Wong Hau Yan Connie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 86-95). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.ix / Contents --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Abbreviations --- p.xvi / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Role of extracellular nucleotides in equine sweat gland epithelial cells --- p.1 / Chapter 1.2 --- Subdivision of P1 and P2 purinoceptor --- p.4 / Chapter 1.3 --- General properties of P2 purinoceptor --- p.5 / Chapter 1.3.1 --- P2X purinoceptor family --- p.5 / Chapter 1.3.2 --- P2Y purinoceptor family --- p.8 / Chapter 1.4 --- The diversity of P2Y purinoceptor --- p.10 / Chapter 1.4.1 --- P2Y1 receptor --- p.10 / Chapter 1.4.2 --- P2Y2 receptor --- p.10 / Chapter 1.4.3 --- P2Y4 receptor --- p.10 / Chapter 1.4.4 --- P2Y6 receptor --- p.10 / Chapter 1.4.5 --- P2Y11 receptor --- p.11 / Chapter 1.5 --- The importance of calcium --- p.13 / Chapter 1.6 --- General aspects of calcium signaling --- p.14 / Chapter 1.7 --- Calcium release from the intracellular calcium stores --- p.15 / Chapter 1.7.1 --- Metabolism of inositol phosphates --- p.15 / Chapter 1.7.2 --- Ca2+ release from the internal calcium store --- p.15 / Chapter 1.8 --- Store-operated calcium channels (SOCC) or Capacitative calcium entry (CCE) --- p.18 / Chapter 1.8.1 --- The nature of the signal for CCE --- p.18 / Chapter 1.8.1.1 --- Conformational coupling --- p.18 / Chapter 1.8.1.2 --- Diffusible messenger --- p.21 / Chapter 1.9 --- Mechanism of intracellular calcium measurement --- p.25 / Chapter 1.10 --- Background of E92/3 cell line --- p.28 / Chapter Chapter 2: --- Materials and methods --- p.29 / Chapter 2.1 --- Cell culture --- p.29 / Chapter 2.2 --- Preparation of the simultaneous measurement --- p.31 / Chapter 2.2.1 --- Cell seeding --- p.31 / Chapter 2.2.2 --- Dye loading --- p.33 / Chapter 2.3 --- The setup of simultaneous measurement --- p.36 / Chapter 2.4 --- Statistical analysis --- p.40 / Chapter Chapter 3: --- Results --- p.41 / Chapter 3.1 --- Major domain of Ca2+ influx is from the basolateral side --- p.41 / Chapter 3.1.1 --- Effect of store depletion by apical ATP --- p.41 / Chapter 3.1.2 --- Effect of store depletion by basolateral ATP --- p.43 / Chapter 3.1.3 --- Effect of store depletion by thapsigargin --- p.47 / Chapter 3.2 --- Differential effect of apical and basolateral nucleotides on [Ca2+]i and Isc --- p.51 / Chapter 3.2.1 --- Basolateral ATP activates an increase in [Ca2+]i but not Isc --- p.51 / Chapter 3.2.2 --- Apical and basolateral ATP activated distinct but partially overlapped internal Ca2+ pool --- p.51 / Chapter 3.2.3 --- "Dose-dependent effect of apical or basolateral ATP, UDP and UTP on [Ca2+]i i and Isc" --- p.54 / Chapter 3.3 --- P2Y receptors subtypes on the basolateral membrane --- p.60 / Chapter 3.3.1 --- "Possible involvement of P2X, P2Y1 and P2Y11 purinoceptors on the basolateral membrane" --- p.60 / Chapter 3.3.2 --- "Cross-desensitization of experiments of UTP, ATP and UDP" --- p.60 / Chapter 3.4 --- The ATP-activated Ca2+ pool and thapsigargin-activated Ca2+ pool are partially overlapped --- p.68 / Chapter 3.5 --- Anion secretion activated by Ca2+ -independent pathway --- p.74 / Chapter Chapter 4: --- Discussion --- p.76 / Chapter 4.1 --- The major membrane for the CCE is from the basolateral side --- p.76 / Chapter 4.2 --- Basolateral P2Y receptors --- p.80 / Chapter 4.3 --- Differential effects of apical and basolateral ATP --- p.82 / Chapter 4.3.1 --- Apical and basolateral ATP release Ca2+ from different pools --- p.83 / Chapter 4.3.2 --- Ca2+ -independent mechanism --- p.83 / Chapter 4.3.3 --- Other potential signaling molecules --- p.84 / Chapter Chapter 5: --- Reference --- p.86

by Psyche, Sui-Ki Fong. / Thesis submitted in: December 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 129-135). / Abstract also in Chinese. / Abstract in Chinese --- p.1 / Abstract in English --- p.2 / Chapter Chapter I. --- Introduction --- p.4 / Chapter I.1. --- Structure and functions of the uterus --- p.4 / Chapter I.1.1. --- Uterus: Gross structure and functions --- p.4 / Chapter I.1.2. --- Uterine wall: functional layers --- p.6 / Chapter a. --- Myometrium --- p.6 / Chapter b. --- Endometrium --- p.6 / Chapter I.1.3. --- Uterine functions : regulatory systems --- p.10 / Chapter a. --- Nervous regulation --- p.10 / Chapter b. --- Hormonal regulation --- p.11 / Chapter I.1.4. --- Uterotrophic agents : selected examples --- p.12 / Chapter