Thesis (M.A.)--Humboldt State University, 2007. / Includes bibliographical references (leaves 68-77). Also available via Humboldt Digital Scholar.

Thesis (Ph.D.) OGI School of Science & Engineering at OHSU, September 2007. / Includes bibliographical references (leaves 186-211).

The investigation into the mechanisms by which compounds such as butyrate induce differentiation in HT-29 cells, is lacking. The colonic carcinoma cell line, HT-29, undergoes differentiation induction in the presence of butyrate and acetoacetate. The Caco-2 cell line spontaneously differentiates on contact inhibition. In this study, a signal transduction pathway involving ATP, cAMP, Ca2+ and the transcriptional factor CREB was investigated following suggestions that the energy state of the cell and diffferentiation are linked. The activity of the MAP kinase cascade, including possible crosstalk that may exist between these pathways was determined. The HT-29 cells were exposed to 5 mM acetoacetate, butyrate, DMSO and propionate. The results of this differentiation induction were compared to Caco-2 and HeLa cells, which are cervical carcinoma cells. It was found that ATP levels are decreased on differentiation induction in HT-29 cells, which, in turn affected the cAMP concentrations. Theoretically, the inducers do not have any effect on PDE 4 activity, and may facilitate the interaction between cAMP and PKA. Influx of Ca2+ into the cells was inhibited to a degree by the inducers, which was possibly overcome by crosstalk between the cAMP and Ca2+ pathways. CREB activation, lineage-specific gene expression, ERK activity and c-myc expression were all dependent on both the inducers used and the cell-type. PKA played a major role in CREB activation in acetoacetate- and butyrate -induced HT-29, Caco-2 and HeLa cells, while a2+/Calmodulin-dependent kinases I/IV may have a secondary role. Alkaline phosphatase expression in HeLa cells was independent of CREB. Evidence that crosstalk between the MAP kinase cascade and the REBactivation pathways exist, was illustrated by increased CREB activation on ERK inhibition in acetoacetate- and butyrate-induced HT-29 and HeLa cells. Also, the role that ERK played in the cells differed with inducer and cell-type. The dependence of cmyc expression on c-jun and c-fos, appeared to be differentiation induction- and celltype specific. Results from this study indicate the potential use of acetoacetate and butyrate as anti-cancer compounds.

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Nous avons utilisé la lignée épithéliale intestinale de crypte de rat IEC-6 afin de mieux comprendre le rôle des C/EBPs dans la différenciation et la prolifération de l'intestin. Nous avons vérifié l'expression des ARNm de C/EBP$\alpha$, C/EBP$\beta$ et C/EBP$\delta$ suite à la stimulation des cellules IEC-6 par la forskoline et l'IBMX qui stimulent la PKA, le TPA qui stimule la PKC et la thapsigargine qui augmente la concentration de Ca$\sp{2+}$ cytoplasmique. La forskoline et l'IBMX induisent les niveaux d'ARNm des trois C/EBPs avec différentes cinétiques tandis que le TPA induit seulement les niveaux d'ARNm de C/EBP$\alpha$ et de C/EBP$\beta.$ La thapsigargine induit les niveaux d'ARNm des trois isoformes par un mécanisme de régulation post-transcriptionnel sans affecter les niveaux de protéines. Les variations des niveaux d'ARNm, induits par les trois voies de signalisation, étaient indépendantes d'une synthèse nouvelle de protéines. Les analyses des niveaux de protéine par Western nous ont permis de constater une induction des trois isoformes en accord avec celle des ARNm suite à une stimulation par la forskoline et l'IBMX. Nous avons observé que le patron d'expression des protéines C/EBP$\alpha$ et C/EBP$\beta$ concorde avec celui des ARNm après une stimulation par le TPA. La capacité de liaison à l'ADN des C/EBPs n'est pas affectée significativement par les voies de la PKA, de la PKC et du Ca$\sp{2+}.$ Les voies de la PKA et de la PKC peuvent moduler l'expression de certains gènes de la réponse inflammatoire comme l'haptoglobine et le thiostatin. La stimulation de ces différentes voies inhibe la prolifération cellulaire des cellules IEC-6 en absence et en présence de sérum. Finalement, nous avons observé que ces différentes voies pouvaient induire à des niveaux variables l'ARNm de p21, un gène très important dans l'arrêt de la prolifération cellulaire. [Résumé abrégé par UMI] [Symboles non conformes]

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2001-2003 / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

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