Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 137-156). Also available in electronic version.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line. / AFRIKAANSE OPSOMMING: Insulien is 'n mitogeen vir baie selle en gelei na binding aan die insulien reseptor, intrasellulêre seine via die klassieke, mitogeniese Raf-MEK-ERK of die metaboliese PB-kinase seintransduksie pad. 'n Insulien gebrek of tipe I diabetes veroorsaak osteopenie. Vetsugtige pasiënte met insulien weestandigheid of tipe II diabetes, 'n siekte wat geassosieer word met foutiewe insulien seintransduksie en hoë vlakke van sirkuierende insulien, het verhoogde of normale been mineraal digtheid (BMD). Die vraag of hiper insulin ernie 'n verlies aan beenmassa teëwerk word dikwels gevra. Teenstrydigheid oor die rol van insulien as 'n osteo-anaboliese stof bestaan egter steeds en hierdie vraag bly dus onbeantwoord. Dat insulien seintransduksie wel 'n kritiese rol speel in beenvormende osteoblaste is onlangs bevestig in studies met muise waarvan die geen vir IRS-l uitgeslaan is. Hierdie muise ontwikkel 'n lae omset osteopenie weens verswakte proliferasie en differensiasie. fn hierdie studie is gevind dat insulien wel in vitro as 'n osteoblast mitogeen kan funksioneer. Dit is in drie relatief onvolwasse (MBA-15.4, -15.6 muis en MG-63 mens) sellyne geillistreer, deur betekenisvolle verhogings in insulien-geaktiveerde proliferasie. In MBA-15.4 preosteoblaste is die persentasie verhoging in insulien-gestimuleerde proliferasie vergelykbaar met dié van die bekende mitogeniese forbolester, TPA. Die UMR-I06 sellyn het kenmerke van gedifferensieerde osteoblaste, en was baie minder responsief op insulien behandeling. Die verskil in die proliferasie vermoë van die verskillende sellyne kan die gevolg wees van verskille wat bestaan tussen spontaan getransformeerde sellyne of die stadium van sel differensiasie. 'n MEK 1/2 inhibitor, UO126 en 'n PB-kinase inhibitor, wortmannin, is gebruik om die insulien seintransduksie pad noodsaaklik vir die aktivering van ERK en osteoblast proliferasie te bepaal. In MBA-1S.4 muis pre-osteoblaste, was fetale kalf SenlTI1(FKS)-geinduseerde DNA sintese totaal afhanklik van MEK. Beide die MBA-15.4 en die meer volwasse MBA-15.6 muis osteoblaste was weerstandig teen die inhibitors op hulle eie, of in kombinasie. Verhoogde MEK-inhibitor konsentrasies het geen verdere effek gehad nie en in verskeie eksperimente is 'n verhoging in preliferasie selfs waargeneem met MEK-inhibisie. Hierdie resultate dui aan dat die klassieke insulien mitogeniese pad nie betrokke is in MBA-I5.4 gestimuleerde selproliferasie nie. Wortmannin het geen effek gehad op insulien- of20% FKS-gestimuleerde DNA sintese nie, maar het wel die aktivering van PB-kinase se metaboliese teiken, AktJPKB geinhibeer. Insulien seintransduksie aktiveer dus ERK deur beide MEK en PB-kinase, maar het geen effek op proliferasie gehad nie. FKS-gestimuleerde ERK aktivering en proliferasie was totaal afhanlik van MEK-ERK aktivering. Insulien-geaktiveerde DNA sintese in die mens MG-63 osteoblaste was gedeeltelik afhanklik van beide MEK en PB-kinase. Alhoewel IPA ook PB-kinase kon aktiveer, was dit totaal afhanklik van MEK vir DNA sintese. Dit dui aan dat daar 'n PB-kinase stroom-op van 'n dominante MEK-ERK seintransduksie pad voorkom. Die verskille wat ons dus waargeneem het in insulien mitogeniese seintransduksie tussen muis en mens, kan aandui dat insuliengestimuleerde seintranduksie paaie kan verskil van spesie tot spesie. Dit is bevestig met die muisselle wat onafhanklik is en mens selle wat afhanklik is van MEK aktivering vir insuliengeaktiveerde DNA sintese. Kroniese, langtermyn steroied behandeling kan beenverlies veroorsaak en die effek van glukokortikoide (GK) op die insulien mitogeniese pad in osteoblaste is dus ook ondersoek. Natrium-ortovanadaat, 'n proteien tirosien fosfatase (PIP) inhibitor het GK-verlaagde proliferasie in repons tot beide IPA- en FKS behandeling herstel in MBA-lSA en MG-63 preosteoblaste. PIPs soos SHP-l en PIP-l B funksioneer deur gefosforileerde kinases te defosforileer en dus te inaktiveer. Beide SHP-l and PIP-lB kon assosieer met kinases in die mitogeniese insulien seintransduksie pad van vinnig groeiende MBA-IS A preosteoblaste in 10% FKS. Verder het SHP-I ook geko-immunopresipiteer met aktiewe, tirosien-gefosforileerde ERK, wat aandui dat SHP-I met ERK assosieer om dit te defosforileer en inaktiveer. Die MEKERK of PB-kinase paaie is nie belangrik vir insulien-geaktiveerde seintransduksie in muis osteoblaste nie. Dit is dus onwaarskynlik dat die PIPs 'n rol sal speel in die negatiewe regulering van hierdie seintransduksie paaie. Die ontdekking dat vanadaat nie glukokortikoiedverlaagde insulien-geaktiveerde DNA sintese kan herstel nie, toon dat vanadaat-sensitiewe PIPs nie 'n rol speel in insulien-geaktiveerde proliferasie in muisselle nie. Hierdie bevinding het verder bevestig dat 'n nuwe insulien mitogeniese pad in die MBA-ISA, maar nie die MG-63 selle moontlik bestaan.

