Developing a low-cost, simple-to-use electrochemical sensor for the detection of circulating tumour DNA in human fluids

It is well-known that two major issues, preventing improved outcomes from cancer are late diagnosis and the evolution of drug resistance during chemotherapy, therefore technologies that address these issues can have a transformative effect on healthcare workflows. In this work we present a simple, low-cost DNA biosensor that was developed specifically to detect mutations in a key oncogene (KRAS). The sensor employed was a screen-printed array of carbon electrodes, used to perform parallel measurements of DNA hybridisation. A DNA amplification reaction was developed with primers for mutant and wild type KRAS sequences which amplified target sequences from representative clinical samples to detectable levels in as few as twenty cycles. High levels of sensitivity were demonstrated alongside a clear exemplar of assay specificity by showing the mutant KRAS sequence was detectable against a significant background of wild type DNA following amplification and hybridisation on the sensor surface. The time to result was found to be 3.5 h with considerable potential for optimisation through assay integration. This quick and versatile biosensor has the potential to be deployed in a low-cost, point-of-care test where patients can be screened either for early diagnosis purposes or monitoring of response to therapy

Urban Biodiversity and Landscape Ecology: Patterns, Processes and Planning

Effective planning for biodiversity in cities and towns is increasingly important as urban areas and their human populations grow, both to achieve conservation goals and because ecological communities support services on which humans depend. Landscape ecology provides important frameworks for understanding and conserving urban biodiversity both within cities and considering whole cities in their regional context, and has played an important role in the development of a substantial and expanding body of knowledge about urban landscapes and communities. Characteristics of the whole city including size, overall amount of green space, age and regional context are important considerations for understanding and planning for biotic assemblages at the scale of entire cities, but have received relatively little research attention. Studies of biodiversity within cities are more abundant and show that longstanding principles regarding how patch size, configuration and composition influence biodiversity apply to urban areas as they do in other habitats. However, the fine spatial scales at which urban areas are fragmented and the altered temporal dynamics compared to non-urban areas indicate a need to apply hierarchical multi-scalar landscape ecology models to urban environments. Transferring results from landscape-scale urban biodiversity research into planning remains challenging, not least because of the requirements for urban green space to provide multiple functions. An increasing array of tools is available to meet this challenge and increasingly requires ecologists to work with planners to address biodiversity challenges. Biodiversity conservation and enhancement is just one strand in urban planning, but is increasingly important in a rapidly urbanising world

Metrology and Molecular Diagnosis of Infection

Metrology, the study of measurement, is an emerging concept within molecular diagnosis of infection. Metrology promotes high-quality, reproducible data to be used in clinical management of infection, through characterisation of technical error and measurement harmonisation. This influences measurement accuracy, which has implications for setting thresholds between healthy and disease states, monitoring disease progression, and establishing cures. This thesis examines the placing of metrology in molecular diagnosis of infectious diseases. Sources of experimental error in advanced methodologies – dPCR and MALDI-TOF MS – that can influence measurement accuracy for RNA, DNA and protein biomarkers were investigated for HIV-1, methicillin-resistant Staphylococcus spp and organisms associated with hospital transmission. Measurement error introduced at different stages of a method can directly impact upon clinical results. A 30% bias was introduced between dPCR and qPCR quantification of HIV-1 DNA in clinical samples, owing to instability in the qPCR calibration material. In addition, experimental variability was found to influence classification of protein profiles which can limit the resolution of MALDI-TOF MS for strain typing bacteria. This thesis also addresses the prospective role of these advanced methods in supporting accurate clinical measurements. dPCR offers precise measurements of RNA and DNA targets and could be used to support qPCR, or for value assignment of reference materials to harmonise inter-laboratory results. MALDI-TOF MS demonstrated potential for strain typing Acinetobacter baumannii; results correlated with epidemiological data and WGS, although were not consistent with reference typing. Further work should examine the extent to which MALDI-TOF MS can support or replace contemporary strain typing methods for identifying nosocomial outbreaks. Molecular approaches possess a crucial role in the detection, quantification and characterisation of pathogens, and are invaluable tools for managing emerging diseases. Supporting accuracy and reproducibility in molecular measurements could help to strengthen diagnostic efforts, streamline clinical pathways and provide overall benefit to patient care

