Ph.D. Theses
Permanent URI for this collection
Browse
Browsing Ph.D. Theses by Author "Bilge, Kuyaş Buğra."
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Negative regulation of Müller reprogramming by SIK2(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Uğurlu, Aslı.; Bilge, Kuyaş Buğra.In fish it is well known that Müller cells proliferate, gain stem cell characteristics and generate new neurons upon injury. Proliferation is as essential step in retina regeneration and in mammals its tight regulation in Müller cells as well as age-dependent restriction of cell cycle re-entry has been proposed to limit retinal regeneration. Müller glia can be stimulated to proliferate by exogenous growth factors such as FGF2 in mammals, however the efficiency of neurogenesis remains highly limited. In this study MIO-M1 cells, which can be stimulated with FGF2 treatment to transdifferentiate into neurons, were used as the model system to better characterize FGF2 dependent MIO-M1 transdifferentiation process and to provide clues for the potential contribution of SIK2, as a negative regulator of FGF2 dependent Müller cell proliferation, to Müller cell reprogramming. Formation of nestin-positive neurospheres with self-renewal capacity confirmed transdifferentiation potential of MIO-M1 cells. Upregulation in Pax6 and Chx10 expression showed that MIO-M1 cells gain progenitor properties. Downregulation in vimentin expression with higher calretinin and Prox1 expression verified that MIO-M1 cells lose their glia characteristics and differentiate into neurons, particularly horizontal cells. Notch signaling, as in other Müller reprogramming events, might have dual roles; it promotes progenitor characteristics and amplification of progenitors in the early phase and involved in horizontal cell fate decision in the late phase. Our results indicate that ERK activity is required for increase in Müller population that will dedifferentiate into progenitors, but an unknown signaling pathway might regulate amplification of progenitor pool. Significant decrease in SIK2 protein level in early phase of the process is required for Müller glia to re-enter cell cycle. Overexpression of SIK2 blocked ERK activation, cell cycle entry and further transdifferentiation process. In the light of these findings, we suggest a novel role for SIK2 in Müller reprogramming through downregulation of ERK activity.Item SIK2: a key player in FGF2-induced proliferation and insulin-induced survivale/hyperglycemia-dependent apoptosis in müller cells(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2011., 2011.) Küser, Gamze.; Bilge, Kuyaş Buğra.Salt inducible kinase 2 (SIK2), a serine/threonine kinase, is primarily expressed in insulin-responsive tissues. It was suggested that SIK2 might be involved in type 2 diabetes by phosphorylating S789 residue of IRS1. In the initial experiments we observed rapid and transient increase of SIK2 activity upon FGF2 stimulation, confirming that the kinase is part of this pathway. Subsequently it was demonstrated that FGF2 dependent ERK and Akt activation was reduced significantly by SIK2 overexpression. SIK2 silencing, on the other hand, enhanced the intensity and the duration of active ERK and Akt levels, and led to a dramatic increase in FGF2-dependent Müller cell proliferation. Gab1 phosphorylation by SIK2 was verified in vitro, their interaction was revealed by coimmunoprecipitation. pSer of Gab1 was diminished significantly by SIK2 silencing. In the same time frame, pTyr levels and binding partner associations (p85/Gab1, Shp2/Gab1, Grb2/Gab1) were increased. Based on this data we propose that SIK2 involved in the negative feedback mechanisms acting on FGF/Ras/ERK and FGF/PI3K/Akt pathways and Müller cell proliferation at the level of Gab1 serine phosphorylation. In the context of insulin pathway we established enhanced tyrosine phosphorylation of IRS1, followed by Akt activation resulting in increased Müller cell survival. SIK2 knockdown leads to considerably earlier FGF2-dependent Akt activation. Its overexpression, hampers Akt activation and cell survival. Coimmunoprecipitation studies indicated IRS1 as an in vivo SIK2 substrate. We observed lower IRS1 expression and tyrosine phosphorylation, enhanced SIK2 activity/expression and downregulation of both basal and insulin-induced Akt activation in MIO-M1 cells under hyperglycemia. SIK2 gene silencing under hyperglycemia restored pAkt level, under normoglycemia in cells overexpressing SIK2, Akt phosphorylation was decreased and apoptosis increased. Based on these observations, we suggest that SIK2 is a negative modulator of insulin-induced IRS1/Akt mediated Müller glial survival pathway, which may contribute to the glial death observed in diabetes.