Doctoral Degrees (Physiological Sciences)

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Browsing Doctoral Degrees (Physiological Sciences) by Author "Gudagudi, Kirankumar"

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    Fibroblast growth factors, A potential game plan for regeneration of skeletal muscle
    (Stellenbosch : Stellenbosch University, 2019-12) Gudagudi, Kirankumar; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Adult mammalian tissue regeneration recruits progenitor stem cells. In skeletal muscle, these are primary satellite cells. Primary satellite cells can be harvested from muscle tissue to investigate or even use as potential therapeutic application. Satellite cells exist in quiescence in the muscle tissue and only become activated following an insult. Most studies investigating satellite cells in vitro use already activated satellite cells, called myoblasts. Fibroblast Growth Factors (FGFs) are fundamental in embryonic development but also in adult skeletal muscle regeneration from injury or pathology. Understanding the role of specific members of this growth factor family could assist in improving the understanding of their influence on the regeneration sequence in skeletal muscle. Methods: Isolated satellite cells from human muscle biopsies were expanded in vitro creating primary human myoblast (PHM) clones. In order to distinguish the rate of proliferation between different PHM clones, a comparative index (CI) was established using the cell cycle and total RNA data of the two PHM clones. Two distinct index calculation models were also presented to determine if these may distinguish between the two clones with greater sensitivity. Secondly, the quiescent state is an integral part of stem cell regulation, therefore choosing the right protocol for inducing quiescence is important. In this study, two developed protocols were assessed, and a new blended protocol addressing the limitations of both protocols was established. This method involved the use of suspension culture (SuCu) with knock out serum replacement (KOSR). Finally, FGF6 and FGF2, both individually and sequentially, were used to treat quiescent myoblasts to determine their involvement in activation and proliferation with the use of cell cycle analysis and mRNA assessment of ki67, p21, myf5, and MyoD. Results and conclusion: The development of the CI was successful in determining the difference in proliferation rate for the different clones. Suspension culture with KOSR, the blended protocol method, resulted in reduced ki67 expression and improved quiescence compared to both the SuCu or KOSR alone. Unlike FGF2, individual treatment with FGF6 was adequate to activate the quiescent PHMs and aid their re-entry into cell cycle with consistency in all three PHM clones by upregulating ki67 expression. However, FGF2 did impede the cell cycle inhibition factor p21, indirectly influencing proliferation. Sequential treatment of FGF6 and FGF2 allowed to determine whether the sequence of treatment would be important. The potential for significantly improving proliferation was found for the sequence: FGF6 followed by FGF2. The inverse sequential treatment order did not demonstrate any significant effect on both activation and proliferation of the quiescent cells. In conclusion, using clones that were distinctly different as assessed by the comparative index, this thesis illuminates that the two FGF family members investigated, act on cell cycle in different ways, thus would influence their utilization in experimental or therapeutic applications.