The gene encoding the glyphosate-tolerant EPSP synthase from Anabaena variabilis

Glyphosate is a broad-spectrum, non-selective, post-emergence herbicide, active against a variety of weed and crop species. The primary site for herbicidal action is 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, the penultimate enzyme of the shikimate pathway. In higher plants and microorganisms, this pathway provides a metabolic route to the synthesis of the aromatic amino acids - phenylalanine, tyrosine and tryptophan - plus other aromatic compounds. EPSP synthase has been purified from various sources to investigate its kinetic characteristics and the inhibition properties of glyphosate. The cyanobacteria are the largest, most diverse and widely distributed group of photosynthetic prokaryotes. The physiological and biochemical effects of glyphosate on certain cyanobacterial strains have been examined. The filamentous, nitrogen fixing strain Anabaena variabilis ATCC 29413, like other cyanobacteria, is tolerant to glyphosate. Tolerance is due to an EPSP synthase that is uninhibited by the herbicide. A detailed kinetic and molecular study of this enzyme has been hindered by the consistently low yield of EPSP synthase protein purified from A. variabilis cells. As such, the purpose of the work described in this thesis was to isolate the A. variabilis EPSP synthase gene (termed aroA) and overexpress the encoded enzyme. A number of cloning methods were undertaken in an attempt to reach this goal. The polymerase chain reaction was employed to amplify a defined segment of the aroA gene from A. variabilis genomic DNA using different sets of degenerate oligonucleotide primers. These primers were designed from conserved regions of EPSP synthase sequence from various plants and microorganisms. A truncated fragment of an aroA gene was synthesised, however, this piece of DNA did not originate from A. variabilis DNA. The source of this contaminating PCR product has yet to be identified. The design of the PCR primers may have affected the specificity of the amplification reaction and could explain why other primer sets failed to amplify the sequence of interest. Subsequently, a library of A. variabilis genomic DNA was constructed in the phagemid vector, pBluescript SK-. After evaluating its size and quality, the library was screened with an aroA probe from the unicellular, non-nitrogen fixing cyanobacterium Synechocystis sp. PCC 6803 to isolate the clone of interest. Prior to screening. Southern blot experiments had demonstrated that the Synechocystis probe hybridised to specific fragments of restriction digested A. variabilis DNA. This heterologous probe was, therefore, considered suitable for screening purposes. Persistent problems with non-specific hybridisation between the probe and the genetic material of the host cell harbouring the library frustrated the attempts made to locate an aroA clone. Reducing the level of background hybridisation required a slightly different approach. It was established that plaque hybridisation was more sensitive than colony hybridisation. Consequently, another A. variabilis library was made, but on this occasion the phage, lambda FIX II, was used as the vector system. Heterologous aroA probes from Synechocystis, E. coli and pea were employed to maximise the possibility of finding the A. variabilis EPSP synthase clone. Despite control experiments signifying the good quality of the newly constructed library, not one of the heterologous probes pulled out the clone of interest. The final strategy involved isolating the A. variabilis EPSP synthase gene by phenotypic complementation of an aroA- auxotrophic mutation of the E. coli strain AB2829. A control experiment showed that expression of the E. coli EPSP synthase from cloned DNA complemented the deficiency of the host and enabled the mutant to grow on selective medium. The E. coli mutant was then transformed with a library of A. variabilis genomic DNA made from EcoRI restriction fragments thought to contain the entire aroA coding sequence. Succeeding experiments showed that such a library could not complement the aroA mutation of E. coli AB2829. The advantages and disadvantages of each of the above techniques are discussed in detail with specific regard to cloning the A. variabilis EPSP synthase gene. Other gene cloning strategies not used in this work are described and the practical reasons for not employing these techniques are debated. The possible exploitation of the EPSP synthase gene from A. variabilis by genetic engineers for the construction of a glyphosate-tolerant crop plant is discussed, as is the contribution the gene sequence could have made to the debate regarding the cyanobacterial origin of higher plant and algal plastids

Acute stroke CDS: automatic retrieval of thrombolysis contraindications from unstructured clinical letters

