by Hiu-ming Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 81-88). / SUMMARY --- p.1 / Chapter CHAPTER 1. --- INTRODUCTION --- p.3 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.7 / Chapter 2.1. --- Retinal pigment epithelium --- p.7 / Chapter 2.1.1. --- Embryology / Chapter 2.1.2. --- Anatomy and histology / Chapter 2.1.3. --- Growth and aging / Chapter 2.1.4. --- Macular region / Chapter 2.2. --- The photoreceptor outer segment --- p.12 / Chapter 2.3. --- Lipofuscin --- p.13 / Chapter 2.4. --- Lipofuscin in retinal pigment epithelium and retinal photoreceptor disc shedding --- p.14 / Chapter 2.5. --- Possible mechanism for lipofuscin formation in the RPE --- p.22 / Chapter 2.6. --- Age-related lipofuscin accumulation in the RPE --- p.22 / Chapter 2.7. --- Racial difference of RPE lipofuscin concentration --- p.25 / Chapter 2.8. --- RPE lipofuscin and age-related macular degeneration --- p.26 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.28 / Chapter 3.1. --- Histologic Specimens --- p.28 / Chapter 3.2. --- Measuring equipment --- p.28 / Chapter 3.3. --- Software of measurements --- p.31 / Chapter 3.4. --- Light source and filters --- p.31 / Chapter 3.5. --- Control --- p.31 / Chapter 3.6. --- Measurement of autofluorescent Intensity --- p.32 / Chapter 3.7. --- Bleaching (oxidation) of melanin --- p.39 / Chapter CHAPTER 4. --- RESULTS --- p.42 / Chapter 4.1. --- Bleaching test --- p.42 / Chapter 4.2. --- RPE autofluorescence observation in different age --- p.43 / Chapter 4.3. --- RPE autofluorescence observation within individual eyes --- p.45 / Chapter 4.4. --- Topographic distribution of lipofuscin --- p.49 / Chapter 4.5. --- Lipofuscin content at the Foveola --- p.50 / Chapter 4.6. --- The relationship of age and lipofuscin content in total RPE --- p.51 / Chapter 4.7. --- The relationship of age and lipofuscin content in the macular RPE --- p.57 / Chapter 4.8. --- Relationship of age and lipofuscin content in the posterior pole of RPE --- p.59 / Chapter 4.9. --- Relationship of age and lipofuscin content in the temporal RPE --- p.61 / Chapter 4.10. --- Relationship of age and lipofuscin content in the nasal RPE --- p.63 / Chapter 4.11. --- Age related topographic changes --- p.65 / Chapter 4.12. --- The relationship of age and lipofuscin content in the RPE of male --- p.66 / Chapter 4.13. --- The relationship of age and lipofuscin content in the RPE of female --- p.68 / Chapter 4.14. --- Relationship of lipofuscin content in different sex --- p.70 / Chapter CHAPTER 5. --- DISCUSSION --- p.71 / Chapter 5.1. --- Evaluation of method --- p.71 / Chapter 5.2. --- RPE lipofuscin content in different age --- p.71 / Chapter 5.3. --- Topographic distribution of lipofuscin --- p.74 / Chapter 5.4. --- Lipofuscin and age-related macular degeneration in Chinese --- p.78 / REFERENCES --- p.81

