Thesis (M.Phil.)--City University of Hong Kong, 2005. / "Submitted to Department of Biology and Chemistry in partial fulfillment of the requirements for the degree of Master of Philosophy" Includes bibliographical references (leaves 124-141)

Physiological electrical fields (EFs) can direct some important angiogenic responses of endothelial cells such as directional migration, orientation and proliferation. It has been reported that human umbilical vein endothelial cells (HUVEC) and human microvasculature endothelial cells (HMEC) migrate in opposite directions; to anode and cathode, respectively. Although, in the present study both cell types migrated toward the cathode, HUVEC directedness started at 50mV/mm while HMEC directedness started at 100mV/mm. These results suggest that EFs can promote wound healing by directing endothelial cells to the wound site since EFs of 40 to 100 mV/mm are present in normal healing wounds. EFs also increased cell proliferation and orientated the cleavage plane of dividing cells perpendicular to the EF vector in both endothelial cell lines. The present study showed for the first the time that EFs upregulated the expression of the chemokine receptors CXCR4 and CXCR2 as well as upregulating the levels of phosphorylation of both chemokines in HUVEC and HMEC. It also showed differences of chemokine receptors used by HUVEC and HMEC cells in the early stages of electrotaxis. Ionizing radiation has been shown to directly phosphorylate VEGF receptors in the absence of its ligand VEGF. A question was raised: in the absence of the ligands are EFs able to directly phosphorylate the chemokine receptors? Results showed that in starved HUVEC cells EFs had no effect on the phosphorylation levels of CXCR4 and CXCR2 however in starved HMEC cells an EF may have a direct effect on the phosphorylation levels of CXCR4 and CXCR2. Therefore, EFs represent a physical stimulus that could directly phosphorylate proteins in the absence of its ligand. This work substantiate the importance of endogenous EFs in directing endothelial cells and suggests that EFs might be developed as a component in the clinic to control angiogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Rap1a and Rap1b are two closely related members of the Ras family of small GTPases. Despite their high sequence similarity, the two proteins serve non-redundant functions in cells and organs. Rap1a plays critical roles during mouse development, and both Rap1a and Rap1b are required for angiogenesis. In glioblastoma cells, however, Rap1b plays a more unique role in tumor cell invasion. Loss of rap1a in mice resulted in 40% embryonic lethality, and caused cardiac defects in mouse embryos and cardiac hypertrophy in adult mice. These phenotypes, distinct from those of the rap1b knockout mice, suggest differential roles of the two GTPases during mouse development. Angiogenesis, the formation of new blood vessels by endothelial cells, is impaired by the loss of rap1. Blood vessel growth into FGF2-containing Matrigel plugs was absent from rap1a-/- mice and aortic rings derived from rap1a-/- mice failed to sprout primitive endothelial tubes in response to FGF2 when embedded in Matrigel. Knocking down of either rap1a or rap1b in human micro-vascular endothelial cells (HMVECs) confirmed that Rap1 plays key roles in endothelial cell function. The knockdown of rap1a or 1b resulted in decreased adhesion to extracellular matrices and impaired cell migration. Rap1 deficient endothelial cells failed to form 3-D tubular structures when plated on Matrigel in vitro. The activation of ERK, p38, and Rac, important signaling molecules in angiogenesis, were all reduced in response to FGF2 when either Rap1 protein was depleted. In U373 human glioblastoma multiforme cells, depletion of rap1b, but not rap1a drastically reduced tumor cell invasion by decreasing the activity of secreted matrix metalloproteinase 2 (MMP2). The adhesion of cells to the extracellular matrices collagen or fibronectin, but not to vitronectin, was decreased upon rap1b depletion. However, a mild increase in proliferation associated with elevation in ERK1/2, p38, Akt and ribosomal S6 protein activation was observed in cells depleted of either rap1a or rap1b. When an MEK1/2 inhibitor U0126 was used, the phosphorylation of p38, Akt and S6 were decreased, however, to various levels, suggesting complex regulatory pathways mediate Rap1 action in glioblastoma cells.

