A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the Degree of Master of Science (Medicine) / Classical epithelio-mesenchymal interactions are said to result in root development. These interactions may be regulated by a number of growth factors. Fibroblast growth factors (FGF’s), members of a highly conserved family of polypeptides, the heparin binding growth factors (HBGF’s) are known to play a crucial role during the development of certain vertebrate organs, including the tooth. Previously, FGF-2, 3, 4, and 8 have been shown to play a role in crown development. The aim of this study was therefore to elucidate the spatial and temporal expression of FGF-2 in the developing root. Parasagittal sections of the maxillary and mandibular arches of six age groups of post-natal mice (days 9, 10, 12, 16, 20 and 24) were cut and the developing roots of the incisor and molar teeth identified. Immunocytochemistry utilizing anti-FGF-2 was performed on sections of teeth from all stages using the strept-avidin biotin technique. Appropriate positive, negative and absorption controls were performed to ensure the specificity of the antibody. FGF-2 was immunolocalized in the cytoplasm and nuclei of the odontoblasts, fibroblasts of the periodontal ligament and pulp chamber, as well as in the osteoblasts surrounding developing bone at all the stages examined. Intense staining for FGF-2 was observed in differentiating odontoblasts at the apical end and the furcation zone of the developing root. FGF-2 localization was also observed in the cytoplasm of the ameloblasts on days 9, 10 and 12 and in cementoblasts on day 16, 20 and 24. The spatio-temporal expression pattern of FGF-2 in the developing mouse tooth root suggests that FGF-2 with other signaling molecules previously reported such as bone morphogenetic proteins-2, 3 and 7 (BMP-2, 3 and 7) participate in the signaling network during the tooth root development. / IT2018

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13452号 / 論医博第2245号 / 新制||医||1054(附属図書館) / (主査)教授 椛島 健治, 教授 森信 暁雄, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The majority of bovine mammary gland research focuses on the main cell types - mammary epithelial cells and fibroblasts. However, the extracellular matrix (ECM) within the mammary gland is also of importance for its ability to regulate cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in immortalized bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. However, the experiment was unable to confirm of reject this hypothesis. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary epithelial cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands. / Ph. D.

Yes / Polymer coatings that support epithelial cell culture have been developed. Ozonolysis and subsequent work up of poly(butyl methacrylate-co-butadiene) copolymers is used to form oligomers with carboxylic acid end groups, which are then further reacted with diamines to provide poly(butyl methacrylate)s with primary amine end groups. The polymers are cast as films and used as cell culture substrates for human dermal fibroblasts and human renal epithelial cells. Fibroblast and epithelial cells adhere and proliferate on acid functional materials but on amine functional films epithelial cells show greater viability than fibroblasts.

<p>Heparan sulfate (HS) belongs to the glycosaminoglycan family of polysaccharides and is found attached to protein cores on cell surfaces and in the extracellular matrix. The HS backbone consists of alternating hexuronic acid and glucosamine units and undergoes a number of modification reactions creating HS chains with alternating highly and low modified domains, where high degree of modification correlates with high negative charge. Fibroblast growth factors (FGFs) and their receptors (FRs) both bind to HS, which affect formation of the FGF–FR complexes on the cell surfaces. Activated FRs can trigger several intracellular signaling pathways leading thereby to diverse cellular responses. </p><p>Work presented in this thesis focuses on the effect of HS and its structures on FGF–FR complex formation and FGF-induced signaling. Studies with short, highly modified oligosaccharides and FGF1 and 2 combined with FR1c, 2c, 3c or 4 showed a correlation between the overall degree of modification and amount/stability of FGF–FR complexes. Our findings imply that several HS structures, differently modified but with the same negative charge density are equal in their ability to support complex formation. Co-application of oligosaccharides with FGF2 to HS-deficient cells and investigation of the thereby induced cell signaling confirmed our findings with a cell-free system. The oligosaccharide with the highest modification degree displayed the biggest impact on cell signaling, which was FGF2 concentration dependent. Studies with long HS polysaccharides with preserved high and low modified domains suggest that the proportion between these two types of domains and also the structure of the low modified domains are of importance for the FGF–HS–FR complex formation and cell activation capacity. </p><p>This work illuminates several aspects in how HS structure influences the interplay between FGFs and FRs and contributes to the understanding of what factors affect a cell’s response following FGF stimulation.</p>

Der spezifische Knockdown von RhoA in neonatalen kardialen Rattenfibroblasten führte auf molekularem Level zu einer Reduktion des Myofibroblastenmarkers α-Glattmuskelaktin und zu einem Anstieg im modifizierten acetylierten Tubulin. Auf subzellulärer Ebene konnte ein Verlust von Stressfasern, Aktinstrukturen höherer Ordnung und eine erhöhte Dichte des Golgi-Apparats beobachtet werden. Außerdem waren die Fokaladhäsionen kürzer und zufällig verteilt, was auf einen Verlust der Zellpolarität hinweist. Auf dem zellulären Level erhöhte der Knockdown von RhoA die Zellfläche aber nicht das Volumen. Diese Veränderungen führten zu einer schnelleren Adhäsion unabhängig vom Substrat, eine Reduktion der Migration in 2D und im Gegensatz dazu eine verbesserte Migration durch eine poröse Membran. Außerdem war die mitogene Antwort der Zellen auf einen Serumstimulus stark reduziert. Eine Veränderung in Zellviabilität konnte zudem nicht beobachtet werden. Die Expression und Sekretion des Fibrose-assoziierten Faktors CTGF war in gehungerten Zellen mit einer Reduktion in RhoA Expression signifikant vermindert, was jedoch in der Anwesenheit eines Serumstimulus aufgehoben werden konnte. Auf einer heterogenen multizellulären Ebene verringerte der Knockdown von RhoA die kontraktile Funktion von generierten künstlichen Herzgeweben unter Kalziumstimulation. Dies ging einher mit einer Reduktion der Expression von α-Glattmuskelaktin und Calsequestrin. Durch die Verwendung spezifischer Inhibitoren der Rho-assoziierten Kinase (ROCK) und HDAC6 konnten einige dieser zellulären Veränderungen imitiert und demensprechend einem Effektorprotein zugeordnet werden. Der ROCK Inhibitor Fasudil konnte die morphologischen Veränderungen und die reduzierte Migrationskapazität in Wildtyp-Fibroblasten abbilden, wobei eine Reduktion der Proliferation nach der Verwendung des HDAC6 Inhibitors Tubastatin A beobachtet wurde.

