Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

Remembrance of the ancestors in contemporary paganism: Lineage, identity, and cultural belonging in the Irish context

This article explores some of the ways of remembering and honouring the ancestors in contemporary Pagan religious traditions, with a focus on the Irish context. An overview is provided of how the "ancestors" are conceptualised within Paganism, as well as where they are believed to be located in the afterlife or Otherworld. Veneration of ancestral peoples is a significant part of many Pagan rituals. Some methods of honouring the dead, and contacting the dead, through ritual practices are described. Remembering and honouring the dead, whether distant forebears or more recent relatives, is particularly important during the Pagan celebration of the festival of Samhain, feast of the dead, on October 31. Issues around ancestors, lineages and ethnicity are significant in many Pagan traditions, and attention is paid to these factors in terms of the Irish Pagan community's sense of cultural belonging as well as their sense of place in a physical respect in relation to the landscape, proximity of sacred sites, and other features of their geographical location.