Lai, Tsz Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 121-136). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Content / List of tables / List of figures / List of abbreviations / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Introduction of food market trends in Hong Kong and mushroom productivity in the world --- p.1 / Chapter 1.1.1 --- Agrocybe aegerita --- p.1 / Chapter 1.1.2 --- Pleurotus spp --- p.2 / Chapter 1.1.3 --- Pholiota nameko --- p.3 / Chapter 1.2 --- Objectives --- p.5 / Chapter Chapter 2: --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.6 / Chapter 2.1 --- Introduction --- p.6 / Chapter 2.1.1 --- Reactive oxygen species (ROS) --- p.6 / Chapter 2.1.1.1 --- Definition of ROS --- p.6 / Chapter 2.1.1.2 --- Sources of ROS --- p.6 / Chapter 2.1.1.2.1 --- Endogenous sources of ROS --- p.6 / Chapter 2.1.1.2.2 --- Exogenous sources of ROS --- p.8 / Chapter 2.1.1.3 --- Damaging effects of ROS --- p.8 / Chapter 2.1.2 --- Antioxidants --- p.10 / Chapter 2.1.2.1 --- Mechanism of action --- p.10 / Chapter 2.1.2.2 --- Sources of antioxidants --- p.11 / Chapter 2.1.2.2.1 --- Dietary antioxidants --- p.11 / Chapter 2.1.2.2.2 --- Antioxidants in edible mushrooms --- p.12 / Chapter 2.1.2.2.3 --- Phenolic compounds in mushrooms --- p.13 / Chapter 2.2 --- Materials and Methods --- p.16 / Chapter 2.2.1 --- Materials --- p.16 / Chapter 2.2.1.1 --- Mushroom fruiting bodies --- p.16 / Chapter 2.2.2 --- Principles of Methods and Experimental Protocols --- p.17 / Chapter 2.2.2.1 --- Sample preparation --- p.17 / Chapter 2.2.2.2 --- Evaluation of antioxidant capacity --- p.18 / Chapter 2.2.2.2.1 --- DPPH radical scavenging activity --- p.18 / Chapter 2.2.2.2.2 --- Superoxide anion scavenging activity --- p.19 / Chapter 2.2.2.2.3 --- Hydroxyl radical scavenging activity --- p.20 / Chapter 2.2.2.2.4 --- Hydrogen peroxide scavenging activity --- p.22 / Chapter 2.2.2.3 --- Determination of phenolic compounds --- p.24 / Chapter 2.2.2.3.1 --- Total phenolic content --- p.24 / Chapter 2.2.2.3.2 --- Identification of phenolic acids --- p.25 / Chapter 2.2.3 --- Statistical analysis --- p.27 / Chapter 2.3 --- Results and Discussion --- p.28 / Chapter 2.3.1 --- Extraction yield --- p.28 / Chapter 2.3.2 --- Evaluation of antioxidant capacity --- p.29 / Chapter 2.3.2.1 --- DPPH radical scavenging activity --- p.29 / Chapter 2.3.2.2 --- Superoxide anion scavenging activity --- p.31 / Chapter 2.3.2.3 --- Hydroxyl radical scavenging activity --- p.33 / Chapter 2.3.2.4 --- Hydrogen peroxide scavenging activity --- p.35 / Chapter 2.3.2.5 --- Comparison of the effective concentrations (EC50) of mushroom water extracts in different antioxidant assays --- p.37 / Chapter 2.3.3 --- Determination of phenolic compounds --- p.38 / Chapter 2.3.3.1 --- Total phenolic content --- p.38 / Chapter 2.3.3.2 --- Identification of phenolic acids --- p.39 / Chapter 2.4 --- Summary --- p.45 / Chapter Chapter 3: --- Anti-angiogenic properties of the Aa water extract --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.1.1 --- Angiogenesis --- p.46 / Chapter 3.1.1.1 --- Process of angiogenesis --- p.46 / Chapter 3.1.1.2 --- Regulations of angiogenesis --- p.47 / Chapter 3.1.1.2.1 --- Fibroblast growth factor (bFGF) --- p.47 / Chapter 3.1.1.2.2 --- Vascular endothelial growth factor (VEGF) --- p.48 / Chapter 3.1.2 --- Tumor angiogenesis --- p.49 / Chapter 3.1.2.1 --- ROS generation in tumor cells --- p.50 / Chapter 3.1.2.2 --- Hydrogen peroxide and VEGF --- p.51 / Chapter 3.1.2.3 --- Previous studies on tumor angiogenesis --- p.52 / Chapter 3.1.2.3.1 --- ROS and endothelial cells proliferation --- p.52 / Chapter 3.1.2.3.2 --- VEGF and endothelial cells functions --- p.53 / Chapter 3.1.3 --- Use of antioxidants in cancer treatment --- p.53 / Chapter 3.1.3.1 --- Antioxidant use of cancer therapy --- p.53 / Chapter 3.1.3.2 --- Antioxidant and endothelial cells functions --- p.54 / Chapter 3.1.3.3 --- Anti-angiogenic effects of polyphenols --- p.56 / Chapter 3.1.3.3.1 --- Phenolic acids --- p.56 / Chapter 3.1.3.3.2 --- Tea catechin --- p.57 / Chapter 3.1.3.3.3 --- Resveratrol --- p.57 / Chapter 3.1.3.3.4 --- Genistein --- p.58 / Chapter 3.2 --- Principles of Methods and Experimental Protocols --- p.60 / Chapter 3.2.1 --- Sample preparation --- p.60 / Chapter 3.2.2 --- Toxicity of the Aa water extract --- p.60 / Chapter 3.2.2.1 --- Limulus amebocyte lysate (LAL) test --- p.60 / Chapter 3.2.2.2 --- Toxicity towards normal cells --- p.61 / Chapter 3.2.2.2.1 --- Cell line and its subculture --- p.61 / Chapter 3.2.2.2.2 --- Colorimetric (MTT) assay --- p.62 / Chapter 3.2.3 --- Effect of the Aa water extract on cancer cells --- p.63 / Chapter 3.2.3.1 --- Cell line and its subculture --- p.63 / Chapter 3.2.3.2 --- Redox status --- p.63 / Chapter 3.2.3.3 --- VEGF secretion --- p.65 / Chapter 3.2.4 --- In vitro cell culture anti-angioenesis analysis --- p.66 / Chapter 3.2.4.1 --- Cell line and its subculture --- p.66 / Chapter 3.2.4.2 --- Endothelial cells proliferation --- p.67 / Chapter 3.2.4.3 --- Endothelial cells migration --- p.68 / Chapter 3.2.4.3.1 --- Wound healing assay --- p.68 / Chapter 3.2.4.3.2 --- Transwell culture insert assay --- p.69 / Chapter 3.2.4.4 --- Endothelial cells tubule formation --- p.71 / Chapter 3.2.5 --- In vitro organ culture anti-angiogenesis analysis --- p.72 / Chapter 3.2.5.1 --- Aortic ring assay --- p.72 / Chapter 3.2.6 --- Statistical analysis --- p.74 / Chapter 3.3 --- Results and Discussions --- p.75 / Chapter 3.3.1 --- Toxicity of the Aa water extract --- p.75 / Chapter 3.3.1.1 --- Limulus amebocyte lysate (LAL) test --- p.75 / Chapter 3.3.1.2 --- Toxicity towards normal cells --- p.75 / Chapter 3.3.2 --- Effect of the Aa water extract on cancer cells --- p.77 / Chapter 3.3.2.1 --- Redox status --- p.77 / Chapter 3.3.2.2 --- VEGF secretion --- p.79 / Chapter 3.3.2.3 --- Relationship between intracellular ROS and VEGF secretion detected --- p.80 / Chapter 3.3.3 --- Effect of the Aa water extract on angiogenesis --- p.82 / Chapter 3.3.3.1 --- Endothelial cells proliferation --- p.82 / Chapter 3.3.3.2 --- Endothelial cells migration --- p.84 / Chapter 3.3.3.2.1 --- Wound healing assay --- p.84 / Chapter 3.3.3.2.2 --- Transwell culture insert assay --- p.87 / Chapter 3.3.3.3 --- Endothelial cells tubule formation --- p.90 / Chapter 3.3.3.4 --- Aortic ring assay --- p.97 / Chapter 3.3.4 --- Effect of phenolic acids on endothelial cells --- p.101 / Chapter 3.3.4.1 --- Endothelial cells proliferation --- p.101 / Chapter 3.3.4.2 --- Endothelial cells migration --- p.102 / Chapter 3.3.4.2.1 --- Wound healing assay --- p.102 / Chapter 3.3.4.2.2 --- Transwell culture insert assay --- p.105 / Chapter 3.3.4.3 --- Endothelial cells tubule formation --- p.106 / Chapter 3.3.4.4 --- Aortic ring assay --- p.112 / Chapter 3.4 --- Summary --- p.116 / Chapter Chapter 4 --- Conclusions and future works --- p.118 / References --- p.121

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Includes bibliographical references.

Cilengitide was anticipated to be a highly promising potential anti-angiogenic small molecule drug but unfortunately it failed phase III clinical trial in early 2013 (survival rate not passed). Today, there is still not a small molecule αvβ3 integrin drug in use in the clinic, new antagonists were urgently needed to advance the treatment of the αvβ3 integrin related diseases. / Peptide-type antagonists possess unique advantages such as non-accumulative, bio-friendly etc. comparing to other types of antagonists (peptidomimetics, non-peptidic etc.). Cilengitide is one of the peptide-type antagonists and it remains the most successful drug candidate demonstrated. Reinvestigation of the binding between Cilengitide and the αvβ3 integrin is therefore still meaningful. Specifically, the present study will concentrate on revealing the factors responsible for higher binding affinity of Cilengitide, comparing to the parent compound c(RGDfV), c(RADfV) and other N-methylated derivatives of c(RGDfV). Note that c(RADfV) could be also viewed as a methylation product of c(RGDfV) (methylation of side chain of the -G- residue). It is intended that the results of this investigation could provide clear guidance towards future efforts in the design of new peptide αvβ3 integrin antagonists. / Utilizing a novel but successful computational protocol, namely an integrated docking, molecular dynamics simulation and MM_GBSA free energy calculation method, the present thesis found that the binding profile between Cilengitide and the αvβ3 integrin is unique when compared to bindings of other methylation compounds. On the one hand, structural analysis (such as RMSD, ligand structure, hydrogen bond, backbone and side chain orientation) revealed that only c(RGDfV) and Cilengitide substantially retain the binding mode of the parent compound c(RGDfV) (Cilengitide is closer than c(RGDfV)). On the other hand, calculated binding energy results revealed that only c(RGDfV) and Cilengitide bind more strongly than c(RGDfV) to the αvβ3 integrin. Since in experiments only Cilengitide binds more strongly than c(RGDfV), it is therefore believed that both structural and energetic factors are responsible for the high binding affinity of Cilengitide. / Other energetic