生長滯後是包括炎症性腸病在內的炎症疾病引起的併發症。實驗表明，炎症使肝臟對生長激素(GH)的作用變得不敏感或引起生長激素抵抗。生長激素抵抗會引起胰島素生長因子-1 (IGF-I)的表達下降，並且會啟動一系列的代謝反應。多年來的研究證明炎症因子白介素-6 (IL-6)，腫瘤壞死因子 -α (TNF-α)和白介素-1β(IL-1β)參與肝臟生長激素抵抗的病理過程。然而這些炎症因子調控生長激素通路的具體機理尚不清楚。通過用人肝癌細胞系Huh-7和慢性炎症及急性炎症兩種老鼠模型，我們發現: 1) TNF-α和IL-1β抑制生長激素受體(GHR)的表達; 2) IL-6誘導細胞因子信號轉導抑制因子-3 (SOCS3)的高表達; 3) IL-6-SOCS3途徑對GH-IGF-I信號通路的抑制作用依賴于GHR的表達量，當TNF-α及IL-1β升高而使GHR的表達量下降後，IL-6就不再對GH-IGF-I信號通路有抑制作用。以上結果表明IL-6， TNF-α和IL-1β抑制肝臟生長激素信號通路的機制是不一樣的，這些結果或許對臨床上治療青少年中炎症引起的生長激素抵抗疾病有一定的指導意義。 / 成纖維細胞生長因子(FGF) 通過結合和啟動成纖維細胞生長因子受體(FGFR)而參與許多生理過程。FGF19屬於FGF15/19亞家族，這個亞家族還包括FGF21和FGF23。FGF19調節肝臟中膽汁酸的穩態及蛋白和糖原的合成。FGF19通過與FGFR4及共受體β-klotho結合來啟動信號通路。研究表明，TNF-α通過抑制共受體β-klotho的表達來抑制脂肪細胞中的FGF21信號通路。然而IL-6，TNF-α和IL-1β在調節肝臟FGF19信號通路中的作用尚不清楚。我們的體外細胞和體內動物實驗結果表明，IL-1β通過JNK和NF-κB通路抑制肝臟中β-klotho的表達。IL-6與TNF-α不調節Huh-7細胞中β-klotho的表達。 / 綜上所述，IL-6，TNF-α及IL-1β在肝臟生長激素及FGF19通路中起不同的調節作用。 / Growth failure is a major complication of inflammatory diseases including inflammatory bowel disease. Evidence suggests that during inflammation, the liver becomes resistant to growth hormone (GH) actions, leading to downregulation of the anabolic gene IGF-I and the activation of catabolic processes. Decades of studies demonstrated that pro-inflammatory cytokines IL-6, TNF-α and IL-1β are involved in the pathogenesis of hepatic GH resistance. However, the exact mechanisms used by these individual cytokines to regulate GH signaling are not defined. Using Huh-7 human hepatoma cells and mouse models of chronic and acute inflammation, we show that TNF-α and IL-1β but not IL-6 inhibited hepatic GH receptor (GHR) expression, and that IL-6 but not TNF-α and IL-1β stimulated expression of suppressor of cytokine signaling-3 (SOCS3). TNF-α/IL-1β and IL-6 acted primarily at GHR and SOCS3 respectively to inhibit the GH-IGF-I pathway. While TNF-α/IL-1β exerted a tonic inhibition on hepatic GH signaling, IL-6 activity is dependent on the active GH pathway. IL-6 lost its inhibition on the GH-IGF-I pathway when GHR expression was blocked as the inflammation progressed. These results reveal previously undefined distinct mechanisms used by TNF-α/IL-1β and IL-6 to inhibit the hepatic GH pathway. Our results may provide a new guidance for clinical practice in treating pediatric infammation-induced GH resistance. / Fibroblast growth factors (FGFs) play critical roles in many physiological processes by binding to and activating FGF receptor (FGFR) family. FGF19 belongs to FGF15/19 subfamily of FGFs that includes FGF15/19, FGF21 and FGF23. FGF19 has been shown to regulate bile acid homeostasis, and protein and glycogen synthesis in the liver. FGF19 binds FGFR4 and the co-receptor β-klotho to initiate signaling. Studies have shown that proinflammatory cytokines such as TNF-α can impair FGF21 signaling in adipose cells by repressing the expression of β-klotho. However, little is known about the effects of IL-6, TNF-α and IL-1β on regulating hepatic FGF19 signaling. In the present study, we found that IL-1β inhibited β-klotho expression both in vitro and in vivo, and this inhibition required JNK and NF-κB pathways. IL-6 and TNF-α did not inhibit β-klotho expression in Huh-7 cells. / Taken together, our results demonstrate that IL-6, TNF-α and IL-1β play different roles in regulating the GH and FGF-19 pathways in the liver. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yueshui. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-182). / Abstracts also in Chinese.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains x, 148 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 136-148).