Dissertations / Theses on the topic 'Bones – Cytology'

Background: Bone-grafting materials are required in orthopaedic surgery to treat bone defects. Bone formation assessment is required for the development of new strategies and approaches and for quality assurance and quality control of currently available materials. Approaches to the assessment of bone formation are yet to be systematically established, quantified and standardized. Aims: the overall aim of this study was to establish a set of comprehensive quantitative approaches for the assessment of bone formation and to evaluate the role of osteoblastic cells, growth factors, and scaffolds on this process. Materials & methods: both in vitro and in vivo parameters for osteoblast phenotype and bone formation were tested in osteosarcoma cell lines, Saos-2 and U2OS cells, mesenchymal cell line, C2C12 cells, primary adipose derived stromal cells (ADSCs), platelet rich plasma (PRP), and morselized bone grafts. The in vitro parameters used were measurement of alkaline phosphatase (ALP) activity, detection of bone nodules and biomineralization, and quantification of immunocytochemistry and conventional RT-PCR of osteoblast genotyping. In vivo parameters involved ectopic bone formation in nude mice and nude rats and a tibial defect model in nude rats. Histomorphometric and quantitative immunohistochemical analyses were also performed. Results: The in vitro characterization and ectopic bone formation capabiltity of Saos-2 and U2OS cells have been established. Saos-2 cell line, which presents many osteoblast genotype and phenotype, is a stable positive control for both in vitro and in vivo bone formation assessments. The measurement of ALP activity in both solid and liquid phases has been standardized. Both the genotype and phenotype of osteoblast lineage cells has been quantitatively assessed during the capability testing of ADSCs and PRP. Quantitative assessment of new bone formation and related protein markers in vivo has been successfully established through the testing of the biological properties of gamma irradiated morselized bone grafts. Conclusion: A comprehensive knowledge of the assessment of bone regeneration and formation in vitro and in vivo has been integrated and developed through years of study. A whole set of in vitro and in vivo approaches for the assessment of bone formation has been modified and standardized to best suit the different clinical applications. This thesis provides an outline of both in vitro and in vivo bone formation assessment and their clinical applications.

Le cartilage est principalement composé de collagène de type II sécrété par les chondrocytes. Nous avons montré que l'épissage alternatif de son gène Col2a1 était régulé de manière opposée par la BMP-2 et le TGF- 1 dans des chondrocytes embryonnaires de souris cultivés sur plastique. Par ailleurs, le traitement de chondrocytes à la BMP-2 nous a conduit à identifier un gène par RT-PCR différentielle, Bat (BMP-2 activated transcript), dont l'expression est stimulée par ce facteur de croissance. L'expression de ce gène favorise le phénotype des chondrocytes en culture, mais bloque leur hypertrophie. En outre, nous avons montré que la protéine Bat, très conservée au cours de l'évolution, joue un rôle dans la mise en place de l'axe dorso-ventral au cours du développement embryonnaire chez le poisson-zèbre, Danio rerio. Ce travail aura mis en évidence deux implications de la protéine Bat : dans la chondrogenèse et dans le développement précoce

Les conséquences de l’évolution démographique vers une population de plus en plus âgée ont un impact énorme sur l’économie en termes de santé. Vieillir en bonne santé est devenu un enjeu majeur et il est devenu primordial d’instaurer des politiques de prévention. La nutrition apparait comme l’une des stratégies préventives les plus prometteuses pour contrer la perte de masse osseuse liée à l’âge. Le couple Calcium/vitamine D ayant des effets limités,il est important de développer d’autres alternatives afin de préserver le capital osseux.L’étude de l’effet du collagène hydrolysé (H C) sur le tissu osseux est devenue une thématique montante ces dernières années. Plusieurs études in vivo ont permis de démontrer que l’ingestion de collagène hydrolysé améliorait le développement de la masse osseuse. L’objectif de cette thèse a été d’étudier la pertinence de l’utilisation du H C dans la mise au point de stratégies nutritionnelles pour la prévention de l’ostéoporose.Après avoir mis en évidence l’intérêt du H C sur l’activité d’ostéoblastes murins MC3T3-E1, nous avons évalué son effet in vivo sur des souris ovariectomisées : modèle d’ostéoporose post-ménopausique liée à une carence oestrogénique. Nous avons montré que le collagène hydrolysé d’origine bovine (B H C) contribue à limiter la perte osseuse induite par l'ovariectomie. Cette étude in vivo nous a permis de constater que la limitation de la perte osseuse induite est liée à un effet spécifique du B H C et dépendante de la longueur du peptide.La deuxième partie de nos travaux a consisté à mettre en lumière le rôle des métabolites de collagène sur l’activité des cellules osseuses. Ainsi, le BHC favorise la survie et la différenciation ostéoblastique et inversement pour les ostéoclastes en modulant le ratio OPG / RANKL.En résumé, nos résultats confirment le potentiel du collagène hydrolysé dans la prise en charge de la perte osseuse liée à l’âge et soulignent l’intérêt du collagène hydrolysé dans la conception de stratégies novatrices pour le management des troubles ostéoporotiques