a. --- Adrenaline and noradrenaline --- p.12 / Chapter b. --- Prostaglandin E2 and F2α --- p.15 / Chapter I.2. --- Endometrial epithelium and uterine fluid composition --- p.19 / Chapter I.2.1. --- Uterine fluid composition --- p.19 / Chapter I.2.2. --- Regulation of uterine fluid volume and composition --- p.19 / Chapter I.2.3. --- Role of endometrial epithelium in the regulation of uterine fluid composition --- p.20 / Chapter I.3. --- Pioneering works on ion transport across the endometrium --- p.21 / Chapter I.4. --- Objectives of study --- p.23 / Chapter Chapter II --- Materials and Methods --- p.24 / Chapter II.1. --- Materials --- p.24 / Chapter II.1.1. --- Culture media and enzyme --- p.24 / Chapter II 1.2. --- Drugs --- p.24 / Chapter II 1.3. --- Chemicals --- p.25 / Chapter II 1.4. --- Animals --- p.25 / Chapter II.2. --- Methods --- p.25 / Chapter II.2.1. --- Preparation of permeable support for cell culture --- p.25 / Chapter II.2.2. --- Preparation of culture medium for cell culture --- p.26 / Chapter II.2.3. --- Cell isolation and culture --- p.28 / Chapter II.2.4. --- Preparation of electrodes --- p.29 / Chapter II.2.5. --- Preparation of solutions --- p.29 / Chapter II.2.6. --- The short-circuit current technique --- p.31 / Chapter a. --- Experimental setup --- p.32 / Chapter b. --- Transepithelial conductance and resistance measurements --- p.37 / Chapter c. --- Experimental procedure --- p.37 / Chapter II.2.7. --- Statistics --- p.38 / Chapter Chapter III. --- Results --- p.39 / Chapter III.1. --- Electrogenic ion transport across the cultured mouse endometrial epithelium --- p.39 / Chapter III.2. --- Stimulation of anion secretion by β-adrenoceptors --- p.54 / Chapter III.3. --- Regulation of anion secretion by prostaglandin E2 --- p.78 / Chapter III.4. --- Cellular mechanisms of adrenaline-stimulated anion secretion --- p.98 / Chapter Chapter IV. --- General Discussion --- p.123 / Chapter Chapter V. --- Reference --- p.129

p16INK4a (p16) traps the cell at the restriction point of the cell cycle by binding to cyclin-dependent kinase 4/6 thus preventing the phosphorylation of the retinoblastoma protein (pRB). As p16 accumulates the cell stops dividing and becomes senescent. This study investigates the modulation of p16 function by the putative membrane protein ABCB5 and a group of five putative oncogenic microRNAs (oncomiRs). ABCB5 is a poorly characterised member of the B-subfamily of human ATP Binding Cassette transporters. ABCB5 is reportedly transcribed into four transcripts, one of which could potentially encode a full-length transporter (ABCB5fl) whilst a second could encode a half-transporter (ABCB5β). The other two transcripts (ABCB5α and ABCB5γ) could only encode short polypeptides. Exogenous expression of ABCB5fl and ABCB5β was achieved in HEK293T cells, but the recombinant protein expressed poorly and localised to the endoplasmic reticulum. Point mutations introduced into the ATP catalytic domain failed to improve expression levels suggesting that protein function was not deleterious to the cell. Exogenous expression in HEK293T cells also allowed commercial antibodies purportedly raised against ABCB5 isoforms to be tested. Several were found not to recognise ABCB5 necessitating re-interpretation of published data. However, one antibody recognised both ABCB5fl and ABCB5β, and was subsequently used to evaluate protein expression levels in other cell types.siRNA knockdown of ABCB5 in human mammary epithelial cells (HMECs) caused a concomitant reduction in p16 expression and an increase in cellular proliferation. Differential siRNAs and RT-qPCR analyses demonstrated ABCB5β to be the relevant transcript with respect to the reduction in p16 expression; however, no native ABCB5β protein was detected in HMECs. Together these data lead to the hypothesis that the ABCB5β transcript may act as a long noncoding RNA to regulate p16. Exogenous expression of each of five distinct putative oncomiRs in HMECs was found to increase cellular proliferation and, surprisingly, increase p16 expression. These results mirror a phenotype commonly observed in p16-positive basal-like breast cancer (BLBC), an aggressive form of breast cancer with poor prognosis and few treatment options. Bioinformatic analysis of the predicted target genes for these oncomiRs identified multiple transcriptional regulators of pRB. These predictions, together with the work performed in a cellular model of p16-positive BLBC, suggest that the oncomiRs may cause unrestricted cell proliferation by indirectly reducing transcription of the pRB gene, RB1. In the absence of pRB, p16 expression is induced via a previously reported oncogeneinduced senescence-like positive feedback loop. These data, and previously published observations, suggest that a similar mechanism may explain the basis of p16-positive BLBC.

