3,827 research outputs found

    Engineering Systems of Anti-Repressors for Next-Generation Transcriptional Programming

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    The ability to control gene expression in more precise, complex, and robust ways is becoming increasingly relevant in biotechnology and medicine. Synthetic biology has sought to accomplish such higher-order gene regulation through the engineering of synthetic gene circuits, whereby a gene’s expression can be controlled via environmental, temporal, or cellular cues. A typical approach to gene regulation is through transcriptional control, using allosteric transcription factors (TFs). TFs are regulatory proteins that interact with operator DNA elements located in proximity to gene promoters to either compromise or activate transcription. For many TFs, including the ones discussed here, this interaction is modulated by binding to a small molecule ligand for which the TF evolved natural specificity and a related metabolism. This modulation can occur with two main phenotypes: a TF shows the repressor (X+) phenotype if its binding to the ligand causes it to dissociate from the DNA, allowing transcription, while a TF shows the anti-repressor (XA) phenotype if its binding to the ligand causes it to associate to the DNA, preventing transcription. While both functional phenotypes are vital components of regulatory gene networks, anti-repressors are quite rare in nature compared to repressors and thus must be engineered. We first developed a generalized workflow for engineering systems of anti-repressors from bacterial TFs in a family of transcription factors related to the ubiquitous lactose repressor (LacI), the LacI/GalR family. Using this workflow, which is based on a re-routing of the TF’s allosteric network, we engineered anti-repressors in the fructose repressor (anti-FruR – responsive to fructose-1,6-phosphate) and ribose repressor (anti-RbsR – responsive to D-ribose) scaffolds, to complement XA TFs engineered previously in the LacI scaffold (anti-LacI – responsive to IPTG). Engineered TFs were then conferred with alternate DNA binding. To demonstrate their utility in synthetic gene circuits, systems of engineered TFs were then deployed to construct transcriptional programs, achieving all of the NOT-oriented Boolean logical operations – NOT, NOR, NAND, and XNOR – in addition to BUFFER and AND. Notably, our gene circuits built using anti-repressors are far simpler in design and, therefore, exert decreased burden on the chassis cells compared to the state-of-the-art as anti-repressors represent compressed logical operations (gates). Further, we extended this workflow to engineer ligand specificity in addition to regulatory phenotype. Performing the engineering workflow with a fourth member of the LacI/GalR family, the galactose isorepressor (GalS – naturally responsive to D-fucose), we engineered IPTG-responsive repressor and anti-repressor GalS mutants in addition to a D-fucose responsive anti-GalS TF. These engineered TFs were then used to create BANDPASS and BANDSTOP biological signal processing filters, themselves compressed compared to the state-of-the-art, and open-loop control systems. These provided facile methods for dynamic turning ‘ON’ and ‘OFF’ of genes in continuous growth in real time. This presents a general advance in gene regulation, moving beyond simple inducible promoters. We then demonstrated the capabilities of our engineered TFs to function in combinatorial logic using a layered logic approach, which currently stands as the state-of-the art. Using our anti-repressors in layered logic had the advantage of reducing cellular metabolic burden, as we were able to create the fundamental NOT/NOR operations with fewer genetic parts. Additionally, we created more TFs to use in layered logic approaches to prevent cellular cross-talk and minimize the number of TFs necessary to create these gene circuits. Here we demonstrated the successful deployment of our XA-built NOR gate system to create the BUFFER, NOT, NOR, OR, AND, and NAND gates. The work presented here describes a workflow for engineering (i) allosteric phenotype, (ii) ligand selectivity, and (iii) DNA specificity in allosteric transcription factors. The products of the workflow themselves serve as vital tools for the construction of next-generation synthetic gene circuits and genetic regulatory devices. Further, from the products of the workflow presented here, certain design heuristics can be gleaned, which should better facilitate the design of allosteric TFs in the future, moving toward a semi-rational engineering approach. Additionally, the work presented here outlines a transcriptional programming structure and metrology which can be broadly adapted and scaled for future applications and expansion. Consequently, this thesis presents a means for advanced control of gene expression, with promise to have long-reaching implications in the future.Ph.D

