The structure/function relationships of the murine Leydig tumour (MA10) cell, luteinising hormone receptor

The LH/CG receptor is a member of the G-protein coupled receptor family. It is found expressed on testicular Leydig cells and ovarian granulosa-luteal and thecal cells. On binding its ligand LH, the activated LH/CG receptor initiates a cascade of intracellular signalling which results in the production of androgens and oestrogens. In this thesis the structural and functional relationships of the LH/CG receptor and in particular the process of LH/CG receptor desensitisation have been investigated. One of the aims of this work was to establish whether, when desensitised, the LH/CG receptor is phosphorylated. In order to achieve this it was first necessary to establish an immunoprecipitation protocol using antibodies which had previously been developed in this laboratory (Pallikaros et al. 1995). We used two different methods of immunoprecipitation and different LH/CG receptor antibodies, however, no immunoprecipitation of the LH/CG receptors from a solubilized membrane preparation occurred. Subsequently it was demonstrated that the LH/CG receptor antibodies did detect the LH/CG receptor in Leydig cells and ovarian preparation using immuncytochemical and confocal techniques and Western blotting. However, variations in the sensitivity of detection were observed depending on the method of fixation, nature of the tissue and the LH/CG receptor antibodies used. It was concluded, therefore, that the lack of immunoprecipitation may have been due to the inaccessibility of the antigenic sites of the LH/CG receptor to the antibodies (e.g. because of aggregation or degradation). Previous work from this laboratory had used an antisense strategy to produce C-terminally truncated LH/CG receptors in intact Leydig (MA10) tumour cells, some of which did not undergo ligand-stimulated desensitisation (West and Cooke 1991). We intended to use this antisense strategy to establish which regions of the LH/CG receptor underwent phosphorylation or were involved in coupling to Gs. However, because we could not reproduce the original results, we investigated the use of a cell free system. We successfully established that the rat LH/CG receptor could be synthesized in a coupled transcription/translation cell-free system. However, when the antisense oligonucleotides previously reported to cause truncated LH/CG receptors, were included in this cell-free system, they were found to have no effect on the size or level of LH/CG receptor synthesis. Preliminary studies indicated that antisense oligonucleotides targeted to the start site of translation may be more effective in preventing LH/CG receptor synthesis. In parallel with the antisense work, we amplified and cloned the LH/CG receptor from the MA10 cells so that we could generate truncated cDNAs by the use of restriction enzymes. These truncated cDNAs could then be expressed in a mammalian cell line and used to establish whether truncated LH/CG receptors could undergo ligand-induced desensitisation. The amplification and cloning of the whole LH/CG receptor proved difficult and so the amplification reaction was divided into two. The amplification and cloning of the C-terminal region of the MA10 LH/CG receptor was successfully performed. Sequence analysis confirmed that it was identical to that of the murine LH/CG receptor. However despite extensive optimisation procedures, it was not possible to amplify the first 1.1 kb of the receptor. Analysis of the predicted secondary structure formed by the mRNA indicated a considerable level of hairpin bend formation suggesting that this may have been responsible for preventing adequate primer hybridisation to the template. The putative kinases responsible for phosphorylation of the activated LH/CG receptor have not, as yet, been identified. In order to investigate the possible role of PKC, the effects of three PKC inhibitors (staurosporine, GF109203X and RO 31-8220) on LH-, cholera toxin- and forskolin-stimulated cAMP production in MA10 cells were investigated. It was found that staurosporine markedly increased, and GF109203X and RO 31-8220 decreased, the cAMP levels stimulated by these compounds. The effects of staurosporine were not via effects on LH binding, phosphodiesterase or cell viability. It is concluded that because the GF and RO compounds are more specific PKC inhibitors than staurosporine, that PKC-mediated phosphorylation may not be involved in LH-induced desensitisation. It is suggested that staurosporine may act via inhibition of CaM-kinase II

Measuring Faculty Teaching Effectiveness Using Conditional Fixed Effects

Using a dataset of 48 faculty members and 88 courses over 26 semesters, the authors estimate Student Evaluation of Teaching (SET) ratings that are conditional on a multitude of course, faculty, and student attributes. They find that ratings are lower for required courses and those where students report a lower prior level of interest. Controlling for these variables substantially alters the SET ratings for many instructors. The average absolute value of the difference between the faculty ratings controlling just for time effects and fully conditional ratings is nearly one-half of a standard deviation in the students’ rating of how much they learned. This difference produces a change in quartile rank for over half the sample across two summary course evaluation measures

The Skaergaard trough layering: sedimentation in a convecting magma chamber.

