No description available.

No description available.

Finally, as the first step to study the regulation of Kit system, we found that IGF-I was a potent regulatory factor that up-regulated the expression of kitlga in zebrafish follicle cells. The stimulation involved transcription but not translation, indicating that the kitlga gene is a direct downstream target of IGF-I. The effect of IGF-I on kitlga was exerted via PI3K-Akt but not MAPK pathway. In contrast, the MAPK pathway may play a negative role in controlling kitlga expression. / Kit ligand (also named stem cell factor, SCF) is a pleiotropic growth factor with diverse biological functions. It exerts effects on target cells by binding to its cognate tyrosine kinase receptor, Kit. In mammals, accumulated evidence has demonstrated important roles for Kit ligand and Kit in gametogenesis, melanogenesis and haematopoiesis. However, very little is known about Kit system in other vertebrates. In the present study, we used zebrafish as the model to investigate the expression, regulation and function of the Kit system in the ovary. / On the other hand, cAMP is involved in regulating the expression of kitlga in zebrafish follicle cells. Two cAMP-activated effectors, PKA and Epac, have reverse effects. PKA promotes but Epac inhibits the expression of kitlga, which was identified by the respective activator. The effect of forskolin and H89 on IGF-I-induced expression of kitlga suggests a cross-talk between the two signaling pathways. Both hCG and PACAP inhibited IGF-I-induced kitlga expression, indicating that they may have negative regulation through cAMP signaling pathways in the full-grown follicles. (Abstract shortened by UMI.) / The zebrafish has two homologues of Kit ligand (kitlga and kitlgb) and Kit (kita and kitb ) instead of one copy for each as in mammals. The present study proposed the origin of these homologues in the zebrafish by phylogenetic and chromosome synteny analyses, and provided further evidence for neo- or subfunctionalization for both Kit ligands and Kit receptors in the zebrafish ovary. All four Kit system members exhibited distinct and significant changes in mRNA expression during folliculogenesis, particularly in the periovulatory period before and after final oocyte maturation and ovulation. / Then we further studied the spatial localization of each member within the follicle. The present study demonstrated that kitlga and kitb are exclusively expressed in the follicle layer, while kitlgb and kita only in the oocyte. Using CHO cell line as a bioreactor, we produced recombinant zebrafish Kitlga and Kitlgb. Analysis in mammalian COS-1 cells and zebrafish primary follicle cells confirmed their biological activity and binding specifity. Two opposite paracrine pathways of Kit system in the zebrafish ovary have been shown. Kitlga from the follicle cells preferably activates Kita in the oocyte in spite of the weak response of Kitb to it. Kitlgb from the oocyte, however, exclusively activates Kitb in the follicle cells without any effects on Kita. / Yao, Kai. / Adviser: Ge Wei. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 136-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20999号 / 医博第4345号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 武田 俊一, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Murine embryonic stem (ES) cells are derived from the inner cell mass of the developing embryonic blastocyst. These cells can self renew which allows them to be propagated indefinitely in the laboratory and they can differentiate into cell types derived from all three germ layers. Manipulation of the mouse genome using gene targeting techniques in conjunction with ES cell technology has provided valuable insights into embryonic development and cell lineage specification. KIT is a trans-membrane receptor tyrosine kinase (RTK) that dimerises upon binding to its ligand, stem cell factor (SCF) resulting in the auto-phosphorylation of intracellular kinase domains. This activity is crucial for the transmission of signals from the cell surface to the nucleus. KIT is expressed on stem and progenitor cells of many lineages and defects in the SCF/KIT signaling pathway causes detrimental effects at both the cellular and physiological level. This project aimed to investigate the role of the SCF/KIT signalling pathway during murine ES cell differentiation and survival. To assess the role of SCF/KIT signalling in ES cell proliferation and survival, we knocked out the c-kit gene in mouse ES cells to produce heterozygous (KitW-lacZ/+) and KIT Null (KitW-lacZ/W-lacZ) cell lines. The self renewal and differentiation profile of these cell lines revealed an auxiliary role for SCF/KIT during ES cell self renewal and an absolute role in survival upon in vitro differentiation. This phenotype of apoptosis upon differentiation was recapitulated in wild type E14 ES cells treated with a KIT neutralising antibody (ACK2). Wild type cells that were treated with the JNK inhibitor, SP600125 had a comparable phenotype to KIT null cells indicating that this could be one of the mediators of KIT signalling that has a protective role in the survival of differentiating ES cells. We hypothesised that blocking classical apoptotic pathways might prevent the death on differentiation observed in KIT null cells. However, neither blocking the pro-apoptotic P38 pathway with the chemical inhibitor PD169316 nor over-expressing the pro-survival protein BCL2 in KIT Null cells could prevent their apoptosis upon differentiation phenotype. This strongly suggests that these pathways are not involved in KIT mediated survival of differentiating ES cells. Although compensatory mechanisms are thought to exist for defective KIT signaling in vivo, an absolute role is assigned to KIT during ES cell differentiation. Further analysis of micro array data comparing gene expression from wild type E14 and KIT Null cell lines may reveal the specific mechanisms of KIT mediated survival during differentiation onset.

