Lun Samantha Wei Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 142-174). / Abstracts in English and Chinese. / Contents --- p.ii / Acknowledgements --- p.vii / Abstract --- p.viii / 摘要 --- p.xi / Chapter Section 1. --- Introduction / Chapter 1.1 --- General review of the epididymis --- p.1 / Chapter 1.1.1 --- Structure of the epididymis --- p.1 / Chapter 1.1.2 --- Function of the epididymis --- p.3 / Chapter 1.1.3 --- Regulation of the epididymal function --- p.5 / Chapter 1.2 --- Vascular endothelial growth factor (VEGF) --- p.8 / Chapter 1.2.1 --- VEGF peptides --- p.8 / Chapter 1.2.2 --- Biological activities of VEGF --- p.10 / Chapter 1.2.3 --- Hormonal regulation of VEGF --- p.11 / Chapter 1.3 --- VEGF receptors --- p.12 / Chapter 1.3.1 --- Flt-1 or VEGFR1 --- p.12 / Chapter 1.3.2 --- Flk-1 or VEGFR2 --- p.13 / Chapter 1.4 --- Caveolae --- p.15 / Chapter 1.4.1 --- Overview of caveolae --- p.15 / Chapter 1.4.2 --- Caveolins/caveolin-1 --- p.16 / Chapter 1.4.3 --- Caveolae and VEGF --- p.18 / Chapter 1.4.4 --- Caveolae and the epididymis --- p.20 / Chapter 1.5 --- VEGF/ VEGF receptors in the epididymis --- p.20 / Chapter 1.6 --- Aims of study --- p.22 / Chapter Section 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.2 --- Animal surgery --- p.35 / Chapter 2.2.1 --- Animals --- p.35 / Chapter 2.2.2 --- Castration and hemi-castration --- p.35 / Chapter 2.2.3 --- Efferent duct ligation (EDL) --- p.36 / Chapter 2.2.4 --- Tissue collection --- p.37 / Chapter 2.3 --- Epididymal cell culture --- p.38 / Chapter 2.4 --- Sample preparation --- p.40 / Chapter 2.4.1 --- Collection of epididymal plasma and sperm --- p.40 / Chapter 2.4.2 --- Purification of caveolae fraction --- p.41 / Chapter 2.5 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.43 / Chapter 2.5.1 --- Preparation of RNA from epididymal tissues --- p.43 / Chapter 2.5.2 --- Quantitation of total RNA --- p.44 / Chapter 2.5.3 --- Reverse transcription (RT) and polymerase chain reaction (PCR) --- p.44 / Chapter 2.5.4 --- Purification and authenticity confirmation of PCR products --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.53 / Chapter 2.6.1 --- Preparation of protein --- p.53 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Western immunoblotting --- p.55 / Chapter 2.7 --- Immunohistochemistry --- p.56 / Chapter 2.7.1 --- Preparation of tissue sections --- p.56 / Chapter 2.7.2 --- Immunohistochemical staining of tissue sections --- p.57 / Chapter 2.7.3 --- Immunostaining of cultured cells --- p.59 / Chapter 2.8 --- Enzyme Linked Immunosorbant Assay (ELISA) --- p.59 / Chapter 2.9 --- Statistical analyses --- p.60 / Chapter Section 3. --- Results / Chapter 3.1 --- Expression and localization of VEGF in the rat epididymis --- p.62 / Chapter 3.1.1 --- RT-PCR of VEGF in the rat epididymis --- p.62 / Chapter 3.1.2 --- Western immunoblot of VEGF in the rat epididymis --- p.63 / Chapter 3.1.3 --- Developmental changes in VEGF expression in the rat epididymis --- p.66 / Chapter 3.1.4 --- Immunolocalization of VEGF in the rat epididymis --- p.66 / Chapter 3.1.5 --- Summary of the localization and expression of VEGF in the rat epididymis --- p.72 / Chapter 3.2 --- Expression and localization of VEGF receptors in the rat epididymis --- p.74 / Chapter 3.2.1 --- RT-PCR of Flt-1 and sFlt-1 in the rat epididymis --- p.74 / Chapter 3.2.2 --- Western immunoblot of Flt-1 in the rat epididymis --- p.74 / Chapter 3.2.3 --- Immunolocalization of Flt-1 in the rat epididymis --- p.75 / Chapter 3.2.4 --- RT-PCR of Flk-1 in the rat epididymis --- p.75 / Chapter 3.2.5 --- Western immunoblot of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.6 --- Immunolocalization of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.7 --- Summary on the localization and expression of VEGF receptors in the rat epididymis --- p.83 / Chapter 3.3 --- Detection of VEGF immunoreactivity in epididymal plasma and sperm lysate collected from cauda epididymidis --- p.83 / Chapter 3.4 --- Effect of castration on VEGF and VEGF receptor expression in the rat epididymis --- p.84 / Chapter 3.4.1 --- Effect of castration with or without testosterone replacement on VEGF expression in the rat epididymis --- p.84 / Chapter 3.4.2 --- Effect of castration with or without testosterone replacement on Flt-1 expression in the rat epididymis --- p.94 / Chapter 3.4.3 --- Effect of castration with or without testosterone replacement on Flk-1 expression in the rat epididymis --- p.98 / Chapter 3.5 --- Effect of efferent duct ligation and hemicastration on VEGF peptide levels in the rat epididymis --- p.102 / Chapter 3.5.1 --- Effect of efferent duct ligation on VEGF expression in the rat epididymis --- p.102 / Chapter 3.5.2 --- Effect of hemi-castration on VEGF expression in the rat epididymis --- p.106 / Chapter 3.6 --- "Summary on the effects of castration, efferent duct ligation, and hemicastration on the epididymal weight, and VEGF/VEGF receptor expression in the rat epididymis" --- p.107 / Chapter 3.6.1 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on the epididymal weight" --- p.107 / Chapter 3.6.2 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on VEGF and VEGF receptor expression in the rat epididymis" --- p.112 / Chapter 3.7 --- Localization and expression of caveolin-1 and 226}0ؤ2 in the rat epididymis --- p.113 / Chapter 3.7.1 --- RT-PCR of caveolin-1 and caveolin-2 in the rat epididymis --- p.113 / Chapter 3.7.2 --- Western immunoblot of caveolin-1 and caveolin-2 in the rat epididymis --- p.114 / Chapter 3.7.3 --- Immunolocalization of caveolin-1 in the rat epididymis --- p.117 / Chapter 3.7.4 --- Summary on the localization and expression of caveolin-1 and -2 in the rat epididymis --- p.119 / Chapter 3.8 --- Co-localization of VEGF receptors with caveolae in the rat epididymis --- p.119 / Chapter Section 4. --- Discussion --- p.124 / Chapter 4.1 --- VEGF expression and localization --- p.124 / Chapter 4.2 --- VEGF receptors expression and localization --- p.129 / Chapter 4.3 --- Possible VEGF action in the rat epididymis --- p.133 / Chapter 4.4 --- Regulation of VEGF and its receptor expression by androgen and/or other testicular factors --- p.136 / References

Yung, Kam Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 107-129). / Abstracts in English and Chinese. / ABSTRACT --- p.ii / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / PUBLICATION --- p.ix / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- "The biology, diagnostics and management of lung cancer" --- p.2 / Chapter 1.1 --- "Basic biology, classification and diagnostics" --- p.2 / Chapter 1.1.1 --- Epidemiology and etiology of lung cancer --- p.2 / Chapter 1.1.2 --- Clinical Presentation and Diagnostics of Lung Cancer --- p.3 / Chapter 1.2 --- Treatment of lung cancer --- p.9 / Chapter 1.2.2 --- Radiotherapy --- p.10 / Chapter 1.2.3 --- Chemotherapy --- p.11 / Chapter CHAPTER 2: --- Epidermal Growth Factor Receptor Mutations in Lung Cancer --- p.13 / Chapter 2.1 --- The Epidermal Growth Factor Receptor --- p.13 / Chapter 2.2 --- Overexpression of EGFR in NSCLC --- p.14 / Chapter 2.3 --- The development of EGFR inhibitors --- p.15 / Chapter 2.3.1 --- Monoclonal Antibodies --- p.16 / Chapter 2.3.2 --- Small-molecule inhibitors --- p.17 / Chapter 2.3.2.1 --- Gefitinib --- p.17 / Chapter 2.3.2.2 --- Erlotinib --- p.19 / Chapter 2.3.2.3 --- Other small-molecule inhibitors --- p.20 / Chapter 2.4 --- Mutations of EGFR in NSCLC --- p.21 / Chapter 2.4.1 --- Activating Mutations conferring sensitivity to tyrosine kinase inhibitors --- p.21 / Chapter 2.4.2 --- Secondary mutations associated with resistance to tyrosine kinase inhibitors --- p.23 / Chapter 2.5 --- EGFR gene amplification --- p.24 / Chapter 2.6 --- Detection of EGFR mutations --- p.25 / Chapter 2.7 --- Aim of the thesis --- p.31 / Chapter SECTION II: --- DETECTION OF EGFR MUTATIONS IN TUMOR AND PLASMA SAMPLES BY MASS