published_or_final_version / abstract / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Low molecular weight gelators (LMWGs) are interesting materials whose applications are as diverse and wide ranging as their molecular structures. These materials self-assemble through the formation of non-covelent intermolecular forces and interactions to form supramolecular assemblies that trap solvent within their matrices. Because of the non-covalent nature of the forces of self-assembly, the gelation process is typically thermally reversible. In addition, low molecular weight gelators can also be modified to respond to various stimuli, such as change in pH, presence of enzymes or metal cations, or exposure to light. The design of low molecular weight gelators is often difficult, and most new classes of low molecular weight gelators are discovered by serendipity. As such, it is often useful to use structural templates in the design of LMWGs. Biomolecules, such as steroids, amino acids and peptides, and carbohydrates make excellent templates due to their inherent propensity to self assemble. A review of the current literature regarding the use of biomolecules as templates for the design and synthesis of LMWGs will be presented in chapter 1. Our research group has been active in the research of carbohydrate-based LMWGs for several years, and these results are also briefly reviewed in the related chapters. The synthesis and characterization of ester derivatives of D-galactose, D-glucose, and amide derivatives of D-glucosamine will be discussed in chapters 2-4, along with their evaluation for gelation in aqueous and organic solvents, such as hexane, ethanol, water, and aqueous DMSO or ethanol mixtures.

Sepsis and septic shock are conditions, with severe outcome or in many cases death. Sepsis is a systemic inflammatory response trigger by bacteraemia but systemic inflammatory response can also be triggered by major trauma, major surgery, pancreatitis, severe burns etc. The systemic inflammatory reaction initiating the evolvement of septic organ dysfunction can be modelled using endotoxin, a Gram-negative bacterial lipopolysaccharide. This thesis used a porcine experimental sepsis model to examine timing of the inflammatory response due to endotoxin infusion (Paper I) and the influence of steroid treatment on the inflammatory response in endotoxemic pigs (Paper II). Timing of steroid treatment and the role of neutrophil granulocyte activation was evaluated with pig specific NGAL assessing neutrophil activation (Paper III). A clinical observational study was performed with the aim to differentiate between sepsis and other inflammatory conditions (e.g. trauma due to major surgery) evaluated by calprotectin as a marker of neutrophil activation (Paper IV). There was a dose-dependency in endotoxin tolerance which was measured with TNF-a. Pre-exposure to endotoxin did not reduce the pulmonary response to endotoxemic challenge. In fact, both PaO2 / FiO2 and static pulmonary compliance were reduced in this group when pre-treated with endotoxin at low dose. Endotoxemic animals treated with hydrocortisone were more stable in circulatory variables than those without such treatment. This was not explained by an ability of steroids to modulate the production of NO (Nitric oxide), which has been suggested to be a mechanism of steroids in this aspect. Pre-treatment with hydrocortisone attenuated the neutrophil granulocyte response and consequently diminished the release of NGAL in plasma. Circulatory derangement was associated with high plasma NGAL levels. Urine NGAL levels did not differ among the four groups. Plasma calprotectin levels on ICU admission is a sensitive marker of systemic inflammation and are markedly increased in patients with sepsis and patients with systemic inflammatory response. Plasma Calprotectin performed better than any of the other inflammatory variables in predicting mortality at 30 days, except from the composite mortality prediction score, SAPS 3.