Due to demographic changes healthy aging has become a major issue and interest in prevention has raised accordingly. In this context, nutrition has become one of the most promising strategies to counter age-related complications, including bone loss. Effects of hydrolyzed collagen (HC) on the bone tissue have focused attention in recent years. Several in vivo studies have demonstrated that hydrolyzed collagen ingestion may preserve bone mass. The aim of this thesis was to study the relevance of HC use in the development of nutritional strategies for osteoporosis prevention. The interest of HC was first investigated on the activity of murine osteoblasts MC3T3-E1. Then, we evaluated itseffect on ovariectomized mice: a postmenopausal osteoporosis model related to estrogenic deficiency. We have shownthat bovine hydrolyzed collagen (BHC) helps reducing bone loss induced by ovariectomy and that observed effect wasspecific and not related to protein content and that it was dependent on the mean molecular weight of the peptide. The second part of our work emphasizes the role of collagen metabolites on bone cell activity. We demonstrated that enreiched serum in BHC promotes osteoblast survival and differentiation, conversely inhibits osteoclastogenesis andmodulates the OPG / RANKL ratio. In summary, our results confirm the benefit of hydrolyzed collagen on bone and point out the value of hydrolyzed collagen in the design of innovative strategies for the management of osteoporotic disorders

Bone marrow mesenchymal stem cells (BM-MSCs) are pluripotent cells that can differentiate into a variety of non-hematopoietic tissues. They also maintain healthy heamatopoiesis by providing supportive cellular microenvironment into BM. In this thesis, MSCs were characterized in terms of their morphological, immunophenotypical and differentiation properties. Then, they were examined by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy together with hierarchical clustering, and FTIR microspectroscopy. In the first part of this study, global structural and compositional changes in BM-MSCs during beta thallasemia major (

Indiana University-Purdue University Indianapolis (IUPUI)
Communication between bone cells is important for the maintenance of bone mass. Although osteocytes are deeply embedded within the mineralized matrix, they are essential for the regulation of osteoblast and osteoclast functions. However, the intracellular proteins that control the morphology and function of osteocytes, and their ability to communicate with other bone cells are still unknown. Kalirin is a novel multi-domain GTP exchange factor (GEF) protein that activates the RhoGTPases. Recently, we found that 14 week old female Kalirin knockout (Kal-KO) mice exhibit a 45% decrease in trabecular bone density and have significantly lower cortical area, perimeter, thickness and polar cross-sectional moment of inertia (-12.6%, -7.2%, -7.6% and -21.9%, respectively) than WT mice. Kalirin was found to be expressed in osteoclasts and osteoblasts but its expression and function in osteocytes is currently unclear. We examined the role of Kalirin on the morphology and function of osteocytes. Primary osteocytes were isolated by sequential collagenase digestions from long bones (femurs and tibias) of 10-week old WT and Kal-KO mice. Immunofluorescent staining revealed Kalirin was localized to the perinuclear region of primary osteocytes and MLO-Y4 cells, and was detected along the cytoplasmic processes of primary osteocytes. We also examined primary osteocytes isolated from the long bones of Kal-KO and WT mice for changes in the length and number of cytoplasmic processes. Kal-KO osteocytes were found to express significantly fewer cytoplasmic processes per cell (3.3±0.21) than WT osteocytes (4.7±0.3). In addition, the cytoplasmic processes of Kal-KO osteocytes were shorter (79.5±4.6 µm) than those observed for WT osteocytes (85.4±3.6 µm) (p <0.01). Quantitative PCR revealed the expression of mRNA for the three major Kalirin isoforms (Kal-7, Kal-9, Kal-12) in primary osteocytes and in MLO-Y4 cells. Moreover, the mRNA levels of osteoprotegerin (OPG) and SOST, which are important for controlling osteoclast differentiation and Wnt signaling leading to bone formation, respectively, were reduced in Kal-KO osteocytes. Next, the role of Kalirin in osteocyte morphology and function was further examined. Treatment of MLO-Y4 cells for 5 days with nerve growth factor, which is known to activate Kalirin in neurons, or over-expression of the Ser-Thr kinase domain of Kal-12, promoted cytoplasmic process elongation and upregulated phosphorylated ERK and RhoA levels. Together, these results suggest that Kalirin controls osteocyte morphology and function in part by regulating cytoskeletal remodeling and the activity of ERK and RhoA. Furthermore, Kalirin may control the bone remodeling cycle by regulating osteocyte signaling to osteoclasts and osteoblasts.

