published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

In sea turtles, the study of the etiology and development of fibropapillomatosis is not fully understood. Sea turtle fibropapillomatosis is a disease characterized by the proliferation of skin fibropapillomas and occasional internal fibromas. In this study, sea turtle fibropapilloma tumor and healthy tissue samples were used to look at VEGF, BCL-2 and Bax expression. Cancer tumors have a well established pattern of protein expression that involves overexpression of vascular endothelial growth factor (VEGF), responsible for the growth of new blood vessels, and a high BCL-2 to Bax ratio that leads to uncontrolled cell proliferation. Real time PCR was used to analyze VEGF expression, and Western blot techniques were used to measure BCL-2 and Bax expression. The results indicated that expression of VEGF was not significantly higher in tumor vs. skin tissue. For the differential expression of BCL-2 and Bax, the results were not in agreement with the established levels found in cancer studies, showing no significant change in BCL-2 expression and significantly higher levels of Bax in tumor vs. healthy tissue. / by Angela Bancalari-Schmidlapp. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Wong Chun Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 149-160). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Abbreviations --- p.v / Acknowledgement --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General review of angiogenesis --- p.1 / Chapter 1.1.1 --- Angiogenesis in development and growth --- p.1 / Chapter 1.1.2 --- The process of angiogenesis --- p.2 / Chapter 1.1.3 --- Types of factors controlling angiogenesis --- p.3 / Chapter 1.2 --- Roles of VEGF and its receptors in the regulation of angiogenesis --- p.6 / Chapter 1.2.1 --- VEGF --- p.6 / Chapter 1.2.2 --- VEGF receptors --- p.8 / Chapter 1.2.3 --- Regulation of VEGF expression by hypoxia and nitric oxide… --- p.10 / Chapter 1.2.4 --- Signal transduction mechanisms of VEGFR-1 and VEGFR-2 --- p.12 / Chapter 1.2.5 --- Anti-apoptotic effect ofVEGF on endothelial cells as a result of signal transduction of VEGFR-2 --- p.14 / Chapter 1.3 --- Angiopoietins --- p.15 / Chapter 1.3.1 --- Angiopoietin 1 (Ang-1) --- p.16 / Chapter 1.3.2 --- Angiopoietin 2 (Ang-2) --- p.19 / Chapter 1.3.3 --- Angiopoietins 3 and 4 (Ang-3 and Ang-4) --- p.24 / Chapter 1.4 --- "Interaction among VEGF, angiopoietin and Tie in the maintenance of vasculature" --- p.25 / Chapter 1.5 --- Tyrosine kinase with immunoglobulin and EGF factor homology domains - Tie 1 and Tie 2 --- p.28 / Chapter 1.6 --- Angiopoietin expression in female reproductive tissues (ovary) --- p.33 / Chapter 1.7 --- Testicular angiogenesis --- p.37 / Chapter 1.8 --- Aims of the present study --- p.38 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Preparation of primary cells from rat testes --- p.40 / Chapter 2.1.1 --- Sertoli cell preparation --- p.40 / Chapter 2.1.2 --- Germ cell preparation --- p.41 / Chapter 2.1.3 --- Interstitial cell and Leydig cell preparation --- p.43 / Chapter 2.2 --- Cell cultures --- p.45 / Chapter 2.2.1 --- Reagents and cell lines --- p.45 / Chapter 2.2.2 --- Cell lines of mouse TM3 Leydig cells and TM4 Sertoli cells --- p.45 / Chapter 2.2.3 --- Mouse MLTC-1 Leydig tumour cells --- p.46 / Chapter 2.2.4 --- Rat R2C Leydig tumour cells --- p.46 / Chapter 2.2.5 --- Rat LC540 Leydig tumour cells --- p.47 / Chapter 2.2.6 --- "Rat C6 glioma cells.............," --- p.47 / Chapter 2.3 --- "Analyses of Angiopoietin 1, Angiopoietin 2, Angiopoietin3, Tie 1 receptor, and Tie 2 receptor mRNA in testicular cell lines and testicular tissues" --- p.48 / Chapter 2.3.1 --- Extraction of total RNA from testicular cell lines and testicular tissues --- p.48 / Chapter 2.3.2 --- Quantitation of total RNA --- p.50 / Chapter 2.3.3 --- First strand cDNA synthesis by reverse transcription (RT) --- p.51 / Chapter 2.3.4 --- Normalization of the amounts of cDNA usedin polymerase chain reaction (PCR) --- p.52 / Chapter 2.3.5 --- Polymerase chain reaction (PCR) --- p.53 / Chapter 2.3.6 --- Purification of PCR products --- p.65 / Chapter 2.3.7 --- Confirmation of PCR product authenticity by automated DNA sequencing --- p.66 / Chapter 2.4 --- Western blot analysis --- p.68 / Chapter 2.4.1 --- Preparation of cell lysates from primary testicular cells and testicular cell lines --- p.68 / Chapter 2.4.2 --- Preparation of mouse testicular tissue and adult rat testicular tissue lysates --- p.68 / Chapter 2.4.3 --- Determination of protein concentration --- p.69 / Chapter 2.4.4 --- Reagents for Western blot analysis --- p.70 / Chapter 2.4.5 --- Preparation of protein samples and markers for Western blot analysis --- p.71 / Chapter 2.4.6 --- Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.72 / Chapter 2.4.7 --- Transfer of proteins to membrane --- p.74 / Chapter 2.4.8 --- Blocking of the membrane --- p.74 / Chapter 2.4.9 --- Immunoblotting --- p.75 / Chapter 2.5 --- "Immunohistochemical staining for Ang-1, Ang-2，Ang-3, Tie 1 and Tie 2 in rat testes" --- p.78 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of Ang-1 and Ang-1 alternatively spliced transcripts in the testis and other testicular cell types --- p.81 / Chapter 3.1.1 --- Detection of Ang-1 expression in the testis and and testicular cell types by nested PCR --- p.81 / Chapter 3.1.2 --- Detection of Ang-1 expression in testicular cell lines by nested PCR --- p.82 / Chapter 3.1.3 --- Sequence analysis of Ang-1 transcript amplified from adult rat testis --- p.84 / Chapter 3.1.4 --- Detection of alternatively spliced species of Ang-1 mRNA in the testis and other testicular cell lines --- p.87 / Chapter 3.2 --- Expression of Ang-2 and Ang-2 isoforms in the testis and various testicular cell types --- p.94 / Chapter 3.2.1 --- Detection of Ang-2 expression in the testis and testicular cell types by nested PCR --- p.94 / Chapter 3.2.2 --- Detection of Ang-2 expression in testicular cell lines by nested PCR --- p.96 / Chapter 3.2.3 --- Sequence analysis of Ang-2 transcript amplified from adult rat testis --- p.98 / Chapter 3.2.4 --- Detection of the expression of Ang-2 isoforms in adult rat testis --- p.99 / Chapter 3.3 --- Expression of Ang-3 in the testis and testicular cell types --- p.103 / Chapter 3.3.1 --- Detection of Ang-3 expression in the testis and primary testicular cells by RT-PCR --- p.103 / Chapter 3.3.2 --- Detection of Ang-3 expression in testicular cell lines by RT-PCR --- p.105 / Chapter 3.3.3 --- Sequence analysis of Ang-3 transcripts amplified from TM4 mouse Sertoli cells and adult rat testis --- p.105 / Chapter 3.4 --- Expression of Tie 1 and Tie 2 in the testis and testicular blood vessel --- p.110 / Chapter 3.4.1 --- Detection of Tie 1 and Tie 2 expression in the testis and rat testicular blood vessel by RT-PCR --- p.110 / Chapter 3.4.2 --- Sequence analysis of Tie 1 transcripts amplified from adult rat testis and rat testicular blood vessel --- p.113 / Chapter 3.4.3 --- Sequence analysis of Tie 2 transcript amplified from rat testicular blood vessel --- p.113 / Chapter 3.5 --- "Western blot analysis of Ang-1 and Ang-2 expression in testicular tissues, primary testicular cells and cell lines" --- p.116 / Chapter 3.6 --- "Localization of Ang-1，Ang-2, Ang-4, Tie 1 and Tie2 proteins in adult rat testis by immunohistochemistry" --- p.122 / Chapter 3.7 --- "Comparison of angiopoietin expression patterns in testis using RT-PCR, Western immunoblotting and immunohistochemistry" --- p.128 / Chapter 3.8 --- Comparison of Tie 1 and Tie 2 expression patterns in testis using RT-PCR and immunohistochemistry --- p.128 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- "Expression of Ang-1 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.131 / Chapter 4.2 --- "Expression of Ang-2 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.136 / Chapter 4.3 --- "Expression of Ang-3 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.141 / Chapter 4.4 --- Expression of Tie 1 and Tie 2 mRNAs and proteinsin adult rat testis and rat testicular blood vessel --- p.143 / Chapter 4.5 --- Conclusion --- p.145 / Chapter 4.6 --- Future work --- p.146 / Chapter Chapter 5 --- References --- p.149

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. To investigate how mutant p53 impacts transcription, we studied how mutant p53 regulates vascular endothelial growth factor receptor 2 (VEGFR2), one of its strongest target genes that we identified through global gene expression profiling in mutant p53 expressing MDA 468 breast cancer cells. VEGFR2, the primary functional VEGF receptor and clinical target of bevacizumab, mediates endothelial cell neovascularization by promoting increased cellular proliferation, migration, and pro-survival signaling. In breast tumors, VEGFR2 is often aberrantly expressed on the breast tumor epithelia,which correlates with worse overall survival. We identify VEGFR2 as a mutant p53 transcriptional target in multiple breast cancer cell lines. Mutant p53 mediated upregulation of VEGFR2 mediates mutant 53 gain of function including increased cellular growth and migration. In humans, breast tumors with TP53 hotspot mutants have elevated VEGFR2 levels compared to tumors with loss of function mutations. The same class of tumors has significantly upregulated HIF1A and VEGFA compared to TP53 wild type tumors, indicating that mutant p53 containing breast tumors express a neoangiogenic gene signature that may intensify VEGFR2 autocrine signaling. A clinical trial suggests that TP53 mutated breast tumors may specifically respond to anti VEGF therapy, while TP53 wild type tumors may not respond. We suggest that mutant p53 containing breast tumors may be distinctively vulnerable to anti VEGF ntherapies. We investigated how mutant p53 impacts transcription of VEGFR2 using multiple techniques including scanning ChIP, micrococcal nuclease PCR, and in vivo DNase I footprinting by ligation mediated PCR. Mutant p53 was found to bind near the VEGFR2 transcriptional start site, causing the promoter to adopt a transcriptionally active conformation. Using SILAC mass spectrometry, we identified subunits of the SWI/SNF chromatin remodeling complex as mutant p53 interactors. Importantly, re ChIP and immunodepletion ChIP demonstrate that mutant p53 and SWI/SNF co-occupy the VEGFR2 promoter. Depletion of multiple SWI/SNF subunits reduced VEGFR2 RNA expression, and SWI/SNF is required for maximal mutant p53 promoter occupancy. Using RNA sequencing, we report that approximately half of all mutant p53 gene alteration impacts transcription of VEGFR2 as well as myriad other target genes by promoter remodeling through interaction with the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53 expressing tumors be uniquely susceptible to anti VEGF therapies, but restoration of SWI/SNF tumor suppressor function by targeting mutant p53 may have therapeutic potential. Mutant p53 interaction with the SWI/SNF complex may explain how mutant p53 modulates the expression of such a diverse set of genes.

