The aims of this thesis were to determine the relative contribution of Pdx-1, MafA and β2 to the human insulin promoter.  Additionally, characterisation of the conserved CRE element (CRE2) was undertaken to identify the factor(s) that bind CRE2 to mediate affects upon the transcription of insulin.  Finally, the ectopic expression of insulin was investigated using an engineered zinc finger protein (ZFP-INS) that ‘switched on’ the endogenous insulin gene in a non-β cell (HEK293). Consistent with previous findings, Pdx-1, MafA and β2 were shown to synergistically activate the rat insulin 1 promoter.  However due to subtle sequence divergence, similar synergistic interaction was not observed on the human promoter.  Synergistic interactions were re-established in a rat insulin 1-like mutated human insulin construct.  The CRE binding protein activating transcription factor-2 (ATF-2) was shown to bind and stimulate transcription via CRE2 while CRE binding protein-1 (CREB-1) inhibited insulin transcription independently of CRE1 or CRE2.  ZFP-INS was shown to induce the ectopic expression of insulin in HEK293 cells via the ILPR region, inducing modifications of histone proteins at the insulin promoter.  Collectively, the continued characterisation of the human insulin promoter may reveal unique regulatory mechanisms controlling the expression of insulin under normal and diabetic conditions.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

The overall goal of my work has been to gain a better understanding of Axenfeld-Rieger Syndrome (ARS), a human autosomal dominantly inherited mal-development of the anterior segment of the eye that is associated with glaucoma. By studying rare genetic causes of this complex disease we are gaining insight into the initial steps that ultimately lead to blindness. To achieve the goal of better understanding ARS, my research project had two parts. In the first part, I performed a retrospective clinical study in which I analyzed the glaucoma-related clinical presentation of ARS patients with FOXC1 and PITX2 defects. This study showed a good genotype-phenotype correlation which may be important for the physician in dealing with ARS patients. Patients with FOXC1 mutations had the mildest prognosis in glaucoma development, while patients with PITX2 defects and patients with FOXC1 duplication had a more severe prognosis in glaucoma development than patients with FOXC1 mutations. I tried to determine the best treatment for glaucoma in these patients. Unfortunately, in this study, current medical therapies did not successfully lower intraocular pressure or prevent progression of glaucoma in ARS patients with FOXC1 or PITX2 alterations. This clinical study also provided useful diagnostic criteria to identify the gene responsible for ARS. The second part of the project was to study the gene regulatory pathways of the PITX2 gene, mutations of which cause ARS. PITX2 is a transcription factor that regulates the expression of genes in the eye. The discovery of direct downstream targets of PITX2 is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development and underlying heritable human disorders. To find direct target genes of PITX2, I have used a recently developed method: the hormone receptor (HR)-inducible expression system for transcription factors coupled microarray analysis. The results obtained using this method have involved PITX2 in control of cellular stress. Recent investigations have suggested significant roles for cellular stress in glaucoma pathology. Understanding the control of these key aspects of cell function will have profound implications for understanding and treating the glaucoma that is the most clinically serious consequence of mutations of PITX2.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Also available online.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 119-129). Also available for download via the World Wide Web; free to University of Oregon users.