前言：間充質幹細胞容易擴增並且能分化為成骨細胞、軟骨細胞和脂肪細胞，並且能對炎症、感染和損傷做出反應，並且遷移到相應的組織部位。這些特性使間充質幹細胞成為骨骼組織工程學中非常重要的細胞來源。外周血間充質幹細胞是一種存在於血液中的間充質幹細胞，而主要的間充質幹細胞存在與骨髓中，被稱之為骨髓間充質幹細胞。在我們實驗室之前的研究中通過DNA微陣列發現外周血間充質幹細胞中很多基因的表達與骨髓間充質幹細胞有很大區別。這其中的一些基因可能參與調控間充質幹細胞的分化和歸巢，我們從中挑選了三個變化比較明顯的基因--CRBP1, N-cadherin和 SOX11做進一步研究。本研究的目的在於研究CRBP1, N-cadherin和 SOX11在骨髓間充質幹細胞分化和遷移中的作用及相關機理。
方法：培養的骨髓間充質幹細胞來源於6-8周大小的SD大鼠。細胞的表型經過多分化潛能測試（成骨分化，成脂分化和成軟骨分化）和流式細胞儀檢驗。克隆大鼠的CRBP1, N-cadherin和SOX11基因到慢病毒載體。而且還設計了針對CRBP1和 N-cadherin的shRNA及非特異性對照shRNA。慢病毒由暫態轉染293FT細胞產生。細胞遷移實驗採用了BD Falcon的細胞遷移系統（cell culture insert）。實驗採用了定量PCR、免疫共沉澱、western雜交和雙螢光報告檢驗。對於體內實驗，細胞經感染帶有不同基因的病毒後，種植到Si-TCP材料並移植到裸鼠皮下。8周後，收集樣品進行組織學和免疫組織學分析。最後，我們建立了大鼠的股骨開放式骨折模型，並在4天后將SOX11基因修飾的間充質幹細胞通過心臟注射打到大鼠體內。4周後，收集股骨骨折樣品並進行microCT、力學測試和組織學分析。
結果：CRBP1過表達能夠促進骨髓間充質幹細胞的成骨分化潛能，並能抑制其成脂分化。進一步的機理研究表明CRBP1可以通過與RXRα的蛋白相互作用抑制RXRα誘導的β-catenin降解，從而維持β-catenin和磷酸化-ERK1/2在較高的水準，導致間充質幹細胞成骨能力增強；N-cadherin過表達可以促進間充質幹細胞的遷移，但是卻通過下調β-catenin和磷酸化ERK1/2抑制其成骨分化。過表達SOX11可以通過增強BMP信號通路促進三系分化。SOX11還可以通過啟動CXCR4的表達來促進細胞遷移。最後，在大鼠的股骨開放骨折模型上通過系統注射，我們證明穩定過表達SOX11的間充質幹細胞遷移到骨折部位的數量明顯增加。這些細胞到達骨折部位以後可以起始骨痂的鈣化，促進骨折的修復。
結論：本研究證明CRBP1, N-cadherin 和SOX11具有調節骨髓間充質幹細胞遷移和/或分化的功能。這些基因也許會成為幹細胞治療的新靶點。系統注射SOX11基因修飾的骨髓間充質幹細胞對於骨折修復可能具有較好的療效。本研究初步研究了CRBP1, N-cadherin 和SOX11在間充質幹細胞中的作用，為探討以間充質幹細胞為基礎的組織工程的某些新臨床應用提供了一些線索。
Introduction: Mesenchymal stem cells (MSCs) can be easily harvested, expanded, and have the capability of differentiating into osteoblasts, chondrocytes and adipocytes, and they can home to various tissues in response to stimuli such as inflammation, infection and injuries. MSCs are therefore valuable cell source for musculoskeletal tissue engineering. Peripheral blood-derived MSCs (PB-MSCs) are one kind of MSCs that reside in peripheral blood, whereas the main source of MSCs is bone marrow-derived MSCs (BM-MSCs). In our previous study, we found many genes were differentially expressed in the PB-MSCs compared to their counterpart BM-MSCs demonstrated by microarray analysis, among which the effects of CRBP1, SOX11 and N-cadherin on MSCs in terms of migration and differentiation are studied.
Methods: BM-MSCs and PB-MSCs were cultured from 6-8 weeks SD rats. The phenotypes of MSCs were characterized by tri-lineage (adipo-, osteo- and chondrogenic) differentiation and flow cytometry analysis. The genes encoding rat CRBP1, SOX11 and N-cadherin were cloned into lentiviral vectors respectively. shRNAs targeting CRBP1, N-cadherin, and one nonspecific shRNA were designed. Pseudo-lentivirus was produced by transient transfection of 293FT cells. Cell migration was examined using transwell insert culture system. Quantitative RT-PCR, CO-IP, western blot and dual-luciferase assay were employed in the studies. For in vivo study, MSCs transduced with different genes were seeded on Si-TCP scaffolds and implanted subcutaneously in nude mice. 8 weeks later, the samples were collected for histological and immunohistological analysis. Finally, an open femoral fracture model was established in 8-week old SD rats, SOX11-modified MSCs were injected at four days after fracture. At 4-week after MSCs injection, the femurs were collected for microCT, mechanical test and histological analysis.
Results: For CRBP1gene, our results showed that CRBP1 overexpression promoted osteogenic differentiation of BM-MSCs, while inhibited their adipogenic differentiation. We demonstrated that CRBP1 promoted osteogenic differentiation by inhibiting RXRα-induced β-catenin degradation through physical interactions, and maintaining β-catenin and pERK1/2 at higher levels. For N-cadherin gene, we found that N-cadherin overexpression promoted MSCs migration, and suppressed osteogenic potential of MSCs through inhibiting ERK and β-catenin signaling pathways. For SOX11 gene, we demonstrated that SOX11 overexpression enhanced the adipo-, osteo- and chondrogenic differentiation of BM-MSCs, through enhancing BMP signaling pathways. The migration capacity of BM-MSCs was also enhanced when Sox-11 was overexpressed, through activating CXCR4 expression. Finally, in the open femur fracture model we demonstrated that a larger number of SOX11-overexpressing BM-MSCs migrated to the fracture site, initiated earlier callus ossification and improved bone fracture healing quality.