The urothelium is a stratified epithelium that serves as a barrier between the urinary tract and blood. It consists of terminally differentiated umbrella cells, which are specialized for synthesizing and assembling uroplakins into a tough apical plaque and responsible for the barrier function; intermediate cells which are few in number but serve as umbrella cell progenitors; and unipotent basal cells, which populate the majority of the urothelium. The urothelium is one of the most quiescent epithelia in the body but can rapidly regenerate in response to damage. The urothelium is also a source of cells that give rise to bladder cancer. Patients with chronic inflammation caused by indwelling catheters or repeated urinary tract infections have a higher risk of developing bladder cancer. Bladder cancers with squamous histological features are considered to be more aggressive with poor prognosis and the majority are categorized as basal subtype. The expression of Peroxisome Proliferator-Activated Receptor-gamma (PPARG) is strongly down regulated in the basal subtype of bladder cancer, suggesting that its removal might be essential in tumorigenesis. PPARG is a nuclear hormone receptor that was originally described as a master regulator of adipogenesis but could also promote cellular differentiation in a number of epithelium. PPARG also serves as an important regulator in anti-inflammatory activity after a variety of injuries, acting in part by antagonizing the NF-B pathway. In urothelial cells, it has been shown that PPARG promotes urothelial differentiation in vitro, but its function in vivo remains unexplored. To determine the role of PPARG in vivo, we used Cre-Lox recombination to conditionally delete the Pparg gene in the mouse urothelium using the ShhCre driver, which drives recombination in basal and intermediate cells, and their respective daughters. Interestingly, ShhCre;Ppargfl/fl mutants lack umbrella and intermediate cells which normally populate the luminal and sub-luminal layers, and are instead populated with an abnormal cell population negative for classical urothelial markers. The basal compartment, which in wild type mice is largely populated by P63+ KRT5+ basal cells with a small sub-population expressing KRT14; has an increased number of KRT14-expressing cells in the mutants and exhibits squamous features that are not present in the normal urothelium. In wild type animals, urinary tract infection (UTI) with uropathogenic E.coli results in a transient innate immune response, followed by proliferation and repair, which is largely complete within 2 weeks. When ShhCre;Ppargfl/fl mutants were challenged with urinary tract infection, the innate immune response was not resolved even after several weeks, as characterized by persistent NF-B activity, excessive influx of neutrophils and macrophages, and massive granulation tissue in the stroma. In addition, the Pparg-knockout urothelium exhibited squamous metaplasia. The Krt14+ basal cell population, which is considered to be the cells of origin of bladder cancer, greatly expanded in the Pparg-deleted urothelium after infection, and some lesions progressed to acquire invasive features. Together these findings suggest that PPARG is essential for the normal differentiation of the urothelium and is a potent regulator of the inflammatory response after UTI. Understanding the link between the loss of PPARG, chronic inflammation and tumorigenesis in the urothelium could shed light on the urothelial differentiation network and pave the way for the development of therapeutic approaches to various urinary diseases.

Ovaries maintain and produce functional female gametes, oocytes, for fertilisation. Oocytes develop inside cellular assemblies, the ovarian follicles, before birth and can reside there for up to 50 years in the human. Despite recent inroads, the precise mechanisms of initial follicle recruitment and growth remain unclear. Although the pituitary gonadotrophins play a role in this developmental process, locally produced factors have been implicated strongly in initiation of follicle growth. It is known that fibroblast growth factor 2 (FGF2) is a powerful mitogen for follicular granulosa cells in culture and initial studies undertaken in this project were successful in detecting FGF2 gene expression in ovarian biopsies from fertile healthy women. To further elucidate which cells were expressing FGF2, laser microdissection was employed to isolate differentially staged follicle populations. Real-time RT-PCR was used to quantify mRNA in relation to follicle development. Decreasing levels of FGF2 expression were detected as follicles developed. Non-radioactive in situ hybridisation confirmed FGF2 mRNA localisation in granulosa cells of preantral follicles. FGF2 protein localisation was assessed with immunohistochemistry; two primary antibodies raised against different fragments of human FGF2 were used. Both antibodies detected FGF2 in the oocyte cytoplasm of putative non-growing follicles, whereas only one of the antibodies showed additional reactivity to the basement membrane region of these same follicles. These results suggest different isoforms of FGF2 may localise specifically to different cellular sites. Follicle stimulating hormone receptor (FSHR) gene expression was also investigated in follicles using laser microdissection, real-time RT-PCR and in situ hybridisation. FSHR mRNA was detected in all follicle populations, including the smallest putative non-growing follicles. Disparity to other published works was attributed to the position of primer annealing, and thus the ability to detect alternatively spliced transcripts. In conclusion, the work presented here provides evidence that FGF2 and FSHR are present in small follicles and that their actions may be stimulatory or inhibitory to initial follicle recruitment.

