No / Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3’UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

Ng, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-131). / Abstracts in English and Chinese. / Thesis Abstract --- p.i / 論文摘要 --- p.vi / Acknowledgements --- p.x / Abbreviations --- p.xii / Thesis Content --- p.xv / List of Figures --- p.xix / List of Tables --- p.xxi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Haematopoiesis --- p.1 / Chapter 1.2 --- Leukemia --- p.2 / Chapter 1.3 --- Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) --- p.3 / Chapter 1.3.1 --- Epidemiology --- p.4 / Chapter 1.3.2 --- Causes and risk factors --- p.4 / Chapter 1.3.3 --- Clinical features --- p.6 / Chapter 1.3.4 --- Morphology --- p.6 / Chapter 1.4 --- Classification of BCP-ALL --- p.7 / Chapter 1.4.1 --- Immunophenotyping --- p.7 / Chapter 1.4.2 --- Cytogenetics and molecular genetics --- p.9 / Chapter 1.5 --- Prognostic factors --- p.13 / Chapter 1.6 --- Current treatments of BCP-ALL --- p.15 / Chapter Chapter 2 --- Literature Review --- p.18 / Chapter 2.1 --- Cytogenetics abnormalities in BCP-ALL --- p.18 / Chapter 2.1.1 --- Chromosomal translocation --- p.18 / Chapter 2.1.2 --- Aneuploidy --- p.21 / Chapter 2.2 --- Epigenetic aberrations --- p.21 / Chapter 2.2.1 --- DNA methylation --- p.22 / Chapter 2.2.2 --- Mechanism of DNA Methylation in Transcription Repression --- p.23 / Chapter 2.3 --- DNA Methylation in Normal Haematopoiesis --- p.25 / Chapter 2.4 --- DNA Methylation in Haematological Malignancies --- p.26 / Chapter 2.4.1 --- DNA methylation in ALL --- p.26 / Chapter 2.4.2 --- DNA methylation in BCP-ALL --- p.29 / Chapter 2.5 --- Angiogenesis in pathogenesis of acute leukemias --- p.30 / Chapter 2.6 --- Thrombospondin-1 (TSP-1) --- p.32 / Chapter 2.6.1 --- Structure of TSP-1 --- p.33 / Chapter 2.6.2 --- The role of TSP-1 in tumorigenesis --- p.34 / Chapter 2.6.3 --- TSP-1 mediates the activation of TGFβ --- p.36 / Chapter 2.6.4 --- TSP-1 mediates TGFβ-induced Apoptosis --- p.37 / Chapter 2.6.5 --- Association of TGFβ with normal haematopoiesis and haematological malignancies progression --- p.40 / Chapter 2.6.6 --- TSP-1 Induced Apoptosis via its Receptor CD36 --- p.42 / Chapter 2.6.7 --- THBS1 promoter hypermethylation and its association with tumorigenesis --- p.43 / Chapter 2.6.8 --- Effect of THBS1 aberrant methylation on TGFp --- p.45 / Chapter Chapter 3 --- Rationale of Study --- p.47 / Chapter Chapter 4 --- Materials and Methods --- p.52 / Chapter 4.1 --- Patient sample --- p.52 / Chapter 4.2 --- Cell lines --- p.52 / Chapter 4.3 --- Mononuclear cells isolation --- p.53 / Chapter 4.4 --- THBS1 promoter hypermethylation analysis --- p.54 / Chapter 4.4.1 --- DNA extraction from mononuclear cells and cell lines --- p.54 / Chapter 4.4.2 --- Bisulfite conversion --- p.55 / Chapter 4.4.3 --- Methylation specific PCR (MSP) --- p.55 / Chapter 4.5 --- Quantification of THBS1 mRNA expression --- p.57 / Chapter 4.5.1 --- RNA extraction --- p.57 / Chapter 4.5.2 --- Reverse transcription PCR --- p.58 / Chapter 4.5.3 --- Real-time RT-PCR --- p.58 / Chapter 4.6 --- Determination of plasma TSP-1 level --- p.59 / Chapter 4.7 --- TSP-1 treatment --- p.60 / Chapter 4.8 --- Flow cytometry analysis --- p.60 / Chapter 4.8.1 --- Annexin-V analysis --- p.60 / Chapter 4.8.2 --- Cell fixation --- p.61 / Chapter 4.8.3 --- Analysis of Caspase-3 activation --- p.62 / Chapter 4.8.4 --- "Analysis of TGFβ downstream pathway activation: Phosphorylation of Smad2/3, JNK and p38" --- p.62 / Chapter 4.9 --- Determination ofTGF-β expression --- p.63 / Chapter 4.10 --- Statistical analysis methods --- p.64 / Chapter Chapter 5 --- Results / Chapter 5.1 --- THBS1 methylation statuses in BCP-ALL patients and cell lines --- p.66 / Chapter 5.2 --- Correlation of THBS1 methylation statuses and clinico- pathological features in BCP-ALL patients --- p.68 / Chapter 5.3 --- Association of THBS1 methylation and THBS1 mRNA expression --- p.69 / Chapter 5.4 --- Effect of TSP-1 treatment on apoptosis level of BCP-ALL cells --- p.72 / Chapter 5.4.1 --- Annexin-V assay --- p.72 / Chapter 5.4.2 --- Caspase-3 activation assay --- p.75 / Chapter 5.5 --- THBS1 methylation and activation of secreted TGFβ --- p.78 / Chapter 5.6 --- Effect of TSP-1 treatment on activation of TGFβ --- p.80 / Chapter 5.7 --- The involvement ofTGFβ activation in TSP-1 induced apoptosis in BCP-ALL --- p.82 / Chapter 5.8 --- The association of TGFβ signaling pathway activities with THBS1 methylation --- p.86 / Chapter Chapter 6 --- Discussion --- p.91 / Chapter 6.1 --- THBS-1 promoter hypermethylation in BCP-ALL cell lines and patients: Correlation with expression