SPECTROMETRY AND DIGITAL PCR --- p.33 / Chapter CHAPTER 3: --- Detection of EGFR mutations by mass spectrometric methods --- p.34 / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.1.1 --- Principles of Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.34 / Chapter 3.1.2 --- The MassARRAY Homogenous MassEXTEND (hME) assay --- p.35 / Chapter 3.1.3 --- The Single-Allele Base Extension Reaction (SABER) and the Allele-Specific Base Extension Reaction (ASBER) --- p.36 / Chapter 3.2 --- Materials and Methods --- p.36 / Chapter 3.2.1 --- The protocol for the detection of EGFR exon 21 point mutation by Mass Spectrometric Methods --- p.37 / Chapter 3.3 --- Results --- p.42 / Chapter 3.4 --- Discussion --- p.49 / Chapter CHAPTER 4: --- Evaluation of the detection limit and sensitivity of the digital PCR assays --- p.51 / Chapter 4.1 --- Introduction --- p.51 / Chapter 4.1.1 --- The theoretical basis of digital PCR quantification and the relationship with the Poisson distribution --- p.51 / Chapter 4.1.2 --- Assessment of Assay Detection Limit --- p.54 / Chapter 4.1.3 --- Comparing Digital PCR with sequencing after conformation sensitive gel electrophoresis (CSGE) --- p.59 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Design of digital PCR assay for the detection of EGFR exon21 L858R point mutation --- p.59 / Chapter 4.2.2 --- Design of digital PCR assay for the detection of EGFR exon19 deletion --- p.60 / Chapter 4.2.3 --- The protocols of digital PCR assays for EGFR mutation detection --- p.64 / Chapter 4.2.4 --- Single molecule detection test --- p.65 / Chapter 4.2.5 --- Artificial mixtures of mutant and wild-type DNA --- p.66 / Chapter 4.2.6 --- Sequencing after CSGE --- p.66 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Results of the single molecule detection test and artificial mixture analysis --- p.67 / Chapter 4.3.2 --- Results of CSGE and sequencing compared with digital PCR --- p.73 / Chapter 4.4 --- Discussion --- p.75 / Chapter CHAPTER 5: --- Detection of EGFR mutations in prospectively collected tumor samples of NSCLC patients --- p.77 / Chapter 5.1 --- Introduction --- p.77 / Chapter 5.2 --- Materials and Methods --- p.78 / Chapter 5.2.1 --- Sample preparation and DNA extraction of tumor tissues --- p.78 / Chapter 5.3 --- Results --- p.79 / Chapter 5.4 --- Discussion --- p.82 / Chapter CHAPTER 6: --- Detection of EGFR mutations in prospectively collected plasma samples of NSCLC patients --- p.85 / Chapter 6.1 --- Introduction --- p.85 / Chapter 6.2 --- Materials and Methods --- p.87 / Chapter 6.2.1 --- Sample preparation and DNA extraction of plasma samples --- p.87 / Chapter 6.3 --- Results --- p.88 / Chapter 6.3.1 --- Digital PCR analysis of EGFR mutations in plasma samples of NSCLC patient --- p.88 / Chapter 6.3.2 --- Variations in plasma EGFR mutation concentration after TKI treatment --- p.93 / Chapter 6.4 --- Discussion --- p.96 / Chapter SECTION III: --- CONCLUDING REMARKS --- p.100 / Chapter CHAPTER 7: --- Conclusion and future perspectives --- p.101 / Chapter 7.1 --- Mass spectrometric analysis --- p.101 / Chapter 7.2 --- Microfluidics Digital PCR --- p.102 / Chapter 7.3 --- Future perspectives --- p.105 / References --- p.107

1 v. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1994

xxiii, 222 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Insulin-like growth factor-I circulates at high concentrations in blood, mainly complexed with IGF-binding proteins. The main objective of the thesis is to determine the general role played by plasma IGF-binding proteins in the regulation of IGF transfer from blood to tissues. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1994