Every year over 20,000 [3] people die as a result of being in a fire. Although flames have the biggest visual impact, it is usually the smoke produced by the combustion of natural and synthetic materials that causes more damage and claims more lives. The main constituents of smoke, both the particulate matter as well as the hot and toxic gasses, are devastating to the tracheal and lung tissues. The damage caused to the lung and trachea by inhaling this smoke can increase a fire victim's susceptibility to infectious disease significantly [1]. Between 20% and 50% of people who suffer inhalation injury contract pneumonia due to the weakened status of their body's defenses [2] and between 4,800 and 6,400 [1] people die from either pneumonia or other complications. Despite the importance of the inner-lining of the trachea to a burn victim's health and survival, current treatments consist of keeping the patient in a clean environment, supplying fresh oxygen, keeping the airways open, and letting the patient's body heal itself [1]. This treatment is not so much an active healing mechanism; rather it is a passive means of allowing the body to repair itself. The main goal of this work is to develop a minimally invasive technique that will replace lost cells on the inside surface of the trachea as efficiently as possible, actively healing the patient's injury. Ideally, the patient would receive a single treatment and then make a complete recovery on his or her own. The main challenge lies in delivering an even layer of intact cells to the inner-surface of the trachea in such a manner that they will stay in place and will replace the damaged or missing tissue. The overall approach is to spray a suspension, composed of epithelial cells in an aqueous solution of Pluronic F-127 polymer, onto the trachea using a jet atomizer. Because Pluronic F-127 solutions can be liquids at room temperature but gels at body temperature, the role of the polymer will be to immobilize the cells onto the tracheal surface long enough for them to attach and grow.

The photobiomodulation therapy (PBMT) has many demonstrated applications in the health area including anti-inflammatory and wound healing effects. The main objective of this work is to verify if the PBMT causes measurable changes in the mechanical properties of cells, specifically in red blood cells, epithelial cells and fibroblasts. In addition, to contribute to the knowledge of the action mechanisms of the PBMT, this study intends to support applications of the PBMT during invasive procedures, such as the direct photo-treatment of the blood in surgical procedures with cardiopulmonary bypass, regarding security of the cellular integrity. For this analysis, three experimental techniques were used: optical magnetic twisting cytometry (OMTC), defocusing microscopy and confocal laser-scanning microscopy. Human bronchial epithelial cells were evaluated with OMTC. The epithelial cell culture was either photo-treated or not, with red laser (lambda=660 nm), and fixed power and time (power density of 153 mW/cm2, time 300 s). It was not possible to observe significant differences between photo-treated and control epithelial cells, for the hysteresivity (ratio between the cell loss and elastic shear moduli). The defocusing microscopy, similar to a phase contrast microscopy, was used to study human red blood cells from fresh blood. The red blood cells were either photo-treated or not, with red laser (lambda=660 nm), and different powers and times (power densities from 0 to 510 mW/cm2, times from 0 to 180 s). Some morphological and mechanical characteristics of individual red blood cells were evaluated, such as volume, radial profile of cell thickness, lateral and vertical membrane fluctuations, for the photo-treated and control red blood cells. It was not possible to detect differences between the two groups, for any of the parameters analyzed. For both techniques, the absence of detectable differences might be due to several factors, such as the non-action of the PBMT, with the parameters used, in the epithelial cells and red blood