and clinico-pathological profile --- p.93 / Chapter 6.1.1 --- THBS1 promoter hypermethylation status in childhood BCP-ALL --- p.93 / Chapter 6.1.2 --- THBS1 methylation as prognostic markers --- p.94 / Chapter 6.1.3 --- "Association of THBS1 methylation status, mRNA expression and TSP-1 protein expression in childhood BCP-ALL" --- p.96 / Chapter 6.2 --- Study of the correlation of TSP-1 induced apoptosis with the THBS1 promoter methylation status --- p.99 / Chapter 6.3 --- Elucidation of the molecular mechanisms of TSP-1 induced apoptosis: study of the involvement of TGFβ activation --- p.103 / Chapter 6.3.1 --- Latent TGFβ activation by TSP-1 in BCP-ALL and association with THBS1 methylation status --- p.103 / Chapter 6.3.2 --- TSP-1 induced cell death through activation ofTGFβ --- p.105 / Chapter 6.3.3 --- TSP-1 induced apoptotic signals via TGFβ signaling pathway --- p.107 / Chapter 6.4 --- Limitation of study --- p.113 / Chapter 6.5 --- Future studies --- p.114 / Chapter 6.5.1 --- Continuation study in TSP-1 induced TGFβ-mediated pathways --- p.114 / Chapter 6.5.2 --- Microarray analysis --- p.115 / Chapter 6.6 --- TSP-1 in treatment of childhood BCP-ALL --- p.115 / Chapter Chapter 7 --- Conclusion --- p.117 / Reference --- p.118

Angiogenesis is the growth of new blood vessels from existing vasculature; it requires the breakdown of existing blood vessel walls followed by the migration and proliferation of endothelial cells to form the new vessels. It is a complex process that is regulated by many pro- and anti-angiogenic factors and the roles of some of these factors are still unclear. Angiogenesis is a key feature of many pathological conditions including cancer, polycystic ovary syndrome and endometriosis so is an area of great research interest. There are several methods currently available for the study of angiogenesis, both in vitro and in vivo, and whilst all of these methods have enhanced understanding of angiogenesis, they also have limitations. The ovary is an excellent model for the study of angiogenesis as it undergoes intense vascular morphogenesis in a cyclical manner. The female reproductive system is unique as no other healthy adult tissue undergoes spontaneous angiogenesis. The tissues in the ovary undergo constant remodelling during both folliculogenesis and the formation and regression of the corpus luteum. Blood vessels are recruited from the ovarian stroma at the preantral stage to form vascular sheaths, in the thecal layer, which surround the developing follicle and supply nutrients, hormones and allow gaseous exchange. As follicular development progresses to the antral stage, when gonadotrophin-dependence is established, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Previous research into ovarian angiogenesis has focussed on the corpus luteum but the mechanisms of the regulation of angiogenesis during folliculogenesis need further elucidation. The work in this thesis aims to develop and utilise an in vitro angiogenesis assay using the culture of intact preantral and early antral follicles to provide a new approach to the study of follicular angiogenesis. During the course of this thesis this assay was utilised to investigate the effect of various factors on follicular angiogenesis and ovarian function. The role of the putative anti-angiogenic factor thrombospondin-1 (TSP-1) in the regulation of physiological angiogenesis was investigated using the in vitro angiogenesis assay developed during the course of this thesis and the role of TSP-1 in normal ovarian function was investigated using the culture of isolated granulosa cells. The results suggest that TSP-1 is able to inhibit angiogenesis and that it has an extravascular role in the ovary, in vitro. These findings were extended to an in vivo angiogenesis model where follicular angiogenesis was assessed by quantitative immunohistochemistry for bromodeoxyuridine and the endothelial cell marker CD31. The extravascular role for TSP-1 was also further investigated in vivo and was assessed by quantitative immunohistochemistry for activated caspase-3. The results confirmed the findings of the in vitro study, indicating that TSP-1 has antiangiogenic action and acts to clear non-dominant follicles from the ovary through the induction of atresia. Vascular endothelial growth factor (VEGF) is the main factor involved in stimulating angiogenesis and many advances have been made into elucidating the role, and the mechanisms of action, of VEGF on angiogenesis. Angiopoietin-1 (Ang-1) is considered to be one of the main factors acting in concert with VEGF to stabilise new blood vessels and its role in angiogenesis has been the subject of