The insulin-like growth factor (IGF) system is composed of IGF, IGF binding proteins (IGFBP-1 to -10) and the acid labile subunit (ALS). IGF exists as two isoforms, IGF-I and IGF-II. IGF-I is the major circulatory form and is primarily secreted by the liver. It functions to regulate proliferation and differentiation in a number of different cell types and elicits an insulin-like metabolic effect. As well as being regulated at levels of transcription and translation, IGF-I activities are also regulated through formation of complexes in circulation. IGF complexes form as binary complexes, such as the IGFBP-1 complex, and ternary complexes containing IGF-I, IGFBP-3 and ALS. Binary and ternary IGF complexes function to maintain stable pools of bioactive IGF-I. They also function to increase IGF half-life and sequester IGF in the bloodstream. <p> ALS and IGFBP-1 are well characterized and exist as 85 kDa and 32 kDa proteins, respectively. They are expressed primarily in liver hepatocytes. Circulating ALS binds the IGF-I-IGFBP-3 complex and increases IGF half-life from 10 min in the IGFBP-3 binary complex to 10-15 hr in the ternary complex. IGFBP-1 binds IGF-I and increases the half-life from 10 min to 30 min. The ternary complex is the predominant IGF-I binding protein complex found in circulation. The IGFBP-1 complex represents only a small fraction of circulating IGF complexes. <p> In this thesis ALS and IGFBP-1 regulation were investigated in terms of expression related to metabolic modulators and streptozotocin (STZ)-induced diabetes. Results from rat studies showed a decreased liver ALS gene expression in STZ-induced diabetic rats. STZ-treatment in rats mimics type-I diabetes with no change in secreted insulin with increase of circulatory glucose. The administration of insulin into the STZ-induced diabetic rats brought ALS levels to that of the untreated controls. ALS expression was positively regulated by insulin in H4IIE hepatoma cells. Growth hormone (GH), glucose, dexamethasone also positively regulated ALS gene expression while cAMP (2-b-cAMP) acted as a negative regulator in H4IIE cells. HepG2 cells expressing constitutively active protein kinase B (PKB) (HepG2-PKB-CA) increased ALS gene expression to levels 20% higher then parental HepG2. Insulin treatment of these cells unexpectedly increased ALS levels in both parental and PKB-CA HepG2. This may have indicated a partial regulatory role of the mitogen activated protein (MAP) kinase pathway as PKB was thought to be over-expressed therefore rendering the insulin signal redundant. Inhibition of the phosphoinositol-3 (PI-3) kinase and MAP kinase pathways through wortmannin and PD98059 incubation, respectively, suggested a possible interplay or crosstalk between the two pathways in insulin signaling. PKB is known to be activated through the PI-3 kinase pathway. Results suggested possibility that PKB may interact through the MAP kinase pathway in regulation of ALS gene expression. The activity of cAMP on ALS gene expression may occur through interaction with the PI-3 kinase pathway as inhibition enhanced the negative effect of cAMP on ALS expression. <p> The secretion of IGFBP-1 was positively regulated by glucose and GH and negatively regulated by insulin in H4IIE cells. HepG2-PKB-CA cells showed significantly lower IGFBP-1 secretion as compared to parental HepG2 cells. The involvement of the PI-3 and MAP kinase pathways in the modulator-mediated effect on IGFBP-1 secretion were. As observed for ALS expression, the effect of insulin on IGFBP-1 secretion may also be affected through interplay or crosstalk between the PI-3 kinase and MAP kinase pathways. Glucose and GH effected IGFBP-1 expression and secretion independent of these pathways although glucose expression may interact in some way through the PI-3 kinase pathway. Our investigation of hepatic regulation of IGFBP-1 secretion and ALS gene expression has shown regulatory roles for the metabolic hormones tested, especially insulin. Mechanisms of cell signaling have also been approached with the use of pathway inhibiters and HepG2-PKB-CA cells. Much work is yet to be done to fully understand the effects of insulin and other hormones on the secretion and expression of IGFBP-1 and ALS.