Five traditional Chinese medicines were screened for their antiangiogenic activities through zebrafish angiogenic assay. Two of them, Tripterygium wilfordii and Rheum palmatum showed potential in the primary screening. T. wilfordii was selected in further study. / In the further investigation of antiangiogenic activity of triptolide on mammal systems, triptolide showed potent activity in human umbilical vein endothelial cells (HUVECs) assays including proliferation, migration and tube formation assay. It inhibited HUVEC proliferation with IC50 as low as 34 nM. It also showed more potency in HUVEC migration and tube formation assay at as low concentration as nanomolar level than SU5416, a putative VEGFR-2 inhibitor currently in clinic trials. RT-PCR and western blotting analysis showed that the underlying mechanism of triptolide correlated with down-regulation of VEGFR-2 and Tie2 expression and production. Tie2 inhibition appeared to be a later event as compared with VEGFR-2. Tie2 overexpression in HUVEC could attenuate the inhibitory effect of triptolide on HUVEC proliferation. Tie2 knockdown mimicked the inhibition activity of triptolide in tube formation assay. These phenomemon revealed that Tie2 signaling pathway plays a crucial role in triptolide-mediated angiogenesis inhibition. In in vivo Matrigel Plug assay, triptolide showed inhibition effect at as low as 100 nM. / T. wilfordii is an immune-suppressive, anti-inflammatory herb used in China for centuries. Through bioassay-guided purification, three antiangiogenic terpenoids were isolated from the ethyl acetate fraction, namely, celastrol, cangoronine and triptolide. Among them, triptolide manifested the most potent antiangiogenic activities against vessel formation. As low as 0.31microM, triptolide inhibited 20% of vessel formation, and the inhibition reached a plateau of 50% at 1.2 microM. Celatrol reduced vessel formation by more than 30% at 0.62microM, but killed 50% of the embryos at higher concentrations. Cangoronine was much weaker, inhibiting vessel formation by 20% at 2.5microM. These three components all showed stronger antiangiogenic activities than 2-methoxyestradiol, a putative compound currently under clinical trials as an antiangiogenic agent for cancer treatment, as the latter inhibited angiogenesis in zebrafish embryos by 34% at 10microM. The loss of vessel formation in the embryos treated with triptolide was further confirmed using endogenous alkaline phosphatase staining. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt2) and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. / This research revealed that zebrafish model is a promising antiangiogenic model for both the screening of antiangiogenic agents from Chinese herbal medicine and the subsequent discovery for the drug targets. Triptolide, an anti-inflammatory component from T. wilfordii, is a potent angiogenic inhibitor through targeting VEGFR-2 and Tie2 pathways in mammal models whereas targeting ang2-tie2 pathway in zebrafish model. The anti-tumor action of triptolide was demonstrated to be partly through inhibition of tumor angiogenesis. Moreover, the potent antiangiogenic action exerted by triptolide at nanomolar dosage level gives proof that it is a promising lead compound for the development of antiangiogenic drug for cancer treatment. (Abstract shortened by UMI.) / He, Mingfang. / Adviser: Paul Pui-Hey Bot. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0247. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 84-106). / 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. / Abstracts in English and Chinese.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 140-156). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 140-156). Also available in print.

published_or_final_version / Anatomy / Master / Master of Philosophy

Statins induce beneficial vascular effects. How statins induce beneficial vascular effects is yet to be determined. Here we examine Simvastatin and vascular endothelial growth factor (VEGF) acting through the phosphoinositide 3-kinase (PI3K) pathway in human coronary artery endothelial cells (HCAEC). While Simvastatin and VEGF both activated mediators in the PI3K pathway, the proteins and the rates of activation were not always consistent. This suggests that although Simvastatin and VEGF share a common PI3K pathway in HCAEC and similar vascular effects, the agonists diverge in the induction of cellular signaling cascades. Simvastatin also was shown to induce phosphoinositide 3, 4, 5-triphosphate (PIPS) organization and PI3K p110 gamma (y) perinuclear localization. Beneficial, non-lipid lowering effects of statins may occur through the PI3K pathway through activation of distinct mediators from those of VEGF. Better understanding of the pathways associated with statins is necessary for the discovery of better treatments for cardiovascular disease (CVD). / Department of Biology

[Truncated abstract] One of the barriers to understanding and preventing proliferative diabetic retinopathy in humans has been the lack of an appropriate animal model. Historically dog, rat and mouse models of diabetic retinopathy have been studied but none of these exhibit the later changes of proliferative diabetic retinopathy. Animals can be rendered diabetic by surgical pancreatectomy or the use of chemicals such as allozan or streptozotocin or by feeding of a high galactose diet. Alternatively, spontaneous rodent models of diabetes have been examined such as the BB rat, KK mouse or NOD mouse. However, in each case the retinal vascular changes observed are those of early nonproliferative diabetic retinopathy comprising at most saccular microaneurysms, increased thickness of the capillary basement membrane, acellular capillaries and pericyte ghosts. … Fluorecein angiography of this transgenic line clearly demonstrates the presence of leaky new vessels, by the appearance of leakage spots scattered throughout the retina from 1 month of age. These mice constitute a valuable model of diabetic retinopathy. Neovascularization in this animal model is induced by VEGF as in human diabetic retinopathy. The source of VEGF in human diabetic retinopathy is the ischemic inner retina. In this transgenic model the source of VEGF are the photoreceptor cells, which are situated just underneath the inner retina. The neovascularization is not dependent on a particular developmental stage and there is no spontaneous regression of new vessels. Thus any results generated in this model are highly relevant to human diabetic retinopathy.