DEVELOPING FLUOROGEN ACTIVATING PROTEIN-FLUORESCENT PROTEIN FRET PAIRS FOR LIVE CELL IMAGING

Fluorogen activating proteins (FAPs) are genetically encoded tags made from single chain antibody fragments (scFv) designed to bind fluorogens with high specificity. Both the fluorogen and FAP can be modified to provide flexibility in properties such as affinity, membrane permeability, spectra, and quantum yield. The fluorogen Malachite Green (MG) has two excitation peaks, the maximum at 630 nm and a secondary peak at 450 nm. The emission spectra of blue-emitting fluorescence proteins, such as mCerulean (mCer), overlap with the MG secondary peak, generating a FRET pair with large Stokes shift emission. Using 405 nm excitation of mCer, we observe acceptor sensitized emission at wavelengths greater than 650 nm with no spectral crosstalk between the donor and acceptor channels. Additionally, donor only controls can be acquired for all cells as the acceptor is not present until after the addition of the fluorogen, providing intra-cellular control. The FAP-FRET system has been characterized using proof of principle constructs: FAP-mCer-transmembrane (TM) as a positive FRET control and FAP-TM-mCer as a negative FRET control and expressed in HeLa cells. Multiple MG derivatives were compared and imaging parameters were optimized to determine the optimal FRET pair. Analysis was performed using code written in Matlab to mask the cell membrane and quantify FRET efficiencies, based on donor intensity before and after addition of fluorogen. Data from several fluorogen showed high energy transfer efficiency (~30%) with the FAP-mCer-TM construct compared to negligible FRET (~4%) for FAP-TM-mCer. Additional techniques were performed to support the FRET efficiency data, including spectral imaging and FLIM, which also reported FRET efficiency around 30% with the positive constructs and negligible FRET with the negative constructs. The FAP-FRET system is currently being used to study the kinetics of signaling proteins within the FcεRI pathway

From 'Mother Earth' to 'Father Holding the Baby': A Gendered Analysis of Organic Agriculture in Gloucestershire, England

This thesis examines what it means to be a gendered subject in the context of organic agriculture. Specifically, it considers the extent to which the ideological standpoint of the organic agriculture movement facilitates the representation and construction of distinctive gender roles, relations and identities within organic fanning. Three particular features of organic agriculture have been identified that could potentially impact upon gender roles, relations and identities: firstly, the organic movement has non-agrarian roots and has therefore attracted individuals who are not part of the traditional gendered heritage of agriculture; secondly, the basis of the organic ideology suggests a fundamentally different approach to society-nature relations to that which dominates the agro-industrial model; and thirdly, the ideology of the organic agriculture movement makes explicit reference to the social relations inherent to agriculture and the role that organic fanning should take in working towards a production process that is 'socially just'. These three reasons suggest that the construction and representation of gender roles, relations and identities within organic farming may be distinct from agriculture more widely, in which highly 'traditional' hierarchical constructions of masculinity and femininity have been shown to persist. Informed by perspectives within feminist geography, two phases of empirical research were undertaken in order to address the research aim. The first phase involved a content analysis of three publications drawn from the UK organic agriculture movement (and one from conventional agriculture) and explored how gender roles, relations and identities have been represented throughout its history. In the second phase the themes that emerged from the textual analysis were explored in more detail through a series of in-depth, semistructured interviews with forty-one men and women working on organic farms in the county of Gloucestershire, UK, in order to critically assess the ongoing construction and maintenance of gender roles, relations and identities within contemporary organic fanning. The findings of the research show that organic agriculture is largely dominated by 'traditional' representations and constructions of gender roles, relations and identities. However, they also show that organic agriculture does provide a space for alternative configurations of gender roles, relations and identities. Nevertheless, these pose a challenge to feminist understandings of what constitutes 'progressive' gender roles, relations and identities since, paradoxically, they draw upon highly traditional notions which associate women and nature whilst at the same time enabling women and men to assume gender roles and relations that transcend conventional boundaries

Psychological Ownership: Effects and Applications

Psychological ownership (PO) can be an important tool for organizations to encourage productivity and certain desirable employee behaviours. In the organizational literature, PO is the sense of ownership over a “target”: not only physical objects, but a concept, a job, or the whole organization. It can express itself in positive ways, such as higher motivation; but it also has potentially negative effects, such as territoriality. This briefing looks closely at the literature to understand the various dimensions of PO, and how organizations can foster the positive aspects of it to help improve employees’ experiences in the workplace by fostering morale while contributing to productivity. By bringing together the most relevant research on the topic, this briefing also highlights areas that are still underdeveloped, such as collective PO, the role of culture, and the employee perspective

The phenotypic plasticity of whole animal and muscle performance during fast-starts in Cottidae