much discussion and controversy. This thesis investigates the effects of Ang-1 on follicular angiogenesis and development, using the in vitro angiogenesis assay, granulosa cell culture and RNA knockdown experiments. The results have shown that Ang-1 can induce follicular angiogenesis at high doses and that at low doses stimulates prosurvival pathways and inhibits apoptotic mediators. This thesis describes a novel in vitro culture system for the study of angiogenesis in ovarian follicles. Using this system the effects of various factors on follicular angiogenesis and on follicle development and survival have been investigated. Investigations into the mechanisms of action of these factors have also been performed. These studies have improved understanding of the regulation of follicular angiogenesis and have indicated extravascular roles for angiogenic factors in the ovary. Since angiogenesis is a key feature of many pathological conditions, the ability to manipulate angiogenesis and to investigate and quantify the effects of proor anti-angiogenic compounds may have important clinical implications.

Introduction: Annually, an estimated 2.5 million traumatic brain injuries (TBI) occur in the United States, of which, over 50,000 result in deaths. Currently, 5.3 million Americans are living with neurological dysfunction secondary to TBIs leading to a $60 billion dollar cost in medical expenses and productivity losses. To date, there are limited treatments available to cure or ease the morbidity of TBI. Despite preventative efforts, traumatic brain injuries (TBI) occur at a staggering rate and it is estimated that 15-20% of survivors develop persistent post-traumatic neurological impairment. The purposed source of neurological dysfunction is a result of circuit reorganization when the brain rebuilds itself. After diffuse TBI, rodents have been shown to develop a late-onset, gain-of-function sensory sensitivity to whisker stimulation; similar to phonophobia and photophobia experienced by human TBI survivors. This morbidity coincides with evidence of post-TBI circuit reorganization, however the etiology of post-traumatic neurological impairment remains largely unknown. Thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) are heavily expressed during pediatric neuronal synapse development. Expression of TSPs, however declines with age. Mechanistically during development, TSP mediates synaptogenesis via bindingα2δ-1 subunit of the voltage-gated calcium channel receptor (α2δ-1). After neurological insult, re-expression of TSPs has been demonstrated and experimental modulation of the TSP/α2δ-1 interaction has led to changes in morbidity. We therefore hypothesize that experimental diffuse TBI will result in re-expression of TSPs, which will be synchronous with increases in synaptic markers in the thalamocortical whisker circuit. Methods: Adult male Sprague-Dawley rats underwent sham or moderate midline fluid percussion brain injury. At multiple time points over 2-months post-injury, expression of TSPs and synaptic markers were quantified from thalamocortical circuit (ventroposterior medial thalamus (VPM), primary somatosensory barrel fields (S1BF)) biopsies using qPCR and automated capillary westerns, respectively. Results: TSP-1 gene expression and protein levels increase in the VPM during the first week after injury. Gene expression of TSP-1 did not significantly change over time in the S1BF, however, there was a significant increase in protein levels in the first and second weeks after injury. No significant changes were demonstrated in synaptic markers in the VPM over the time course. TSP-1 protein levels demonstrated a similar multimodal response to synaptic markers in the S1BF.Conclusion: Re-expression of TSP-1 and synchronous changes in synaptic marker supports a role for TSP-1 mediated synaptogenesis after experimental diffuse TBI in the S1BF. These data positions us for future investigation of pharmacological inhibition of TSP-mediated synaptogenesis after TBI; which may represent a prophylactic strategy against circuit reorganization and neurological dysfunction after TBI.