Investigation of the role of long non-coding RNAs in oncogene induced cellular senescence

Cellular senescence is a stable arrest of cell proliferation induced by several factors such as activated oncogenes, oxidative stress and shortening of telomeres. Senescence acts as a tumour suppression mechanism to halt the progression of cancer. However, senescence may also impact negatively upon tissue regeneration, thus contributing to the effects of ageing. The eukaryotic genome is controlled by various modes of transcriptional and translational regulation. Focus has therefore centred on the role of long non- coding RNAs (lncRNAs) in regulating the genome. Accordingly, understanding how lncRNAs function to regulate the senescent genome is integral to improving our knowledge and understanding of tumour suppression and ageing. Within this study, I set out to investigate the expression of lncRNAs’ expression within models of senescence. Through a custom expression array, I have shown that expression of multiple different lncRNAs is up-regulated and down regulated in IMR90 replicative senescent fibroblasts and oncogene-induced senescent melanocytes. LncRNA expression was determined to be specific to stable senescence-associated cell arrest and predominantly within the nucleus of senescent cells. In order to examine the function of lncRNA expression in senescence, I selected lncRNA transcript ENST0000430998 (lncRNA_98) to focus my investigations upon. LncRNA_98 was robustly upregulated within multiple models of senescence and efficiently depleted using anti-sense oligonucleotide technology. Characterisation and unbiased RNA-sequencing of lncRNA_98 deficient senescent cells highlighted a list of genes that are regulated by lncRNA_98 expression in senescent cells and may regulate aspects of the senescence program. Specifically, the formation of SAHF was impeded upon depletion of lncRNA_98 expression and levels of total pRB protein expression severely decreased. Validation and recapitulation of consequences of pRB depletion was confirmed through lncRNA_98 knock-out cells generated using CRISPR technology. Surprisingly, inhibition of ATM kinase functions permitted the restoration of pRB protein levels within lncRNA_98 deficient cells. I propose that lncRNA_98 antagonizes the ability of ATM kinase to downregulate pRB expression at a post-transcriptional level, thereby potentiating senescence. Furthermore, lncRNA expression was detected within fibroblasts of old individuals and visualised within senescent melanocytes in human benign nevi, a barrier to melanoma progression. Conversely, mining of 337 TCGA primary melanoma data sets highlighted that the lncRNA_98 gene and its expression was lost from a significant proportion of melanoma samples, consistent with lncRNA_98 having a tumour suppressor functions. The data presented in this study illustrates that lncRNA_98 expression has a regulatory role over pRB expression in senescence and may regulate aspects of tumourigenesis and ageing.

Learning to construct our identities over the life course: a study with lesbian, gay, bisexual and transgender adults in Scotland

To date, adult educational research has had a limited focus on lesbian, gay, bisexual and transgendered (LGBT) adults and the learning processes in which they engage across the life course. Adopting a biographical and life history methodology, this study aimed to critically explore the potentially distinctive nature and impact of how, when and where LGBT adults learn to construct their identities over their lives. In-depth, semi-structured interviews, dialogue and discussion with LGBT individuals and groups provided rich narratives that reflect shifting, diverse and multiple ways of identifying and living as LGBT. Participants engage in learning in unique ways that play a significant role in the construction and expression of such identities, that in turn influence how, when and where learning happens. Framed largely by complex heteronormative forces, learning can have a negative, distortive impact that deeply troubles any balanced, positive sense of being LGBT, leading to self- censoring, alienation and in some cases, hopelessness. However, learning is also more positively experiential, critically reflective, inventive and queer in nature. This can transform how participants understand their sexual identities and the lifewide spaces in which they learn, engendering agency and resilience. Intersectional perspectives reveal learning that participants struggle with, but can reconcile the disjuncture between evolving LGBT and other myriad identities as parents, Christians, teachers, nurses, academics, activists and retirees. The study’s main contributions lie in three areas. A focus on LGBT experience can contribute to the creation of new opportunities to develop intergenerational learning processes. The study also extends the possibilities for greater criticality in older adult education theory, research and practice, based on the continued, rich learning in which participants engage post-work and in later life. Combined with this, there is scope to further explore the nature of ‘life-deep learning’ for other societal groups, brought by combined religious, moral, ideological and social learning that guides action, beliefs, values, and expression of identity. The LGBT adults in this study demonstrate engagement in distinct forms of life-deep learning to navigate social and moral opprobrium. From this they gain hope, self-respect, empathy with others, and deeper self-knowledge.