1,041 research outputs found

    Researching animal research: What the humanities and social sciences can contribute to laboratory animal science and welfare

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    Every year around 80 million scientific procedures are carried out on animals globally. These experiments have the potential to generate new understandings of biology and clinical treatments. They also give rise to ongoing societal debate.This book demonstrates how the humanities and social sciences can contribute to understanding what is created through animal procedures - including constitutional forms of research governance, different institutional cultures of care, the professional careers of scientists and veterinarians, collaborations with patients and publics, and research animals, specially bred for experiments or surplus to requirements.Developing the idea of the animal research nexus, this book explores how connections and disconnections are made between these different elements, how these have reshaped each other historically, and how they configure the current practice and policy of UK animal research

    Fanconi anaemia and LINE-1 retrotransposition in the mammalian genome

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    Transposable elements (TEs) are discrete, repetitive sequences of DNA that mobilise within genomes. For decades, TEs were dismissed as “junk DNA”, however, it is now clear that these elements have the potential to trigger genome instability, cause disease and shape the course of genome evolution. L1 elements constitute the only autonomous elements which remain active in the human genome and comprises approximately 17% of human DNA. As a retrotransposon, L1 canonically mobilises through a “cut and paste” mechanism called target primed reverse transcription (TPRT). Due to the deleterious impacts of L1 activity, mammalian cells have evolved a range of mechanisms to supress the mobilisation of these elements. The interactions between L1 elements and the host factors which regulate them are therefore an area of active research. Several DNA repair genes have shown potential as regulators of L1 activity. Moreover, in cell lines deficient in non-homologous end-joining, L1 has shown the potential to retrotranspose without its ORF2p endonuclease, which is usually a requirement for canonical TPRT. This retrotransposition has been termed endonuclease independent (ENi) retrotransposition, and takes place at unrepaired double stranded breaks in the DNA. Interestingly, several DNA repair factors have also been identified as potential regulators of L1 retrotransposition (both positive and negative), including a number of proteins from the Fanconi Anaemia pathway. The relationship between these factors and L1 has yet to be fully characterised, and it remains to be seen whether L1 can exploit other DNA lesions in the way that it utilises DSBs in ENi retrotransposition. This thesis aims to further investigate the relationship between L1 retrotransposition in the mammalian genome and DNA repair factors, particularly those comprising the Fanconi Anaemia pathway. Using cultured cell retrotransposition assays, I systematically tested a battery of mutant element in cells deficient in different proteins of the FANC pathway. In this way, I establish that ENi retrotransposition can be observed in a FANC background. I also demonstrate that FANC A deficient cells support retrotransposition of several L1 mutants which are immobile in parental cell lines. This includes elements with severe ORF1p mutations, mutations in the ORF2p endonuclease domain and mutations in the ORF2p PIP box. Despite testing a range of cell lines deficient in different DNA repair factors, including cells deficient in a range of FANC proteins, the retrotransposition of ORF1p, PIP and mutants appears to be unique to FANC A. My results are potentially indicative of a unique mechanism of retrotransposition in FANC A cells, a phenomena which has precedence in the ENi pathway of retrotransposition. Mass spectrometry of immunoprecipitated T7-tagged ORF1p, both in FANC A and parental cells, demonstrated that a different selection of host factors interact with ORF1p in the two cell lines. Several of these have not been previously identified as L1 interactors, including YTHDF2, a protein which binds and destabilises m6A-containing RNA. Previous reports suggest that YTHDF2 regulates the stability of RNA:DNA hybrids in vivo, and associates with R loop containing loci. Through co-immunoprecipitation of YTHDF2 with ORF1p, I confirm that the protein interacts with L1 elements in vitro