cells or to the small sensitivity of each technique. Confocal laser-scanning microscopy was used to evaluate the actin filaments of mouse fibroblasts. The fibroblast cell culture was either photo-treated or not, with red (lambda=625 nm) or infrared (lambda=808 nm) light and fixed power and time (power density from 113 to 158 mW/cm2, time 300 s). The nucleus and cell areas increased slightly when comparing photo-treated and control cells. On the other hand, the total actin, total actin density and the number of filaments decreased. These changes were detected for a short time after treatment, however, after 24 h they are not anymore detectable. The total branch length does not seem to suffer any modifications. In summary, with the data acquired with the three techniques, it was found that the PBMT, in the red range, with the parameters used, could not cause noticeable changes in red blood cells and epithelial cells, in vitro. On the other hand, the PBMT in the red and near-infrared range, with the power and times used, cause changes in actin filaments of fibroblasts, in vitro, in particular the decrease of the total actin density. / A terapia por fotobiomodulação tem muitas aplicações na área de Saúde devido a sua ação anti-inflamatória e de reparação tecidual. O objetivo geral desse trabalho é verificar se a terapia por fotobiomodulação provoca mudanças nas propriedades mecânicas de células, em particular em hemácias, células epiteliais e fibroblastos. Além de contribuir com o conhecimento dos mecanismos de ação da terapia por fotobiomodulação, este estudo pretende subsidiar aplicações da terapia por fotobiomodulação durante procedimentos mais invasivos, como a iluminação direta do sangue em procedimentos cirúrgicos com circulação extracorpórea, sob o ponto de vista da segurança quanto à integridade celular. Para essa análise foram utilizadas três técnicas experimentais: citometria óptica magnética de oscilação (OMTC), microscopia de desfocalização e microscopia confocal. Com a técnica de OMTC foram avaliadas células epiteliais brônquicas humanas em cultura, foto-tratadas com laser vermelho (lambda=660 nm), com potência e tempo fixos (densidade de potência de 153 mW/cm2, tempo 300 s). Não foi possível constatar diferenças significativas entre as células epiteliais foto-tratadas e as células controle, para a histerisividade (razão entre os módulos viscoso e elástico das células). Com a técnica de microscopia de desfocalização, semelhante a uma microscopia de contraste de fase, foram estudadas hemácias humanas de sangue recém coletado. As hemácias foram tratadas com laser vermelho (lambda=660 nm), com potências e tempos variados (densidade de potência de 0 a 510 mW/cm2, tempo de 0 a 180 s). Foram avaliadas algumas características morfológicas e mecânicas das hemácias individualmente, como o volume, perfil radial de espessura, flutuações lateral e vertical da membrana, tanto para hemácias foto-tratadas quanto para hemácias controle. Não foi possível detectar diferenças entre as hemácias foto-tratadas e controle para nenhum dos parâmetros avaliados. Para ambas as técnicas, a falta de mudanças observáveis poderia ser devida a diversos fatores, como a não ação da terapia por fotobiomodulação nas células epiteliais e nas hemácias, com os parâmetros aqui empregados, ou à falta de sensibilidade de cada uma das técnicas usadas. A microscopia confocal foi utilizada para avaliar os filamentos de actina de fibroblastos de camundongo em cultura, os quais foram foto-tratados com luz vermelha (lambda=625 nm) ou infravermelha (lambda=808 nm) e potência e tempo fixos (densidade de potência de 113 a 158 mW/cm2, tempo 300 s). Foi possível constatar ligeiro aumento nas áreas nuclear e celular das células foto-tratadas em relação aos fibroblastos controle. Também foi possível verificar a diminuição da quantidade total de actina, densidade de actina e do número de filamentos de actina nos fibroblastos foto-tratados. Essas mudanças são detectadas para tempos curtos após o tratamento, sendo que depois de 24 h elas desaparecem. O tamanho total dos filamentos parece não sofrer alterações. A partir dos dados coletados com as três técnicas, foi possível constatar que a terapia por fotobiomodulação, com os parâmetros utilizados, não consegue provocar mudanças perceptíveis em hemácias e em células epiteliais, in vitro. Porém, causa mudanças nos filamentos de actina de fibroblastos, in vitro, em particular a diminuição da densidade de actina total.