Fimose, definida como a dificuldade em retrair o prepúcio para expor a glande, devido à presença de um anel fibroso, tem sido tradicionalmente tratada por meio da circuncisão. Durante a última década, no entanto, a corticoterapia foi proposta como tratamento clínico da fimose. Apresentamos um estudo duplo cego, para tratamento clínico de fimose em crianças, comparando um creme de furoato de mometasona 0,1%, aplicado topicamente, com um creme hidratante. Crianças de 2 a 13 anos de idade (n=110) apresentando fimose grau 5 de acordo com a escala de retratibilidade de Kikiros et al, todas previamente agendadas para cirurgia, foram incluídas neste trabalho. Após oito semanas de tratamento tópico, tanto com o creme hidratante quanto com o corticóide, os pacientes foram reavaliados e os que não responderam ao tratamento, antes da abertura do estudo duplo cego, foram submetidos a um adicional tratamento com corticóide, por mais oito semanas. No grupo dos pacientes tratados com corticóide, o anel fimótico desapareceu e a glande foi exposta em quarenta e nove pacientes (88%). No grupo placebo, o mesmo aconteceu 28 pacientes (52%) (*p <0,05). Dos 7 pacientes que não responderam ao tratamento inicial, com corticóide, 5 apresentaram boa resposta, ao tratamento adicional, enquanto que dos 26 pacientes do grupo placebo, 22 apresentaram exposição da glande, após o tratamento adicional (*p <0,05). Dois meninos, do grupo tratado com corticóide e quatro do grupo placebo, ainda continuaram com o anel fimótico, mesmo após o tratamento adicional com corticóide, e foram submetidos à postectomia. Concluímos que o uso de corticóide tópico, durante oito semanas, representa uma boa opção para o tratamento da fimose, principalmente na idade pré-escolar. / Phimosis, defined as the inability to retract the prepuce in order to expose the glans, due to the presence of a preputial fibrotic ring, is surgically treated in 1% of children. During the last decade, however, topical steroid treatment was proposed for phimosis. We herein present a double blind study comparing 0,1% mometasone furoate topical cream vs moisturizing cream (placebo) treatment for phimosis. Children aged 2 to 13 year old (n=110) presenting with phimosis (retractability grade 5 Kikiro´s classification) and scheduled for circumcision were included in this trial. The patients were evaluated after eight weeks topical treatment with moisturizing cream (n=54) or steroid cream (n=56). In the steroid group, the ring disappeared and glans was exposure obtained in forty nine patients (87,5%) vs twenty eight patients (52%) in the placebo group (*p<0,05). Non responders from both groups received an additional eight weeks steroid cream treatment. In the steroid group, five of seven patients were finally cured vs twenty two out of twenty six in the placebo group (*p<0,05). Two children with persisting phimosis (Kikiro´s retractability grade 5 and appearance grade 3 in the steroid group (4%) vs four children in the placebo group (7%) ended up receiving circumcision. We conclude that eight weeks topical steroid cream treatment represents a very good option for phimosis management in this age group.

by Linda Shiou-mei Ooi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 95-109). / Abstract --- p.i / Acknowledgements --- p.iii / List of Figures --- p.viii / Chapter Chapter I. --- Introduction --- p.1 / Objectives --- p.4 / Chapter Chapter II. --- Literature Review --- p.5 / Chapter II. 1. --- Lipoprotein-Lipids --- p.5 / Chapter II.1.1. --- General Concept and Metabolism of Lipoprotein-lipids --- p.5 / Chapter II. 1.2. --- Factors affecting plasma lipoprotein-lipids --- p.11 / Chapter II. 1.2.1. --- Ageing --- p.11 / Chapter II. 1.2.2. --- Obesity --- p.12 / Chapter II. 1.2.3. --- Diet --- p.13 / Chapter II. 1.2.4. --- Alcohol --- p.13 / Chapter II. 1.2.5. --- Cigarette smoking --- p.14 / Chapter II. 1.2.6. --- Exercise --- p.14 / Chapter II. 1.2.7. --- Gender differences --- p.14 / Chapter II.2. --- Sex