The insulin-like growth factor (IGF) system is composed of IGF, IGF binding proteins (IGFBP-1 to -10) and the acid labile subunit (ALS). IGF exists as two isoforms, IGF-I and IGF-II. IGF-I is the major circulatory form and is primarily secreted by the liver. It functions to regulate proliferation and differentiation in a number of different cell types and elicits an insulin-like metabolic effect. As well as being regulated at levels of transcription and translation, IGF-I activities are also regulated through formation of complexes in circulation. IGF complexes form as binary complexes, such as the IGFBP-1 complex, and ternary complexes containing IGF-I, IGFBP-3 and ALS. Binary and ternary IGF complexes function to maintain stable pools of bioactive IGF-I. They also function to increase IGF half-life and sequester IGF in the bloodstream. <p> ALS and IGFBP-1 are well characterized and exist as 85 kDa and 32 kDa proteins, respectively. They are expressed primarily in liver hepatocytes. Circulating ALS binds the IGF-I-IGFBP-3 complex and increases IGF half-life from 10 min in the IGFBP-3 binary complex to 10-15 hr in the ternary complex. IGFBP-1 binds IGF-I and increases the half-life from 10 min to 30 min. The ternary complex is the predominant IGF-I binding protein complex found in circulation. The IGFBP-1 complex represents only a small fraction of circulating IGF complexes. <p> In this thesis ALS and IGFBP-1 regulation were investigated in terms of expression related to metabolic modulators and streptozotocin (STZ)-induced diabetes. Results from rat studies showed a decreased liver ALS gene expression in STZ-induced diabetic rats. STZ-treatment in rats mimics type-I diabetes with no change in secreted insulin with increase of circulatory glucose. The administration of insulin into the STZ-induced diabetic rats brought ALS levels to that of the untreated controls. ALS expression was positively regulated by insulin in H4IIE hepatoma cells. Growth hormone (GH), glucose, dexamethasone also positively regulated ALS gene expression while cAMP (2-b-cAMP) acted as a negative regulator in H4IIE cells. HepG2 cells expressing constitutively active protein kinase B (PKB) (HepG2-PKB-CA) increased ALS gene expression to levels 20% higher then parental HepG2. Insulin treatment of these cells unexpectedly increased ALS levels in both parental and PKB-CA HepG2. This may have indicated a partial regulatory role of the mitogen activated protein (MAP) kinase pathway as PKB was thought to be over-expressed therefore rendering the insulin signal redundant. Inhibition of the phosphoinositol-3 (PI-3) kinase and MAP kinase pathways through wortmannin and PD98059 incubation, respectively, suggested a possible interplay or crosstalk between the two pathways in insulin signaling. PKB is known to be activated through the PI-3 kinase pathway. Results suggested possibility that PKB may interact through the MAP kinase pathway in regulation of ALS gene expression. The activity of cAMP on ALS gene expression may occur through interaction with the PI-3 kinase pathway as inhibition enhanced the negative effect of cAMP on ALS expression. <p> The secretion of IGFBP-1 was positively regulated by glucose and GH and negatively regulated by insulin in H4IIE cells. HepG2-PKB-CA cells showed significantly lower IGFBP-1 secretion as compared to parental HepG2 cells. The involvement of the PI-3 and MAP kinase pathways in the modulator-mediated effect on IGFBP-1 secretion were. As observed for ALS expression, the effect of insulin on IGFBP-1 secretion may also be affected through interplay or crosstalk between the PI-3 kinase and MAP kinase pathways. Glucose and GH effected IGFBP-1 expression and secretion independent of these pathways although glucose expression may interact in some way through the PI-3 kinase pathway. Our investigation of hepatic regulation of IGFBP-1 secretion and ALS gene expression has shown regulatory roles for the metabolic hormones tested, especially insulin. Mechanisms of cell signaling have also been approached with the use of pathway inhibiters and HepG2-PKB-CA cells. Much work is yet to be done to fully understand the effects of insulin and other hormones on the secretion and expression of IGFBP-1 and ALS.

