developed. The first measure, the projected mean intercept length (PMIL), allows extraction of the total bone surface (BS/TV) and the mean intercept length (MIL) from projections of trabecular structure. The second measure, the line projection deviation (LPD), is a technique that quantifies the preferential alignment of trabecular bone from projections of the trabecular structure. Hence, in combination, the PMIL and LPD allow non-invasive extraction of BS/TV and more detailed preferential alignment from projections of the trabecular structure. In this thesis the PMIL and LPD are introduced and their properties explored. The PMIL and LPD are used to examine the anisotropy and architectural properties of a number of human vertebral body trabecular bone samples. When used in combination with clinical densitometry, these measures improve explanation of the variance in strength, elastic modulus and toughness of vertebral body trabecular bone samples by up to 40% when compared to densitometric values alone. While µCT can provide the information needed to access trabecular architecture, it cannot be used in clinical settings since its high radiation dose makes it only applicable to small objects ex-vivo. At present, clinically available CT does not provide sufficient resolution to resolve trabecular structures. Thus, the methods described in this thesis will allow estimates of structural parameters from plain X-rays, providing for the first time, the possibility of clinical use of such estimates. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311218 / Thesis (Ph.D.) -- School of Medical Sciences, 2008

La cirrosis biliar primaria es una enfermedad hepática que se manifiesta por un patrón clínico, bioquímico e histológico de colestasis crónica. Su curso puede complicarse con el desarrollo de una patología metabólica ósea cuyo tipo, frecuencia y mecanismos patogenéticos no están bien establecidos. Esta tesis se ha realizado con el fin de analizar la prevalencia y tipo de enfermedad metabólica ósea que se asocia a la cirrosis biliar primaria (CBP) e investigar los factores que influyen en su desarrollo. 1) PACIENTES Y MÉTODOS Se han estudiado 20 pacientes con CBP (18M, 2 V) con una edad media de 48.6 +/- 10.6 años. A todos ellos se ha practicado: estudio del grado de colestasis biológica y de la función hepatocelular, además de determinaciones bioquímicas y hormonales (25-hidroxivitamina D; 1,25 dihidroxivitamina D y parathormona N-terminal) del metabolismo mineral. En 16 pacientes se determinó la capacidad de absorción intestinal de calcio. A todos los pacientes se practicaron radiografías de la columna vertebral y biopsia ósea, por vía transilíaca, tras doble "marcaje" con tetraciclinas, para análisis histomorfométrico en especimen óseo no decalcificado. El estudio histomorfométrico se realizó mediante el método semiautomático y se analizaron parámetros estáticos y dinámicos. 2) RESULTADOS Siete pacientes tenían una osteoporosis al presentar una reducción del volumen trabecular. Tres de ellos tenían asociado un trastorno moderado de la mineralización ósea que no cumplía criterios de osteomalacia. Quince pacientes (5 con osteoporosis) tenían una disminución del grado de formación ósea y en 19 casos la reabsorción ósea era normal o estaba disminuida. Los pacientes con osteoporosis tenían una duración de la CBP más prolongada (5.4 +/- 2.8) que los pacientes sin osteoporosis (2.0 +/- 2.1 a p= 0.07). Además, la osteoporosis fue significativamente más frecuente en las mujeres postmenopáusicas, ya que 6 de los 7 pacientes con osteoporosis (86%) pero sólo 3 de los 11 sin osteoporosis (27%) eran mujeres postmenopaúsicas (p= 0.02). Por otro lado, los pacientes con osteoporosis tenían con mayor frecuencia una malabsorción intestinal de calcio (80%) que los pacientes sin osteoporosis (18%) (p= 0.03). Aunque la severidad de la colestasis no se relacionó con la presencia de osteoporosis, sí se halló una relación lineal inversa entre la absorción intestinal de calcio y la concentración plasmática de sales biliares (r= -0.55, p < 0.05) y el nivel sérico de la fosfatasa alcalina (r= -0.5, p < 0.05). Cuatro pacientes, dos de ellos con trastornos de la mineralización ósea, tenían niveles séricos bajos de 25-hidroxivitamina D. Sin embargo, los niveles séricos del metabolitos 1,25-dihidroxivitamina D fueron normales en todos los casos. 3) CONCLUSIONES Los resultados de este estudio indican que: 1.- La osteoporosis es la enfermedad metabólica ósea que comúnmente se asocia a la cirrosis biliar primaria. Su prevalencia fue del 35% en nuestra serie analizada. 2.- Los pacientes con cirrosis biliar primaria de nuestro medio no desarrollan una osteomalacia, aunque no es infrecuente que presenten un trastorno moderado de la mineralización ósea. Un 15% de los pacientes desarrollaron este trastorno. 3.