The influence of ageing and culture conditions on limbal epithelial cells

PhD ThesisIntroduction. The use of limbal stem cells as a cellular therapy has expanded over the last decade. Currently, the success rate of limbal stem cell transplantations is around 76% with advanced donor age being a possible reason for this figure. Medium and oxygen concentration could also have a detrimental effect on the growth of these cells for transplantation. With age the limbus undergoes a topological change which could alter the stem cells surroundings. A mouse model of corneal ageing was also used to complement the human study and avoid culturing and tissue storage artefacts. Aims. The main aim of this thesis was to assess the effect of age on corneal epithelial progenitor cells. This then led to the question; is the mouse cornea affected by age and can interventions mediate these outcomes? And finally do culture conditions affect the growth of corneal epithelial stem cell containing cultures, and how does this relate to changes to the niche with age? Methods. Limbal derived progenitor cells were extracted from human corneosclearal disks donated for research. Cells were cultured either in LEM or DKSFM and either in hypoxia (3%) or normoxia (21%). Immunofluorescence, qPCR, TRAP assay, morphological and clonal analysis where used to assess progenitor composition. The mice used were strain B6.129S-Tert, tm1Yjc/J. Old/AL and DR mice were 22 months old, young were 3 months old. Rapamycin treated mice were 16 months old and were treated for four months, starting at 12 months of age. Dietary restriction was implemented for a period of 16 months from 6 months of age, additionally in the mouse eye a DNA damage marker was also assessed, both telomeric and non-telomericly. Results and Conclusions. Age has a detrimental effect on the ability to culture a limbally derived clonal population. However age did not affect the levels of positive and negative gene expression markers or ΔNp63 protein level. Interestingly the time that corneal tissue is stored for did not affect the ability to isolate these progenitor cells. The growth of cells in hypoxia decreased negative marker KRT3 and senescence marker p21 regardless of culture medium. In the mouse model, age had a detrimental effect on the cornea with decreased TAp63, increased ɣH2A.X and TAFs. DR however, tended to have a beneficial effect on the mouse cornea. Interestingly rapamycin seems to be detrimental to the mouse cornea, with similarities with the effects of age.Biomedical Research Centre (BRC) and Newcastle upon Tyne Hospitals NHS Foundation Trust (NUTH)

Political brand identity: an examination of the complexities of Conservative brand and internal market engagement during the 2010 UK General Election campaign

This paper seeks to build an understanding of the importance of internal communications when building a strong political brand. Using Kapferer’s brand prism as a conceptual framework, the paper explores UK Conservative Party members’ attitudes towards the development of the Conservative brand as personified by David Cameron. There are clear implications for political strategists as the findings suggest that it is crucial to engage the internal market in the co-creation of the marketing communications strategy for as brand evangelists they interpret the brand promise at the local level

Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids

Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine

Reading Maud’s remains: Tennyson, geological processes, and palaeontological reconstructions

As Tennyson's “little Hamlet ,” Maud (1855) posits a speaker who, like Hamlet, confronts the ignominious fate of dead remains. Maud's speaker contemplates such remains as bone, hair, shell, and he experiences his world as one composed of hard inorganic matter, such things as rocks, gems, flint, stone, coal, and gold. While Maud's imagery of “stones, and hard substances” has been read as signifying the speaker's desire “unnaturally to harden himself into insensibility” (Killham 231, 235), I argue that these substances benefit from being read in the context of Tennyson's wider understanding of geological processes. Along with highlighting these materials, the text's imagery focuses on processes of fossilisation, while Maud's characters appear to be in the grip of an insidious petrification. Despite the preoccupation with geological materials and processes, the poem has received little critical attention in these terms. Dennis R. Dean, for example, whose Tennyson and Geology (1985) is still the most rigorous study of the sources of Tennyson's knowledge of geology, does not detect a geological register in the poem, arguing that by the time Tennyson began to write Maud, he was “relatively at ease with the geological world” (Dean 21). I argue, however, that Maud reveals that Tennyson was anything but “at ease” with geology. While In Memoriam (1851) wrestles with religious doubt that is both initiated, and, to some extent, alleviated by geological theories, it finally affirms the transcendence of spirit over matter. Maud, conversely, gravitates towards the ground, concerning itself with the corporal remains of life and with the agents of change that operate on all matter. Influenced by his reading of geology, and particularly Charles Lyell's provocative writings on the embedding and fossilisation of organic material in strata in his Principles of Geology (1830–33) volume 2, Tennyson's poem probes the taphonomic processes that result in the incorporation of dead remains and even living flesh into the geological system

Human iPSC disease modelling reveals functional and structural defects in retinal pigment epithelial cells harbouring the m.3243A > G mitochondrial DNA mutation.

The m.3243A > G mitochondrial DNA mutation was originally described in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The phenotypic spectrum of the m.3243A > G mutation has since expanded to include a spectrum of neuromuscular and ocular manifestations, including reduced vision with retinal degeneration, the underlying mechanism of which remains unclear. We used dermal fibroblasts, from patients with retinal pathology secondary to the m.3243A > G mutation to generate heteroplasmic induced pluripotent stem cell (hiPSC) clones. RPE cells differentiated from these hiPSCs contained morphologically abnormal mitochondria and melanosomes, and exhibited marked functional defects in phagocytosis of photoreceptor outer segments. These findings have striking similarities to the pathological abnormalities reported in RPE cells studied from post-mortem tissues of affected m.3243A > G mutation carriers. Overall, our results indicate that RPE cells carrying the m.3243A > G mutation have a reduced ability to perform the critical physiological function of phagocytosis. Aberrant melanosomal morphology may potentially have consequences on the ability of the cells to perform another important protective function, namely absorption of stray light. Our in vitro cell model could prove a powerful tool to further dissect the complex pathophysiological mechanisms that underlie the tissue specificity of the m.3243A > G mutation, and importantly, allow the future testing of novel therapeutic agents

James Hutton’s geological tours of Scotland : romanticism, literary strategies, and the scientific quest

This article explores a somewhat neglected part of the story of the emergence of geology as a science and discourse in the late eighteenth century – James Hutton’s posthumously published accounts of the geological tours of Scotland that he undertook in the years 1785 to 1788 in search of empirical evidence in support of his theory of the Earth and that he intended to include in the projected third volume of his Theory of the Earth of 1795. The article brings some of the assumptions and techniques of literary criticism to bear on Hutton’s scientific travel writing in order to open up new connections between geology, Romantic aesthetics and eighteenth-century travel writing about Scotland. Close analysis of Hutton’s accounts of his field trips to Glen Tilt, Galloway and Arran, supplemented by later accounts of the discoveries at Jedburgh and Siccar Point, reveals the interplay between desire, travel and the scientific quest and foregrounds the textual strategies that Hutton uses to persuade his readers that they share in the experience of geological discovery and interpretation as ‘virtual witnesses’. As well as allowing us to revisit the interrelation between scientific theory and discovery, this article concludes that Hutton was a much better writer than he has been given credit for and suggests that if these geological tours had been published in 1795 they would have made it impossible for critics to dismiss him as an armchair geologist

Potential virus-mediated nitrogen cycling in oxygen-depleted oceanic waters

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Gazitua, M. C., Vik, D. R., Roux, S., Gregory, A. C., Bolduc, B., Widner, B., Mulholland, M. R., Hallam, S. J., Ulloa, O., & Sullivan, M. B. Potential virus-mediated nitrogen cycling in oxygen-depleted oceanic waters. Isme Journal, (2020), doi:10.1038/s41396-020-00825-6.Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ viruses might impact nitrogen (N) cycling via AMGs. Identified viral genomes encoded six N-cycle AMGs associated with denitrification, nitrification, assimilatory nitrate reduction, and nitrite transport. The majority of these AMGs (80%) were identified in T4-like Myoviridae phages, predicted to infect Cyanobacteria and Proteobacteria, or in unclassified archaeal viruses predicted to infect Thaumarchaeota. Four AMGs were exclusive to anoxic waters and had distributions that paralleled homologous microbial genes. Together, these findings suggest viruses modulate N-cycling processes within the ETSP OMZ and may contribute to nitrogen loss throughout the global oceans thus providing a baseline for their inclusion in the ecosystem and geochemical models.We thank Sullivan Lab members and Heather Maughan for comments on the paper, Bess Ward for her contribution in the N-cycle context of our story, Kurt Hanselmann for his assistance in the calculations of the Gibbs-free energies, and the scientific party and crew of the R/V Atlantis (grant OCE-1356056 to MRM) for the sampling opportunity and support at sea. This work was funded in part by awards from the Agouron Institute to OU and MBS, a Gordon and Betty Moore Foundation Investigator Award (#3790) and NSF Biological Oceanography Awards (#1536989 and #1829831) to MBS, and the Millennium Science Initiative (grant ICN12_019-IMO) to OU. The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231

Potential Virus-Mediated Nitrogen Cycling in Oxygen-Depleted Oceanic Waters

Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ viruses might impact nitrogen (N) cycling via AMGs. Identified viral genomes encoded six N-cycle AMGs associated with denitrification, nitrification, assimilatory nitrate reduction, and nitrite transport. The majority of these AMGs (80%) were identified in T4-like Myoviridae phages, predicted to infect Cyanobacteria and Proteobacteria, or in unclassified archaeal viruses predicted to infect Thaumarchaeota. Four AMGs were exclusive to anoxic waters and had distributions that paralleled homologous microbial genes. Together, these findings suggest viruses modulate N-cycling processes within the ETSP OMZ and may contribute to nitrogen loss throughout the global oceans thus providing a baseline for their inclusion in the ecosystem and geochemical models

Jurassic sedimentation in the Cleveland Basin : a review

This review combines two Presidential Addresses (2005, 2006) and aims to provides an up-to-date overview of the stratigraphy and sedimentation of the Jurassic sequence of the Cleveland Basin (Yorkshire), including poorly known data from the western outcrop. These fascinating rocks have been the focus of geological research since the 18th Century and have had a profound influence on the development of the geological sciences. Throughout the 20th Century, the excellent coastal exposures have acted as a magnet for palaeontologists, stratigraphers, sedimentologists and geochemists, as a natural geological laboratory, and in recent decades, the coastal exposures received increased scientific interest as a result of their analogy with hydrocarbon source and reservoir rocks in the North Sea. Designation of the international Global Stratotype Section and Point (GSSP) for the Sinemurian–Pliensbachian stage boundary in Robin Hood's Bay, the establishment of the Dinosaur Coast, and development of the Rotunda Museum in Scarborough have all given the regional geology additional importance. The Lias Group (Hettangian–Toarcian age; 199.6–175.6 Ma), exposed in the well known coastal sections, is illustrated by the fully cored Felixkirk Borehole, located at the western margin of the outcrop, and is one of the best examples of shallow marine sedimentation in an epeiric shelf-sea setting. It comprises two large-scale, upward coarsening cycles, namely the Redcar Mudstone to Staithes Sandstone cycle, followed by the Cleveland Ironstone to Blea Wyke Sandstone cycle. Within this broad pattern, smaller scale transgressive–regressive cycles are described from stratigraphically expanded and reduced successions. Detailed ammonite biostratigraphy provides a finely calibrated temporal framework to study the variations in sedimentation, which include storm-generated limestones and sandstones (‘tempestites’) interbedded with mudstone deposited during fair-weather periods. Hemipelagic mud, occasionally organic-rich, reflects deeper-water anoxic events that may indicate a response to global climate change. In cores, the tempestite beds (Hettangian–Sinemurian) are characterized by sharp bases that, at outcrop, are often masked by downward penetrating burrows. Cyclicity on a centimetre scale in the overlying Pliensbachian ‘Banded Shales’ may be the result of orbitally induced, climatic cycles. Gradational upward coarsening to the Staithes Sandstone Formation marks a transition to sand-rich tempestite deposits, characterized by low angle and swaley cross-lamination, interbedded with sand-starved units (striped siltstones). The sands were probably deposited from sediment-laden, storm-surge and ebb currents in inner- and mid-shelf settings; the sandy substrate was, at some levels, extensively bioturbated by deposit feeding organisms that produced a spectacular range of trace fossil assemblages characteristic of shoreface, inner-, mid-, and outer-shelf settings. Intrabasinal tectonics was a controlling factor during deposition of both the Staithes Sandstone and the overlying Cleveland Ironstone (Late Pliensbachian). The influx of sand is attributed to hinterland uplift and increased sediment flux. More marked intraformational uplift during deposition of the Cleveland Ironstone is manifested in a much attenuated succession in the west of the basin (Felixkirk); southwards, towards the Market Weighton High, the Pecten/Main Seam of the ironstone oversteps unconformably onto progressively older beds to rest on the lower part of the Redcar Mudstone Formation. Ironstone, in the form of berthierine ooids and sideritic mud, was deposited during 5–6 cycles (in coastal exposures) of high sea-level stands that cut off siliciclastic influx from the low-gradient hinterland; regressive, upward-shoaling intervals are marked by interbedded, bioturbated siltstone and fine-grained sandstone. The Toarcian succession (Whitby Mudstone and Blea Wyke Sandstone formations) continues the second upward coarsening cycle in response to increased subsidence, rising sea-level, and an influx of siliciclastic sand. Oxygenated, open marine mud was deposited during the initial deepening phase, followed by bituminous mud, attributed to ocean-water stratification and the establishment of anoxic bottom conditions; in the west of the basin an upward shoaling sequence suggests that water depths were not as great. Recent research on the geochemistry and stable isotope signatures across this early Toarcian interval indicates a widespread, global anoxic event, possibly attributed to the release of methane hydrate on the ocean floor. The Alum Shale Member represents increasingly oxygenated bottom conditions and an upward coarsening motif with passage to the Blea Wyke Sandstone Formation, which is preserved only in the Peak Trough, an actively subsiding graben. Basin uplift accompanied by gentle folding in late Toarcian to Aalenian times removed much of the late Toarcian succession so that the Middle Jurassic Dogger Formation (Aalenian), a complex, condensed, shallow water unit rests unconformably on beds as low as the Alum Shale over much of the basin. Deep boreholes and revision mapping by the British Geological Survey (BGS) in the west of the outcrop have allowed a fuller, basin-wide synthesis of the palaeoenvironments and the influence of intra-Jurassic tectonics during Mid- to Late Jurassic times. During Mid-Jurassic times the low-lying, paralic coastal plain, typified by braided and meandering fluvial systems and lacustrine deposits was invaded by marine incursions from the south and east. Each transgressive event was different in its geographical penetration across the coastal plain, resulting in varied lithofacies and palaeoenvironments including ooidal ironstone and lime mud (Eller Beck Formation), peloid and ooid carbonate shoals (Lebberston Member), and tidal sand bars, pelloidal limestones and nearshore marine muds (Scarborough Formation). Trace fossils, including dinosaur footprints, and macro-plant fossils tell us much about the palaeoenvironments on the coastal plain, during this time interval (175.6–164.7 Ma) that was characterized by a warm, seasonal climate. The basin wide transgression and marked global sea-level rise represented by the Cornbrash Formation, marks deposition in a shallow marine environment during the Callovian, followed by sand (Osgodby Formation) and deeper water muds (Oxford Clay Formation) that spread northwards from the East Midlands over the Market Weighton High during the Oxfordian. Subsequent shallowing of the basin resulted in the establishment of a carbonate/siliciclastic platform typified by ooidal shoals, coral patch reefs and sponge spicule-rich marine sands (Corallian Group). Their complex sedimentation pattern was influenced by local infra-Oxfordian tectonics related to the Howardian–Flamborough Fault Belt. Although the Ampthill Clay and Kimmeridge Clay formations, the latter representing the most important regional hydrocarbon source rock, are not well-exposed, recent boreholes in the Cleveland Basin have allowed a much better understanding of the hemi-pelagic marine environment (both oxic and anoxic) during this phase of sedimentation which marks a global sea-level rise. Although well-studied by world standards, the Jurassic sediments of the Cleveland Basin continue to throw up surprises and advances in our understanding of the Earth as a dynamic system over a period of c. 30 million years. These studies have directly and indirectly influenced our understanding of the Earth as a system, and have played an important role in educating non-specialists, undergraduates and professional geologists over many decades