    Biomedical and Pharmacological Applications of Marine Collagen

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    Biomimetic polymers and materials have been widely used in a variety of biomedical and pharmacological applications. Particularly, collagen-based biomaterials have been extensively applied in various biomedical fields, such as scaffolds in tissue engineering. However, there are many challenges associated with the use of mammalian collagen, including the issues of religious constrains, allergic or autoimmune reactions, and the spread of animal diseases. Over the past few decades, marine collagen (MC) has emerged as a promising biomaterial for biomedical and pharmacological applications. Marine organisms are a rich source of structurally novel and biologically active compounds, and to date, many biological components have been isolated from various marine resources. MC offers advantages over mammalian collagen due to its water solubility, low immunogenicity, safety, biocompatibility, antimicrobial activity, functionality, and low production costs. Due to its characteristics and physicobiochemical properties, it has tremendous potential for use as a scaffold biomaterial in tissue engineering and regenerative medicine, in drug delivery systems, and as a therapeutic. In this Special Issue, we encourage submissions related to the recent developments, advancements, trends, challenges, and future perspectives in this new research field. We expect to receive contributions from different areas of multidisciplinary research, including—but not restricted to—extraction, purification, characterization, fabrication, and experimentation of MC, with a particular focus on their biotechnological, biomedical and pharmacological uses

    Spatiotemporal analysis of transcription dynamics

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    Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins

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    During embryonic development, dramatic cell shape changes and movements reshape the embryonic body plan. These require robust but dynamic linkage between the cell–cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by mechanosensitive multiprotein complexes assembled via multivalent connections. Here we combine genetic, cell biological, and modeling approaches to define the mechanism of action and functions of an important player, Drosophila polychaetoid, homologue of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell–cell junctions during these events. The cadherin–catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction–cytoskeletal interface, suggesting different proteins localize and function in distinct ways, perhaps in distinct subcomplexes, but combine to produce robust connections

    Development of polarization-resolved optical scanning microscopy imaging techniques to study biomolecular organizations

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    Light, as electromagnetic radiation, conveys energy through space and time via fluctuations in electric and magnetic fields. This thesis explores the interaction of light and biological structures through polarization-resolved imaging techniques. Light microscopy, and polarization analysis enable the examination of biological entities. Biological function often centers on chromatin, the genetic material composed of DNA wrapped around histone proteins within cell nuclei. This structure's chiral nature gives rise to interactions with polarized light. This research encompasses three main aspects. Firstly, an existing multimodal Circular Intensity Differential Scattering (CIDS) and fluorescence microscopy are upgraded into an open configuration to be integrated with other modalities. Secondly, a novel cell classification method employing CIDS and a phasor representation is introduced. Thirdly, polarization analysis of fluorescence emission is employed for pathological investigations. Accordingly, the thesis is organized into three chapters. Chapter 1 lays the theoretical foundation for light propagation and polarization, outlining the Jones and Stokes-Mueller formalisms. The interaction between light and optical elements, transmission, and reflection processes are discussed. Polarized light's ability to reveal image contrast in polarizing microscopes, linear and nonlinear polarization-resolved microscopy, and Mueller matrix microscopy as a comprehensive technique for studying biological structures are detailed. Chapter 2 focuses on CIDS, a label-free light scattering method, including a single point angular spectroscopy mode and scanning microscopy imaging. A significant upgrade of the setup is achieved, incorporating automation, calibration, and statistical analysis routines. An intuitive phasor approach is proposed, enabling image segmentation, cell discrimination, and enhanced interpretation of polarimetric contrast. As a result, image processing programs have been developed to provide automated measurements using polarization-resolved laser scanning microscopy imaging integrated with confocal fluorescence microscopy of cells and chromatin inside cell nuclei, including the use of new types of samples such as progeria cells. Chapter 3 applies a polarization-resolved two-photon excitation fluorescence (2PEF) microscopy to study multicellular cancerous cells. A homemade 2PEF microscope is developed for colon cancer cell analysis. The integration of polarization and fluorescence techniques leads to a comprehensive understanding of the molecular orientation within samples, particularly useful for cancer diagnosis. Overall, this thesis presents an exploration of polarization-resolved imaging techniques for studying biological structures, encompassing theory, experimental enhancements, innovative methodologies, and practical applications
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