- La osteoporosis asociada a la cirrosis biliar primaria es de bajo "turnover" óseo y su base fisiopatológica es un déficit de la formación ósea. El 71% de los pacientes con osteoporosis tenían un déficit de la formación ósea, alteración que también presentaba el 83% de los pacientes sin osteoporosis. Ello permite sugerir que un elevado porcentaje de pacientes con cirrosis biliar primaria y masa ósea normal están en alto riesgo de desarrollar una osteoporosis. 4,- Los factores de riesgo implicados en el desarrollo de osteoporosis en la cirrosis biliar primaria son: duración de la hepatopatía, estado postmenopáusico y malabsorción intestinal de calcio. 5,- El desarrollo de un trastorno moderado de la mineralización ósea es más frecuente en los pacientes con déficit de 2S-hidroxivitamina D. Sin embargo, el "status" deficitario de vitamina D no es exclusivo de los pacientes con trastorno de la mineralización ósea.

[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.

Made available in DSpace on 2014-06-11T19:29:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-22Bitstream added on 2014-06-13T18:59:46Z : No. of bitstreams: 1 000748628.pdf: 1456061 bytes, checksum: b5ef0d0192e00742b4d0ca806b84a009 (MD5) / Avaliar a densidade mineral óssea (DMO) e o conteúdo mineral ósseo (CMO) de adolescentes do sexo feminino, usuárias de anticoncepcional oral combinado (AOC) de baixa dosagem padronizado (EE 20 μg/ Desogestrel 150 μg), por período de um ano de seu uso e comparar os dados obtidos aos de adolescentes saudáveis da mesma faixa etária, não usuárias. Trata-se de um estudo controlado paralelo não randomizado. Sessenta e sete adolescentes, de 12 a 20 anos de idade, foram divididas em grupo de usuárias de AOC (n=41) e grupo controle (n=26). As adolescentes pertencentes aos dois grupos foram submetidas a exame físico geral e especial para obtenção de peso, estatura, índice de massa corpórea (IMC), avaliação dos caracteres sexuais secundários (critérios de Tanner), obtenção de idade óssea (IO) pelo método de Greulich & Pyle, ingestão de cálcio obtida pelo recordatório de 3 dias e obtenção da idade do evento menarca. As usuárias de AOC foram submetidas ao exame de Densitometria Óssea por atenuação de raio x de dupla energia (DXA), no momento de inclusão no trabalho; 6 e 12 meses depois, para obtenção de CMO (g) e DMO (g/cm2) em região lombar (L1-L4), fêmur proximal total, corpo total e corpo subtotal. O grupo controle foi avaliado através da DXA, no momento inicial; 12 meses depois, para obtenção de DMO e CMO, nos mesmos locais. A comparação entre as variáveis dos grupos de não usuárias e usuárias de AOC, no momento zero, foi realizada através do teste de Mann-Whitney, fixado o nível de significância de 5% ou utilizado o p-valor correspondente, enquanto para a comparação evolutiva dos grupos utilizou-se a variação das porcentagens das medianas das variáveis relativas à massa óssea, nos momentos inicial e final. Não houve diferenças estatísticas nas comparações entre as idades cronológicas (IC) e IO, entre as variáveis antropométricas e as resultantes do... / To evaluate bone mineral density (BMD) and bone mineral content (BMC) in female adolescents taking a standard low dose (EE 20μg/ Desogestrel 150μg) combination oral contraceptive (COC) over a one year period and comparing them to healthy adolescents from the same age group not taking COC’s. A non-randomized parallel control study with 67 adolescents from 12 to 20 years of age divided into user (COC; n=41) and control (n=26) groups. Both groups were submitted to a general physical and specific examination for weight, height, body mass index (BMI), secondary sexual characteristics evaluation (Tanner criteria), bone age (BA) by the Greulich & Pyle method, calcium intake by 3 days diet recording, and obtaining age at menarche. COC users underwent bone density exam by dual-energy X-ray absorptiometry (DXA) at time of inclusion in the study and at 6 and 12 months, to obtain BMC (g) and BMD (g/cm2) in the lumbar (L1-L4) and total proximal femur regions, for whole body and subtotal whole body. The Control group underwent DXA at inclusion and 12 months to obtain BMD and BMC in the same locations. Comparisons between groups at moment zero was through the Mann-Whitney test with significance level fixed at 5% or corresponding p value; evolutive group comparisons used variations in median percentages for bone mass variables at start and final moments. There were no statistical differences in chronological (CA) and BA, anthropometric variables, and bone densitometry results at the initial moment between COC and control groups. However after 12 months follow-up, COC users presented low bone mass acquisition in the lumbar spine BMD and BMC median variation percentages between initial and final moments (+2.07% and +1.57% respectively) while the control group presented expressive variations (+12.16% and +16.84% respectively). Total body BMD and BMC presented similar behaviour; variation in the COC group was +0.84% and +1.22%, considerably lower ...

