A thesis submitted to the Faculty of Humanities, University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the degree of Doctor of Philosophy December 2015 / Introduction South Africa is characterised by numerous dichotomies and diversities, within which its two-tier healthcare system operates. An under-resourced state sector serves a majority of the population and a resource-intensive private sector serves a small minority. Within the constitutional framework of human rights and distributive justice there are nevertheless expectations of fair and equal access to healthcare services. There is furthermore an expectation of quality care across the health system, in spite of a number of systemic challenges related to staff and equipment shortages, unrealistic working hours and poor working conditions. Organ transplant is available to different degrees within the South African healthcare sector. Whilst transplant programmes are burgeoning internationally, cadaver transplant numbers in South Africa have decreased over recent years as donor organs have become increasingly scarce. Current research suggests that these challenges to transplant in South Africa arise from aspects of personal and cultural beliefs, illegal transplant practices and resource constraints - which all serve to compromise the ethical implementation of transplant services in the two-tier healthcare system. The impact of interprofessional communication and transplant professional–patient communication has not been previously researched in South Africa. However, research into other healthcare issues has shown that communication is vital to the ethical provision of healthcare services, especially those which involve patient-centeredness and multidisciplinary interaction. Transplant involves a significant amount of communication within a particularly large network of recipients and their families, cadaver donor families, living donors and a range of transplant professionals. This communication seems a vital part of the transplant process, disseminating information which role-players need in order to promote favourable outcomes. Given the extensive networks involved in the transplant process, communication would seem to be a fertile area for research. This study aimed to explore communication in organ transplant in Gauteng province, South Africa. It considered both interprofessional communication and communication with patients as this took place within the hierarchical healthcare system and throughout the transplant process. An ethics of care framework was utilised in order to account for the expectations of care which South Africans confer upon their health system. Methods The study took place in the Gauteng province of South Africa across six healthcare institutions. Both the state and the private sector were equally represented. Altogether, thirty in-depth interviews with transplant professionals, two focus groups with transplant coordinators, two interviews with cadaver donor families, and one focus group with living kidney donors, were conducted. Thematic analysis and triangulation of the data utilising Braun and Clarke’s (2006) principles revealed three main themes relating to context, communication with patients, and interprofessional communication Findings The South African transplant context is complex and multifaceted, shaped by both the patients’ expectations of care and the transplant professionals’ perceptions of care. These expectations and perceptions are influenced by personal beliefs, suspicions of biomedicine, the media, and resource inequalities which pose challenges to accessing transplant services. The transplant context is characterised by ethical dilemmas relating to distributive justice, as questions about resource distribution and allocation of donor organs are raised. Transplant communication is influenced by context and varies depending upon role-players in transplant and the different phases of