Conclusions: This study demonstrated that CRBP1, N-cadherin and SOX11 gene can regulate the migration and/or differentiation potentials of BM-MSCs. These genes may become new therapeutic targets in stem cell therapy applications. Systemic administration of genetically modified SOX11-overexpressing BM-MSCs may be useful in promoting fracture healing. Overall, this study defined some unknown functions of CRBP1, N-cadherin and SOX11 in MSCs and shed the lights on some novel therapeutic implications for MSCs-based tissue engineering.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Xu, Liangliang.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 128-144).
Abstract also in Chinese.
Declaration --- p.i
Abstract --- p.ii
摘要 --- p.v
Acknowledgements --- p.vii
Chapter 1 --- p.1
Introduction --- p.1
Chapter 1.1 --- Mesenchymal stem cells --- p.2
Chapter 1.1.1 --- Characteristics of mesenchymal stem cells --- p.2
Chapter 1.1.2 --- Bone marrow- and peripheral blood-derived MSCs --- p.4
Chapter 1.1.3 --- Other tissue-derived MSCs --- p.5
Chapter 1.2 --- Adipogenesis of MSCs --- p.6
Chapter 1.3 --- Chondrogenesis of MSCs --- p.7
Chapter 1.4 --- Osteogenesis of MSCs --- p.8
Chapter 1.4.1 --- Regulators of osteogenesis --- p.9
Chapter 1.4.2 --- Stratergies for improving bone tissue engineering --- p.11
Chapter 1.5 --- Signaling pathways involved in osteogenesis --- p.13
Chapter 1.5.1 --- ERK signaling pathway --- p.14
Chapter 1.5.2 --- Wnt signaling pathway --- p.15
Chapter 1.5.3 --- BMP signaling pathway --- p.17
Chapter 1.6 --- Migration of MSCs --- p.20
Chapter 1.7 --- Fracture healing --- p.22
Chapter 1.8 --- Clinical application of MSCs --- p.23
Chapter 1.8.1 --- BM-MSCs vs. PB-MSCs --- p.24
Chapter 1.8.2 --- Autologous vs. Allogeneic MSCs transplantation --- p.25
Chapter 1.9 --- Scope of the present study --- p.26
Chapter 1.9.1 --- CRBP1 --- p.26
Chapter 1.9.2 --- N-cadherin --- p.27
Chapter 1.9.3 --- SOX11 --- p.27
Chapter 1.10 --- Experimental scheme --- p.29
Chapter 2 --- p.31
Comparison between PB-MSCs and BM-MSCs --- p.31
Chapter 2.1 --- Chapter introduction --- p.32
Chapter 2.2 --- Materials and methods --- p.33
Chapter 2.2.1 --- Cell culture --- p.33
Chapter 2.2.2 --- Flow cytometry --- p.33
Chapter 2.2.3 --- Adipogenic differentiation --- p.34
Chapter 2.2.4 --- Osteogenic differentiation --- p.34
Chapter 2.2.5 --- RNA Extraction and Real-time PCR --- p.34
Chapter 2.3 --- Results --- p.35
Chapter 2.3.1 --- Morphology of PB-MSCs --- p.35
Chapter 2.3.2 --- Cellular surface markers of BM-MSCs and PB-MSCs --- p.36
Chapter 2.3.3 --- Multi-differentiation potential of BM-MSCs and PB-MSCs --- p.38
Chapter 2.3.4 --- Target genes expression in BM-MSCs and PB-MSCs --- p.39
Chapter 2.4 --- Discussion and future work --- p.40
Chapter 3 --- p.41
Role of CRBP1 in Differentiation and Migration of MSCs --- p.41
Chapter 3.1 --- Chapter introduction --- p.42
Chapter 3.2 --- Materials and methods --- p.46
Chapter 3.2.1 --- Chemicals --- p.46
Chapter 3.2.2 --- Isolation and culture of BM-MSCs --- p.46
Chapter 3.2.3 --- RNA Extraction and Real-time PCR --- p.47
Chapter 3.2.4 --- Plasmid construction, transfection, production of lentivirus and infection --- p.48
Chapter 3.2.5 --- Osteogenic differentiation --- p.50
Chapter 3.2.6 --- Adipogenic differentiation --- p.50
Chapter 3.2.7 --- Western blot --- p.51
Chapter 3.2.8 --- Immunofluorescence labeling and fluorescence microscopy --- p.52
Chapter 3.2.9 --- Cell migration assay --- p.52
Chapter 3.2.10 --- Ectopic bone formation assay --- p.52
Chapter 3.2.11 --- Statistical analysis --- p.53
Chapter 3.3 --- Results --- p.53