Chapter 1. Fast-starts are used by most fish species in order to capture prey and escape predators. An introduction to this mode of fish locomotion and the structure and function of the muscle powering swimming movements, is given. Temperature has the potential to alter fast-start behaviour at various levels of organisation ranging from the whole animal to the molecular and can act over time scales extending from the immediate to the evolutionary. The thermal dependence of fast-start performance is discussed. Chapter 2. The effects of acclimation and acute temperature on the kinematics of the escape response in two species of maidne Cottidae, the short-horn sculpin (Myoxocephalus scorpius L.) and the long-spined sea scorpion (Taurulus bubalis Euphr.) were examined. Hypotheses were formulated based on relevant studies and the natural history of the fish to test the idea that seasonal temperature acclimation conferred a fitness advantage and to examine whether acclimation responses were constant through development. Chapter 3. The effect of seasonal thermal acclimation on the in vivo strain and power output of the fast muscle fibres during escape responses in the short- horn sculpin was examined. Chapter 4. The kinematics, in vivo muscle action, power output and energetics of escape and prey capture responses in the short-horn sculpin are discussed. Fast-starts were filmed using high speed video synchronised with sonomicrometry and EMG. The in vivo muscle strain and activation recordings were abstracted for use in work loop experiments. Changes in the metabolic substrates following work loops from the two different types of fast-starts were analysed using high performance liquid chromatography (HPLC). Chapter 5 The velocity of the wave of curvature passing down the fish and the power requirements during fast-start escape responses were calculated non-invasively. This was carried out on both cottid species acclimated to 5 and 15 ° and filmed using high speed cinematography at 0.8, 5.0, 15.0 and 20.0 °. The power requirements for the contralateral contraction were 20 W. kg-1 muscle in 5 °-acclimated fish escaping at 5 ° and 58 W. kg-1 muscle in 15 °-acclimated fish swimming at 15 °. Comparative values of power output measured from work loop experiments in Chapter 3 were 33 and 66 W.kg-1 respectively

From 'Mother Earth' to 'Father Holding the Baby': A Gendered Analysis of Organic Agriculture in Gloucestershire, England

This thesis examines what it means to be a gendered subject in the context of organic\ud agriculture. Specifically, it considers the extent to which the ideological standpoint of the\ud organic agriculture movement facilitates the representation and construction of distinctive\ud gender roles, relations and identities within organic fanning. Three particular features of\ud organic agriculture have been identified that could potentially impact upon gender roles,\ud relations and identities: firstly, the organic movement has non-agrarian roots and has\ud therefore attracted individuals who are not part of the traditional gendered heritage of\ud agriculture; secondly, the basis of the organic ideology suggests a fundamentally different\ud approach to society-nature relations to that which dominates the agro-industrial model;\ud and thirdly, the ideology of the organic agriculture movement makes explicit reference to\ud the social relations inherent to agriculture and the role that organic fanning should take in\ud working towards a production process that is 'socially just'. These three reasons suggest\ud that the construction and representation of gender roles, relations and identities within\ud organic farming may be distinct from agriculture more widely, in which highly\ud 'traditional' hierarchical constructions of masculinity and femininity have been shown to\ud persist.\ud Informed by perspectives within feminist geography, two phases of empirical research\ud were undertaken in order to address the research aim. The first phase involved a content\ud analysis of three publications drawn from the UK organic agriculture movement (and one\ud from conventional agriculture) and explored how gender roles, relations and identities\ud have been represented throughout its history. In the second phase the themes that emerged\ud from the textual analysis were explored in more detail through a series of in-depth, semistructured\ud interviews with forty-one men and women working on organic farms in the\ud county of Gloucestershire, UK, in order to critically assess the ongoing construction and\ud maintenance of gender roles, relations and identities within contemporary organic\ud fanning. The findings of the research show that organic agriculture is largely dominated by\ud 'traditional' representations and constructions of gender roles, relations and identities.\ud However, they also show that organic agriculture does provide a space for alternative\ud configurations of gender roles, relations and identities. Nevertheless, these pose a\ud challenge to feminist understandings of what constitutes 'progressive' gender roles,\ud relations and identities since, paradoxically, they draw upon highly traditional notions\ud which associate women and nature whilst at the same time enabling women and men to\ud assume gender roles and relations that transcend conventional boundaries

The inflammatory potential of the diet in childhood is associated with cardiometabolic risk in adolescence/young adulthood in the ALSPAC birth cohort

PURPOSE: This study examined the association between a Dietary Inflammatory Score adapted for children (cDIS) and Cardiometabolic Risk (CMR) score in adolescence/early adulthood in the Avon Longitudinal Study of Parents and Children (ALSPAC). METHODS: The cDIS was calculated at 7, 10 and 13 years using diet diary data. Anthropometric and biochemical data at 17 (N = 1937) and 24 (N = 1957) years were used to calculate CMR scores at each age [mean sex-specific z-scores from triacylglycerol, HDL-cholesterol, LDL-cholesterol, mean arterial blood pressure (MAP), homeostatic model assessment of insulin resistance (HOMA-IR) and fat-mass index (FMI)]. Multivariable linear regression models examined associations between cDIS at 7, 10 and 13 years and a continuous CMR z-score and individual CMR markers at 17 and 24 years. RESULTS: In fully adjusted models, a higher cDIS (more pro-inflammatory diet) at 7 years was associated with an increase in CMR z-score at 17 years (β 0.19; 95% CI 0.03–0.35 for third versus first cDIS tertile) and at 24 years (β 0.28; 95% CI 0.11,0.44 for third versus first cDIS tertile). There was a weak association between a higher cDIS at 10 years and an increase in CMR z-score at 17 years (β 0.16; 95% CI − 0.003, 0.32 for third versus first cDIS tertile). No other clear associations were evident. FMI, MAP and HOMA-IR were the main CMR factors contributing to these associations. CONCLUSION: A more pro-inflammatory diet during childhood was associated with a worse cardiometabolic profile in late adolescence/early adulthood. A childhood diet abundant in nutrients with anti-inflammatory properties could help reduce development of CMR factors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00394-022-02860-9