The epithelial sodium channel (ENaC) is the central component of the sodium absorption pathway in epithelia. It is critical for sodium homeostasis and blood pressure control, which is demonstrated by rare genetic disorders such as Liddle�s syndrome and pseudohypoaldosteronism type I, that are associated with hyper- and hypotension, respectively. ENaC is mainly regulated by mechanisms that control the expression of active channels at the cell surface. Ubiquitin ligases of the Nedd4-like family, such as Nedd4 and Nedd4-2 decrease epithelial sodium absorption by binding to and targeting ENaC for endocytosis and degradation. This is most likely achieved by catalyzing the ubiquitination of ENaC. Conversely the serum- and glucocorticoid regulated kinase (SGK) increases ENaC activity. This effect is partly mediated by the interaction of SGK with the ubiquitin ligases Nedd4 and Nedd4-2. SGK is able to bind to both Nedd4 and Nedd4-2, however only Nedd4-2 is phosphorylated by SGK. The phosphorylation of Nedd4-2 inhibits its interaction with ENaC, thus reducing ENaC ubiquitination, thereby increasing surface expression and sodium absorption. Nedd4-like proteins interact with ENaC via their WW-domains. These domains bind PY-motifs (PPXY) present in ENaC subunits. Nedd4 and Nedd4-2 both have four highly similar WW-domains. Previous studies have shown that interaction between Nedd4 and ENaC is mainly mediated by WW-domain 3. SGK also has a PY-motif; therefore it was analyzed whether the WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK. Here, it is shown that single or tandem WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK and that, despite their high similarity, different WW-domains of Nedd4 and Nedd4-2 are involved. These data also suggest that WW-domains 2 and 3 of Nedd4-2 mediate the interaction with SGK in a concerted manner, and that in vitro the phosphorylation of SGK at serine residue 422 increases its affinity for the WW-domains of Nedd4-2. The stimulatory effect of SGK on ENaC activity is partly mediated via Nedd4-2 and will decrease if competition between Nedd4 and Nedd4-2 for binding to SGK occurs. Here it is shown that Nedd4 and Nedd4-2 are located in the same subcellular compartment and that they compete for binding to SGK. Besides its function in the proteasomal degradation pathway ubiquitination is involved in the regulation of membrane protein trafficking, including their endocytosis. ENaC was shown previously to be ubiquitinated. Here, we provide evidence that ENaC can be ubiquitinated differentially depending on its cellular location. Channels residing in the plasma membrane are multiubiquitinated and we suggest that this serves as an internalization signal for ENaC and a control for further trafficking. Cytosolic ENaC is mainly polyubiquitinated, and therefore probably targeted for proteasomal degradation. However, mono- and multiubiquitination of ENaC located within the cytosol is very likely to occur as well. In addition, it is shown that both proteasomal and lysosomal pathways are involved in the regulation of ENaC.