    Advances in Micro- and Nanomechanics

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    This book focuses on recent advances in both theoretical and experimental studies of material behaviour at the micro- and nano-scales. Special attention is given to experimental studies of nanofilms, nanoparticles and nanocomposites as well as tooth defects. Various experimental techniques were used. Magneto- and thermoelastic coupling were considered, as were nonlocal models of thin structures

    Advances in Binders for Construction Materials

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    The global binder production for construction materials is approximately 7.5 billion tons per year, contributing ~6% to the global anthropogenic atmospheric CO2 emissions. Reducing this carbon footprint is a key aim of the construction industry, and current research focuses on developing new innovative ways to attain more sustainable binders and concrete/mortars as a real alternative to the current global demand for Portland cement.With this aim, several potential alternative binders are currently being investigated by scientists worldwide, based on calcium aluminate cement, calcium sulfoaluminate cement, alkali-activated binders, calcined clay limestone cements, nanomaterials, or supersulfated cements. This Special Issue presents contributions that address research and practical advances in i) alternative binder manufacturing processes; ii) chemical, microstructural, and structural characterization of unhydrated binders and of hydrated systems; iii) the properties and modelling of concrete and mortars; iv) applications and durability of concrete and mortars; and v) the conservation and repair of historic concrete/mortar structures using alternative binders.We believe this Special Issue will be of high interest in the binder industry and construction community, based upon the novelty and quality of the results and the real potential application of the findings to the practice and industry

    Vulnerability of the Nigerian coast and communities to climate change induced coastal erosion

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    Improving coastal resilience to climate change hazards requires understanding past shoreline changes. As the coastal population grows, evaluation and monitoring of shoreline changes are essential for planning and development. Population growth increases exposure to sea level rise and coastal hazards. Nigeria, where the study is situated, is among the top fifteen countries in the world for coastal population exposure to sea level rise. This study provided a novel lens in establishing a link between social factors and the intensifying coastal erosion along the Akwa Ibom State study coast. The mixed-method approach used in the study to assess the vulnerability of the Nigerian coast and communities to climate change-induced coastal erosion proved to be essential in gathering a wide range of data (physical, socio economic, participatory GIS maps and social learning) that contributed to a more robust and holistic assessment of coastal erosion, which is a complex issue due to the interplay between the human and natural environments. Remotely sensed data was used to examine the susceptibility and coastal evolution of Akwa Ibom State over 36 years (1984 -2020). Longer-term (1984- 2020) and short-term (2015-2020) shoreline change analyses were used to understand coastal erosion and accretion. From 1984-2020, the total average linear regression rate (LRR) was - 2.7+0.18m/yr and from 2015-2020, it was -3.94 +1.28m/yr, demonstrating an erosional trend along the study coast. Although the rate of erosion varies along the study coast, the linear regression rates (LRR) results show a predominant trend of erosion in both the short and longer term. According to the 2022 Intergovernmental Panel on Climate Change report, loss of land, loss of assets, community disruption and livelihood, loss of environmental resources, ecosystem, loss of life, or adverse health impact are all potential risks along the African coast due to climate change – this study shows that these risks are already occurring today. To quantify the anticipated future coastal erosion risk by 2040 along the study coast, the findings in this study show an overall average LRR of -2.73+ 0.99 m/yr which anticipates that coastal erosion will still be prevalent along the coast by 2040. And, given the current global climate change situation, should be expected to be much higher than the current forecasting. This study re-conceptualised the European Environmental Agency Driver-Pressure StateImpact-Response (DPSIR) model to show Hazard-Driver-Pressure-State-Impact ResponseObservation causal linkages to coastal erosion hazards. The results showed how human activities and environmental interactions have evolved through time, causing coastal erosion. Removal of vegetation cover/backstop for residential and agricultural purposes, indicate that human activities significantly contribute to the study area's susceptibility, rapid shoreline changes, and vulnerability to coastal erosion, in addition to oceanic and climate change drivers such as sea level rise and storminess. Risk perception of coastal erosion in the study area was analysed using the rhizoanalytic method proposed by Deleueze. The method demonstrates how connections and movements can be related and how data can be used to show multiplicity, mark and unmark ideas, rupture pre-conceptions and make new connections. This study shows that coastal erosion awareness is insufficient to build a long-term management plan and sustain coastal resilience. The Hino's conceptual model which provides in-depth understanding on planned retreat was used to illustrate migratory and planned retreat for the study coast where relocation has already occurred due to coastal erosion. The result fell within the Self-Reliance quadrant, indicating that people left the risk zone without government backing or retreat plans. Other coastal residents who have not relocated fell within the Hunkered Down quadrant, showing that they are willing to stay in the risk zone and cope with the threat unless the government/environmental agencies relocate them. This study shows that coastal resilience requires adaptive capacity and government support. However, multilevel governance has inhibited government-community dialogue and involvement, increasing coastal erosion vulnerability. The coastal vulnerability index to coastal erosion was calculated using the Analytical Hierarchy Process weightings. It revealed that 67.55% of the study coast falls within the high-very high vulnerability class while 32.45% is within the very low-low vulnerability class. This study developed and combined a risk perception index to coastal erosion (RPIerosion) and participatory GIS (PGIS) mapping into a novel coastal vulnerability index called the integrated coastal erosion vulnerability index (ICEVI). The case study evaluation in Akata, showed an improvement in the overall vulnerability assessment to reflect the real-world scenario, which was consistent with field data. This study demonstrated not only the presence and challenges of coastal erosion in the research area but also the relevance of involvement between the local stakeholders, government and environmental agencies. Thus, showing the potential for the perspectives of the inhabitants of these regions to inform the understanding of the resilience capacity of the people impacted, and importantly to inform future co-design and/or selection of effective adaptation methods, to better support coastal climate change resilience in these communities. Overall, the study provides a useful contribution to coastal erosion vulnerability assessments in data-scarce regions more broadly, where the mixed-methods approach used here can be applied elsewhere