transplant. Demands for care by those hoping to receive an organ had a noticeable influence on transplant professional-potential recipient communication in the pre-transplant phase, a period when emotions of desperation and uncertainty were prominent. By the time recipients had received their organ and entered the more stable post-transplant phase, a relationship of trust developed in which communication was regular and caring roles seemed fulfilled. The opposite trend was evident in communication between transplant professionals and donor families. This was characterised by notions of care in the pre-transplant phase, contrasting with a perception amongst donor families that care was sometimes overlooked in the post-transplant phase - a time often imbued with chronic uncertainty. Even in the pre-transplant phase numerous ethical issues surrounding autonomy, decision-making and informed consent proved to complicate and challenge transplant communication. Interprofessional communication was shaped by hierarchical institutional organisation, a lack of continuity of care, and resource constraints, all of which challenged transplant professionals seeking to provide care, and sometimes resulted in aggressive interchanges. The pressure to procure an organ timeously – which could result in patient care and professional respect being somewhat disregarded – could so compromise interprofessional communications that moral distress was created. Furthermore, as a result of miscommunications, an ethical vacuum where the best interests of patients in the transplant process were not, apparently, a foremost consideration, was identified. Conclusion Transplant is a highly complex process requiring a number of different communication styles and skills and accompanied by intricate ethical challenges. Although transplant professionals seemed cognisant of the need for careful communication, inequalities, resource scarcity and conflict intervened to create a space for moral distress and uncertainty in which communication was affected, and the provision of care was the casualty. Appraising results within an ethics of care framework suggests that transplant in Gauteng cannot be considered to be a process fully informed by the imperative of care. The ethics of care proved to be a helpful framework for understanding transplant communication in Gauteng because of the way it accounts for interpersonal relationships - fundamental to the transplant process - whilst also emphasising the importance of resources necessary to provide good care. It was concluded that in the current environment, where there is little legal direction or political buy-in, transplant in Gauteng will be unable to reach its full potential. / MT2016

INTRODUCTION Cardiovascular diseases (CVD) are more common in kidney transplant recipients (KTRs) than in the general population. The high incidence of CVD in the KTRs can be attributed to traditional risk factors, additional risk factors associated with graft dysfunction and those specifically related to transplantation. Carotid intima-media thickness (cIMT) is a proven surrogate of atherosclerosis; it correlates with vessel pathology and is precisely imaged using ultrasound technology. This study was aimed at determining the prevalence and predictors of cardiovascular risk among KTRs at the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and to examine the relationship between cardiovascular risk factors and carotid intima media thickness. METHODS Patients aged 18 years and above who received a kidney transplant at the CMJAH between January 2005 and December 2009 were recruited. A questionnaire that captured cardiovascular risk factors was administered. Patients records were assessed for information on their post transplant follow up. All patients had echocardiography and carotid doppler done for measurement of intima-media thickness. The Framingham Risk Score was used to categorize patients into low, moderate, high