目前，骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展，採用核酸干擾策略進行治療的很多問題已被解決。然而，小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大，其系統給藥後可能對其他非骨組織產生副作用。所以，亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統，以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞，使其具有高度的細胞選擇性。在該研究中，我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。 / 靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統：首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備（天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑，ζ電位，包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。 / 實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右，其包封率可高達80%。該遞送系統較穩定，可使攜載的小干擾核酸具有較高的基因沉默效率，而且沒有明顯的細胞毒性。體內試驗表明，該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因，且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞，避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp，Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。 / 靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統：將針對大鼠成骨細胞（ROS 17/2.8細胞系）進行正向篩選，大鼠肝細胞（BRL-3A細胞系）和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑，ζ電位，包封率和形態學進行考察。在體外評價實驗中，考察了該遞送系統的選擇性，細胞毒性，基因沉默效率以及細胞攝取機制。在體內實驗中，對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。 / 實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm，其電勢為-23 ± 2 mV，包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系（靶向細胞）中的攝取， 然而在BRL-3A 細胞系（非靶向細胞）中攝入很少。該遞送系統沒有明顯細胞毒性，在10 nM小干擾核酸的低濃度下，體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈，降低其被肝組織的攝取。在肝组织冰凍切片中，肝血竇和肝細胞中沒有明顯的小干擾核酸分佈，進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞，避免被前破骨細胞/破骨細胞吞噬。 / 重要意義：本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑，實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下，首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具，為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。 / Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study. / (AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models. / The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo. / L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo. / The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo. / Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-142). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.vi / Table of contents --- p.ix / Publications --- p.xiv / List of tables --- p.xvi / List of figures --- p.xvii / List of abbreviations --- p.xxi / Chapter One Introduction --- p.1 / Chapter 1.1 --- Great challenges in skeletal disorders --- p.2 / Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3 / Chapter 1.2.1 --- Mechanism of RNAi --- p.3 / Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4 / Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7 / Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11 / Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12 / Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13 / Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15 / Chapter 1.3.2.1 --- Physiological property --- p.15 / Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16 / Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18 / Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18 / Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20 / Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20 / Chapter 1.4.2.2 --- Polymers --- p.22 / Chapter 1.4.2.3 --- Targeted delivery system --- p.23 / Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24 / Chapter 1.6 --- Objective of present study --- p.25 / Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29 / Chapter 2.2.3 --- Development of formulation --- p.30 / Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30 / Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30 / Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30 / Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33 / Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33 / Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33 / Chapter 2.2.5.3 --- Stability in serum --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34 / Chapter 2.3.2 --- Development of formulation --- p.37 / Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38 / Chapter 2.3.4 --- Stability in serum --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusion --- p.42 / Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Biological evaluation in vitro --- p.46 / Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46 / Chapter 3.2.2.2 --- Cell culture --- p.46 / Chapter 3.2.2.3 --- Cellular uptake --- p.47 / Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47 / Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48 / Chapter 3.2.3 --- Cytotoxicity --- p.49 / Chapter 3.2.4 --- Tissue distribution --- p.50 / Chapter 3.2.4.1 --- Experimental design --- p.50 / Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50 / Chapter 3.2.4.3 --- Quantitative Analysis --- p.50 / Chapter 3.2.5 --- Localization of siRNA in liver --- p.51 / Chapter 3.2.5.1 --- Experimental design --- p.51 / Chapter 3.2.5.2 --- Histochemisty analysis --- p.51 / Chapter 3.2.6 --- Gene silencing in tissues --- p.52 / Chapter 3.2.6.1 --- Experimental design --- p.52 / Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52 / Chapter 3.2.6.3 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53 / Chapter 3.2.7.1 --- Experimental design --- p.53 / Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53 / Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54 / Chapter 3.2.8.1 --- Experimental design --- p.54 / Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54 / Chapter 3.2.9 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results --- p.56 / Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56 / Chapter 3.3.2 --- Cellular uptake --- p.57 / Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57 / Chapter 3.3.4 --- Cytotoxicity --- p.59 / Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60 / Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62 / Chapter 3.3.7 --- Localization of siRNA in liver --- p.63 / Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64 / Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65 / Chapter 3.3.10 --- Gene silencing at cellular level --- p.71 / Chapter 3.4 --- Discussion --- p.74 / Chapter 3.5 --- Conclusion --- p.77 / Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Materials --- p.80 / Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80 / Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81 / Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81 / Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82 / Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82 / Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83 / Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83 / Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83 / Chapter 4.2.3 --- Development of formulation --- p.84 / Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84 / Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85 / Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85 / Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86 / Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86 / Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87 / Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87 / Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88 / Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88 / Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88 / Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88 / Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90 / Chapter 4.3.2 --- Formulation development --- p.93 / Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95 / Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.5 --- Conclusion --- p.101 / Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and Methods --- p.103 / Chapter 5.2.1 --- Materials --- p.103 / Chapter 5.2.2 --- Biological evaluation in vitro --- p.104 / Chapter 5.2.2.1 --- Cell culture --- p.104 / Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105 / Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105 / Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105 / Chapter 5.2.3 --- Cytotoxicity --- p.106 / Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106 / Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106 / Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107 / Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107 / Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107 / Chapter 5.2.5.1 --- Experimental design --- p.107 / Chapter 5.2.5.2 --- Tissue distribution --- p.108 / Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108 / Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108 / Chapter 5.2.6 --- Statistical analysis --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109 / Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111 / Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112 / Chapter 5.3.4 --- Cytotoxicity --- p.113 / Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113 / Chapter 5.3.6 --- Tissue distribution --- p.118 / Chapter 5.3.7 --- Localization of siRNA in liver --- p.119 / Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120 / Chapter 5.4 --- Discussion --- p.123 / Chapter 5.5 --- Conclusion --- p.125 / Chapter Chapter Six --- Summary of the study and future research --- p.126 / Chapter 6.1 --- Summary of the study --- p.127 / Chapter 6.2 --- Future research --- p.128 / References --- p.130