Yuen Mong Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 155-169). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.v / Contents --- p.vi / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Developmental biology of the prostate --- p.1 / Chapter 1.1.1 --- Development of the prostate gland in humans and rodents --- p.1 / Chapter 1.1.2 --- Mesenchymal-epithelial interaction --- p.2 / Chapter 1.2 --- Overview of the endocrinology of prostate --- p.3 / Chapter 1.3 --- Estrogen in male and prostate gland --- p.4 / Chapter 1.3.1 --- Stimulating effect of estrogen on prostate gland --- p.4 / Chapter 1.3.2 --- Inhibitory effect of estrogen on prostate gland --- p.5 / Chapter 1.4 --- Study of the role of estrogen receptors in prostate gland with the use of estrogen receptor knockout mice --- p.6 / Chapter 1.4.1 --- The two isoforms of estrogen receptors (ER): ERα and ERβ --- p.6 / Chapter 1.4.2 --- The use of estrogen receptor knockout mice for the study of ER --- p.7 / Chapter 1.5 --- Estrogen as a carcinogen --- p.8 / Chapter 1.5.1 --- Formation of DNA adducts --- p.8 / Chapter 1.5.2 --- Formation of oxidants --- p.9 / Chapter 1.5.3 --- Estrogen as a microtubule-disrupting agent --- p.10 / Chapter 1.6 --- Estrogen carcinogenicity in animal models --- p.11 / Chapter 1.6.1 --- Syrian golden hamster model --- p.11 / Chapter 1.6.2 --- Rat model --- p.12 / Chapter 1.7 --- Animal models of prostate cancer by hormonal induction --- p.12 / Chapter 1.7.1 --- Canine model --- p.13 / Chapter 1.7.2 --- Noble rat model --- p.13 / Chapter 1.7.3 --- Sprague-Dawley rat model --- p.15 / Chapter 1.7.4 --- Wistar and F344 rat model --- p.15 / Chapter 1.8 --- Perinatal estrogen exposure and prostate development --- p.16 / Chapter 1.8.1 --- Prenatal estrogen exposure --- p.15 / Chapter 1.8.2 --- Neonatal estrogen exposure --- p.17 / Chapter 1.9 --- Therapeutic use of synthetic estrogen --- p.18 / Chapter 1.9.1 --- Use of diethylstilbestrol in treating prostate cancer --- p.18 / Chapter 1.9.2 --- Use of diethylstilbestrol during pregnancy --- p.19 / Chapter 1.10 --- Estrogen contamination in food --- p.20 / Chapter 1.10.1 --- Estrogen in milk and dairy products --- p.20 / Chapter 1.10.2 --- Estrogen in meat --- p.21 / Figure 1.1 --- p.23 / Chapter Chapter 2. --- Materials and methods --- p.25 / Chapter 2.1 --- In vitro study of estrogen carcninogenicity in normal prostatic cell line --- p.25 / Chapter 2.1.1 --- NRP-152 cell line --- p.25 / Chapter 2.1.2 --- In vitro estrogen treatment on NRP-152 cells --- p.25 / Chapter 2.1.3 --- Colony formation by soft agar assay --- p.27 / Chapter 2.1.4 --- Determination of growth parameters of estrogen-treated and untreated NRP-152 cells --- p.29 / Chapter 2.1.5 --- Gene expression profiling in estrogen-transformed and untreated parental NRP-152 cells by cDNA microarray --- p.30 / Chapter 2.1.6 --- Immunohistochemistry of cultured cells --- p.34 / Chapter 2.1.7 --- Immunofluorescence on cultured cells --- p.36 / Chapter 2.1.8 --- Electron microscopy of the estrogen-transformed and untreated parental NRP-152 cells --- p.37 / Chapter 2.1.9 --- Tumorigenicity in nude mice --- p.38 / Chapter 2.1.10 --- Protein expressions and Western blottings in estrogen-transformed and untreated parental NRP-152 cells --- p.39 / Chapter 2.2 --- In vivo study of estrorgen carcinogenicity in rat protstate gland --- p.41 / Chapter 2.2.1 --- Origin and supply of Noble rats --- p.41 / Chapter 2.2.2 --- Perinatal estrogen imprinting on male Noble rats with diethylstilbestrol --- p.42 / Chapter 2.2.3 --- Long-term hormonal treatment with sex steroids on male Noble rats at adulthood --- p.43 / Chapter 2.2.4 --- Morphological study of Noble rat prostates --- p.44 / Chapter 2.2.5 --- Protein expressions by immunohistochemistry in estrogen-primed and hormone-treated Noble rat prostates --- p.45 / Tables 2.1 -2.2 --- p.48 / Chapter Chapter 3. --- Results --- p.50 / Chapter 3.1 --- In vitro study --- p.50 / Chapter 3.1.1 --- Dose selection for estrogen treatment