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Lymphocytes, the principal cells of the immune system, perform the immune function throughout the body by their unique capacity to circulate in blood stream and lymphatic vessels and migrate in lymphoid and non-lymphoid tissues. The mechanisms regulating lymphocyte adhesion and migration, interactions with cells and components within the extracellular matrix are not fully understood. The aim of this work has been to elucidate molecular mechanisms governing T lymphocyte adhesion and migration by endogenous molecules. The studies presented in this thesis have shown that thrombospondin-1 (TSP-1) is expressed in T lymphocytes with a high turnover, manifested by variable cell surface expression, and is regulated by SDF-1a, adhesion to fibronectin and collagen type IV. The TSP-1 binding site of calreticulin (CRT), spanning amino acid 19-32, was shown to be a major triggering factor for T cell migration within a three-dimensional collagen type 1 matrix. The chemokine SDF-1a stimulated migration via a calreticulin-TSP-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous TSP-1 elicited a motogenic signal to the T cells through the C-terminal domain of TSP-1 and its cell surface receptor integrin-associated protein (IAP, CD47). Inhibition experiments of ligand binding of CD91 by receptor associated protein (RAP) and small interfering RNA technology indicated that CD91 is an important factor in TSP-1-mediated T cell adhesion and migration. These results unveil an autocrine mechanism of CRT-TSP-1-CD47-CD91 interaction for the control of T cell motility and migration within 3D extracellular matrix substrata. The data demonstrated that T cell adhesion and migration are sequential events governed by a series of interacting cell surface molecules comprising a CRT-TSP-1-CD47-CD91 pathway where endogenous TSP-1 functions as the hub. Ligation of the CD3/T cell antigen receptor complex determines T cell adhesion through this mechanism. CRT interaction with the N-terminal domain of TSP-1 elicits cytoplasmic spreading, and augments adhesion, while a counter-adhesive motogenic pathway, triggering interaction of the C-terminal domain of TSP-1, induces migration. CD91-dependent internalization of TSP-1 is a crucial event of this motogenic pathway. In conclusion, the studies provide a novel mechanism governing T cell adhesion and migration within extracellular matrix substrata.

Abstract Cardiac remodeling is defined as changes in the size, shape and function of the heart, caused most commonly by hypertension-induced left ventricular (LV) hypertrophy and myocardial infarction (MI). It is characterized by changes in cellular and extracellular compartments regulated by e.g. neurohumoral and inflammatory factors. In the present study the expression of novel, load induced factors, thrombospondin (TSP)-1 and -4, matrix Gla protein (MGP), tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14, was investigated during cardiac remodeling. Their expression in the heart was characterized using experimental models of pressure overload, hypertensive hypertrophy and MI, and the effect of hypertrophic agonists and cellular stretch was studied in vitro. The effect of beta-blocker treatment on TSP expression was also examined. TSP-1 and -4 were rapidly upregulated in response to pressure overload, and the induction of TSP-4 gene expression was attenuated in hypertrophied heart. After MI, TSP-1 and -4 mRNA and TSP-1 protein levels were increased, and the induction was attenuated by metoprolol. TSP-1 and -4 expression correlated with natriuretic peptide expression and LV remodeling after MI. In hypertensive hypertrophy, only TSP-4 expression decreased after metoprolol treatment and was correlated with LV remodeling. MGP gene expression was increased in response to pressure overload and MI both in the early and late phase of cardiac remodeling. MGP protein levels were increased in the acute phase of post-MI remodeling and in hypertensive hypertrophy. In vitro, angiotensin II increased MGP gene expression in myocytes and fibroblasts, whereas expression decreased in response to mechanical stretch. In response to increased cardiac load Fn14 expression was upregulated both acutely and chronically while TWEAK expression remained relatively constant. Fn14 localized mainly to fibroblasts in the inflammatory area while TWEAK localized to myocytes and endothelial cells. In myocytes, Fn14 expression was induced by hypertrophic agonists and mechanical stretch in contrast to stabile or decreased TWEAK expression. This study provides new insights into the expression of the studied novel factors in cardiac remodeling. The distinct expression of TSPs in pressure overload and post-MI suggests that TSP-1 and -4 may have unique roles in the remodeling process. The results also imply that MGP is part of the common gene program of hypertrophic remodeling in vivo and contributes to the molecular basis of cardiac hypertrophy. Finally, the study demonstrates differential regulation of TWEAK and Fn14 expression in the heart and emphasizes the importance of Fn14 as a mediator of TWEAK/Fn14 signaling and as a potential target of therapeutic interventions.