Made available in DSpace on 2014-06-11T19:22:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-12-19Bitstream added on 2014-06-13T19:49:04Z : No. of bitstreams: 1 brock_rs_me_botfm.pdf: 602525 bytes, checksum: bd2115da27f5dda49225e5afda53918e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Defeitos ósseos decorrentes de traumas, ressecções de tumores ou mesmo malformações congênitas, são encontrados com freqüência na prática médica. O tratamento destas deformidades é feito mediante reconstruções cirúrgicas, principalmente na cirurgia plástica, proporcionando aos pacientes melhor qualidade de vida. Os defeitos ósseos são corrigidos preferencialmente com enxertos ósseos autológos por não causarem rejeição, mas estes apresentam como desvantagens a morbidade das áreas doadoras e a grande porcentagem de absorção dos enxertos, com diminuição ou perda do resultado final. Outros métodos de reconstrução, como o uso de materiais aloplásticos, são utilizados mas, muitas vezes, evoluem com rejeição e extrusão ou infecção, e necessitam ser retirados. Retalhos livres, compostos de osso com músculo ou derme e subcutâneo, em casos graves, representam a melhor opção. Entretanto, este método requer preparo específico da equipe cirúrgica, maior tempo de cirurgia e, muitas vezes, apresenta trombose vascular e perda do retalho. Avaliar a viabilidade e a formação óssea em retalho gáleoperiostal preenchido com pó de osso em calota craniana de coelhos. Foram estudados 40 coelhos divididos em dois grupos, o primeiro com retalho gáleo-periostal e o segundo com o mesmo retalho, porém preenchido com pó de osso. Os resultados demonstraram neoformação óssea em ambos, mas com diferenças na estrutura e conformação óssea. O retalho gáleo-periosteal preenchido com pó de osso em calota craniana de coelhos é viável. A formação óssea ocorreu em ambos os grupos, preenchido ou não com pó de osso. A maturidade do tecido ósseo foi maior nos retalhos preenchidos com pó de osso / Osseus defects from traumas, tumor ressections or congenital malformations are usual in medical practice. The treatment of these deformities has been made with reconstructive surgeries, specially in plastic surgery, to give the patients better quality of life. The osseus defects are usually corrected with autologous bone grafts. These grafts are used because they do not cause rejection. However, they have disadvantages like the donnor site morbidity, the high number of absorption of these grafts and the final result partial or total lost. Other reconstruction methods like alloplastic materials are used, but they have high percentage of rejection and extrusion or even infection of these materials, which need to be taken off. Flaps of bone and muscle or dermis and subcutaneous are considered the best choice in difficult cases. However, this method needs specific training of the medical group, longer surgeries and, sometimes, presents the flap necrosis after vascular thrombosis. To study the viability and bone neoformation in a vascularized galea and periosteum flap filled with bone fragments. Fourty rabbits were studied, and divided into two groups. One had a simple galea and periosteum flap done and the other had the same flap done but filled with bone fragments of the calvaria. The results demonstrated bone formation in both groups, but with differences in the bone form and structure. The galea-periosteum flap filled with bone dust at rabbit’s calvaria is viable. The bone formation happened in both groups, with or without bone dust. Bone maturity was higher in the flaps filled with bone dust

Orientador: Silvia Regina Brandalise / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T21:43:20Z (GMT). No. of bitstreams: 1 Aguiar_SimonedosSantos_D.pdf: 43958145 bytes, checksum: b9dafbbd99ad0e567b3c1f03a0c7b37e (MD5) Previous issue date: 2006 / Resumo: Introdução Os osteossarcomas (OS) são tumores agressivos, primários de osso, com prognóstico reservado. As deleções dos genes supressores de tumor, RBl e P53, localizados nos cromossomos 13 e 17 respectivamente, são freqüentemente encontradas neste tipo de tumor. As alterações citogenéticas encontradas nos OS são de alta complexidade, porém nenhuma delas é recorrente, não podendo caracterizá-lo. A técnica da Hibridização Genômica Comparativa (CGH) é uma ferramenta muito precisa para o estudo das deleções e amplificações gênicas ocorridas neste tumor. Materiais e Métodos. Tecido tumoral de 41 crianças com OS foi analisado pela técnica de CGH para pesquisar possíveis ganhos e/ou perdas gênicas . A técnica da Hibridização In Situ Fluorescente (FISH) foi realizada para estudar as deleções dos genes P53 e RBl. Vinte e quatro pacientes eram do sexo feminino e 17 do sexo masculino, com mediana de 12 anos e 4 meses. Resultados. As anormalidades cromossômicas observadas com a técnica de CGH foram diversas e variadas, especialmente ganhos nos cromossomos lp, 2p, 3q, 5q,5p e 6p e, perdas nos cromossomos 14q (50% no - 14q 11.2), 15q e 16p. Alto índice de perdas foi observado no cromossomo 21 (26 de 41 casos; p=0,008), sendo a região mais freqüentemente afetada a 21ql 1.2. Com relação ao estudo dos supressores tumorais, a deleção do P53 ocorreu em 68,3% dos casos (p=0,02) e do RBl em 87,5% dos casos (p=0,000001). Conclusão. Apesar de ambos supressores (PS3 e RBl) estarem deletados na maioria dos pacientes, este evento parece não estar associado ao