Hormones --- p.14 / Chapter II.2.1. --- General concepts of sex hormone-production and the metabolism of sex hormones --- p.15 / Chapter II.2.1.1. --- Biosynthesis of testosterone --- p.16 / Chapter II.2.1.2. --- Metabolism of testosterone --- p.17 / Chapter II.2.1.3. --- Dihydrotestosterone (DHT) --- p.18 / Chapter II.2.1.4. --- Androstenedione --- p.18 / Chapter II.2.1.5. --- Biosynthesis of estrogen in men --- p.18 / Chapter II.2.1.6. --- Metabolism of estrogen --- p.19 / Chapter II.2.2. --- Factors affecting sex hormone levels in plasma --- p.19 / Chapter II.2.2.1. --- Sex hormone binding globulin (SHBG) --- p.19 / Chapter II.2.2.2. --- Sampling time --- p.19 / Chapter II.2.2.3. --- Stress and acute or chronic non-endocrine illnesses --- p.20 / Chapter II.2.2.4. --- Ageing --- p.21 / Chapter II.2.2.5. --- Diet and nutrition --- p.21 / Chapter II.2.2.6. --- "Medication, drugs and alcohol" --- p.22 / Chapter II.2.2.7. --- Body composition and obesity --- p.22 / Chapter II.2.2.8. --- Variations in states of sleep-wake cycle --- p.23 / Chapter II.2.2.9. --- Levels of physical and sympathetic nervous system activity --- p.23 / Chapter II. 3. --- The relationship between sex hormones and lipoproteins --- p.24 / Chapter II.3.1. --- "Gender difference in sex hormones, menopause and lipoprotein-lipids" --- p.24 / Chapter II.3.2. --- "Interventional study, exogenous sex hormone and lipoprotein-lipidsin men" --- p.25 / Chapter II.3.3. --- The relationships of endogenous sex hormones and lipoprotein-lipids --- p.26 / Chapter Chapter III. --- Materials and Methods --- p.28 / Chapter III.l. --- Subjects and Sampling Methods --- p.28 / Chapter III.2. --- Quantitation of serum lipoprotein-lipids --- p.29 / Chapter III.2.1 --- Determination of cholesterol and triglyceride --- p.29 / Chapter Table III.2.1.A. --- Intra-assay variations for cholesterol and triglyceride --- p.30 / Chapter Table III.2.1.B. --- Inter -assay variations for Cholesterol and Triglyceride --- p.30 / Chapter III.2.2. --- Determination of HDL-Cholesterol and its subfractions --- p.31 / Chapter Table III.2.2. --- Intra- and inter-assay variation for HDL-cholesterol --- p.31 / Chapter III.2.3. --- Determination of VLDL-C and LDL-C --- p.32 / Chapter III.2.4. --- Quantitative determination of serum apolipoproteins and Lp(a) --- p.32 / Chapter III.2.4.1. --- Determination of Apolipoproteins A-I and B --- p.33 / Chapter III.2.4.2. --- Determination of Lipoprotein (a) --- p.33 / Chapter III. 3. --- Quantitative determination of sex hormones --- p.33 / Chapter III.3.1. --- For urinary unconjugated and serum total testosterone --- p.34 / Chapter III.3.1.1 --- Experimental Procedures --- p.35 / Determination of the optimal antibody titre --- p.35 / Establishment of a standard curve and quality controls --- p.35 / Preparation of standards --- p.35 / Purification of radioactively-labelled 3H-testosterone --- p.37 / Preparation of charcoal- stripped urine as zero calibrator (blank) --- p.37 / Preparation of spiked urine or plasma --- p.38 / Preparation of samples for RIA --- p.38 / RIA --- p.39 / Calculation --- p.40 / Chapter III.3.1.2. --- Characteristics of the radioimmunoassay for testosterone --- p.40 / Sensitivity --- p.40 / Precision studies --- p.42 / Within- and between- batch imprecisions --- p.42 / Chapter Table III.3.1.2.A. --- Within-run variation --- p.42 / Chapter Table III.3.1.2.B. --- Between-run variation --- p.42 / Recoveries --- p.43 / Chapter Table III.3.1.2.C. --- "The recoveries of known amounts of testosterone added to charcoal-stripped urine, between immunoassays" --- p.43 / Test of linearity --- p.43 / Comparison with another procedure --- p.43 / Cross reactivity of the antiserum --- p.44 / Procedure --- p.46 / Chapter Table III.3.1.2.D. --- Cross reactivity of some naturally occurring steroids with testosterone antiserum --- p.47 / Chapter III.3.2 --- For urinary total testosterone --- p.47 / Test of linearity and recovery --- p.48 / Chapter III.3.3. --- For