Autism spectrum disorders (ASDs) are a diverse class of neurodevelopmental disorders with various aberrant cellular phenotypes such as dysfunctional neurotransmission and irregular neuronal morphology. ASDs have a broad underlying genetic background with many genes linked to their etiology. UBE3A has been identified as a top gene candidate associated with ASD, and overexpression of UBE3A via copy-number variation confers hallmark ASD behaviors in humans and transgenic mice. Our previous work revealed that synapse formation was negatively affected in the Ube3A-ASD mouse model (Ube3A 2X Tg, or simply “2X Tg”). However, the cellular and molecular mechanisms underlying the synaptic dysregulation remain unknown. We sought to identify a cell-type specific mechanism by which these morphological changes were conferred. We found that selective overexpression of Ube3A in neurons failed to induce changes in dendritic spine maturation. In contrast, overexpression of Ube3A in astrocytes resulted in alterations in spines and synapses. Further, we identified thrombospondin-2 (TSP2), a secreted astrocytic glycoprotein promoting synaptogenesis and spinogenesis, is involved in the defective spine maturation. Ube3A overexpression confers a loss of transcriptional down-regulation of TSP2 in astrocytes, and the medium of astrocyte cultures with Ube3A overexpression was sufficient to trigger spine changes similar to that observed in mixed cultures that globally overexpress Ube3A. Importantly, depletion of TSP2 promoted similar loss of dendritic spine maturation, whereas supplement of TSP2 to 2X Tg astrocyte media was able to rescue the spine defects. Furthermore, overexpression of Ube3A in an astrocyte-specific manner recapitulated aberrant dendritic spine maturation as well as autism-like behaviors displayed in 2X Tg mice. Collectively, these findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels.

No description available.

Leung, Winnie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 155-164). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / Abbreviations --- p.xii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Clinical management of Cancer --- p.2 / Chapter 1.2 --- Multidrug resistance --- p.8 / Chapter 1.3 --- Aim of study --- p.14 / Chapter Chapter 2 --- Identification of gene contributing to multidrug resistance in human uterine sarcoma cells --- p.16 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Material and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Cell lines --- p.20 / Chapter 2.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.20 / Chapter 2.2.1.3 --- Gene expression assay reagents --- p.22 / Chapter 2.2.1.4 --- Western blotting reagents --- p.24 / Chapter 2.2.1.5 --- MTT assay reagents --- p.29 / Chapter 2.2.1.6 --- Apoptosis analysis by flow cytometry reagents --- p.29 / Chapter 2.2.2 --- Metho --- p.ds / Chapter 2.2.2.1 --- Cell Culture --- p.31 / Chapter 2.2.2.2 --- MTT assay --- p.32 / Chapter 2.2.2.3 --- Gene expression essay (RT-PCR) --- p.33 / Chapter 2.2.2.4 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.37 / Chapter 2.2.2.5 --- Quantification of doxorubicin uptake by flow cytometry --- p.40 / Chapter 2.2.2.6 --- Apoptosis analysis by flow cytometry --- p.41 / Chapter 2.3 --- Results --- p.4 / Chapter 2.3.1 --- Cytotoxicity of doxorubicin on SA and DX5 cells --- p.43 / Chapter 2.3.2 --- mRNA expression of multidrug resistance related genes in SA and DX5 cells --- p.46 / Chapter 2.3.3 --- P-glycoprotein expression in SA and DX5 cells --- p.49 / Chapter 2.3.4 --- Doxorubicin (Dox) uptake by SA and DX5 cells --- p.51 / Chapter 2.3.5 --- Doxorubicin induced Apoptosis in SA and DX5 cells --- p.54 / Chapter 2.4 --- Discussion --- p.61 / Chapter 2.5 --- Conclusion --- p.65 / Chapter Chapter 3 --- Alternation in P-glycoprotein expression in DX5_Ninjl cells --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials / Chapter 3.2.1.1 --- Cell lines --- p.70 / Chapter 3.2.1.2 --- "Cell culture medium, supplements and buffers" --- p.70 / Chapter 3.2.1.3 --- Gene expression assay reagents --- p.70 / Chapter 3.2.1.4 --- Western blotting reagents --- p.72 / Chapter 3.2.1.5 --- Plasmid DNA extraction --- p.75 / Chapter 3.2.1.6 --- Transient transfection --- p.76 / Chapter 3.2.1.7 --- MTT reagents --- p.76 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell culture --- p.78 / Chapter 3.2.2.2 --- Gene expression essay (RT-PCR) --- p.79 / Chapter 3.