of NRP-152 cells from cell proliferation assay --- p.50 / Chapter 3.1.2 --- Colony formation in soft agar --- p.50 / Chapter 3.1.3 --- Morphology of NRP-152 cells and the estrogen-transformed clones --- p.51 / Chapter 3.1.4 --- Study of growth parameters --- p.52 / Chapter 3.1.5 --- CDNA array analysis of differentia] gene pattern --- p.53 / Chapter 3.1.6 --- Immunohistochemistry of untreated parental and estrogen- transformed NRP-152 cells --- p.55 / Chapter 3.1.7 --- Electron microscopy --- p.58 / Chapter 3.1.8 --- Tumorigenicity of NRP-152 cells and the estrogen-transformed clones --- p.59 / Chapter 3.1.9 --- Western blottings --- p.59 / Chapter 3.2 --- In vivo study --- p.52 / Chapter 3.2.1 --- Survival of male Nobel rats during perinatal and long-term hormone treatment --- p.62 / Chapter 3.2.2 --- Histological studies of Noble rat prostates --- p.63 / Chapter 3.2.3 --- Immunohistochemistry of the hormone-treated and control Noble rat prostates --- p.65 / Figure 3.1.1 -3.1.44 --- p.73 / Figure 3.2.1 - 3.2.50 --- p.97 / Table 3.1 -3.4 --- p.117 / Chapter Chapter 4. --- Discussions --- p.121 / Chapter 4.1 --- The study on the transformation of cells and soft agar assay --- p.121 / Chapter 4.2 --- Growth patterns of the estrogen-transformed clones --- p.123 / Chapter 4.3 --- Altered differential gene expression --- p.124 / Chapter 4.3.1 --- TUBA --- p.124 / Chapter 4.3.2 --- PTEN --- p.125 / Chapter 4.3.3 --- RAP 1A --- p.126 / Chapter 4.3.4 --- BRCA2 --- p.126 / Chapter 4.4 --- Ultrastructural study in the estrogen-transformed and untreated parental NRP-152 cells --- p.127 / Chapter 4.5 --- Neoplastic lesions induced in prostates of estrogen-imprinted and long-term combined hormone treated Noble rats --- p.129 / Chapter 4.6 --- Altered protein expressions in estrogen-transformed NRP-152 cells and estrogen-imprinted and hormone-treated Noble rat prostates --- p.132 / Chapter 4.6.1 --- Alteration in steroid hormone receptors --- p.132 / Chapter 4.6.2 --- Alternation in cytoskeleton (tubulin-α) --- p.138 / Chapter 4.6.3 --- Alternation in PTEN --- p.141 / Chapter 4.6.4 --- Alternation in Rap1 --- p.143 / Chapter 4.6.5 --- Alternation in BRCA2 --- p.145 / Chapter 4.6.6 --- "Altered in scavenger enzyme (Superoxide dismutase, SOD-1)" --- p.147 / Chapter Chapter 5. --- Summary --- p.150 / Reference --- p.155

Tsang Lai-Ling Angel. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 136-145). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgment --- p.vi / Table of Content --- p.vii / List of Publications --- p.xii / List of Figures --- p.xiv / List of Tables --- p.xvii / Abbreviations --- p.xviii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- The Human Uterus Vs Rat Uterus --- p.1 / Chapter 1.1.1 --- Myometrium --- p.1 / Chapter 1.1.2 --- Endometrium --- p.1 / Chapter 1.2 --- The Human Endometrium Vs Rat Endometrium --- p.2 / Chapter 1.2.1 --- The structure of Human Endometrium --- p.2 / Chapter 1.2.2 --- Cyclic Changes in the Endometrium --- p.4 / Chapter 1.2.3 --- Physiological Roles of the Endometrium --- p.7 / Chapter 1.2.4 --- Uterine Fluid Volume and its Composition --- p.7 / Chapter 1.2.4.1 --- Regulation of Uterine Fluid Volume and Composition --- p.7 / Chapter 1.2.4.2 --- Role of Endometrial Epithelium in the Regulation of Uterine Fluid Volume --- p.9 / Chapter 1.3 --- Epithelial Ion Channels --- p.9 / Chapter 1.3.1 --- Epithelial CI- Channels in Secretory Epithelia --- p.11 / Chapter 1.3.1.1 --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.13 / Chapter 1.3.2 --- Epithelial Na+ Channel (ENaC) in Absorbing Epithelia --- p.18 / Chapter 1.3.3 --- ENaC and CFTR in Endometrial Epithelia --- p.26 / Chapter 1.4 --- Hormonal Regulation of the Endometrial Epithelium --- p.29 / Chapter 1.4.1 --- Estrogen and Progesterone --- p.29 / Chapter 1.4.2 --- Aldosterone --- p.32 / Chapter 1.5 --- Aim of Study --- p.35 / Chapter Chapter2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- Culture Medium and Enzymes --- p.38 / Chapter 2.1.2 --- Drugs --- p.38 / Chapter 2.1.3 --- Molecular Biology --- p.39 / Chapter 2.1.4 --- Experimental Tissues and Animals --- p.39 / Chapter 2.2 --- Preparations --- p.39 / Chapter 2.2.1 --- Pervious Support for Cell Growth --- p.39 / Chapter 2.2.2 --- Growth Medium --- p.40 / Chapter 2.2.3 --- Culture of Mouse Endometrium Epithelial Cells --- p.43 / Chapter 2.2.4 --- Solutions for the Short-Circuit Current Measurement --- p.44 / Chapter 2.2.5 --- Electrodes for the Short-Circuit Current Measurement --- p.44 / Chapter 2.2.6 --- Solutions for Molecular Biology Experiment --- p.44 / Chapter 2.2.6.1 --- Diethyl Pyrocarbonate (DEPC)-treated Water --- p.44 / Chapter 2.2.6.2 --- lx TAE (DNA gel electrophoresis and its running buffer) --- p.45 / Chapter 2.2.6.3 --- 5x MOPS (RNA gel electrophoresis and its running buffer) --- p.45 / Chapter 2.2.6.4 --- Formaldehyde Gel-loading Buffer --- p.45 / Chapter 2.3 --- Protocols --- p.46 / Chapter 2.3.1 --- Effect of Ovarian Hormones and Aldosterone on CFTR and ENaC Expression --- p.45 / Chapter 2.3.2 --- Possible Interaction between CFTR and ENaC upon Hormones Stimulation --- p.47 / Chapter 2.4 --- Methods of Measurement --- p.48 / Chapter 2.4.1 --- The Short-Circuit Current Technique --- p.48 / Chapter 2.4.1.1 --- The Short-Circuit Current Setup --- p.48 / Chapter 2.4.1.2 --- Experimental Procedures --- p.52 / Chapter 2.4.1.3 --- Data Analysis --- p.55 / Chapter 2.4.2 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.55 / Chapter 2.4.2.1 --- RNA Isolation --- p.55 / Chapter 2.4.2.2 --- RNA Gel Electrophoresis --- p.56 / Chapter 2.4.2.3 --- Reverse Transcription (RT) --- p.57 / Chapter 2.4.2.4 --- Primer used for the Polymerase Chain Reaction (PCR) --- p.58 / Chapter 2.4.2.5 --- General Procedure of PCR and Competitive RT-PCR --- p.59 / Chapter 2.4.2.6 --- DNA Gel Electrophoresis --- p.61 / Chapter 2.4.3 --- Capillary Electrophoresis - Laser Induced Fluorescence (CE-LIF) --- p.62 / Chapter 2.4.3.1 --- Capillary Tube --- p.54 / Chapter 2.4.3.2 --- Detection System --- p.65 / Chapter 2.4.3.3 --- Experimental Procedures --- p.65 / Chapter 2.4.3.4 --- Data Analysis --- p.66 / Chapter 2.4.4 --- Statistical Analysis / Chapter Chapter3 --- Results --- p.68 / Chapter 3.1 --- Influence of Ovarian Hormones on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Epithelial Na+ Channel (ENaC) Expression in Mouse Endometrial Epithelium --- p.68 / Chapter 3.2 --- Culture Condition on Expression and Function of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Mouse Endometrial Epithelial Cells --- p.92 / Chapter 3.3 --- Expression Regulation of Endometrial Epithelial Na+ Channel (ENaC) Subunits and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Na+ Diet During the Estrus Cycle in Mice --- p.98 / Chapter 3.4 --- Enhanced Epithelial Na+ Channel (ENaC) Activity in Mouse Endometrial Epithelium by Upregulation of γ-ENaC Subunit --- p.114 / Chapter Chapter4 --- General Discussion --- p.127 / Appendix --- p.132 / Chapter A. --- RNA Isolation --- p.132 / Chapter B. --- Reverse Transcription (RT) --- p.133 / Chapter C. --- Polymerase Chain Reaction (PCR) --- p.134 / Chapter D. --- Sequences and Conditions of All Primers --- p.135 / References --- p.136

The FHIT gene is located on the most fragile site in the human genome. FHIT gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissue. Previous work in mouse and cell culture models established FHIT to be an authentic tumor suppressor. Re-expression of FHIT in cell culture causes cell death via initiation of apoptosis, but the precise mechanism underlying this process is unclear. It is well established that cellular transition from normal to transformed occurs in multiple steps and requires the accumulation of several genetic changes. Relying on the compelling phenotype of tumor development in FHIT knockout mice, this project aimed to elucidate a mechanism through which FHIT-deficient cells are primed to survive multiple genetic and environmental stresses, and promote progression of cancer. My