urinary unconjugated and serum total 17β-Estradiol --- p.50 / Chapter III.3.3.1 --- Experimental procedure --- p.50 / Determination of the optimal antibody titre --- p.50 / Establishment of a standard curve and quality controls --- p.52 / Preparation of standards --- p.52 / Preparation of tracer 3H-estradiol and construction of a standard curve --- p.52 / Preparation of spiked control urine or plasma --- p.52 / Preparation of samples for RIA --- p.53 / Chapter III.3.3.2. --- Characteristics of the radioimmunoassay for E2 --- p.53 / Sensitivity --- p.54 / Precision studies --- p.54 / Within- and between- batch imprecisions --- p.54 / Chapter Table III.3.3.2.A. --- Within-run variation of known amount of E2 added to charcoal- stripped urine --- p.54 / Chapter Table III.3.3.2.B. --- Between-run variation of known amount of e2 added to charcoal- stripped urine --- p.54 / Recoveries --- p.56 / Chapter Table III.3.3.2.C. --- "The recoveries of known amount of e2 added to charcoal- stripped urine, between immunoassays" --- p.56 / Test of linearity --- p.56 / Comparison with another procedure --- p.56 / Chapter III.3.4. --- For urinary total estradiol --- p.58 / Test of linearity and recovery --- p.58 / Chapter III.4. --- Determination of serum sex hormone-binding globulin (SHBG) --- p.60 / Chapter III.5. --- Determination of urinary unconjugated Cortisol --- p.60 / Chapter III.6. --- Statistical methods --- p.62 / Chapter III.6.1. --- Biological Variations --- p.62 / Chapter III.6.2. --- Univariate and multivariate correlations --- p.62 / Chapter Chapter IV. --- Results --- p.64 / Chapter IV. 1. --- The characteristics of the experimental subjects and their lipoprotein-lipids profiles --- p.64 / Chapter Table IV. 1. A. --- The anthropometric and biochemical characteristics of the experimental male subjects --- p.64 / Chapter Table IV.1.B. --- The lipoprotein-lipids profiles in 46 healthy Hong Kong Chinese men --- p.65 / Chapter IV. 2. --- "Levels of sex hormones in serum and urine, and urinary free Cortisol" --- p.65 / Chapter Table IV.2. --- The sex hormones at serum and urinary levels and urinary free Cortisol in 46 healthy Hong Kong Chinese men --- p.66 / Chapter Table IV.2.A. --- Formula for the indirect calculation of unbound (free) testosterone levels in plasma --- p.67 / Chapter Table IV.2.B. --- Formula for the indirect calculation of unbound (free) 17β-estradiol levels in plasma --- p.68 / Chapter IV. 3. --- Biological variations --- p.69 / Chapter Table IV. 3. --- "The biological variations of serum lipoprotein-lipids, serum sex hormones and urinary sex hormones and Cortisol in 46 healthy Hong Kong Chinese men" --- p.69 / Chapter Table IV.3.A. --- Correlations of serum lipoprotein-lipids between short-term (3- week) variations --- p.70 / Chapter Table IV.3.B. --- Correlations of serum and urinary sex hormones and urinary unconjugated Cortisol between short-term (3-week) variations --- p.70 / Chapter IV. 4. --- Univariate correlation --- p.71 / Chapter Table IV.4. --- The univariate correlation table --- p.72 / Chapter IV.4.1. --- Inter-relationship among serum and urinary sex hormones --- p.73 / Chapter IV.4.2. --- Urinary free Cortisol and sex hormones and serum lipoprotein-lipids --- p.73 / Chapter IV.4.3. --- Correlation between urinary sex hormones and serum lipoprotein- lipids --- p.73 / Chapter IV.4.4. --- Correlations among serum lipoprotein-lipids --- p.74 / Chapter IV.4.5. --- Correlations between serum lipoprotein-lipids and sex hormones --- p.74 / Chapter IV.4.6. --- "Correlations between anthropometric variables, sex hormones and lipoprotein-lipids" --- p.75 / Chapter IV.4.7. --- Correlation of the ratio of HDL2 and HDL3 and other variables --- p.76 / Chapter IV. 5. --- Multiple linear stepwise regression --- p.77 / Chapter Table IV.5. --- "Stepwise multiple linear regression of lipoprotein-lipids on BMI, W/H Ratio, and Age" --- p.77 / Chapter Table IV.5. A. --- "Stepwise multiple linear regression of