Siu, Yeung Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 65-75). / Abstracts in English and Chinese. / Title Page --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v-viii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi-xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6 / Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1 / Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2 / Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3 / Chapter 1.1.4 --- Other comitogens --- p.4 / Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5 / Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12 / Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7 / Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8 / Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9 / Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12 / Chapter 1.3 --- Objective of this study --- p.13-15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell cultures --- p.16 / Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18 / Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17 / Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17 / Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17 / Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20 / Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18 / Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19 / Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19 / Chapter 2.3.2.2 --- Total protein assay --- p.19 / Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20 / Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22 / Chapter 2.4.1 --- Dialysis --- p.21 / Chapter 2.4.2 --- Temperature treatment --- p.21 / Chapter 2.4.3 --- Proteolysis --- p.22 / Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26 / Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22 / Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23 / Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23 / Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23 / Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24 / Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24 / Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25 / Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25 / Chapter 2.5.7 --- Visualization of autoradiography --- p.26 / Chapter 2.5.8 --- Visualization by silver staining --- p.26 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30 / Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27 / Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29 / Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39 / Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31 / Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36 / Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38 / Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44 / Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49 / Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45 / Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50 / Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51 / Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52 / Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53 / Chapter 4.5 --- Possible directions for future investigation --- p.55 / Chapter 4.6 --- Conclusions --- p.56 / Chapter Chapter 5 --- Appendices / Chapter 5.1 --- Reagents and solutiuons --- p.57-64 / Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57 / Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59 / Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60 / Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61 / Chapter Chapter 6 --- References --- p.65-75

No description available.

published_or_final_version / Molecular Biology / Master / Master of Philosophy

It is now well established that cold-triggered calcium influx mediates cold-induced gene expression and development of freezing tolerance (cold acclimation). In this thesis, cold signaling events both upstream and downstream of calcium influx were examined. / First, it was shown that the studies on calcium mediation of cold acclimation in alfalfa cell suspension cultures could be applied to intact seedlings of Arabidopsis. Calcium chelators and channel blockers caused a strong reduction in the cold-induced accumulation of kin1 and kin2 transcripts, suggesting that calcium influx was an essential event during cold signaling and that the source of calcium for this influx was largely the calcium-rich cell wall. Evidence suggesting the involvement of calcium-dependent protein kinases (CDPKs) was also obtained. / Second, the nature of events upstream of calcium influx was explored. For this study, transgenic Brassica napus seedlings possessing both the endogenous cold-inducible BN115 gene and the coding part of beta-glucuronidase (GUS) gene placed under the control of the BN115 promoter were used. Thus cold-activation of the BN115 promoter drove the expression of both BN115 at the transcriptional level and the GUS enzyme activity at the translational level. Cold-activation of BN115 was inhibited by chemicals which cause membrane fluidization, cytoskeletal stabilization and inhibition of Ca2+ influx, and mimicked at 25°C by chemicals causing membrane rigidification, cytoskeletal destabilization and Ca2+ influx. Inhibitors of protein and lipid kinases prevented cold-activation of BN115, but inhibition of protein phosphatases activated BN115 at 25°C. / Third, given the increasing importance of mitogen-activated protein kinases (MAPKs) in signal transduction, the nature of molecular mechanisms that lead to cold-activation of a previously reported MAPK, SAMK, was investigated. During this study, the first plant MAPK activated by heat shock was discovered and named HAMK (Heat-shock-activated MAPK). It was shown that cold-activation of SAMK is mediated by cold-induced membrane rigidification, whereas the heat shock-activation of HAMK occurs through heat shock-induced membrane fluidization. Whereas activation of both SAMK and HAMK is blocked by an actin microfilament stabilizer, it is mimicked at 25°C by chemical destabilizers of microtubules or actin microfilaments. All of these events are inhibited by blocking the influx of extracellular Ca 2+. Cold-activation of SAMK and heat-activation of HAMK was prevented by treatment of cells with inhibitors of CDPKs. Thus, cold and heat shock are sensed by structural changes in the plasma membrane, which transduces the signal via cytoskeletal rearrangements to the opening of calcium channels, leading to Ca2+ influx, activation of CDPKs and activation of distinct MAPK cascades.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [85]-99).

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains x, 160 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic insulin-secreting beta cell failure is central to the development of diabetes. Therapeutic applications targeted at understanding and manipulating beta cell destruction mechanisms should enhance the preservation of functional beta cell mass and prevent diabetes. To this end, we have demonstrated that diabetogenic assaults (e.g., endoplasmic reticulum stress, glucolipotoxicity, and pro-inflammatory cytokines) attenuate the activation of beta cell pro-survival signaling pathways via a stress-inducible molecule called Mitogen-inducible gene 6 (Mig6). We discovered that the overabundance of Mig6 exacerbates stress-induced beta cell apoptosis and inhibits insulin secretion. Conversely, the deficiency of Mig6 partially protected beta cells from DNA damage-induced cell death. Further, we established that Mig6 haploinsufficient mice retained islet integrity and function and exhibited greater beta cell mass recovery following treatment with multiple low doses of the beta cell toxin streptozotocin. These data suggest that Mig6 may be a therapeutic target for beta cell preservation in diabetes.