work indicates that FHIT expression is required for the normal cellular response to oxidative stress, and presents evidence that in the absence of FHIT, an oxidative stress response pathway is superinduced. When FHIT is depleted from cells exposed to cigarette smoke, the expression of a subset of oxidative stress response genes is enhanced. Enhanced activation of these genes can occur as an adapative response to stress induced by reactive oxygen species production, and is frequently detected in cancer. Investigation into the mechanism underlying the enhanced gene expression determined that FHIT loss is associated with decreased levels of the transcriptional repressor Bach1. In this manner, we propose that loss of Fhit supports an antioxidant program that is pivotal in establishing and maintaining carcinogenic transformation.

Dans de nombreux tissus épithéliaux, les cellules ne migrent pas sur des substrats plats mais font au contraire partie d’une monocouche bidimensionnelle courbée. C'est le cas par exemple pour les tubules rénaux, les acini du sein, les alvéoles pulmonaires ou bien les cryptes du côlon et de l’intestin. Cependant, malgré l’omniprésence de cette courbure hors plan in vivo, peu d’études expérimentales s’interrogent sur son influence sur le développement et la migration cellulaire. En effet, le comportement collectif des cellules au sein d’un épithélium à deux dimensions a été principalement étudié sur des substrats plans, négligeant ainsi l'influence de la topologie de l’environnement de culture sur le développement cellulaire. Dans ce manuscrit de thèse, nous exposons les différentes expériences menées afin de caractériser et de quantifier l’influence de cette courbure hors du plan sur le développement, la migration ainsi que les propriétés mécaniques des tissus épithéliaux. Un dispositif expérimental a été mis au point afin d’obtenir des courbures et des conditions initiales reproductibles. Afin de découpler l’effet du à la courbure de ceux dus au confinement latéral, nous avons reproduit les mêmes expériences sur des substrats plats où les cellules sont confinées dans des bandes adhésives de largueurs comparables au périmètre des tubes.En mettant en parallèle nos résultats sur ces deux types de substrats, nous suggérons que forcer les cellules à croître sur des cylindres de diamètre pertinent biologiquement induit de nouvelles propriétés biologiques qui sont à dissocier de l'effet du au confinement latéral du tissu. / That the mechanics of a cell’s microenvironment greatly influences cellular behaviors and phenotypes is well established. For example, the morphology, performance and even fate of a cell adhering to a substrate is highly influenced by the substrate rigidity. Similarly, cytoskeleton organization and cell polarity can be controlled by confining the cell to 2d micro patterns while micro or nano substrate topography influences cell adhesion and orientation. On the other hand, significantly less research exists regarding the effect of out of plane curvature on individual cells and cell assemblies, despite the intrinsic curvature of epithelial sheets which frequently form tubes, cysts, crypts, or villi with radii of curvature on the order of a few cell diameters. This thesis accordingly examines the relationship between the collective properties of epithelial tissue and out of plane curvature by employing micro fabricated environments to deconstruct the response of a cell monolayer to the geometry of its neighborhood. Curved substrates provide a controlled way to study the role of a fixed out of plane curvature on a system otherwise identical to the classic 2D assay. In particular, fibers with curvature radii between 0.5um-100um were populated with MDCK cells from a model epithelial, kidney-derived, cell line and the resulting migration dynamics and cell architecture quantified. The specific cellular behaviors induced by large curvatures provide plausible explanations of certain aspects of tubulogenesis.

Abstract not available