Knowledge Components and Methods for Policy Propagation in Data Flows

Data-oriented systems and applications are at the centre of current developments of the World Wide Web (WWW). On the Web of Data (WoD), information sources can be accessed and processed for many purposes. Users need to be aware of any licences or terms of use, which are associated with the data sources they want to use. Conversely, publishers need support in assigning the appropriate policies alongside the data they distribute. In this work, we tackle the problem of policy propagation in data flows - an expression that refers to the way data is consumed, manipulated and produced within processes. We pose the question of what kind of components are required, and how they can be acquired, managed, and deployed, to support users on deciding what policies propagate to the output of a data-intensive system from the ones associated with its input. We observe three scenarios: applications of the Semantic Web, workflow reuse in Open Science, and the exploitation of urban data in City Data Hubs. Starting from the analysis of Semantic Web applications, we propose a data-centric approach to semantically describe processes as data flows: the Datanode ontology, which comprises a hierarchy of the possible relations between data objects. By means of Policy Propagation Rules, it is possible to link data flow steps and policies derivable from semantic descriptions of data licences. We show how these components can be designed, how they can be effectively managed, and how to reason efficiently with them. In a second phase, the developed components are verified using a Smart City Data Hub as a case study, where we developed an end-to-end solution for policy propagation. Finally, we evaluate our approach and report on a user study aimed at assessing both the quality and the value of the proposed solution

Online Analysis of Dynamic Streaming Data

Die Arbeit zum Thema "Online Analysis of Dynamic Streaming Data" beschäftigt sich mit der Distanzmessung dynamischer, semistrukturierter Daten in kontinuierlichen Datenströmen um Analysen auf diesen Datenstrukturen bereits zur Laufzeit zu ermöglichen. Hierzu wird eine Formalisierung zur Distanzberechnung für statische und dynamische Bäume eingeführt und durch eine explizite Betrachtung der Dynamik von Attributen einzelner Knoten der Bäume ergänzt. Die Echtzeitanalyse basierend auf der Distanzmessung wird durch ein dichte-basiertes Clustering ergänzt, um eine Anwendung des Clustering, einer Klassifikation, aber auch einer Anomalieerkennung zu demonstrieren. Die Ergebnisse dieser Arbeit basieren auf einer theoretischen Analyse der eingeführten Formalisierung von Distanzmessungen für dynamische Bäume. Diese Analysen werden unterlegt mit empirischen Messungen auf Basis von Monitoring-Daten von Batchjobs aus dem Batchsystem des GridKa Daten- und Rechenzentrums. Die Evaluation der vorgeschlagenen Formalisierung sowie der darauf aufbauenden Echtzeitanalysemethoden zeigen die Effizienz und Skalierbarkeit des Verfahrens. Zudem wird gezeigt, dass die Betrachtung von Attributen und Attribut-Statistiken von besonderer Bedeutung für die Qualität der Ergebnisse von Analysen dynamischer, semistrukturierter Daten ist. Außerdem zeigt die Evaluation, dass die Qualität der Ergebnisse durch eine unabhängige Kombination mehrerer Distanzen weiter verbessert werden kann. Insbesondere wird durch die Ergebnisse dieser Arbeit die Analyse sich über die Zeit verändernder Daten ermöglicht

Urban geoscience report : capacity for 3D urban modelling

This report considers opportunities for future 3D urban geology modelling at the BGS. A total of 42 towns and cities in Great Britain were considered in this study, selected based on expected growth areas, e.g. Leeds, Oxford, and/or regionally important urban centres e.g. London, Glasgow. The selected areas also include 13 ‘Cohort 1’ towns identified by the UK Governments Towns Fund, such as Blackpool and Middlesbrough. The review reflects on recent and current 3D urban modelling approaches; considers the nature and complexity of the geology of British towns and cities; evaluates the availability of geological data for 3D modelling and suitable 3D modelling software; and highlights priority areas for innovation. It concludes by providing a series of recommendations for urban geology modelling

Characterizing the RNA Editing Specificityof ADAR Isoforms and Deaminase Domains In Vitro

