You play your part – participatory media and older women.

This chapter will examine participatory and collaborative production strategies that create opportunities for older women to participate in media production. It draws on practice-led research in participatory and community-based media in the Govan area of Glasgow, Scotland. In particular, it examines the production process of a participatory documentary I produced and directed with senior citizens who are members of the Govan Seniors Film Club, based at the Portal Arts Centre in Govan.You Play Your Part is a 20-minute documentary about campaigning women in and around Govan.

I film therefore I am: process and participation, networks and knowledge – examples from Scottish community media projects.

This chapter examines community media projects in Scotland as social processes that nurture knowledge through participation in production. A visual and media anthropology framework (Ginsburg, 2005) with an emphasis on the social context of media production informs the analysis of community media. Drawing on community media projects in the Govan area of Glasgow and the Isle of Bute, the techniques of production foreground “the relational aspects of filmmaking” (Grimshaw and Ravetz, 2005: 7) and act as a catalyst for knowledge and networks of relations embedded in time and place. Community media is defined here as a creative social process, characterised by an approach to production that is multi-authored, collaborative and informed by the lives of participants, and which recognises the relevance of networks of relations to that practice (Caines, 2007: 2). As a networked process, community media production is recognised as existing in collaboration between a director or producer, such as myself, and organisations, institutions and participants, who are connected through a range of identities, practices and place. These relations born of the production process reflect a complex area of practice and participation that brings together “parallel and overlapping public spheres” (Meadows et al., 2002: 3). This relates to broader concerns with networks (Carpentier, Servaes and Lie, 2003; Rodríguez, 2001), both revealed during the process of production and enhanced by it, and how they can be described with reference to the knowledge practice of community media.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Compliance in food consumption of young and long-lived elderly of a city in southern Brazil

Introduction: Life expectancy is increasing and becoming a characteristic phenomenon of developed countries and, increasingly, of developing countries, such as Brazil. The aging process causes changes of some physiological functions such as loss of smell, taste, loss of appetite, among other things that end up changing the food intake of these individuals. Objectives: This study aimed to assess food consumption of the young and long-lived elderly in a city in southern Brazil. Methods: A cross-sectional survey conducted through home visits in Palmeira das Missões - RS, Brazil. The sociodemographic, anthropometrical and dietary data were collected through questionnaires and 24-hour recall. The adequacy of nutrients was assessed according to the Dietary Reference Intakes. Data were analyzed using SPSS 18.0 software. Results: The study included 424 older adults, 84,4% (n = 358) aged less than 80 years old and 15,6% (n = 66) older than 80. The intake of energy and protein was insufficient for both young elderly and the oldest. The consumption of vitamins and minerals has been insufficient in all seniors except for iron, which presented an excessive intake. There was a statistically significant difference between the elderly and oldest only for the consumption of lipids and vitamin B12. Conclusion: The majority of studies with elderly corroborate the results found in this article. An inadequate intake of nutrients can develop nutritional deficiencies, and consequently it can result in physiological and pathological changes which would compromise the functional capacity of the elderly. Energy consumption was insufficient and macronutrients were inadequate, both for the young elderly as for the oldest. Additionally, the consumption of vitamins and minerals was insufficient to everyone except the iron, which presented excessive intake for young and oldest elderly.