lipoprotein-lipids on BMI, W/H Ratio, Age, SHBG, and serum and urinary Sex Hormones" --- p.80 / Chapter Table IV.5.B. --- "Stepwise multiple linear regression of lipoprotein-lipids on BMI, W/H Ratio, Age, SHBG, Triglyceride and serum and urinary Sex Hormones" --- p.81 / Chapter Chapter V. --- Discussion --- p.82 / Chapter V. l. --- Experimental subjects and their lipoprotein-lipids profiles --- p.82 / Chapter V. 2. --- Levels of sex hormones in serum and urine --- p.84 / Chapter Table V.2. --- Values of sex steroids in 46 healthy Hong Kong Chinese men compared to others cited in literature --- p.85 / Chapter V. 3. --- "17β-Estradiol,atherogenic lipoprotein-lpids and HDL3" --- p.88 / Chapter V. 4. --- "Testosterone, and HDL-C and its subfractions" --- p.90 / Chapter Chapter VI. --- Conclusions --- p.92 / References --- p.95 / Appendices --- p.110

by Lai-pang Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 148-182). / ABSTRACT / LIST OF ILLUSTRATIONS / LIST OF TABLES / ACKNOWLEDGEMENTS / ABBREVIATIONS / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter CHAPTER II --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- The uterus and its changes in the normal menstrual cycle / Chapter 2.2 --- Anatomy and physiology of normal myometrium / Chapter 2.3 --- Clinical features and management of uterine leiomyoma / Chapter 2.4 --- Pathology of human uterine leiomyoma / Chapter 2.5 --- The relationship between growth fractions and ER in breast carcinoma / Chapter 2.6 --- Steroid receptors and epidermal growth factor receptor / Chapter 2.6.1 --- Steroid receptors / Chapter 2.6.2 --- Epidermal growth factor receptor / Chapter 2.7 --- "Structures of oestrogen receptor, progesterone receptor, Ki-67 and epidermal growth factor receptor" / Chapter 2.7.1 --- The structure of oestrogen receptor / Chapter 2.7.2 --- The structure of progesterone receptor / Chapter 2.7.3 --- The structure of Ki-67 / Chapter 2.7.4 --- The structure of epidermal growth factor receptor / Chapter 2.8 --- "Antibodies to steroid receptors, monoclonal Ki-67 and epidermal growth factor receptor" / Chapter 2.8.1 --- Steroid receptors / Chapter 2.8.2 --- Monoclonal Ki-67 / Chapter 2.8.3 --- Epidermal growth factor receptor / Chapter 2.9 --- "Functions of steroid receptors, Ki-67 and epidermal growth factor receptor" / Chapter 2.9.1 --- The functions of steroid receptors / Chapter 2.9.2 --- The functions of Ki-67 / Chapter 2.9.3 --- The functions of epidermal growth factor receptor / Chapter 2.10 --- Cell cycle / Chapter 2.11 --- Immunohistochemistry / Chapter 2.11.1 --- Introduction / Chapter 2.11.2 --- Methodology of immunostaining / Chapter 2.11.3 --- Avidin-biotin-peroxidase complex technique / Chapter 2.11.4 --- Chromogens / Chapter 2.11.5 --- Enhancement methods / Chapter 2.11.6 --- Fixation for immunohistochemistry / Chapter CHAPTER III --- MATERIALS AND METHODS --- p.63 / Chapter 3.1 --- Reagents and chemicals / Chapter 3.1.1 --- Primary monoclonal antibodies / Chapter 3.1.2 --- Secondary antibodies / Chapter 3.1.3 --- Avidin-biotin complex / Chapter 3.1.4 --- DAB solution / Chapter 3.1.5 --- Buffers / Chapter 3.1.6 --- Miscellaneous / Chapter 3.2 --- Patients and specimens / Chapter 3.2.1 --- Specimen collection / Chapter 3.2.2 --- Preparation of specimens / Chapter 3.3 --- Immunohistochemical staining / Chapter 3.3.1 --- Slide preparation / Chapter 3.3.2 --- Antigen retrieval / Chapter 3.3.3 --- Procedures of immunohistochemical staining / Chapter 3.3.4 --- Interpretation of immunostaining / Chapter CHAPTER IV --- RESULTS --- p.80 / Chapter 4.1 --- Clinical information / Chapter 4.2 --- Oestrogen receptor / Chapter 4.3 --- Progesterone receptor / Chapter 4.4 --- Epidermal growth factor receptor / Chapter 4.5 --- Ki-67 / Chapter CHAPTER V --- DISCUSSION --- p.120 / Chapter 5.1 --- Methods and interpretation of the results / Chapter 5.1.1 --- The advantages of the immunohistochemical staining technique / Chapter 5.1.2 --- Interpretation and reporting of