The efficiency of a number of experimental techniques for the extraction of total RNA from various cells and tissues (including MDCK strain I cells) was assessed, and the optimal conditions for hybridisation of Na,K-ATPase isoform-specific DNA probes to this RNA were determined. The specificity of hybridisation of DNA probes for the Na,K-ATPase al, a2, a3, and beta1 isoforms was assessed using RNA isolated from rat tissues. The relative abundance of isoform mRNA's in rat kidney, brain, lung, and myocardial tissues was determined by Northern blotting. The abundance of Na,K-ATPase isoforms was also determined in the myocardial tissues of the Milan rat, a hypertensive animal model. Significant differences between the abundance of Na,K-ATPase isoform mRNA's in hypertensive rats and their age and sex matched controls were found. The relative abundance (per ng of total RNA) of al, a3, and beta1 mRNA's in left ventricle and, that of a1, and beta1 mRNA's in right ventricle were significantly decreased in hypertensive rats. The relative abundance (per mug of total RNA) of a2 and beta1 mRNA's in atria was significantly increased in hypertensive rats. These differences found in ventricles and atria were further accentuated by expression of the results per gram wet weight of tissue. The results from ventricular tissues were in contrast to those previously reported by Herrera et al. (1988) who found either increases or no change in the abundance of a1 and beta1 mRNA's in hypertensive rat aorta, skeletal muscle and left ventricle. The differences between these results may be related to the deoxy-corticosterone treatment and high salt diet of the hypertensive rat model used by Herrera et al. (1988). Na,K-ATPase isoform-specific DNA restriction endonuclease fragments were used to investigate the expression of the isoform mRNA's in MDCK strain I cells. Only a1 and beta1 mRNA's was detected on Northern blots, with no detectable a2 or a3 isoform mRNA signals being found in this cell line. [3H]-ouabain binding to cells was used, as an estimate of the cell surface expression of Na,K-ATPase. Possible factors affecting the expression of Na,K-ATPase during the normal cell growth of MDCK strain I cells were investigated. Factors such as cell seeding density, cell growth substrate and the volume of growth medium used, were all found to affect both the level and pattern of expression of Na,K-ATPase during the normal cell growth or culturing cycle. After 2 days of culture the large increases in the expression of Na,K-ATPase assayed in low density compared to high density seeded cells, were not correlated with concomitant changes in the relative abundance of Na,K-ATPase a subunit mRNA. These results indicate that the large changes in cell surface expression of Na,K- ATPase found during cell growth are probably controlled by post transcriptional processes. The effect of certain hormones or their agonists (aldosterone, deoxy-corticosterone, corticosterone, dexamethasone, and tri-iodo thyronine), on the expression of Na,K-ATPase in MDCK strain I cells was also briefly investigated. Under the conditions used, hormone treatment was not found to induce any measurable expression of a2 or a3 mRNA's. The mineralocorticoid aldosterone, and the glucocorticoid corticosterone, both produced small but significant increases in the level of Na,K-ATPase present on the cell membrane, however these increases were not correlated with similar increases in the abundance of both Na,K-ATPase a1 and beta1 mRNA's. The small size of increases in Na,K-ATPase enzyme abundance after hormone treatments and the inability of those treatments to induce consistent increases in Na,K-ATPase mRNA's further suggests that changes in the cell surface expression of Na,K-ATPase in MDCK cells is the result of regulation at a post transcriptional level.

This thesis is a study of the biology of Drosophila imaginal disc cells growing as continuous cell lines. Their morphological characteristics and cellular properties were analysed in order to characterise the cells in vitro. Morphological analysis of the cells revealed several cell types within the original cell lines perhaps representing a diversity in the cellular origin of these cells. A new cloning technique was devised which enabled a single imaginal disc cell to give rise to new cell line and indicated that a single cell could give rise to the different cell types seen in culture. This indicates that the diversity in morphology of the cells in culture was an indication of the conditions in culture rather than as a result of cellular diversity. The original cell lines and the newly derived cloned cell lines were subjected to the insect moulting hormone 20-HE in order to ascertain the degree of differentiation that was possible in culture. The cells showed a dramatic morphological response to hormone, they elongated, began to aggregate and threw out cell processes. This is combined with concomitant biochemical changes in the cell lines, including the induction of chitin synthesis and acetylcholinesterase. The cells in culture show a characteristic pattern of aggregation which was studied at the ultrastructural level using electron microscopy. These studies and also immunofluorescence of cell aggregates indicated the prevalence of cell processes and a role was postulated for their action in bringing about these aggregates. Aggregation was also correlated with the expression of PS integrins, which are well characterised Drosophila adhesion molecules. The adhesive properties of the cells were further characterised with reaggregation experiments in different media as a prelude to setting up cell sorting assays between wing and leg cell lines. This proved somewhat inconclusive but pointed to some sorting out occurring between wing and leg cells.