risk and very high risk groups. Results were analyzed using statistical package for social sciences (SPSS) version 17, p value of 0.05 was considered significant. RESULTS One hundred (KTRs) 63 male (63%) and 37 female (37%) were recruited ranging in age from 19 to 70 years, with a mean age of 42.2 ± 12.42. Thirty six patients (36%) were found to have high cardiovascular risk. Multiple regression showed proteinuria (p = 0.022), higher cumulative steroid dosage (p = 0.028), elevated serum triglycerides (p = 0.04) and the presence of plaques in the carotid artery (p = 0.012) as predictors of higher cardiovascular risk.Carotid intima-media thickness correlates with higher CVD risk. Fourteen patients (14%) had a carotid artery plaque. Twenty five patients (25%) had cIMT of >0.7 mm. CONCLUSION Kidney transplant recipients in CMJAH were found to have high cardiovascular risk (36%) and carotid intima-media thickness correlates with this high CVD risk. Routine follow up of KTRs should include measurement of cIMT as it provides a simple non-invasive assessment of subclinical atherosclerosis.

No description available.

Introduction : l’infection par le cytomégalovirus (CMV) humain est la plus fréquente des infections après greffe d'organe. Des effets indirects à long terme sont fortement suspectés mais restent encore largement incompris. Notre travail de thèse s’est intéressé à mieux comprendre les conséquences de l’infection du donneur par le CMV sur la réponse immunitaire du receveur et sur le devenir de son greffon. Résultat : nous avons initialement rapporté que l'infection du donneur (D+) par le CMV est un facteur de risque indépendant de perte de fonction du greffon rénal particulièrement si le receveur est également séropositif avant la greffe (D+R+ comparé aux D+R-). Le risque est fortement majoré en cas de mésappariement complet en HLA de classe I entre le receveur et son donneur. Puis nous avons analysé le rôle du greffon infecté dans le développement de la réponse lymphocytaire anti-CMV. Nous avons rapporté pour la première fois que la superinfection CMV entraine une augmentation du nombre de LT CD8 répondeurs spécifiques du CMV à distance de la transplantation, à condition que le donneur et le receveur partagent des identités HLA-I. De plus nous avons montré chez le sujet D+R- que l'expansion des lymphocytes T CD8 anti CMV restreints par le HLA-A2 nécessite l'expression de ce HLA par le donneur. Ces résultats ensemble indiquent le rôle des cellules du donneur dans l’inflation des LT CD8 anti-CMV à distance de la greffe. Dans un troisième travail, nous avons montré qu’un polymorphisme du gène de Programmed Cell Death 1 (PD-1.3) influe sur la survie des greffons rénaux et pulmonaires D+, les patients porteurs de l’allèle variant A ayant un meilleur pronostic que les patients homozygotes GG. Nos données indiquent aussi que les patients homozygotes AA ont un plus grand nombre de lymphocytes anti-CMV producteurs d'IFN-ɣ, suggérant que ce polymorphisme pourrait être associé à une dysfonction de la réponse immunitaire spécifique anti-CMV. Conclusion : ensemble ces données suggèrent pour la première fois que la qualité de la réponse lymphocytaire cytotoxique anti-CMV pourrait être importante pour contrôler la réplication virale dans le greffon et les lésions induites par cette dernière. Ainsi nous proposons deux mécanismes à l’origine du développement des lésions liées à l'infection à CMV dans le rein: défaut de reconnaissance des cellules allogéniques infectées en cas de mésappariement complet en HLA de classe I et une dysfonction LT CD8 anti-CMV. / Background: cytomegalovirus (CMV) is the leading cause of viral infection after solid organ transplantation. Despite a large body of literature, the effects of chronic cytomegalovirus (CMV) infection on graft outcome remain controversial.Results: we first reported that donor CMV infection (D+) was an independent risk factor of kidney graft loss, especially