Adenosine deaminases acting on RNA (ADARs) convert adenosine-to-inosine in double stranded RNA. Selectivity of editing sites depends on the sequence of the RNA as well as the secondary structure. Identification of sites of ADAR editing by editome analysis is skewed due to the different abundance of each adenosine-containing triplet, as well as the presence of complex RNA structures. To determine the editing specificity of each ADAR protein, a high throughput sequencing based assay was developed to measure editing in a synthetic dsRNA substrate with one of each of the 16 different adenosine-containing triplets, so that each possible editing site was equally represented. The ADAR1- and ADAR2-deaminases, as well as the full-length ADAR2 and isoforms ADAR1- p150 and ADAR1-p110, were purified and activity was measured for each, so that the inherent activity of the deaminase domains could be characterized and then compared to the editing patterns seen for the longer proteins containing dsRNA binding domains and Z-RNA binding domains. The ADAR1 deaminase was found to skew slightly to favoring 5’A editing sites, while ADAR2- deaminase favored 5’U. From homology modelling of ADAR1 onto the ADAR2 crystal structure, this difference in editing specificity could be due to the ADAR1 protein having a weaker interaction with the orphan base of the RNA substrate. Characterization of full-length ADAR1-p110 and ADAR2 found that each full-length protein had less editing of the UAG triplet than the respective deaminases, while ADAR1-p110 increased editing of the AAG triplet and ADAR2 increased editing of the CAG triplet. Comparing ADAR1 isoforms p110 and p150 found no significant differences in editing specificity. The in vitro assay was also used to confirm the inactivity of the ADAR3 deaminase, and probe and characterize the editing specificity of the ADAR3 deaminase mutant A389V, which rescued editing activity. The pattern of editing was similar to that of the ADAR1-deaminase, despite ADAR3 sharing sequence similarity with ADAR2. Insights into the different patterns of substrate selectivity by ADAR deaminases and the likely causes of these differences, can provide insight for future development of ADAR deaminase constructs for site-directed RNA editing. A pilot experiment for characterizing in vitro editing in complex RNA was also performed, using HEK 293T total RNA and reovirus T1L RNA as substrates for the purified ADAR constructs. An adapted sequencing library preparation was successfully used to identify and count individual editing events in reovirus T1L RNA, and further improvements are required to generate enough editing sites to compare editing frequencies in both substrates to the 50bp in vitro substrate. The goal of characterizing editing in these complex substrates will be to identify RNA secondary structures which are differentially edited by the full-length ADAR1-p110 and ADAR2, when compared to the dsRBD-lacking deaminase constructs

Engineering a feedback-based synthetic gene circuit for targeted continuous evolution of a gene in E. coli

Directed evolution is an invaluable technique for engineering proteins to possess desired physical and chemical properties when very little structural and functional information is known. It is divided into two sequential steps: generating a library of protein variants using mutagenic techniques; and applying a screening or selection strategy to scan the library for variants displaying desired properties. Library generation is performed using either in vitro or in vivo techniques, while screening or selection typically occurs in a suitable host cell. Currently, in vitro methods like error-prone PCR are popular for library generation. However, these techniques can be labour intensive, prone to mutation biases, and generate limited library sizes for screening. In vivo mutagenic techniques overcome these limitations by enabling simultaneous library generation and selection within cells. By generating random mutations in the gene-of-interest within one cell cycle, each cell in a batch culture potentially represents a library variant. Such a continuous evolution system can run for weeks with minimal human intervention, greatly expanding the genetic search space for protein engineering. The challenge lies in developing a mutator system that specifically generates mutations in the target gene, while maintaining the cell’s genomic fidelity. With this goal in mind, a mutator system was engineered in E. coli that introduces targeted cytidine deamination damage and subsequently performs error-prone DNA repair by hijacking the base excision repair pathway. The targeted damage occurs via activation induced cytidine deaminase fused to T7 RNA polymerase, while the error-prone DNA repair is performed by a three-protein fusion comprising a 5’-3’-exonuclease, an AP-endonuclease and an error-prone DNA polymerase. The mutagenic characteristics of this system was tested by knocking out GFP expression and analysing the mutant library using next generation sequencing techniques. The system was also experimentally shown to generate functionally active mutations that reverted inactivated β-lactamase gene variants to confer ampicillin resistance.Open Acces