immunohistochemical results / Chapter 5.1.3 --- Interpretation of the results by semi- quantitative assessment and statistical analysis / Chapter 5.2 --- The status of steroid receptors in uterine leiomyoma / Chapter 5.2.1 --- ER status in uterine leiomyoma and normal myometrium / Chapter 5.2.2 --- PR status in uterine leiomyoma and normal myometrium / Chapter 5.3 --- EGF-R status in uterine leiomyoma / Chapter 5.4 --- Ki-67 status in uterine leiomyoma and normal myometrium / Chapter 5.5 --- "The relationship between steroid receptors, Ki-67, EGF-R and uterine leiomyoma growth" / Chapter 5.6 --- Biological indices in the assessment of tumor / Chapter 5.7 --- Microwave technology in immunohistology for surgical pathology / Chapter CHAPTER VI --- CONCLUSIONS --- p.144 / REFERENCES --- p.148

Substance abuse and mental health disorders are a leading source of years lost to disability from medical causes worldwide. Unfortunately, for most neurological disorders it is unclear how underlying genetic predispositions govern behavioral response to environmental stressors. Owing to their convenience, genetic tractability, and small brains, the fruit fly, Drosophila melanogaster, has become an invaluable model in which to dissect the neurological basis of conserved complex behaviors. Here, I characterized the respective roles of two genes in alcohol response and sleep behavior. Steroid hormones profoundly influence behavioral response to alcohol, yet the role of unconventional non-genomic steroid signaling in this process is unknown. I discovered that Drosophila DopEcR, a G-protein coupled receptor (GPCR) activated by dopamine or the major insect steroid hormone ecdysone, plays a critical role in ethanol-induced behaviors. DopEcR mutants took longer to sedate when exposed to ethanol vapor, and post-eclosion expression of DopEcR-RNAi phenocopied mutant resistance. DopEcR was necessary in particular neuronal subsets, including cholinergic and peptidergic neurons, and promoted ethanol sedation by suppressing epidermal growth factor/extracellular signal-regulated kinase signaling. In adult flies, ecdysone negatively regulated DopEcR-mediated ethanol-induced sedation. We also found that DopEcR inhibits ethanol-induced locomotion, a conserved dopaminergic behavior. Together, these findings provide novel insight into how an unconventional steroid GPCR interacts with multiple downstream signaling cascades to fine tune behavioral response to alcohol. Despite an established link between epilepsy and sleep behavior, it remains unclear how epileptogenic mutations affect sleep and seizure susceptibility. To address this, I examined the rest/wake behavior of two fly models of epilepsy with paralytic voltage-gated sodium channel mutations known to cause human generalized epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS). GEFS+ and DS flies display heat-induced seizure susceptibility, but at normal temperatures I found that both mutants had exaggerated nighttime sleep behavior. GEFS+ sleep was more resistant to pharmacologic and genetic reduction of GABA transmission as compared to control’s response. This finding is consistent with augmented GABAergic suppression of wake-promoting pigment-dispersing factor (PDF) neurons in GEFS+ mutants. Contrastingly, DS sleep was almost completely resistant to pharmacologic GABA reduction, suggesting that PDF neurons are incapable of functioning despite disinhibition. The sleep of both GEFS+ and DS flies was largely suppressed, but not eliminated, by scotophase light, highlighting the importance of light stimulus and circadian signals in the manifestation of their phenotypes. Following sleep deprivation, GEFS+ and DS mutants failed show to homeostatic rebound. Sleep loss also unexpectedly reduced the seizure susceptibility of GEFS+ flies. This study revealed the sleep architecture of Drosophila voltage-gated sodium channel mutants and provides a unique platform in which to further study the sleep/epilepsy relationship.