in pretransplant infected recipients (R+). In addition, we observed that full HLA-I mismatching was an important determinant of this risk. In a second study, we focused on effect of donor CMV infection on anti-CMV specific immune response. We reported that CMV superinfection greatly increased the number of anti-CMV IFN-ɣ-producing T cells, provided that donor and recipient shared at least one HLA-I identity. Then in D+R- HLA-A2-expressing recipients, we compared the number of anti-CMVpp65 CD8+T cells restricted by HLA-A2 depending on whether the donor expressed or not HLA-A2. Patients who received non-HLA-A2 kidneys developed very few anti-CMVpp65 T-cells restricted by HLA-A2 as compared to those who received an HLA-A2-expressing kidney. This result indicated that presentation of CMV peptides by donor cells was crucial to stimulate the expansion of pp65-specific memory CD8 T cells. Finally, we established that a SNP in the Programmed Cell Death 1 gene (PD-1.3) influenced D+ kidney and lung transplants survival, while it was also associated with the level of anti-CMV specific T-cell response. Conclusion: taken together, these data suggest that anti-CMV specific immune response is pivotal to control infection within the graft and prevent subsequent organ damages. We propose two mechanisms to explain effect of donor CMV infection on graft outcome: (1) inability of anti-CMV CD8 T cells to recognize donor-infected cells in case of full HLA-I mismatching, (2) dysfunction of anti-CMV CD8 T cells after transplantation in some patients, highlighted by our genetic study.

Lau Fung Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 107-130). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Pang Sai Yau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves [87-90]). / Chapter chapter one： --- introduction / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- biology of cancellous bone grafts --- p.2 / Chapter 1.2.1 --- Biology of bone graft incorporation --- p.2 / Chapter 1.2.1.1 --- Osteogenesis --- p.2 / Chapter 1.2.1.2 --- Vascularization --- p.3 / Chapter 1.2.1.3 --- Osteoinduction --- p.3 / Chapter 1.2.1.4 --- Osteoconduction --- p.4 / Chapter 1.2.2 --- Histological changes of bone grafts after bone transplantation --- p.4 / Chapter 1.2.2.1 --- Histologic pictures of cancellous autograft --- p.4 / Chapter 1.2.2.2 --- Histologic pictures of cancellous bone allograft --- p.5 / Chapter 1.2.2.3 --- Summary of the histologic changes of bone grafts --- p.5 / Chapter 1.3 --- application of cancellous bone grafts --- p.6 / Chapter 1.3.1 --- Principles of graft incorporation --- p.6 / Chapter 1.3.1.1 --- Operative site --- p.6 / Chapter 1.3.1.2 --- Graft material --- p.7 / Chapter 1.3.1.2.1 --- Autogenic cancellous bone --- p.8 / Chapter 1.3.1.2.2 --- Autogenic cortical bone --- p.9 / Chapter 1.3.2.2.3 --- Vascularized autogenic bone grafts --- p.9 / Chapter 1.3.2.2.4 --- Bone allografts --- p.10 / Chapter 1.3.2.2.5 --- Graft adjuncts and substitutes --- p.11 / Chapter 1.3.2.3 --- Systemic factors influencing gaft incorporation --- p.13 / Chapter 1.3.2.4 --- Local factors influencing graft incorporation --- p.13 / Chapter 1.3.3 --- Bone graft complications --- p.13 / Chapter 1.3.4 --- Placement of a graft --- p.14 / Chapter 1.3.5 --- Bone graft harvesting --- p.15 / Chapter 1.3.5.1 --- Iliac bone graft --- p.15 / Chapter 1.3.5.2 --- Femoral head bone allograft --- p.16 / Chapter 1.4 --- Application of flow conductance concept in a cancellous bone graft --- p.17 / Chapter 1.4.1 --- Physical structure of cancellous bone --- p.17 / Chapter 1.4.2 --- Porosity of cancellous bone --- p.17 / Chapter 1.4.3 --- Flow conductance concept --- p.18 / Chapter chapter two： --- material and method / Chapter 2.1 --- Transplantation of cancellous bone graft - Rabbit model --- p.19 / Chapter 2.1.1 --- Preparation of porcine cancellous bone graft --- p.19 / Chapter 2.1.1.1 --- Bone drilling --- p.19 / Chapter 2.1.1.2 --- Defat and freeze-dry --- p.20 / Chapter 2.1.2 --- Flow conductance measurement --- p.21 / Chapter 2.1.2.1 --- Porosity measurement --- p.21 / Chapter 2.1.2.2 --- Conductance measurement --- p.24 / Chapter 2.1.3 --- Rabbit model --- p.26 / Chapter 2.1.4 --- Methods of assessment --- p.29 / Chapter 2.1.4.1 --- Intraosseous pressure measurement --- p.29 / Chapter 2.1.4.2 --- Histologic study --- p.30 / Chapter 2.1.4.3 --- Blood flow study - use of tracer microspheres --- p.30 / Chapter 2.2 --- Flow conductance measurement of human cancellous bone --- p.34 / Chapter chapter three： --- results / Chapter 3.1 --- Results of the effects of various conductance of the grafts on bone healing in animal model --- p.38 / Chapter 3.1.1 --- Intraosseous pressure measurement --- p.38 / Chapter 3.1.2 --- Histological study --- p.40 / Chapter 3.1.3 --- Blood flow study of cancellous bone grafts --- p.52 / Chapter 3.2 --- Human specimens study --- p.62 / Chapter chapter four： --- discussion / Chapter 4.1 --- Discussion of the results in vivo study --- p.66 / Chapter 4.1.1 --- Intraosseous pressure measurement - a baseline study --- p.66 / Chapter 4.1.2 --- Effects of flow conductance of porcine cancellous grafts on bone regeneration --- p.67 / Chapter 4.1.2.1. --- Threshold conductance --- p.67 / Chapter 4.1.2.2. --- Histological score --- p.68 / Chapter 4.1.3 --- Discussion of graft healing from the blood flow study --- p.70 / Chapter 4.1.3.1 --- Tibia blood supply in relation to bone healing --- p.70 / Chapter 4.1.3.2 --- Effect of different flow conductance on blood flow changes in the tibia-graft structure --- p.72 / Chapter 4.1.4 --- "Comparison of length, porosity and conductance as the parameter on graft healing" --- p.74 / Chapter 4.2 --- Discussion on human bone specimens study --- p.76 / Chapter 4.3 --- General discussion --- p.78 / Chapter 4.3.1 --- The limitation of the animal model --- p.78 / Chapter 4.3.2 --- Some problems related to the clinical aspects --- p.79 / Chapter chapter five： --- conclusion --- p.81

by Leung Kwok-sui. / Thesis (M.D.)--Chinese University of Hong Kong, 1991. / Bibliography: leaves 112-121. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xvii / CHAPTER / Chapter I --- Introduction --- p.1 / Chapter II --- The Evolution of the Fixation Devices for Peritrochanteric Fractures --- p.13 / Chapter II.1 --- Patho-anatomy and Biomechanics of Peritrochanteric Fractures --- p.14 / Chapter II.2 --- A Review of the Implants Available for Peritrochanteric Fractures --- p.19 / Chapter III --- Methodology --- p.39 / Chapter III.1 --- Biomechanical Analysis of the Gamma Nail and the Dynamic Hip Screw --- p.40 / Chapter III. 1.1 --- The Testing Machine and Equipments --- p.40 / Chapter III. 1.2 --- The Design of the Testing Jig --- p.41 / Chapter III. 1.3 --- The Test of the Sliding Characteristics of the Gamma Lag Screw --- p.43 / Chapter III. 1.4 --- The Biomechanical Behaviour of Gamma Nail Fixation and the Dynamic Hip Screw Fixation in Cadaveric Femora --- p.47 / Chapter III.2 --- Randomized Prospective Trial of Gamma Nail and Dynamic Hip Screw in the Treatment of Peritrochanteric Fractures Among Geriatric Patients --- p.51 / Chapter III.3 --- Anthropometric Study of Chinese Femora with Respect to the Design of the Gamma Nail and the Application of the Anthropometric Data for the Modification of the Gamma Nail --- p.55 / Chapter III.4 --- Method of Statistical Analysis --- p.61 / Chapter IV --- Results --- p.62 / Chapter IV. 1 --- The Biomechanical Analysis of the Gamma Nail and the Dynamic Hip Screws --- p.63 / Chapter IV. 1.1 --- The Sliding Characteristics of Gamma Lag Screw --- p.63 / Chapter IV. 1.2 --- The Biomechanical Behaviours of Gamma Nail and the Dynamic Hip Screw --- p.65 / Chapter IV.2 --- Clinical Studies --- p.70 / Chapter IV.2.1 --- Randomised Prospective Trial of Gamma Nail and Dynamic Hip Screw in the Treatment of Peritrochanteric Fractures --- p.70 / Chapter