Background: The p63 gene is a member of the p53 transcription factor family and can produce six different proteins using two promoters and differential splicing. Expression of p63 is required for proper formation of epithelial tissues. Studies on the transcriptional control of specific genes involved in cell survival, proliferation, differentiation and adhesion have revealed the contributions of p63 to the continuously renewing stratified epithelium. In this thesis, the aim was to improve our understanding of the roles of p63 in epithelial homeostasis by investigating expression of p63 in normal and benign hyper-proliferative epithelia and exploring the influence of p63 deregulation on cancer progression. Materials and methods: Using quantitative real time RT-PCR and immunohistochemistry, we first examined the expression of different p63 isoforms in patients diagnosed with psoriasis - a benign hyper-proliferative and inflammatory skin disease. Afterwards, we investigated responses of p63 in psoriatic epidermis upon Narrowband-UVB (NB-UVB) phototherapy. At the same time, we studied the potential impact of p63 in carcinogenesis by searching for p63 transcriptional targets in a cell line derived from squamous cell carcinoma of the head and neck (SCCHN) - the sixth most common cancer worldwide with over-expression of the ∆Np63α protein as a common feature. p63 gene silencing and microarray were used to identify p63 regulated genes. Real time RT-PCR, western blot, immunohistochemistry, chromatin immunoprecipitation, transient transfection and reporter assays were performed to confirm specific genes as direct p63 targets. Results: Significant down-regulation of p63 mRNA levels was found in psoriatic lesions compared to patients’ own clinically normal skin. Moreover, a trend of decreased TAp63 mRNA levels was seen in patients’ normal skin compared to age- and sex-matched healthy controls. Following NB-UVB phototherapy, an effective first line therapy for psoriasis, expression of p63 was not significantly affected. However, significant changes in p53, FABP5, miR-21 and miR-125b were found. Surprisingly, location and expression levels of p63 proteins detected by immunohistochemistry were similar under all skin conditions. A direct transcriptional regulation of TRAF4 by p63 was seen in the SCCHN cell line and we further found that the localization of the TRAF4 protein was associated with histological differentiation of SCCHN cells. However, unlike its over-expression in SCCHN, similar TRAF4 mRNA expression levels were seen in psoriatic lesions as compared to healthy controls. Besides TRAF4, a total of 127 genes were identified as potentially p63 regulated in the SCCHN cell line and strikingly, about 20% of these genes are involved in cell adhesion or migration. Conclusions: Dysregulation of p63 isoforms in psoriatic epidermis, especially decreased TAp63 expression, and their resistance to NB-UVB phototherapy implicated a contribution of p63 to the psoriasis phenotype. Transcriptional regulation of genes involved in multiple biological pathways indicated that over-expression of p63 in SCCHN might account for altered cell differentiation, adhesion and migration, thus contributing to SCCHN. In conclusion, our studies have found additional mechanisms through which p63 guarded homeostasis of the established epithelium. Deregulation of p63 might play a role in distinct pathological conditions by participating in diverse cellular pathways under different microenvironments.

Aged-related macular degeneration (AMD) is a common eye condition among people older than 65 years and is a leading cause of vision loss. It gradually destroys the macula, the part of the eye that provides sharp, central vision needed for seeing objects clearly. This study aims to test the hypothesis that the morphology of retina pigment epithelium, a key site of AMD pathology, can reflect the various stresses aging and AMD progression impose. We first identify and separate the young and old age group for mouse eyes. Then we classify, the mouse eyes using two genotypes (C57BL/6L, RD10), and two age group (young, old).We show that without dimensional reduction, the cell area and shape measures do not provide good classification of the mouse eyes. But with the dimension reduction at the eye level, the cell area and shape measures provide excellent classification for mouse genotype and age.

Thesis--M. Med. Sc., University of Hong Kong, 1982.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Epidemiological studies have described a link between chronic inflammatory conditions, such as diabetes or obesity, and EOC suggesting that systemic inflammation may increase the risk of the disease. The purpose of this study was to identify the impact of prolonged exposure to low-grade inflammation on EOC tumorigenicity. We hypothesized that exposure to this inflammation would accelerate ovarian tumor growth. In vitro, normal and transformed ovarian epithelial cells had limited responsiveness to inflammatory cytokines. In vivo, LPS-induced low-grade chronic systemic inflammation accelerated EOC progression primarily through enhanced angiogenesis. Evaluation of the relationships between chronic systemic inflammation and EOC may provide a role for anti-inflammatory treatment in combinational EOC therapies. Additionally, as the rate of metabolic disorders increases in the Western world the results from this work may facilitate the advancement of complimentary therapeutic interventions for other cancers that are influenced by inflammation.

Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.