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Gestational trophoblastic disease (GTD) is arised from the neoplastic trophoblasts in placenta. Trophoblasts have the characteristic of proliferation and invasion. GTD is classified as partial hydatidiform mole (PHM), complete hydatidiform form mole (CHM), invasive hydatidiform mole (IHM), choriocarcinoma (CCA), placental site trophoblastic tumour (PSTT), epithelioid trophoblastic tumour (ETT), exaggerated placental site trophoblastic reaction (EPSR) and placental site nodule (PSN). HM has the potential to develop into malignant trophoblastic disease, and metastasis to other parts of body. FoxD3 gene belongs to Forkhead family. Its protein acts as embryonic stem cell transcription factor and plays an important role in neural crest and placenta development. Previous studies from our team have reported that other embryonic stem cell transcription factors, such as Nanog, Sox2 Oct4 and Stat3, are related with pathogenesis of GTD. This study aim is to investigate the protein expression profile of FoxD3 in different types of GTD using immunohistochemistry method. In this study, 70 formalin fixed paraffin embedded tissue blocks from 16 normal first trimester placenta, 38 CHM, 9 CCA, 5 PSTT and 2 ETT were retrieved. Paraffin sections were prepared and stained with FoxD3 antibody by using immunohistochemistry method. Compared with normal placentas, there was significantly increased expression of FoxD3 in trophoblasts of CM and PSTT (p<0.05). In CCA, there was high expression of FoxD3 in syncytiotrophoblasts and intermediate trophoblasts (p<0.05). In ETT, the immunoreactivity of FoxD3 is not significantly increased when compared with intermediate trophoblasts (p=0.07). To conclude, FoxD3 was found to be over-expressed in GTD. FoxD3 may contribute to pathogenesis of GTD. Further investigations are needed to discover the relationship with other embryonic transcription factors and genes to improve the diagnosis, prognosis and treatment of GTD. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Objectives: Forkhead box protein P1 (FOXP1) is a transcription factor, and a member of the P-subfamily of forkhead box transcription factor and regulate transcription of a subset of genes that involved in various cellular events. It plays a critical role in regulating cell growth and proliferation, differentiation, embryogenesis, adult tissue homeostasis, and possibly tumorigenesis. Predominant nuclear localisation of FOXP1 protein is commonly expressed at low level in normal tissues and upregulated in proliferative cells. Studies have demonstrated that the loss of FOXP1 expression and cytoplasmic mis-localisation is significantly associated with various malignant cancers, including breast cancer. FOXP1 can act either as a tumor suppressor or as an oncogenic protein in cell-type specific functions. It has been shown to be a co-regulator of estrogen receptor alpha and can modify a specific subset of forkhead box transcription factor class O (FOXO)-target genes. We hypothesise that there is association between FOXP1 expression and patient survival, and explore the potential role of FOXP1 expression as a prognostic marker in breast cancer. Methods: One hundred and twenty breast cancer samples in tissue microarray blocks were examined for FOXP1 expression by immuno-histochemistry. Nuclear and cytoplasmic FOXP1 expression patterns were analysed with clinico-pathological parameters. Statistical analysis was performed using SPSS software to determine the correlation between FOXP1 expression and clinico-pathological parameters. The correlation between subcellular FOXP1 expression and survival was evaluated by COX regression analysis. Results: Nuclear or cytoplasmic FOXP1 expression showed no association with clinico-pathological parameters. However, our results showed that there was significant association with estrogen receptor and progesterone receptor when nuclear and cytoplasmic scores were combined as total FOXP1 score (p=0.022 and p=0.028 respectively). In univariate analysis, high nuclear and cytoplasmic FOXP1 expression had no significant correlation with poor survival, while high total FOXP1 expression was associated with poor overall and disease-specific survival (p=0.045). Tumor stage and lymph-node involvement were significantly related to poorer overall and disease-specific survival, while other clinico-pathological parameters did not. In breast cancer with advanced tumor grade and lymph-node involvement, overall and disease-specific survival are significantly associated with high FOXP1 expression (p=0.041 and p=0.015 respectively). Conclusion: Unlike previous reports, our findings show that increased nuclear and cytoplasmic FOXP1 expression were both observed and high total FOXP1 expression was associated with poorer survival, particularly in cases of advance tumor grade and with lymph node metastases. These finding are supported by a recent report that showed that FOXP1 can up-regulate its own expression by binding to the promoter of FOXP1 and promote cell survival of breast cancer cells by suppressing FOXO-induced apoptosis. It may be possible that FOXP1 expression is up-regulated in a positive feedback loop in breast cancer cells such that there is both increased nuclear transcriptional activity and cytoplasm localisation of FOXP1. Further investigation is necessary to understand the role of FOXP1 in the progression of breast cancer and determine its potential use as a prognostic marker. / published_or_final_version / Pathology / Master / Master of Medical Sciences

The mouse pelage hair consists of three types of hair coined primary (guard), secondary (awls and auchenes) and tertiary (zigzag) hair. They display distinct morphologies and are induced consecutively during hair morphogenesis. Previously two identified regulatory mouse mutants, Yellow submarine (Ysb) and Light coat and circling (Lcc) which the chromosomal rearrangements have disrupted the cis-acting regulatory elements of Sox2; resulting in the loss of Sox2 expression in the inner ear. The mutants displayed lighter hair coat color due to a reduction in the proportion of secondary hair and increased proportion of tertiary hair. Sox18 null mutants display darker coat colour and reduced proportion of zigzag hair. To dissect the underlying mechanisms of the phenotypes in hair type specification in 〖Sox2〗^Ysb and 〖Sox2 〗^Lcc mutants and the role of Sox2 and Sox18 in regulating the process; the expression of Sox2 in the hair follicle and the change in the density of hair types in mutants were analyzed. I have identified the expression pattern of Sox2 in the dermal papilla (DP) of the hair follicle and verified its down-regulation in 〖Sox2〗^Ysband 〖Sox2 〗^Lcc mutants. The DP at the base of hair follicle is the signaling center for the regulation of hair development. Sox2 is specifically expressed in the DP of primary and secondary but not in tertiary hair while Sox18 is expressed in the DP of all hair types. Analysis of Sox2 mutants showed that the number of secondary hair was normal at induction but was reduced and accompanied by an increase in tertiary hair in adult mice. The number of tertiary hair was reduced in Sox18 null mutants. To gain insight into the molecular basis of hair type specification and potential targets of Sox2 in the regulation, gene expression profile in DP cells of 〖Sox2 〗^(EGFP/+)and 〖Sox2 〗^(EGFP/Ysb) mice was examined; the data suggests that genes in the Wnt and BMP signalling pathway were down-regulated in Sox2 mutants; while Runx3 and Corin may act downstream of Sox2 in regulating hair type specification and pigmentation. Hair follicles enter cycles of growth and regression throughout life during the hair cycle. Sox2 was only expressed in the growth phase while Sox18 was persistently expressed throughout the hair cycle. I further asked if Sox2 and Sox18 regulate post-natal hair development by analysing the expression pattern of Sox2 and Sox18 in wildtype mice and mutants throughout the hair cycle and the progression of hair growth in the mutants. The growth phase of the first hair cycle was extended in Sox2 mutants while the hair cycle in Sox18 null mutants was normal. Cell proliferation was compromised during hair regeneration leading to a delay in hair regeneration in Sox2 mutants. Sox2 and Sox18 showed overlapping expression in the DP and both regulate hair type specification. To test if Sox2 and Sox18 synergistically regulate hair development, the 〖Sox2〗^(Ysb/Ysb);〖Sox18〗^(-/-) mutants have been generated. Hair morphogenesis and differentiation were impaired; while the number of tertiary hair was increased with reduced number of secondary hair, which phenocopied that of Sox2 mutants. In conclusion, the results suggest that Sox2 and Sox18 functions synergistically on the regulation of hair growth and differentiation. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy