Implications of miRNA expression pattern in bovine oocytes and follicular fluids for developmental competence

Developmental competence determines the oocyte capacity to support initial embryo growth, but the molecular mechanisms underlying this phenomenon are still ill-defined. Changes in microRNA (miRNA) expression pattern have been described during follicular growth in several species. Therefore, aim of this study was to investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) is associated with the acquisition of developmental competence. Samples were collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H- and L-ovaries) because previous studies demonstrated that this parameter is a reliable predictor of oocyte competence. After miRNA deep sequencing and bioinformatic data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were differentially expressed between H- and L-ovaries. Overall, our results indicate that miRNA levels both in FF and in the ooplasm must remain within specific thresholds and that changes in either direction compromising oocyte competence. Some of the miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-451) where already known to regulate follicle growth and their expression pattern indicate that they are also involved in the acquisition of developmental competence. Some miRNAs were differentially expressed in both compartments but with opposite patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene Ontology analysis showed that the predicted gene targets of most differentially expressed miRNAs are part of a few signalling pathways. Regulation of maternal mRNA storage and mitochondrial activity seem to be the processes more functionally relevant in determining oocyte quality. In conclusion, our data identified a few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte developmental competence. This provides new insights that could help with the management of cattle reproductive efficiency

Expression of tumour necrosis factors during chick lens development

During development of the lens, epithelial cells at the lens equator begin a differentiation process to become secondary fibre cells. The differentiating cells elongate and migrate towards the centre of the lens where they envelop the older, central fibre cells. Differentiation into fibre cells is accompanied by the breakdown of all organelles, such as the mitochondria. All organelle degradation is completed and denucleation occurs at the border of the organelle free zone (OFZ) which contains the central, terminally differentiated, fibre cells. The differentiation pathway is not well characterised, though it is believed to have similarities to an attenuated form of apoptosis supported by the identification of apoptosis related genes, such as TNF, in the lens. This study continues the search for and characterisation of apoptosis related genes expressed during lens development, focusing on TNFs and their extended family. Reverse Transcriptase-(RT-) PCR was carried out, identifying a number of TNF and extended family member genes in the chick lens, expression studies established novel, statistically significant differential expression for TRAF2 and TRAF3. TRAF2 protein expression from western blotting, similar to RT-PCR expression was found to decline as the lens developed. TRAF2 localisation studies showed limited expression in the equatorial region but there was extensive signalling found in the developing iris, a region in the corneal-scleral boundary and some staining was also detected in the ciliary body. TRAF3 protein and RT-PCR expression were similar, with increasing expression as the lens developed. Western blotting identified two bands and subcellular fractionation confirmed different localisation for the two isoforms. Immunofluorescence identified increasing TRAF3 staining in the cortical fibre cells, this staining was found to be similar to proteins that were reported to be involved in lens fibre cell remodelling and maintenance, suggesting a possibly similar role for TRAF3. Following interest in TRAIL as a gene therapy for Posterior Capsule Opacification (PCO) its expression was examined using RT-PCR and Western blotting which showed low, similar levels of expression throughout the stages of lens development studied. Peroxidase staining showed interesting staining in the equatorial epithelial cells and those just beginning to differentiate at the transition zone. Novel nuclear staining was identified at all time points in both epithelial and fibre cells containing nuclei. Characterisation of whole lens culture was undertaken to discover the optimum culture system for the whole chick lens. Of the published research using whole chick lens culture none stated the basic morphology of the developing lens in organ culture, though each lab had their preferred methodology. The characterisation resulted in the preference of E10 chick lenses being grown with vitreous attached in medium containing glucose. Understanding the morphology of lenses in culture will be invaluable when undertaking the functional studies required to clarify the roles in the lens of the newly identified genes, specifically TRAF2 and TRAF3.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Capital and culture : an investigation into New Labour cultural policy and the European Capital of Culture 2008