[Truncated abstract] Steroid receptors belong to the superfamily of nuclear receptors, and include the androgen receptor (AR), estrogen receptors (ER[alpha] and ER[beta], glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and the progesterone receptors (PRA and PRB). Before binding ligand, the receptor undergoes biochemical and structural modifications through a series of interactions with molecular chaperones and cochaperones all within a receptor heterocomplex. The mature receptor complexes with the major chaperone Hsp90, the stabilising cochaperone p23, and one member of a group of cochaperones termed immunophilins. Steroid receptor-associated immunophilins include the cyclophilin, CyP40, two FK506-binding proteins, FKBP51 and FKBP52, and the protein phosphatase, PP5. Immunophilins are characterised by the presence of TPR domains which compete directly for the TPR-acceptor site within Hsp90. This leads to mutually exclusive, immunophilin-containing receptor complexes. While PP5 contains a C-terminal phosphatase domain, CyP40, FKBP51 and FKBP52 each contain an N-terminal peptidyl prolyl isomerase (PPIase) domain, which catalyses the cis/trans isomerisation of prolyl peptide bonds. FKBP52 has been demonstrated to potentiate the ligand-dependent activity of AR, GR and PR, but not ER[alpha]. Knowing that CyP40 is the preferred immunophilin associated with the ER[alpha] heterocomplex, it was hypothesised that this immunophilin plays a role in ER[alpha] function. ... As all mutants maintained this potentiating activity it was concluded that the five altered residues found within gpGR do not contribute to the altered interaction of FKBP52 and receptor. However, it cannot be discounted that FKBP51 is more competitive for gpGR. Immunophilins are hormonally regulated, with FKBP52 found to be essential for female fertility in mice. It was hypothesised that levels of immunophilins, associated with steroid receptors important in the menstrual cycle, would be regulated to reflect hormonal activity within cycling endometrium. Human pre-menopausal endometrial sections taken from different phases of the menstrual cycle were examined immunohistochemically for expression of CyP40, FKBP51, FKBP51 and PP5. Immunophilin levels peaked at the mid-secretory phase correlating with stromal decidualization, a process essential for eventual blastocyst implantation. The importance of immunophilins to steroid receptor action was therefore reinforced by the observation that immunophilins appear to be hormonally regulated in cycling pre-menopausal human endometrium. Further studies into the effects of immunophilin loss and knockdown on steroid receptor-mediated responses in specific mouse tissues, knockout-derived mouse embryo fibroblasts and cancer cell lines may contribute to our understanding of the receptor-selective and tissue-specific actions of the immunophilins. Elucidation of the mechanisms through which they modulate receptor function may provide opportunities for therapeutic intervention in steroid-related disease.

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a co-activator that enhances the hormone-dependent transactivation of estrogen (ER) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that specifically overexpress either wild-type human E6-AP (E6-APWT) or the ubiquitin-protein ligase defective mutant E6-AP (E6-APC833S) in the mammary gland. Here we show that overexpression of E6-APWT results in impaired mammary gland development. In contrast, overexpression of E6-APC833S or loss of E6-AP (E6-APKO) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are in large part due to the alteration of PR-B protein levels. RNAi-mediated knockdown of E6-AP in T47D breast cancer cells increased PR-B protein levels and stability. In vitro ubiquitination assay using purified E6-AP and PR-B reinforce these conclusions and demonstrate that E6-AP promotes PR-B turnover in an ubiquitin-dependent manner. Furthermore, we also show that E6-AP regulates progesterone-induced Wnt-4 expression by modulating the steady state level of PR-B in both mice and in human breast cancer cells. This novel mechanism appears to regulate normal physiology of the mammary gland and its dysregulation may prove to contribute importantly to mammary cancer development and progression. To test this hypothesis, we examined the E6-AP transgenic mice for tumor formation over a period of 6, 9, 12, 18 and 24 months. Our data shows that, unlike the E6-APWT mice that show normal phenotype, the E6-APC833S mice develop mammary hyperplasia at high penetrance (80%); with a median latency of 18 months. Our findings indicate that the inactivation of the E3-ligase function of E6-AP is sufficient to initiate the process of mammary tumor development. These findings strongly suggest that E6-AP may act as a tumor suppressor by down regulating the ER-alpha, PR-B and thereby their signaling pathways.

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