prognóstico. Anormalidades ainda não reportadas presentes no cromossomo 21 nos OS pediátricos, sugerem que a seqüência mapeada nesta região cromossômica possa estar envolvida na patogênese deste tumor / Abstract: Background. Osteosarcomas (OS) are aggressive bone tumors and often have a poor prognosis. It is already known that abnormalities in chromosomes 13 and 17 are frequently observed in OS patients, being also expected a deletion of RBI and P53 genes. The tumors exhibit karyotypes with a high degree of complexity, that has made it difficult to determine if any recurrent chromosomal aberrations could characterize OS. To address inherent difficulties associated with classical cytogenetic analysis, comparative genomic hybridization (CGH) has been applied to OS tissue. Patients and Methods. Forty one pediatric OS specimens were analyzed by CGH techniques, and the expression of RBI and P53 were analyzed by FISH . Twenty four patients were girls and 17 boys. Median age was 12 years and 4 months.Results. Chromosomal abnormalities were highly diverse and variable specially gains in chromosome lp, 2p, 3q, 5q , 5p and 6p and losses in chromosome 14q (50% in - 14q 11.2), 15q and 16p. High level of losses in chromosome 21 were present (26 of 41 cases; p-0,008), being 21 q 11.2 region the most frequent one. Concerning about genes expression, P53 is deleted in 68,3% of the cases (p=0,02) and RBI in 87,5% (p=0,000001) .Conclusion. Although both oncogenes (P53 and RBI) are deleted in OS population, it remains impossible to determine if this abnormality is a prognostic factor. These new and unreported findings in chromosome 21 of pediatric OS tumors, suggest that specific sequences mapping these chromosomal regions, would likely to play a role in the development of OS / Doutorado / Pediatria / Mestre em Saude da Criança e do Adolescente

Several hundred inherited disorders of the skeleton have been delineated. Individually these conditions are rare, but as a group they cause much crippling and hardship. Several factors, including the rarity and complexity of the manifestations of these conditions, as well as semantic overlap, impede the accurate diagnosis which is essential for effective treatment. In this regard, the adoption of microcomputers warrants evaluation as a high technology aid. Microcomputers have developed tremendous capabilities during recent years. The state of the art has become such that a diagnostic aid facility on such a device has been demonstrated in various disciplines of medicine and may also be feasible in the area of inherited skeletal disorders. The study which forms the basis of this thesis, concerns the investigation of this feasibility and has led to the development of an effective working model which sets the basis for microcomputer-aided diagnosis. The design features followed in this project are similar to those conventionally employed for "Expert systems" on mainframe computers. A comprehensive knowledge base consisting of over 200 skeletal disorders and 700 radiographic and clinical manifestations, has resulted. Furthermore, the application is capable of "learning", although inference as employed by the inference engines of real expert systems, is not employed. In this context learning implies that the knowledge base, with the passage of time, improves considerably when used by experts. Serendipitous findings in this regard are: • 1) Considerable improvement of existing profile descriptions can occur without any increased demands on computer memory and storage space; • 2) Growth of the knowledge base in the form of additional disease profiles can be effected with very modest inroads on memory and storage resources. The computerized diagnostic aid which resulted from this thesis, has been demonstrated to be successful in both the Department of Human Genetics of the University of Cape Town and the Department of Paediatrics of the Johannes Gutenberg University in Mainz. Evaluated both in terms of efficiency and utility, the system provides an enhancement to the specialist genetic diagnostician. These achievements have been effected by means of a unique newly developed application of compressed bit-mapping, attained by writing the applicable programs in Turbo Pascal and 8086- assembler languages. Calculations indicate that much larger data bases may possibly be implemented on present-day microcomputers by means of the methods developed in this project.