No description available.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010. / Härtill 4 uppsatser.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-03Bitstream added on 2014-06-13T20:30:21Z : No. of bitstreams: 1 oliveira_lcf_me_araca.pdf: 916072 bytes, checksum: a0dad11c3aa9513daedea50b01cd4ff9 (MD5) / O objetivo deste estudo foi avaliar o mecanismo envolvido na produção de TGF-β, FGF-2 e CCL3/MIP-1α induzida por Stem cell factor (SCF) em células da musculatura lisa de traqueia (CMLT) e as vias de transdução de sinalização ativadas. Traqueias de camundongos normais foram coletadas, fragmentadas e colocadas em garrafas contendo meio de cultura DMEM com 10% de Soro Fetal Bovino. CMLT foram estimuladas com SCF (1, 10 and 100 ng/mL) e avaliadas após 1, 6 e 24 horas. Características fenotípicas das CMLT foram analisadas por imunofluorescência para α-actina de músculo liso (α-AML), α-citoqueratina e α-proteína de ativação de fibroblastos (α-FAP). Ativação de c-kit em CMLT estimuladas por SCF foi avaliada por citometria de fluxo. A expressão de RNAm para TGF-β foi observada pela reação de polimerase em cadeia-transcriptase reversa (RT-PCR) e a produção da proteína, por ensaio de imunoabsorção enzimática (ELISA). A produção de FGF-2 foi avaliada por immunoblot e a produção de CCL3/MIP-1α por ELISA. Em outro conjunto de experimentos, CMLT foram pré-tratadas com inibidores de MAPK p42/44(PD 98059 [PD]), p38(SB 202190[SB]), e JNK (SP 600125 [SP]) por 30 minutos seguidos de estimulação com SCF (10 ng/mL) por 24 horas. Células pré-tratadas com anticorpos específicos não revelaram qualquer marcação para citoqueratina nem para α-FAP, contudo, ocorrendo a marcação para α-AML, indicando a pureza da linhagem celular primária. SCF induziu a expressão de receptores c-kit em CMLT. CMLT estimuladas por 10 ng/mL de SCF expressaram TGF-β mRNA e produziram TGF- β proteína, FGF-2 e CCL3/MIP-1α após 24 horas. O pré-tratamento com SB, PD e SP inibiu estas produções. Estas produções foram mediadas pela ativação das vias p42/44, p38, e JNK. CMLT parecem ser importantes células residentes... / The aim of this study was to evaluate the mechanism involved in SCF-induced TGF-β, FGF-2 and CCL3/MIP-1α production in tracheal smooth muscle cells (tSMC) and the activated signaling transduction pathway. Normal mouse tracheas were collected, fragmented and placed in bottles containing the culture medium DMEM with 10% Fetal Bovine Serum. tSMC primary cultures were stimulated with SCF (1, 10 and 100 ng/mL) and evaluated at 1, 6 and 24 hours. The phenotypic characteristic of tSMC in primary culture was analyzed using immunofluorescence staining for α-smooth muscle actin (α-SMA), α-cytokeratin and α-Fibroblast Activation Protein (α-FAP). c-Kit activation in SCF-stimulated tSMC was evaluated by flow cytometric. The TGF-β mRNA expression was observed by reverse-transcriptase polymerase chain reaction (RT-PCR) and protein production by enzyme-linked immunosorbent assay (ELISA). FGF-2 production was evaluated by immunoblot and CCL3/MIP-1α production by ELISA. In other set of experiment, tSMC were pretreated p42/44 inhibitor (PD 98059 [PD]), p38 inhibitor (SB 202190[SB]), or JNK inhibitor (SP 600125 [SP]) for 30 minutes followed by stimulation with SCF (10 ng/mL) for 24 hour. Cells treated with specific antibodies, showing neither labeling for cytokeratin nor either FAP, however, labeling for α-SMA indicating purity of the primary cell line. SCF induces c-Kit expression on tSMC. SCF-stimulated tSMC express TGF-β mRNA expression and FGF-2 and CCL3/MIP-1α production at 10 ng/mL after 24 hours. SB, PD and SP pre-treatment inhibited these productions. These productions were mediated by activation pathways p42/44, p38, and JNK. tSMC seems to be important resident cells involved in the cell activation and tissue repair, since they are capable of producing growth factors and chemokines and added new... (Complete abstract click electronic access below)