The upper parts of the floor cumulates of the Skaergaard Intrusion, East Greenland, contain abundant features known as troughs. The troughs are gently plunging synformal structures comprising stacks of crescentic modally graded layers with a sharply defined mafic base that grades upward into plagioclase-rich material. The origin of the troughs and layering is contentious, attributed variously to deposition of mineral grains by magmatic currents descending from the nearby walls, or to in situ development by localised recrystallisation during gravitationally-driven compaction. They are characterised by outcrop-scale features such as mineral lineations parallel to the trough axis, evidence of erosion and layer truncation associated with migration of the trough axis, and disruption of layering by syn-magmatic slumping. A detailed microstructural study of the modal trough layers, using electron backscatter diffraction together with geochemical mapping, demonstrates that these rocks do not record evidence for deformation by either dislocation creep or dissolution-reprecipitation. Instead, the troughs are characterised by the alignment of euhedral plagioclase crystals with unmodified primary igneous compositional zoning. We argue that the lineations and foliations are, therefore, a consequence of grain alignment during magmatic flow. Post-accumulation amplification of the modal layering occurred as a result of differential migration of an unmixed immiscible interstitial liquid, with upwards migration of the Si-rich conjugate into the plagioclase-rich upper part of the layers, whereas the Fe-rich immiscible conjugate remained in the mafic base. Both field and microstructure evidence support the origin of the troughs as the sites of repeated deposition from crystal-rich currents descending from the nearby chamber walls

The contribution of cool and hot executive function to academic achievement, learning-related behaviours, and classroom behaviour

The primary aim of this study was to examine whether individual differences in cool and hot executive functions (EF) were associated with children’s transition to school, in terms of both academic performance and classroom behaviour. Children between 5- and 7-years-of-age (N = 90) completed performance based assessments of cool and hot EF as well as verbal ability. Teachers reported on children’s reading and numeracy performance, learning-related behaviours, hyperactivity, and aggression. Results revealed that EF, in particular working memory, was associated with reading and numeracy performance and that this relation was mediated by learning-related behaviours. EF was not associated with hyperactive or aggressive behaviour. The findings strengthen the evidence base for the importance of EF in early academic performance and underscore its potential to be a beneficial part of early education curriculum and a target for early intervention for successful transition to school

Evaluating the impact of a simulation study in emergency stroke care

Very few discrete-event simulation studies follow up on recommendations with evaluation of whether modelled benefits have been realised and the extent to which modelling contributed to any change. This paper evaluates changes made to the emergency stroke care pathway at a UK hospital informed by a simulation modelling study. The aims of the study were to increase the proportion of people with strokes that undergo a time-sensitive treatment to breakdown a blood clot within the brain and decrease the time to treatment. Evaluation involved analysis of stroke treatment pre- and post- implementation, as well as a comparison of how the research team believed the intervention would aid implementation compared to what actually happened. Two years after the care pathway was changed, treatment rates had increased in line with expectations and the hospital was treating four times as many patients than before the intervention in half the time. There is evidence that the modelling process aided implementation, but not always in line with expectations of the research team. Despite user involvement throughout the study it proved difficult to involve a representative group of clinical stakeholders in conceptual modelling and this affected model credibility. The research team also found batch experimentation more useful than visual interactive simulation to structure debate and decision making. In particular, simple charts of results focused debates on the clinical effectiveness of drugs - an emergent barrier to change. Visual interactive simulation proved more useful for engaging different hospitals and initiating new projects

Can clinical audits be enhanced by pathway simulation and machine learning? An example from the acute stroke pathway

OBJECTIVE: To evaluate the application of clinical pathway simulation in machine learning, using clinical audit data, in order to identify key drivers for improving use and speed of thrombolysis at individual hospitals

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f+ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further

Young Asian males: social exclusion and social injustice in British professional football?

The disproportionately low number of Asian Heritage professional soccer players in Britain is a matter that has been increasingly noted within the game. Indeed, comparisons are now being made between players of Afro-Caribbean Heritage 'breaking in' 35 years ago and the current lack of Asian Heritage players. Through use of questionnaires, this research focuses on the perceptions of Youth/Community Development officers at UK professional football clubs and Asian Heritage males who are involved in playing the game. Comparisons are drawn in particular with the Bains and Patel report (1996) which remains probably the most significant discussion of the issue from the last decade. Findings suggest that there are some encouraging signs of progress in some clubs and that the two groups share views on certain barriers to Asian Heritage players entering professional soccer. However, they remain very polarized in important respects, not least concerning the prevalence and impact of racism

Peroxide-based crosslinking of solid silicone rubber, part II: The counter-intuitive influence of dicumylperoxide concentration on crosslink effectiveness and related network structure

Application of elastomers in general demands the conversion of their soluble networks into crosslinked structures. This abrupt change causes several modifications, both in the atomic/molecular level and at the macro-scale. In this study, solid silicone rubber (high molecular weight poly(dimethylsiloxane)), was crosslinked with dicumylperoxide (DCP), a widely used crosslinking agent by the rubber industry. The changes caused by different DCP concentrations were investigated, aiming to bring attention to the molecular transformations, usually neglected when processing-oriented studies are conducted. DCP concentration showed a limited contribution to the network's molecular dynamics, which was found to be mainly dominated by entanglements. The dominance of entanglements over other molecular constraints, like crosslink points, justifies the threshold and counter-intuitive behavior of tensile and hardness properties. However, differences were found in the crystallization ability after crosslinking, when the more crosslink points were introduced, the lower the crystallinity was and the less stable the PDMS crystallites were. In addition to providing a deeper understanding of an industrially applied rubber system n terms of the effective concentration of DCP, and the reasoning behind such concentration, the findings of this study add to the state-of-the-art comprehension of elastomeric networks, and how they behave on a molecular level