IV.2.2 --- Comparisons between the Clinical Use of Standard and Modified Gamma Nails --- p.75 / Chapter IV.3 --- The Anthropometric Study of the Proximal Chinese Femora and the Application of Anthropometric Data on the Modification of Gamma Nails --- p.78 / Chapter V --- Discussion --- p.85 / Chapter VI --- Conclusion --- p.109 / REFERENCES --- p.112 / APPENDICES --- p.122 / Chapter Appendix 1 --- Data Record Sheet of Retrospective Analysis of Geriatric Fractures Treated in the Prince of Wales Hospital --- p.123 / Chapter Appendix 2 --- Calibration Curve of the Linear Variable Differential Transformer (LVDT) --- p.125 / Chapter Appendix 3 --- Data Record Sheets for the Randomized Prospective Trial of Gamma Nail and Dynamic Hip Screw --- p.126 / Chapter Appendix 4 --- Operative Procedure of Dynamic Hip Screw and Gamma Nail - A Summary and Modifications --- p.132 / Chapter Appendix 5 --- Methodology for the Measurement of the Sliding of the Lag Screw of Gamma Nail on Serial X-ray Films --- p.138 / Chapter Appendix 6 --- Results of X-ray Measurement and Bone Densitometry Measurement of Cadaveric Femora --- p.142 / Chapter Appendix 7 --- Extra Data from the Results of the Randomized Prospective Trial of Gamma Nail and Dynamic Hip Screw --- p.144 / Chapter Appendix 8 --- Results of Anthropometric Study of 30 Chinese Femora --- p.145

幹細胞療法是近年的研究熱點之一，然而幹細胞在組織修復中的實際應用受到移植後幹細胞存活率低的制約，約80% 的幹細胞在移植至組織後不能存活。 人類臍帶血管周皮 (HUCPV) 幹細胞為多功能間充質幹細胞移植提供豐富的細胞來源。 在合適的誘導環境下，它們具有向多種間充質細胞系分化的能力。 與從骨髓或臍帶血中提取的間充質幹細胞比較，人類臍帶血管周皮幹細胞的體外增殖更為容易。 在本研究中，我們從人類臍帶血管周圍組織中分離人類臍帶血管周皮幹細胞，並採用流式細胞技術分選細胞表面標記物CD34、CD45呈陰性同時CD44 、CD90、 CD105、 CD146呈陽性的HUCPV細胞。HUCPV細胞在體外培養以及三維支架的環境下具有分化為骨和軟骨的能力。 / 在本研究中，我們主要研究腦和生殖器官表達基因（BRE）在HUCPV細胞中的功能。 BRE蛋白與其他已知蛋白的同源性均不高，目前尚未鑑定出任何功能性的結構域。 至今為止，BRE基因的已知功能大多數是通過對腫瘤模型的研究發現的。 據報導，BRE能夠提高DNA損傷的腫瘤細胞的存活率，但BRE在幹細胞中的作用仍不清楚。 我們發現，當HUCPV細胞分化後，其BRE的表達水平降低。 此外，利用BRE-siRNA降低HUCPV細胞中BRE基因的表達，能夠促進HUCPV細胞向骨和軟骨分化的進程。 因此，我們假設BRE對維持HUCPV細胞的幹細胞功能具有重要的作用。 由於經過BRE基因沉默處理的HUCPV細胞與對照組相比並無顯著的表型差別，我們採用微陣列（microarray）以及比較蛋白組學的方法研究兩者間的區別，從而找出BRE基因的功能以及可能涉及BRE的信號通路。 / 通過微陣列技術，我們深入地分析了BRE基因表達沉默後HUCPV細胞的轉錄組。 在經過BRE基因沉默處理的HUCPV細胞中，我們發現與維持幹細胞多向分化潛能有關的OCT4、 FGF5和FOXO1A等基因的表達顯著下調。 另外，BRE基因的沉默能夠影響表觀遺傳調控基因以及TGF-β 信號通路組成部件的表達，而TGF -β 信號通路是維持幹細胞自我更新的重要通路。 這些結果提示，BRE作為一個重要的調控因子，在維持HUCPV細胞的多向分化潛能的同時能夠防止細胞分化。 / 在比較蛋白組學的研究中，我們發現BRE基因的沉默能夠降低細胞骨架結合蛋白的表達，例如actin, annexin II 及 tropomyosin。 此外，我們利用免疫共沉澱的方法證明了BRE蛋白與actin及 annexin II蛋白直接結合。 細胞骨架的改變可能為HUCPV細胞的分化提供了一個有利的環境，因而BRE基因的沉默能夠促進HUCPV細胞向骨和軟骨分化。 支持這一推論的其中一個依據是Lim et al., 2000; Solursh, 1989; Zhang et al., 2006，文獻報導肌動蛋白多聚化抑製劑能夠促進軟骨形成的過程。 綜上所述，本研究為進一步研究BRE基因在HUCPV細胞中的功能以及與BRE直接作用的蛋白打下了基礎。 / Stem cells therapy has gained considerable attention in recent years. However, the practical use of stem cells for tissue repair has been hindered due to their low survival rate after grafting into tissues, for approximately 80% of the stem cells died after implantation. Human umbilical cord perivascular (HUCPV) stem cells offer a new and rich resource of multipotent mesenchymal stem cells. These cells possess the ability to differentiate into various mesenchymal cell lineages when induced. HUCPV cells can be more easily amplified in culture than mesenchymal stem cells extracted from bone marrow or umbilical cord blood. In this study, HUCPV cells were isolated from the perivascular regions of human umbilical cords. The HUCPV cells were sorted using flow cytometer for CD34⁻, CD44⁺, CD45⁻, CD90⁺, CD105⁺ and CD146⁺ surface markers. These HUCPV cells were found to be capable of differentiating into osteogenic lineage in monolayer culture and chondrogenic lineage in pellet culture. These cells were also found to be capable of differentiating into osteogenic and chondrogenic lineage in silk fibroin which acted as three-dimensional scaffolds for the cells to grow on. / The function of the Brain and Reproductive Organ-Expressed (BRE) gene in the context of HUCPV cells was investigated. The BRE protein shares no homology with any