Chapter 3.3.1 --- Transducing BM-MSCs with lentivirus carrying CRBP1 or shRNAs --- p.53
Chapter 3.3.2 --- CRBP1 accelerates osteogenesis of BM-MSCs via enhancing ERK1/2 and β-catenin pathways --- p.56
Chapter 3.3.3 --- CRBP1 stabilizes β-catenin by inhibiting RXRα-induced degradation --- p.58
Chapter 3.3.4 --- CRBP1 inhibits adipogenesis of BM-MSCs --- p.61
Chapter 3.3.5 --- CRBP1 overexpression has no effect on MSCs migration potential --- p.63
Chapter 3.3.6 --- CRBP1 promotes ectopic bone formation in vivo --- p.64
Chapter 3.4 --- Discussion --- p.66
Chapter 3.5 --- Future work --- p.73
Chapter 4 --- p.74
Role of N-cadherin in Differentiation and Migration of MSCs --- p.74
Chapter 4.1 --- Chapter introduction --- p.75
Chapter 4.2 --- Materials and methods --- p.78
Chapter 4.2.1 --- Chemicals --- p.78
Chapter 4.2.2 --- Isolation and culture of BM-MSCs --- p.78
Chapter 4.2.3 --- Plasmid construction, transfection, production of lentivirus and infection --- p.79
Chapter 4.2.4 --- Osteogenic differentiation and ALP activity assay --- p.81
Chapter 4.2.5 --- Western blot --- p.81
Chapter 4.2.6 --- Ectopic bone formation assay --- p.82
Chapter 4.2.7 --- Statistical analysis --- p.82
Chapter 4.3 --- Results --- p.83
Chapter 4.3.1 --- Expression of N-cadherin during osteogenesis in MSCs --- p.83
Chapter 4.3.2 --- N-cadherin overexpression inhibits osteogenesis through suppressing β-catein and ERK1/2 signaling pathways --- p.84
Chapter 4.3.3 --- N-cadherin silencing increases osteogenesis through enhancing β-catenin and ERK1/2 signaling pathways --- p.86
Chapter 4.3.4 --- N-cadherin promotes migration of MSCs --- p.87
Chapter 4.3.5 --- Cellular surface markers of SV40-immortalized MSCs --- p.89
Chapter 4.3.6 --- N-cadherin inhibits ectopic bone formation in vivo --- p.89
Chapter 4.4 --- Discussion --- p.91
Chapter 4.5 --- Future work --- p.94
Chapter 5 --- p.96
Role of SOX11 in Differentiation and Migration of MSCs --- p.96
Chapter 5.1 --- Chapter introduction --- p.97
Chapter 5.2 --- Materials and methods --- p.105
Chapter 5.2.1 --- Plasmid construction, transfection, production of lentivirus and infection --- p.105
Chapter 5.2.2 --- Cell culture --- p.106
Chapter 5.2.3 --- Luciferase reporter gene assay --- p.106
Chapter 5.2.4 --- Osteogenic differentiation and ALP activity assay --- p.106
Chapter 5.2.5 --- Adipogenic differentiation --- p.107
Chapter 5.2.5 --- Chondrogenic diffferentiation --- p.107
Chapter 5.2.6 --- Western blot --- p.108
Chapter 5.2.7 --- RNA Extraction and Real-time PCR --- p.108
Chapter 5.2.8 --- Cell migration --- p.110
Chapter 5.2.9 --- Ectopic bone formation --- p.110
Chapter 5.2.10 --- Fracture healing model and analysis --- p.111
Chapter 5.2.11 --- Statistical Analysis --- p.112
Chapter 5.3 --- Results --- p.112
Chapter 5.3.1 --- SOX11 is upregulated during osteogenesis of BM-MSCs --- p.112
Chapter 5.3.2 --- SOX11 promotes adipogenesis in BM-MSCs --- p.113
Chapter 5.3.3 --- SOX11 promotes migration of BM-MSCs --- p.114
Chapter 5.3.4 --- SOX11 promotes osteogenesis in BM-MSCs --- p.115
Chapter 5.3.5 --- SOX11 promotes chondrogenesis of MSCs --- p.117
Chapter 5.3.6 --- Mechanisms of how SOX11 regulates differentiation and migration of MSCs --- p.118
Chapter 5.3.7 --- SOX11-modified MSCs promote bone fracture healing in an open femur fracture rat model --- p.122
Chapter 5.4 --- Discussion --- p.126
Chapter 5.5 --- Future work --- p.131
Appendix --- p.153

Meng, Fanbiao.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 114-138).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Siu, Yeung Tung.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 65-75).
Abstracts in English and Chinese.