No description available.

Lai, Ka Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 114-121). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.x / Chapter Chapter1 --- General introduction --- p.1 / Chapter 1.1 --- Cell differentiation --- p.1 / Chapter 1.1.1 --- S tem cell treatments --- p.2 / Chapter 1.1.2 --- Differentiation therapy for cancer --- p.3 / Chapter 1.2 --- Cyclic adenosine monophosphate (cAMP) signaling involved in cell differentiation --- p.4 / Chapter 1.2.1 --- cAMP -signaling pathways leading to transcription activities --- p.4 / Chapter 1.2.1 --- Regulation of cell differentiation by cAMP/PKA signal --- p.5 / Chapter 1.3 --- Aim of thesis --- p.5 / Chapter Chapter2 --- "Materials, media, buffers and solutions" --- p.7 / Chapter 2.1 --- Mate rials --- p.7 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.12 / Chapter 2.2.1 --- General culture buffers --- p.12 / Chapter 2.2.2 --- Culture medium --- p.12 / Chapter 2.2.3 --- Assay buffers and solutions --- p.13 / Chapter 2.2.3.1 --- Buffers and solutions for RT-PCR --- p.13 / Chapter 2.2.3.2 --- Buffers and solutions for assay of [3H]cAMP production --- p.13 / Chapter 2.2.3.3 --- Buffers and solutions for Western blotting --- p.14 / Chapter 2.2.3.4 --- Buffers and solutions for histamine assay --- p.16 / Chapter 2.2.3.5 --- Buffers and solutions for flow cytometry --- p.17 / Chapter Chapter3 --- Methods --- p.18 / Chapter 3.1 --- Maintenance of rat pheochromocytoma (PC12) cells --- p.18 / Chapter 3.2 --- Dete rmination of AC isoforms expression in PC12 cells by RT-PCR analysis --- p.19 / Chapter 3.2.1 --- RNA isolation --- p.19 / Chapter 3.2.2 --- cDNA synthesis by reverse transcription (RT) --- p.20 / Chapter 3.2.3 --- Semi-quantitative PCR --- p.21 / Chapter 3.3 --- Maintenance of human erythroleukemia (HEL) cells --- p.23 / Chapter 3.4 --- Dete rmination of [3H]cAMP Production in HEL cells --- p.23 / Chapter 3.4.1 --- Principle of assay --- p.23 / Chapter 3.4.2 --- Column preparation --- p.24 / Chapter 3.4.3 --- Measurem ent of [3H]cAMP production in HEL cells --- p.24 / Chapter 3.4.4 --- Data analysis --- p.25 / Chapter 3.5 --- Im munodetection of STAT3 and pTyr705STAT3 by western blotting --- p.25 / Chapter 3.6 --- Harvesting of HE L cells after differentiation treatment --- p.27 / Chapter 3.7 --- Flow cyto metry analysis of HEL cells --- p.27 / Chapter 3.7.1 --- F ITC-conjugated CD41 -antibody staining --- p.28 / Chapter 3.7.2 --- P I staining --- p.28 / Chapter 3.8 --- Determination of extracellular and intracellular histamine of HEL cells --- p.29 / Chapter 3.8.1 --- Sample preparation --- p.29 / Chapter 3.8.2 --- Automated assay of histamine content --- p.30 / Chapter 3.9 --- siRNA mediated knockdown of STAT3 in HEK293 cells --- p.30 / Chapter 3.9.1 --- Culture human embryonic kidney (HEK293) cells --- p.30 / Chapter 3.9.1 --- siRNA transfection --- p.31 / Chapter Chapter4 --- mRNA expression of adenylyl cyclase isoforms during early stage of NGF-induced differentiation of PC12 cells --- p.33 / Chapter 4.1 --- Introduction --- p.33 / Chapter 4.1.1 --- Dif ferentiation of PC12 cells --- p.33 / Chapter 4.1.1.1 --- Induction of neurite outgrowth by NGF in PC12 cells --- p.33 / Chapter 4.1.1.2 --- Effect of cAMP on NGF-induced neurite outgrowth in PC12 cells --- p.34 / Chapter 4.1.1.3 --- Effect of cAMP on NGF-induced neurite outgrowth in PC12 cells --- p.35 / Chapter 4.1.2 --- Enhanced forskolin-stimulated [3H]cAMP productionin NGF-difFerentiated PC12 cells --- p.36 / Chapter 4.1.3 --- Classification of adenylyl cyclases --- p.38 / Chapter 4.1.4 --- Aims of study --- p.39 / Chapter 4.2 --- Results and discussion --- p.40 / Chapter Chapter5 --- Effect of cicaprost on PMA-mediated differentiation of human erythroleukemia (HEL) cells --- p.48 / Chapter 5.1 --- Introduction --- p.48 / Chapter 5.1.1 --- Differentiation of HEL cells --- p.48 / Chapter 5.1.2 --- Prostac yclin (PGI2) and human IP receptors --- p.49 / Chapter 5.1.3 --- Agonists and antagonists of IP receptors --- p.50 / Chapter 5.1.4 --- IP signaling in HEL cells --- p.52 / Chapter 5.1.5 --- Effect of cAMP on megakaryocytic differentiation --- p.52 / Chapter 5.1.6 --- Aims of study --- p.54 / Chapter 5.2 --- Results and discussion --- p.56 / Chapter 5.2.1 --- Preliminar y studies --- p.56 / Chapter 5.2.1.1 --- PMA induced cell adhesion and morphological change --- p.56 / Chapter 5.2.1.2 --- Cell proliferation and protein content --- p.57 / Chapter 5.2.1.3 --- IP signaling in HEL cells --- p.57 / Chapter 5.2.1.4 --- Presence of histaminase in FBS --- p.60 / Chapter 5.2.1.5 --- Summary of preliminary studies --- p.61 / Chapter 5.2.2 --- PMA -induced cell spreading of HEL cells --- p.63 / Chapter 5.2.3 --- PMA -induced DNA synthesis of HEL cells --- p.65 / Chapter 5.2.4 --- PMA -induced cell size and cell complexity of HEL cells --- p.67 / Chapter 5.2.5 --- PMA -induced CD41/CD61 expression of HEL cells --- p.69 / Chapter 5.2.6 --- PMA -induced histamine production of HEL cells --- p.72 / Chapter 5.2.7 --- IP receptor-dependent and IP receptor-independent actions of cicaprost --- p.74 / Chapter 5.2.8 --- STAT3 knockdown by siRNA --- p.75 / Chapter 5.3 --- Role of STAT3 in MK differentiation --- p.76 / Chapter 5.4 --- Summary --- p.78 / Chapter Chapter6 --- General discussions and future study --- p.105 / Chapter 6.1 --- General discussions --- p.105 / Chapter 6.2 --- Future study --- p.111 / References --- p.114

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1989.

No description available.

published_or_final_version / Anatomy / Master / Master of Philosophy

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)(+) progenitor cells that can differentiate to beta cells ex vivo. Here we evaluate the role of Ngn3(+) cells in beta cell expansion in situ. PDL not only induced doubling of the beta cell volume but also increased the total number of islets. beta cells proliferated without extended delay (the so-called 'refractory' period), their proliferation potential was highest in small islets, and 86% of the beta cell expansion was attributable to proliferation of pre-existing beta cells. At sufficiently high Ngn3 expression level, upto 14% of all beta cells and 40% of small islet beta cells derived from non-beta cells. Moreover, beta cell proliferation was blunted by a selective ablation of Ngn3(+) cells but not by conditional knockout of Ngn3 in pre-existing beta cells supporting a key role for Ngn3(+) insulin(-) cells in beta cell proliferation and expansion. We conclude that Ngn3(+) cell-dependent proliferation of pre-existing and newly-formed beta cells as well as reprogramming of non-beta cells contribute to in vivo beta cell expansion in the injured pancreas of adult mice.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 89-107). Also available in print.