other known gene products and contains no known functional domain. To date, most of what we know about the function of this gene has been conducted in the tumor model. It has been reported that BRE can enhance the cellular survival of cancer cells following DNA damage. The role of BRE in stem cells has never been examined. We have established that BRE expression was down-regulated when HUCPV cells started to differentiate. In addition, silencing BRE expression, using BRE-siRNA, in HUCPV cells could accelerate osteogenic and chondrogenic differentiation. Hence, we hypothesized that BRE played an important role in maintaining the stemness of HUCPV cells. Because there was a lack of phenotypic difference between the BRE-silenced HUCPV cells and cells transfected with the control-siRNA, we decided to profile these cells using microarray and proteomic analyses. The aim was to elucidate the function of the BRE gene and establish whether BRE was involved in any signaling pathways. / In the microarray analysis, we examined the transcriptome of HUCPV cells in response to BRE-silencing in depth. Amongst the genes that we identified were significantly down-regulated by BRE-silencing and involved in the maintenance of pluripotency in ES cells were OCT4, FGF5 and FOXO1A. BRE-silencing also altered the expression of epigenetic genes and also components of the TGF-β signaling pathway. This pathway is crucially involved in maintaining stem cell self-renewal. Therefore, we propose that BRE acts like a modulator that promotes stemness and at the same time inhibits the differentiation of HUCPV cells. / In the comparative proteomic study, BRE-silencing resulted in decreased expression patterns of cytoskeletal binding proteins such as actin, annexin II and tropomyosin. In addition, co-immunoprecipitation experiments revealed that the BRE protein can bind directly with actin and annexin II. It is possible that altering the cytoskeleton may provide a favorable environment for HUCPV cells to differentiate. This may explain why we were able to accelerate osteogenic and chondrogenic differentiation following BRE-silencing. In support of the view, it has been reported that chondrogenesis could be enhanced after cells have been treated with actin polymerization inhibitors (Lim et al., 2000; Solursh, 1989; Zhang et al., 2006). In sum, our studies provide an insight into the function of the BRE gene in HUCPV cells and the proteins that BRE can directly act on. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Elve. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 135-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.viii / List of Figures --- p.ix / List of Tables --- p.xiii / Table of Abbreviations --- p.xiv / Contents --- p.xviii / Chapter 1 --- p.1 / Literature Review --- p.1 / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.2 --- Embryonic stem cells (ESCs) --- p.2 / Chapter 1.3 --- Epiblast-derived stem (EpiS) cells --- p.2 / Chapter 1.4 --- Somatic stem cells (SSCs) --- p.3 / Chapter 1.5 --- Induced pluripotent stem (iPS) cells --- p.5 / Chapter 1.6 --- Human umbilical cord perivascular (HUCPV) cells --- p.7 / Chapter 1.7 --- CD146 --- p.8 / Chapter 1.8 --- Stem cell senescence --- p.9 / Chapter 1.9 --- Brain and reproductive organ-expressed (BRE) protein --- p.12 / Chapter 1.10 --- Stem cell self-renewal --- p.14 / Chapter 1.11 --- Apoptosis --- p.16 / Chapter 1.12 --- Stem cell niche --- p.21 / Chapter 1.13 --- Stem cell homing --- p.22 / Chapter 1.14 --- Objective --- p.22 / Chapter 2 --- p.24 / Accelerated osteogenic and chondrogenic differentiation of HUCPV cells by modulating the expression of BRE --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Rationale --- p.27 / Chapter 2.3 --- Materials and Methods --- p.27 / Chapter 2.3.1 --- Extraction of HUCPV cells from umbilical cord --- p.27 / Chapter 2.3.2 --- Cell culture condition --- p.28 / Chapter 2.3.3 --- Flow cytometry analysis and cell sorting --- p.28 / Chapter 2.3.4 --- In vitro osteogenic differentiation --- p.29 / Chapter 2.3.5 --- In vitro chondrogenic differentiation --- p.29 / Chapter 2.3.6 --- Alcian blue staining --- p.29 / Chapter 2.3.7 --- Alizarin red S staining --- p.30 / Chapter 2.3.8 --- Immunofluorescence analysis --- p.30 / Chapter 2.3.9 --- Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) --- p.31 / Chapter 2.3.10 --- Transfection with siRNA --- p.35 / Chapter 2.3.11 --- Microarray --- p.35 / Chapter 2.3.12 --- Cell lysis and immunoprecipitation --- p.36 / Chapter 2.3.13 --- SDS-PAGE and Western blot --- p.36 / Chapter 2.3.14 --- Isoelectric focusing and 2-dimensional gel electrophoresis --- p.37 / Chapter 2.3.15 --- Migration (wound healing) assay --- p.38 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- HUCPV cells were capable to differentiate into osteoblasts and chondrocytes --- p.38 / Chapter 2.4.2 --- BRE expression is down-regulated when HUCPV cells begins to differentiate --- p.40 / Chapter 2.4.3 --- Silencing of BRE expression accelerates induction of osteogenesis and chondrogenesis --- p.40 / Chapter 2.4.4 --- Microarray analysis of BRE-silenced HUCPV cells --- p.42 / Chapter 2.4.4.1 --- Stemness factors --- p.43 / Chapter 2.4.4.2 --- Epigenetic regulation --- p.43 / Chapter 2.4.4.3 --- Signaling pathways crucial for stemness maintenance --- p.44 / Chapter 2.4.4.4 --- TGF-β signaling --- p.44 / Chapter 2.4.4.5 --- FGF signaling --- p.44 / Chapter 2.4.4.6 --- NOTCH signaling --- p.45 / Chapter 2.4.4.7 --- WNT signaling --- p.46 / Chapter 2.4.4.8 --- Homeobox transcription factors (HOX) --- p.46 / Chapter 2.4.4.9 --- Cell cycle regulation --- p.47 / Chapter 2.4.4.10 --- Chemokines and cytokines regulation --- p.48 / Chapter 2.4.4.11 --- Apoptosis --- p.49 / Chapter 2.4.5 --- BRE-silencing alters the cellular proteome of HUCPV cells --- p.50 / Chapter 2.4.5.1 --- BRE-silencing alters the cytoskeletal binding proteins of HUCPV cells --- p.51 / Chapter 2.4.5.2 --- BRE-silencing alters the expressions of stemness-related proteins in HUCPV cells --- p.52 / Chapter 2.4.5.3 --- BRE-silencing alters the expressions of apoptosis-related proteins in HUCPV cells --- p.53 / Chapter 2.5 --- Discussion --- p.86 / Chapter 2.5.1 --- Microarray study discussion --- p.87 / Chapter 2.5.2 --- Proteomic study discussion --- p.89 / Chapter 3 --- p.93 / Replicative senescence alters the transcriptome and proteome of HUCPV cells --- p.93 / Chapter 3.1 --- Introduction --- p.93 / Chapter 3.2 --- Materials and methods --- p.93 / Chapter 3.3 --- Results --- p.93 / Chapter 3.3.1 --- Microarray analysis of aged HUCPV cells --- p.94 / Chapter 3.3.1.1 --- Stemness factors --- p.95 / Chapter 3.3.1.2 --- Epigenetic regulation --- p.96 / Chapter 3.3.1.3 --- Senescence associated markers --- p.96 / Chapter 3.3.1.4 --- Chemokines and cytokines regulation --- p.97 / Chapter 3.3.1.5 --- Matrix metalloproteinases regulation --- p.97 / Chapter 3.3.1.6 --- WNT signaling --- p.98 / Chapter 3.3.1.7 --- Toll-like receptor signaling pathway --- p.98 / Chapter 3.3.2 --- Proteomic profiling of aged HUCPV cells --- p.98 / Chapter 3.4 --- Discussion --- p.117 / Chapter 3.4.1 --- Aging alters the transcriptome of HUCPV cells --- p.117 / Chapter 3.4.2 --- Aging alters the proteome of HUCPV cells --- p.118 / Chapter 4 --- p.121 / Osteogenic and chondrogenic differentiation capacities of HUCPV cells in silk fibroin scaffold --- p.121 / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.2 --- Materials and methods --- p.121 / Chapter 4.2.1 --- Extraction of silk fibroin --- p.121 / Chapter 4.2.2 --- Fabrication of porous silk fibroin scaffold --- p.122 / Chapter 4.2.3 --- Scanning electron microscopy --- p.123 / Chapter 4.2.4 --- Cell culture --- p.123 / Chapter 4.3 --- Results --- p.124 / Chapter 4.4 --- Discussion --- p.132 / Chapter 5 --- p.133 / Conclusions --- p.133 / References --- p.135

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