Title Page --- p.i
Abstract in English --- p.ii
Abstract in Chinese --- p.iii
Acknowledgement --- p.iv
Table of Contents --- p.v-viii
List of Figures --- p.ix
List of Tables --- p.x
Abbreviations --- p.xi-xii
Chapter Chapter 1 --- Introduction
Chapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6
Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1
Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2
Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3
Chapter 1.1.4 --- Other comitogens --- p.4
Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5
Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12
Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7
Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8
Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9
Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12
Chapter 1.3 --- Objective of this study --- p.13-15
Chapter Chapter 2 --- Materials and Methods
Chapter 2.1 --- Cell cultures --- p.16
Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18
Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17
Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17
Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17
Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20
Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18
Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19
Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19
Chapter 2.3.2.2 --- Total protein assay --- p.19
Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20
Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22
Chapter 2.4.1 --- Dialysis --- p.21
Chapter 2.4.2 --- Temperature treatment --- p.21
Chapter 2.4.3 --- Proteolysis --- p.22
Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26
Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22
Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23
Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23
Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23
Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24
Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24
Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25
Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25
Chapter 2.5.7 --- Visualization of autoradiography --- p.26
Chapter 2.5.8 --- Visualization by silver staining --- p.26
Chapter Chapter 3 --- Results
Chapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30
Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27
Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29
Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39
Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31
Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36
Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38
Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44
Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49
Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45
Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47
Chapter Chapter 4 --- Discussion
Chapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50
Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51
Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52
Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53
Chapter 4.5 --- Possible directions for future investigation --- p.55
Chapter 4.6 --- Conclusions --- p.56
Chapter Chapter 5 --- Appendices
Chapter 5.1 --- Reagents and solutiuons --- p.57-64
Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57
Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59
Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60
Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61
Chapter Chapter 6 --- References --- p.65-75

Indiana University-Purdue University Indianapolis (IUPUI)
Pyk2: Potential Regulator of Post-Menopausal Bone Loss H.W. LARGURA1,2*, P. ELENISTE2, S. HUANG2, S. LIU1, M. ALLEN3, A. BRUZZANITI2. 1Indiana University School of Dentistry Department Orthodontics and Oral Facial Development, 2Indiana University School of Dentistry Department of Oral Biology, 3Indiana University School of Medicine Department of Anatomy and Cell Biology, Indianapolis, Indiana, USA Osteoporosis is a pathologic condition of bone, commonly found in post-menopausal women, which occurs from an imbalance between bone formation and resorption. Following menopause, the bone resorbing activity of osteoclasts exceeds bone formation by osteoblasts, resulting in decreased trabecular and cortical bone and a subsequent decrease in bone mass. Reduced bone mass increases the risk of pathologic fracture of bones. Due to adverse effects associated with current treatment protocols for bone loss, alternative treatment modalities with reduced adverse effects are needed. Estrogen plays a role in maintaining balance in the bone remodeling cycle by controlling remodeling activation, osteoblast and osteoclast numbers, and their respective effectiveness in formation and resorption. With declining estrogen levels, this elegantly balanced interaction is altered and bone resorption exceeds bone formation, resulting in bone loss and increased bone fragility. Pyk2 is a protein tyrosine kinase that plays an important role in regulating bone resorption by osteoclasts, as well as osteoblast proliferation and differentiation. Deletion of the Pyk2 gene in mice leads to an increase in bone mass, in part due to dysfunctional osteoclast and osteoblast activity. In this study, we examined the role of Pyk2 in the effects of estrogen on bone mass. We used wild type (WT) and Pyk2 knock-out (KO) mice that had been ovariectomized (OVX) and treated with or without estrogen (E2)-releasing pellets. Control mice included sham OVX surgery receiving placebo pellet. We found that deletion of Pyk2 conferred increased bone mass in sham, OVX and OVX+E2 mice. In addition, Pyk2 KO mice supplemented with 17estradiol exhibited a marked increase in bone volume/trabecular volume, trabecular number, and trabecular thickness, but not cortical bone parameters compared to WT mice. Results of this study provide evidence for the role of Pyk2 in the effects of estrogen on bone mass. Understanding the role of Pyk2 in bone could lead to the development of new pharmaceutical targets for the treatment of bone loss associated with osteoporosis.