This thesis is an investigation into the relationship between culture in New Labour policy and within the competition for the European Capital of Culture 2008. The study interrogates a policy paradigm which it identifies as a 'creative city/urban planning' approach to urban regeneration. It locates this approach within a wider New Labour 'Third Way' politics, in that it attempts to reconcile economic instrumentalism with a rhetorical commitment to a politics of the social. Based on elite interviews and documentary analysis, this thesis argues that this approach to urban regeneration draws on a misappropriation of the work of cultural theorist Raymond Williams. It demonstrates how this misappropriation results in an unbounded anthropological definition, whereby culture colonises all areas of economic and social life. Within this template, culture becomes a surrogate economic and social policy. This is illustrated in the case-study of Liverpool's bidding for, winning of and plans for Capital of Culture 2008. This analysis shows how culture without parameters is usurped within both a neo-liberal economic agenda, and a policy template which recasts social inequality as a personal cultural deficit. Within Liverpool's urban strategy, culture is conceived as a social and economic panacea. However, when culture comes to mean everything, it invariably means nothing. This thesis attempts to put Raymond Williams' 'vague and baggy monster' back in its theoretical cage.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Time-dependent integrity during storage of natural surface water samples for the trace analysis of pharmaceutical products, feminizing hormones and pesticides

Monitoring and analysis of trace contaminants such as pharmaceuticals and pesticides require the preservation of the samples before they can be quantified using the appropriate analytical methods. Our objective is to determine the sample shelf life to insure proper quantification of ultratrace contaminants. To this end, we tested the stability of a variety of pharmaceutical products including caffeine, natural steroids, and selected pesticides under refrigerated storage conditions. The analysis was performed using multi-residue methods using an on-line solid-phase extraction combined with liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) in the selected reaction monitoring mode. After 21 days of storage, no significant difference in the recoveries was observed compared to day 0 for pharmaceutical products, while for pesticides, significant losses occurred for DIA and simazine after 10 days (14% and 17% reduction respectively) and a statistically significant decrease in the recovery was noted for cyanazine (78% disappearance). However, the estrogen and progestogen steroids were unstable during storage. The disappearance rates obtained after 21 days of storage vary from 63 to 72% for the feminizing hormones. Overall, pharmaceuticals and pesticides seem to be stable for refrigerated storage for up to about 10 days (except cyanazine) and steroidal hormones can be quite sensitive to degradation and should not be stored for more than a few days

Pathways of Superoxide (O2-) decay in the Eastern Tropical North Atlantic

Superoxide (O2-: IUPAC name dioxide (•1−)) is an important transient reactive oxygen species (ROS) in the ocean formed as an intermediate in the redox transformation of oxygen (O2) into hydrogen peroxide (H2O2) and vice versa. This highly reactive and very short-lived radical anion can be produced both via photochemical and biological processes in the ocean. In this paper we examine the decomposition rate of O2- throughout the water column, using new data collected in the Eastern Tropical North Atlantic (ETNA) Ocean. For this approach we applied a semi factorial experimental design, to identify and quantify the pathways of the major identified sinks in the ocean. In this work we occupied 6 stations, 2 on the West African continental shelf and 4 open ocean stations, including the CVOO time series site adjacent to Cape Verde. Our results indicate that in the surface ocean, impacted by Saharan aerosols and sediment resuspension, the main decay pathways for superoxide is via reactions with Mn(II) and organic matter

Conservation by trans-border cooperation: population genetic structure and diversity of geoffroy’s bat (Myotis emarginatus) at its north-western european range edge

In the European Union, all bat species are strictly protected and member states must ensure their conservation. However, if populations are genetically structured, conservation units that correspond to whole countries may be too large, putting small populations with specific conservation requirements at risk. Geoffroy’s bat (Myotis emarginatus) has undergone well-documented declines at its north-western European range edge between the 1960 and 1990s and is considered to be negatively affected by habitat fragmentation. Here we analysed the species’ genetic population structure and diversity to identify subpopulations with reduced genetic diversity and to scientifically inform conservation management. We generated 811 microsatellite-based genetic profiles obtained from 42 European nursery colonies and analysed a total of 932 sequences of the hypervariable region II of the mitochondrial control region sampled from across Europe. While two geographically widespread genetic populations were inferred to be present in north-western Europe, both nuclear and mitochondrial genetic diversity were lowest in the areas that had experienced a decline during the last century. A microsatellite-based analysis of demographic history did not permit, however, to unequivocally link that reduced genetic diversity to the population contraction event. Given the large geographic extent of the genetic populations, preserving the connectivity of mating sites requires concerted conservation efforts across multiple political jurisdictions. Genetic monitoring ought to be done on a regular basis to ensure that large-scale connectivity is maintained and further loss of genetic diversity is prevented