Introduction: Thrombolysis treatment for acute ischaemic stroke can lead to better outcomes if administered early enough. However, contraindications exist which put the patient at greater risk of a bleed (e.g. recent major surgery, anticoagulant medication). Therefore, clinicians must check a patient's past medical history before proceeding with treatment. In this work we present a machine learning approach for accurate automatic detection of this information in unstructured text documents such as discharge letters or referral letters, to support the clinician in making a decision about whether to administer thrombolysis. Methods: We consulted local and national guidelines for thrombolysis eligibility, identifying 86 entities which are relevant to the thrombolysis decision. A total of 8,067 documents from 2,912 patients were manually annotated with these entities by medical students and clinicians. Using this data, we trained and validated several transformer-based named entity recognition (NER) models, focusing on transformer models which have been pre-trained on a biomedical corpus as these have shown most promise in the biomedical NER literature. Results: Our best model was a PubMedBERT-based approach, which obtained a lenient micro/macro F1 score of 0.829/0.723. Ensembling 5 variants of this model gave a significant boost to precision, obtaining micro/macro F1 of 0.846/0.734 which approaches the human annotator performance of 0.847/0.839. We further propose numeric definitions for the concepts of name regularity (similarity of all spans which refer to an entity) and context regularity (similarity of all context surrounding mentions of an entity), using these to analyse the types of errors made by the system and finding that the name regularity of an entity is a stronger predictor of model performance than raw training set frequency. Discussion: Overall, this work shows the potential of machine learning to provide clinical decision support (CDS) for the time-critical decision of thrombolysis administration in ischaemic stroke by quickly surfacing relevant information, leading to prompt treatment and hence to better patient outcomes

Partners No More: Relational Transformation and the Turn to Litigation in Two Conservationist Organizations

The rise in litigation against administrative bodies by environmental and other political interest groups worldwide has been explained predominantly through the liberalization of standing doctrines. Under this explanation, termed here the floodgate model, restrictive standing rules have dammed the flow of suits that groups were otherwise ready and eager to pursue. I examine this hypothesis by analyzing processes of institutional transformation in two conservationist organizations: the Sierra Club in the United States and the Society for the Protection of Nature in Israel (SPNI). Rather than an eagerness to embrace newly available litigation opportunities, as the floodgate model would predict, the groups\u27 history reveals a gradual process of transformation marked by internal, largely intergenerational divisions between those who abhorred conflict with state institutions and those who saw such conflict as not only appropriate but necessary to the mission of the group. Furthermore, in contrast to the pluralist interactions that the floodgate model imagines, both groups\u27 relations with pertinent agencies in earlier eras better accorded with the partnership-based corporatist paradigm. Sociolegal research has long indicated the importance of relational distance to the transformation of interpersonal disputes. I argue that, at the group level as well, the presence or absence of a (national) partnership-centered relationship determines propensities to bring political issues to court. As such, well beyond change in groups\u27 legal capacity and resources, current increases in levels of political litigation suggest more fundamental transformations in the structure and meaning of relations between citizen groups and the state

OSUL 2013 Innovation Task Force Final Report

Final report of the Innovation Task Force. Part of the OSUL 2013 Libraries Visioning Task Forces process.The University Archives has determined that this item is of continuing value to OSU's history

Media content created for Museums without Walls: An interactive resource exploring connections between Orkney and the Hudson's Bay Company

During the summer of 2021 the Stromness Museum together with the University of Dundee launched a pilot project to explore around 80 objects in their collections relating to Orkney’s history with the Hudson’s Bay Company in Canada. Our aim was to design an interactive online resource which brought together 3D scans and photographs of the museum’s collections together with insights and stories from different perspectives, including Orcadian, Cree, Métis and Inuit voices. What resulted was a compelling and diverse variety of material offering fresh insights and contemporary connections to this complex period of shared history.  The resource is freely available online and as a touchscreen exhibit inside the Stromness Museum in Orkney. https://www.stromnessmuseum.org.uk/sites/stromnessmuseum.org.uk/files/hbc/index.html Data and file overview The archived material consists of 3D scanned objects from the Museum’s collections in .glb format; photographs of objects and family photographs relating to personal accounts featured in the resource; soundbites from various people interviewed for the resource about their family heritage and the Hudson’s Bay Company; curator written blurbs; and artwork relating to the resource interface. Sharing and access information The material (with the exception of a walk-through screen capture video) is not publicly accessible for download and re-use without requested consent. This is because much of the materials concern Indigenous objects, stories and imagery and we do not wish for this to be re-used elsewhere without prior consent from the individuals represented. All material is available to view (but not download and re-use) on the Stromness Museum’s website: (https://www.stromnessmuseum.org.uk/sites/stromnessmuseum.org.uk/files/hbc/index.html) Methodological information Our approach for generating material as part of this project followed a collaborative co-curation methodology which facilitated curation of the resource to be led by the contributions and engagements of the individuals and communities we worked with. The content maps and walk-through screen capture video are made available under a CC-BY-NC license, for full details of this license copy and paste the following link into your browser, https://creativecommons.org/licenses/by-nc/4.0