Yung, Lai Hang.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 93-115).
Abstract also in Chinese.
Declaration --- p.i
Acknowledgements --- p.ii
Abbreviations --- p.iii
Abstract in English --- p.v
Abstract in Chinese --- p.ix
Contents --- p.xi
Chapter Chapter I - --- Introduction
Chapter 1.1) --- Endothelial cells function --- p.1
Chapter 1.2) --- Oxidative stress in the vascular wall --- p.2
Chapter 1.2.1) --- Sources of ROS --- p.3
Chapter 1.2.2) --- Actions of ROS --- p.3
Chapter 1.2.2.1) --- Impaired endothelium-dependent vasodilatation --- p.3
Chapter 1.2.2.2) --- VSMC migration --- p.4
Chapter 1.2.2.3) --- Programmed cell death (cell apoptosis) --- p.4
Chapter 1.3) --- Endothelial cell apoptosis --- p.7
Chapter 1.3.1) --- Apoptosis and cardiovascular diseases --- p.7
Chapter 1.3.2) --- Mechanisms of endothelial cells apoptosis --- p.7
Chapter 1.3.2.1) --- What are caspases? --- p.8
Chapter 1.3.2.2) --- Death receptor-mediated apoptosis --- p.9
Chapter 1.3.2.3) --- Mitochondria-dependent pathway --- p.9
Chapter 1.3.3) --- Regulations of endothelial cells apoptosis --- p.10
Chapter 1.3.3.1) --- Oxidative stress --- p.10
Chapter 1.3.3.2) --- Shear Stress --- p.11
Chapter 1.3.3.3) --- Growth factors --- p.12
Chapter 1.3.3.4) --- NO --- p.12
Chapter 1.3.3.5) --- Inflammatory mediators --- p.13
Chapter 1.4) --- Mitogen activated kinases signaling in apoptosis --- p.15
Chapter 1.5) --- Bone morphogenic proteins (BMPs) --- p.17
Chapter 1.5.1) --- BMPs functions and cardiovascular system --- p.17
Chapter 1.5.2) --- BMPs signaling pathways --- p.18
Chapter 1.5.2.1) --- Smad-dependent pathway --- p.18
Chapter 1.5.2.2) --- MAPKs and SAPKs pathways --- p.19
Chapter 1.5.2.3) --- Antagonists of BMPs signaling --- p.20
Chapter 1.5.3) --- BMP4 and cardiovascular diseases --- p.20
Chapter 1.6) --- "Justification, long-term significance and objectives of the present project" --- p.23
Chapter Chapter II - --- Methods and Materials
Chapter 2.1) --- Animal handling --- p.24
Chapter 2.2) --- Endothelial cell isolation and culture --- p.24
Chapter 2.2.1) --- Primary culture of rat endothelial cells --- p.24
Chapter 2.2.2) --- Culture of human umbilical cord vein endothelial cells… --- p.25
Chapter 2.3) --- Apoptosis assessment --- p.25
Chapter 2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.25
Chapter 2.3.2) --- Cell death detection ELISA kit --- p.26
Chapter 2.3.3) --- Flow cytometry --- p.27
Chapter 2.4) --- Western blot analysis --- p.28
Chapter 2.4.1) --- Sample preparation --- p.28
Chapter 2.4.2) --- SDS-PAGE and transfer --- p.28
Chapter 2.5) --- DHE fluorescence --- p.29
Chapter 2.6) --- "Drugs, chemicals and other reagents" --- p.30
Chapter 2.6.1) --- Drugs and chemicals used in the present experiments --- p.30
Chapter 2.6.2) --- Reagents for Western blot analysis --- p.30
Chapter 2.6.3) --- Primary antibodies --- p.33
Chapter 2.7) --- Small interfering RNA experiment --- p.34
Chapter 2.8) --- Statistical analysis --- p.34
Chapter Chapter III - --- BMP4 induces endothelial cell apoptosis in ROS related p38 MAPK and JNK mediated caspase-3 dependent pathway
Chapter 3.1) --- Introduction --- p.35
Chapter 3.2) --- Methods and materials --- p.39
Chapter 3.2.1) --- Isolation and culture of endothelial cells --- p.39
Chapter 3.2.2) --- Drugs treatment --- p.39
Chapter 3.2.3) --- Assay for cell apoptosis --- p.40
Chapter 3.2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.40
Chapter 3.2.3.2) --- Cell death detection ELISA kit --- p.41
Chapter 3.2.3.3) --- Flow cytometric analysis --- p.41
Chapter 3.2.4) --- Western blot analysis --- p.41
Chapter 3.2.5) --- Dihydroethidium (DHE) staining --- p.42
Chapter 3.2.6) --- Statistical analysis --- p.42
Chapter 3.3) --- Results --- p.43
Chapter 3.3.1) --- Dose- and time-dependent effect of BMP4 --- p.43
Chapter 3.3.2) --- Role of caspases in apoptosis of RAECs and HUVECs --- p.43
Chapter 3.3.3) --- Roles of BMP4 and ROS in endothelial cell apoptosis --- p.44
Chapter 3.3.3.1) --- Noggin antagonism of BMP4-induced effect --- p.44
Chapter 3.3.3.2) --- NAD(P)H oxidase-mediated ROS production --- p.44
Chapter 3.3.3.3) --- Inhibition of endothelial cell apoptosis by ROS scavengers --- p.45
Chapter 3.3.4) --- Roles of MAPKs/SAPKs in BMP4-induced endothelial cell apoptosis --- p.45
Chapter 3.3.5) --- Relationship between ROS and MAPKs/SAPKs --- p.46
Chapter 3.3.6) --- Relationship between p38 MAPK and JNK --- p.46
Chapter 3.4) --- Discussion --- p.82
Chapter 3.4.1) --- Caspase-dependent pathways --- p.82
Chapter 3.4.2) --- Oxidative stress --- p.85
Chapter 3.4.3) --- Role of MAPKs activation in BMP4-induced endothelial cell apoptosis --- p.87
Chapter 3.4.4) --- ROS mediates BMP4-induced activation of MAPKs --- p.88
Chapter 3.4.5) --- Role of p38 MAPK in the activation of JNK 1 --- p.89
Chapter 3.5) --- Concluding remarks --- p.91
References --- p.93
Publications and Awards --- p.116

Wong Cheuk Ying.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2006.
Includes bibliographical references (leaves 101-124).
Abstracts in English and Chinese.