Liver fibrosis, a major medical problem with significant morbidity and mortality, is considered as a wound-healing response to a variety of chronic stimuli. It is characterized by an excessive deposition of extracellular matrix (ECM) proteins, which disrupts the normal architecture of liver and ultimately leads to pathophysiological damage to liver. Hepatoma-derived growth factor (HDGF), a growth factor originally purified from hepatoma cells, is highly expressed in fetal hepatocytes and hepatoma. It is known to play multifunctional roles in mitogenesis, organogenesis, embryogenesis, and tumorigenesis. Its expression correlates with the proliferating state of hepatocellular carcinoma (HCC) and serves as a prognostic factor. Since liver fibrosis frequently occurs prior to HCC development, the specific aim of this study is to investigate the role of HDGF in the progression of liver fibrosis by using animal models of mice receiving either bile duct ligation surgery or carbon tetrachloride administration. Quantitative real-time PCR and Western blotting analysis showed a significant elevation of HDGF expression in both models. HDGF levels correlated with progression of liver fibrosis in a time-dependent manner as well as paralleled with the expression of other two fibrotic markers, transforming growth factor-b1 (TGF-b1) and pro-collagen type I, in fibrotic livers. Intriguingly, the over-expressed HDGF protein was localized mainly in perivenous hepatocytes of fibrotic livers. Besides, adenovirus-mediated HDGF gene delivery potentiated the production of TGF-b1 and pro-collagen type I, thereby enhancing the intrahepatic collagen matrix deposits as evidenced by Sirius red stain and morphometrical analysis. In cultured hepatocytes, TGF-b1 and HDGF mutually up-regulated their de novo synthesis only when grown on collagen-coated matrix, strongly suggesting that the TGF-b1- and/or HDGF-driven pro-fibrogenic signaling is collagen-dependent and a vicious circle may exist at the initial stage of hepatic fibrogenesis. Moreover, administration with recombinant HDGF stimulated BrdU uptake and synthesis of both a-smooth muscle actin and pro-collagen type I in cultured hepatic stellate cells, implicating that a mode of paracrinal action lies between these two cell types. In conclusion, HDGF plays a pro-fibrogenic role during liver fibrosis and blockade of HDGF pathway may potentially constitute the preventive or therapeutic strategies for chronic liver diseases.

Heparan sulfate (HS) belongs to the glycosaminoglycan family of polysaccharides and is found attached to protein cores on cell surfaces and in the extracellular matrix. The HS backbone consists of alternating hexuronic acid and glucosamine units and undergoes a number of modification reactions creating HS chains with alternating highly and low modified domains, where high degree of modification correlates with high negative charge. Fibroblast growth factors (FGFs) and their receptors (FRs) both bind to HS, which affect formation of the FGF–FR complexes on the cell surfaces. Activated FRs can trigger several intracellular signaling pathways leading thereby to diverse cellular responses. Work presented in this thesis focuses on the effect of HS and its structures on FGF–FR complex formation and FGF-induced signaling. Studies with short, highly modified oligosaccharides and FGF1 and 2 combined with FR1c, 2c, 3c or 4 showed a correlation between the overall degree of modification and amount/stability of FGF–FR complexes. Our findings imply that several HS structures, differently modified but with the same negative charge density are equal in their ability to support complex formation. Co-application of oligosaccharides with FGF2 to HS-deficient cells and investigation of the thereby induced cell signaling confirmed our findings with a cell-free system. The oligosaccharide with the highest modification degree displayed the biggest impact on cell signaling, which was FGF2 concentration dependent. Studies with long HS polysaccharides with preserved high and low modified domains suggest that the proportion between these two types of domains and also the structure of the low modified domains are of importance for the FGF–HS–FR complex formation and cell activation capacity. This work illuminates several aspects in how HS structure influences the interplay between FGFs and FRs and contributes to the understanding of what factors affect a cell’s response following FGF stimulation.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Department of Animal Science, 1994.