study revealed that the high binding affinity of Cilengtide compared to c(RGDfV) originates from Coulombic interaction (sum of electrostatic interaction and polar solvation energy), while van der Waals interaction and non-polar solvation energy was not favorable for Cilengitide binding. For the residue contribution, energy changes on the -R- and -G- residues upon methylation were nearly zero. Changes on the -D- and -f- residues were favorable for Cilengitide binding, while change on the -V- residue was not. Total changes on the five residues are favorable however. Pair-wise analysis suggests that interactions of the -D- residue in Cilengitide were very important for the binding, as suggested by its sizeable coupling energies with other residues. Amongst them the coupling between -D- and Mg688 is the most important pair. / In brief, the present study provides a quantitative understanding towards the binding between Cilengitide series ligand and the αvβ3 integrin. Through comparison with bindings between the methylated analogues of c(RGDfV), important features responsible for high binding affinity of Cilengitide were revealed. Some of the results contained in the thesis are in the process of being reported in "C. Yan, S. C. F. Au-Yeung. Investigation of the 'Methyl Effect' in the Cilengitide Binding Ligand Series and Its Implications on Future Integrin Antagonist Design. J. Med. Chem., in preparation (2014)". / Cilengitide是一种曾被高度寄望成为抗血管增生小分子药物的化合物。遗憾的是，2013年初所述化合物没有通过临床三期测试 (存活率不过关)。由于至今临床上仍然没有αvβ3整合素的小分子药物在使用，新的拮抗剂需要被设计出来以推进αvβ3整合素相关疾病的治疗。 / 相比于其它类型的拮抗剂 (类肽物、非肽类等)，多肽型的拮抗剂具有其独特的优点，譬如不积聚、生物友好等。Cilengitide是一种多肽型的拮抗剂，至今为止其仍是已证明的最成功的药物候选。重新研究Cilengitide与αvβ3整合素的结合情况因此仍然具有意义。具体地，本研究将聚焦于揭示相比于母体化合物c(RGDfV)、c(RADfV) 和c(RGDfV) 其它N-甲基化衍生物Cilengitide高亲和性的因素。c(RADfV) 可视为c(RGDfV) 另一甲基化的产物（-G- 残基侧链甲基化）。本研究的结果希望能给未来新型多肽型αvβ3整合素拮抗剂的设计以清晰的指导。 / 通过采用一种新的但颇成功的计算协议，即一种分子对接、分子动力学和MM_GBSA的综合方法，本论文发现相比于上述非Cilengitide的配体，Cilengitide与 αvβ3整合素的结合模式非常独特。一方面，结构分析 (RMSD、配体结构、氢键和骨架及侧链取向) 显示只有c(RGDfV) 与Cilengitide保留了其母体化合物，即c(RGDfV) 的结合模式（Cilengitide比c(RGDfV) 更接近）。另一方面，结合能计算结果显示只有Cilengitide和c(RGDfV) 与αvβ3整合素的结合比母体化合物c(RGDfV) 更强。由于实验上只有Cilengitide的结合强于c(RGDfV)，因此有理由相信结构和能量因素均对Cilengitide高亲和性负责。 / 其它能量研究显示相比于c(RGDfV)，Cilengitide的高亲和性来源于库仑作用 (静电作用和极性溶剂化能的加和)。范德华作用和非极性溶剂化能则对Cilengitide结合不利。残基贡献方面，甲基化后 -R- 和 -G- 的能量变化基本为零；-D- 和 -f- 残基的能量变化则有利于Cilengitide的结合，而 -V- 能量变化则不利。然而这五个残基的总变化对结合是有利的。配对分析结果显示Cilengitide中 -D- 残基的作用对于结合是非常重要的，这从其与其它残基可观的耦合能可以得知。其中，-D- 与Mg688的耦合是最重要的一对。 / 简言之，本研究提供了对Cilengitide系列配体与αvβ3整合素结合的定量理解。通过c(RGDfV) 甲基化后同类物的结合相互间的比较，揭示了负责Cilengitide高亲和性的重要特征。本论文中的一些研究结果正将报道于： / “严长青，欧阳植勋。关于Cilengitide配体系列中“甲基效应”的研究及其对未来αvβ3整合素拮抗剂设计的启示。药物化学杂志，准备中(2014)” / Yan, Changqing. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 138-146). / Abstracts also in Chinese. / Title from PDF title page (viewed on 18, January, 2017). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Thesis (M. Orth.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 74-95).

published_or_final_version / Dentistry / Master / Master of Orthodontics

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.