Abstract --- p.i
摘要 --- p.iv
Acknowledgement --- p.vi
List of Abbreviations --- p.vii
Chapter Chapter 1. --- General Introduction --- p.1
Chapter Chapter 2. --- Methods --- p.4
Chapter 2.1 --- Measurement of cAMP and cGMP in VSMCs --- p.4
Chapter 2.1.1 --- Cell culture --- p.4
Chapter 2.1.2 --- Enzyme-immunoassay colorimetric measurement for cAMP and cGMP --- p.5
Chapter 2.1.3 --- Statistical analysis --- p.6
Chapter 2.2 --- Measurement of apoptosis in VSMCs and bone marrow-derived stem cells --- p.6
Chapter 2.2.1 --- Cell culture --- p.6
Chapter 2.2.2 --- Hoechst33258 --- p.7
Chapter 2.2.3 --- Cell Death ELISA plus --- p.7
Chapter 2.2.4 --- Protein extraction and Western blot analysis of PKG expression --- p.8
Chapter 2.2.5 --- Statistical analysis --- p.9
Chapter 2.3 --- Measurement of cell proliferation in VSMCs and bone marrow-derived stem cells --- p.9
Chapter 2.3.1 --- Cell culture --- p.9
Chapter 2.3.2 --- Cell count --- p.10
Chapter 2.3.3 --- MTT assay --- p.11
Chapter 2.3.4 --- BrdU-(5`Bromo-2-deoxyuridine) ELISA colorimetric assay --- p.11
Chapter 2.3.5 --- Statistical analysis --- p.12
Chapter Chapter 3. --- Effects of hyperglycemia on CGRP-induced cAMP response in VSMCs
Chapter 3.1 --- Introduction --- p.13
Chapter 3.2 --- Results --- p.18
Chapter 3.3 --- Discussion --- p.22
Chapter Chapter 4. --- Role of cGMP and protein kinase G in regulation of apoptosis in VSMCs
Chapter 4.1 --- Introduction --- p.26
Chapter 4.2 --- Results --- p.30
Chapter 4.3 --- Discussion --- p.44
Chapter Chapter 5. --- Role of protein kinase G in regulation of proliferation in VSMCs
Chapter 5.1 --- Introduction --- p.55
Chapter 5.2 --- Results --- p.58
Chapter 5.3 --- Discussion --- p.67
Chapter Chapter 6. --- Effects of aging and eNOS- and iNOS-gene deletion (using eNOS- and iNOS-knockout mice) on apoptosis of VSMCs
Chapter 6.1 --- Introduction --- p.73
Chapter 6.2 --- Results --- p.76
Chapter 6.3 --- Discussion --- p.79
Chapter Chapter 7. --- Role of protein kinase G in regulation of apoptosis and proliferation of bone marrow stromal stem cells
Chapter 7.1 --- Introduction --- p.81
Chapter 7.2 --- Results --- p.84
Chapter 7.3 --- Discussion --- p.92
Chapter Chapter 8. --- Overall discussion --- p.95
Chapter Chapter 9. --- References --- p.101

Bone morphogenetic proteins (BMPs) are a large family of multi-functional secreted signalling molecules. Previously BMP2/4 were shown to inhibit skin pigmentation by downregulating tyrosinase expression and activity in epidermal melanocytes. However, a possible role for other BMP family members and their antagonists in melanogenesis has not yet been explored. In this study we show that BMP4 and BMP6, from two different BMP subclasses, and their antagonists noggin and sclerostin were variably expressed in melanocytes and keratinocytes in human skin. We further examined their involvement in melanogenesis and melanin transfer using fully matched primary cultures of adult human melanocytes and keratinocytes. BMP6 markedly stimulated melanogenesis by upregulating tyrosinase expression and activity, and also stimulated the formation of filopodia and Myosin-X expression in melanocytes, which was associated with increased melanosome transfer from melanocytes to keratinocytes. BMP4, by contrast, inhibited melanin synthesis and transfer to below baseline levels. These findings were confirmed using siRNA knockdown of BMP receptors BMPR1A/1B or of Myosin-X, as well as by incubating cells with the antagonists noggin and sclerostin. While BMP6 was found to use the p38MAPK pathway to regulate melanogenesis in human melanocytes independently of the Smad pathway, p38MAPK, PI3-K and Smad pathways were all involved in BMP6-mediated melanin transfer. This suggests that pigment formation may be regulated independently of pigment transfer. These data reveal a complex involvement of regulation of different members of the BMP family, their antagonists and inhibitory Smads, in melanocytes behaviour.

BMP signaling has a crucial role in skin development and homeostasis, whereas molecular mechanisms underlying its involvement in regulating gene expression programs in keratinocytes and fibroblasts remain largely unknown. We show here that several BMP ligands, all BMP receptors, and BMP-associated Smad1/5/8 are expressed in human primary epidermal keratinocytes and dermal fibroblasts. Treatment of both cell types by BMP-4 resulted in the activation of the BMP-Smad, but not BMP-MAPK pathways. Global microarray analysis revealed that BMP-4 treatment induces distinct and cell type-specific changes in gene expression programs in keratinocytes and fibroblasts, which are far more complex than the effects of BMPs on cell proliferation/differentiation described earlier. Furthermore, our data suggest that the potential modulation of cell adhesion, extracellular matrix remodeling, motility, metabolism, signaling, and transcription by BMP-4 in keratinocytes and fibroblasts is likely to be achieved by the distinct and cell-type-specific sets of molecules. Thus, these data provide an important basis for delineating mechanisms that underlie the distinct effects of the BMP pathway on different cell populations in the skin, and will be helpful in further establishing molecular signaling networks regulating skin homeostasis in health and disease.