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.81 / Chapter 3.2.2.4 --- DNA plasmid extraction --- p.83 / Chapter 3.2.2.5 --- Transient transfection --- p.84 / Chapter 3.2.2.6 --- MTT assay --- p.85 / Chapter 3.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.86 / Chapter 3.3 --- Results / Chapter 3.3.1 --- mRNA expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.87 / Chapter 3.3.2 --- The protein expression of Ninjurinl (Ninj1) in SA and DX5 cells --- p.89 / Chapter 3.3.3 --- Ninjurin1 (Ninj1) cDNA transfection in DX5 cells --- p.91 / Chapter 3.3.4 --- mRNA expression of MDR1 in Ninjurin1-transfected DX5 cells (DX5_Ninjl) --- p.93 / Chapter 3.3.5 --- P-glycoprotein expression in Ninjurin1-transfected DX5 cells --- p.95 / Chapter 3.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5_Ninjl cells" --- p.97 / Chapter 3.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_Ninjl cells" --- p.99 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.5 --- Conclusion --- p.105 / Chapter Chapter 4 --- Alternation in MDR1 expression in DX5一THBS1 cells --- p.106 / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials / Chapter 4.2.1.1 --- Cell lines --- p.109 / Chapter 4.2.1.2 --- Cell culture medium； supplements and buffers --- p.109 / Chapter 4.2.1.3 --- Gene expression assay reagents --- p.109 / Chapter 4.2.1.4 --- Western blotting reagents --- p.111 / Chapter 4.2.1.5 --- Plasmid DNA extraction --- p.114 / Chapter 4.2.1.6 --- Transient transfection --- p.115 / Chapter 4.2.1.7 --- MTT reagents --- p.115 / Chapter 4.2.2 --- Methods / Chapter 4.2.2.1 --- Cell culture --- p.117 / Chapter 4.2.2.2 --- Gene expression essay (RT-PCR) --- p.118 / Chapter 4.2.2.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein lysate and Western blotting --- p.120 / Chapter 4.2.2.4 --- DNA plasmid extraction --- p.123 / Chapter 4.2.2.5 --- Transient transfection --- p.123 / Chapter 4.2.2.6 --- MTT assay --- p.124 / Chapter 4.2.2.7 --- Quantification of doxorubicin (Dox) uptake by flow cytometry --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1 --- mRNA expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.126 / Chapter 4.3.2 --- The protein expression of Thrombospondinl (THBS1) in SA and DX5 cells --- p.128 / Chapter 4.3.3 --- Thrombospondinl (THBS1) cDNA transfection in DX5 cells --- p.130 / Chapter 4.3.4 --- mRNA expression of MDR1 in Thrombospondinl-transfected DX5 cells (DX5_THBS1) --- p.132 / Chapter 4.3.5 --- P-glycoprotein expression in Thrombospondinl-transfected DX5 cells --- p.134 / Chapter 4.3.6 --- "Cytotoxicity of doxorubicin (Dox) on DX5 control, DX5 vector control and DX5一THBS1 cells" --- p.136 / Chapter 4.3.7 --- "Doxorubicin (Dox) uptake by SA control, DX5 control and DX5_THBS1 cells" --- p.138 / Chapter 4.4 --- Discussion --- p.141 / Chapter 4.5 --- Conclusion --- p.145 / Chapter Chapter 5 --- General discussion --- p.146 / Chapter 5.1 --- Doxorubicin induced multidrug resistance in human uterin sarcoma cells via upregulation of P-glycoprotein --- p.147 / Chapter 5.2 --- The down-regulation of Ninjurin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.148 / Chapter 5.3 --- The down-regulation of Thrombospondin1 in human uterine sarcoma cells contributed to multidrug resistance --- p.150 / Chapter 5.4 --- Conclusion and Future Perspective --- p.153 / Reference --- p.155