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple myeloma (MM) is an incurable malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM) and by multiple osteolytic lesions throughout the skeleton. We previously reported that CD166 is a functional molecule on normal hematopoietic stem cells (HSC) that plays a critical role in HSC homing and engraftment, suggesting that CD166 is involved in HSC trafficking and lodgment. CD166, a member of the immunoglobulin superfamily capable of mediating homophilic interactions, has been shown to enhance metastasis and invasion in several tumors. However, whether CD166 is involved in MM and plays a role in MM progression has not been addressed. We demonstrated that a fraction of all human MM cell lines tested and MM patients’ BM CD138+ cells express CD166. Additionally, CD166+ cells preferentially home to the BM of NSG mice. Knocking-down (KD) CD166 expression on MM cells with shRNA reduced their homing to the BM. Furthermore, in a long-term xenograft model, NSG mice inoculated with CD166KD cells showed delayed disease progression and prolonged survival compared to mice receiving mock transduced cells. To examine the potential role of CD166 in osteolytic lesions, we first used a novel Ex Vivo Organ Culture Assay (EVOCA) which creates an in vitro 3D system for the interaction of MM cells with the bone microenvironment. EVOCA data from MM cells lines as well as from primary MM patients’ CD138+ BM cells demonstrated that bone osteolytic resorption was significantly reduced when CD166 was absent on MM cells or calvarial cells. We then confirmed our ex vivo findings with intra-tibial inoculation of MM cells in vivo. Mice inoculated with CD166KD cells had significantly less osteolytic lesions. Further analysis demonstrated that CD166 expression on MM cells alters bone remodeling by inhibiting RUNX2 gene expression in osteoblast precursors and increasing RANKL to OPG ratio in osteoclast precursors. We also identified that CD166 is indispensable for osteoclastogenesis via the activation of TRAF6-dependent signaling pathways. These results suggest that CD166 directs MM cell homing to the BM and promotes MM disease progression and osteolytic disease. CD166 may serve as a therapeutic target in the treatment of MM.

Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional structural spine deformity with lateral curve and vertebral rotation occurring predominantly in adolescent girls during the peri-pubertal period. The prevalence of AIS is nearly 4% in Hong Kong and 2-3% worldwide. AIS without treatment or with improper treatment may deteriorate progressively and lead to significant cosmetic problems and functional disabilities. In severe cases, increased mortality rate can result from the associated early onset of cardiopulmonary failure. Up to now, the treatment of the AIS is basically passive through bracing and corrective spinal surgery. The current protocol of treatment is not totally satisfactory since the curve may continue to progress with brace treatment and corrective surgery is associated with major surgery and permanent fusion of parts of the spine. This is due to the fact that one is still uncertain about the etiology of AIS and therefore cannot directly treat the AIS. Among the different proposed etiology of AIS, the role of abnormal skeletal growth and development during peri-pubertal period has been one of the main focuses in addition to genetic predisposition in the development of AIS. / It has been well established that girls with idiopathic scoliosis have a tendency to be taller and more slender than their peers. Recently, it has been shown that the trabecular bone mineral density at the spine, hip, and peripheral bones of AIS girls was lower than their healthy peers. Studies from our center have also demonstrated growth discrepancy between anterior and posterior vertebral column using magnetic resonance imaging (MRI) technique. The vertebral bodies were shown to be slender in AIS patients than that in normal controls. The observation pointed to a disproportionate growth of the anterior and posterior spinal column resulting from imbalance in endochondral and membranous ossification. The present study hypothesizes that the abnormality of skeletal growth could be a systemic problem affected by both endochondral ossification and membranous ossification. The degree of abnormal growth could vary with different curve severities. The concurrent finding of abnormal skeletal growth and osteopenia could be related to certain underlying abnormal genetic factors affecting the etiopathogenesis of AIS. The hypothesis leads to the following objectives: (1) To study the anthropometric measurements and the related changes in AIS girls with different curve severity; (2) To document the presence of abnormal systemic growth through endochondral ossification; (3) To document the presence of abnormal membranous ossification through studies of the morphology and bone mineral density of the midshaft of the appendicular skeleton and the skull; (4) To study the association of occurrence of AIS and its related phenotypes with the genes associated with growth and osteopenia. (Abstract shortened by UMI.) / by Yeung Hiu-Yan. / "January 2006." / Adviser: Jack C. Y. Cheng. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6301. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 206-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.