    Ciguatoxins

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    Ciguatoxins (CTXs), which are responsible for Ciguatera fish poisoning (CFP), are liposoluble toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. This book presents 18 scientific papers that offer new information and scientific evidence on: (i) CTX occurrence in aquatic environments, with an emphasis on edible aquatic organisms; (ii) analysis methods for the determination of CTXs; (iii) advances in research on CTX-producing organisms; (iv) environmental factors involved in the presence of CTXs; and (v) the assessment of public health risks related to the presence of CTXs, as well as risk management and mitigation strategies

    Writing Facts

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    »Fact« is one of the most crucial inventions of modern times. Susanne Knaller discusses the functions of this powerful notion in the arts and the sciences, its impact on aesthetic models and systems of knowledge. The practice of writing provides an effective procedure to realize and to understand facts. This concerns preparatory procedures, formal choices, models of argumentation, and narrative patterns. By considering »writing facts« and »writing facts«, the volume shows why and how »facts« are a result of knowledge, rules, and norms as well as of description, argumentation, and narration. This approach allows new perspectives on »fact« and its impact on modernity

    Reasoning in criminal intelligence analysis through an argumentation theory-based framework

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    This thesis provides an in-depth analysis of criminal intelligence analysts’ analytical reasoning process and offers an argumentation theory-based framework as a means to support that reasoning process in software applications. Researchers have extensively researched specific areas of criminal intelligence analysts’ sensemaking and reasoning processes over the decades. However, the research is fractured across different research studies and those research studies often have high-level descriptions of how criminal intelligence analysts formulate their rationale (argument). This thesis addresses this gap by offering low level descriptions on how the reasoning-formulation process takes place. It is presented as a single framework, with supporting templates, to inform the software implementation process. Knowledge from nine experienced criminal intelligence analysts from West Midlands Police and Belgium’s Local and Federal Police forces were elicited through a semi-structured interview for study 1 and the Critical Decision Method (CDM), as part of the Cognitive Task Analysis (CTA) approach, was used for study 2 and study 3. The data analysis for study 1 made use of the Qualitative Conventional Content Analysis approach. The data analysis for study 2 made use of a mixed method approach, consisting out of Qualitative Directed Content Analysis and the Emerging Theme Approach. The data analysis for study 3 made use of the Qualitative Directed Content Analysis approach. The results from the three studies along with the concepts from the existing literature informed the construction of the argumentation theory-based framework. The evaluation study for the framework’s components made use of Paper Prototype Testing as a participatory design method over an electronic medium. The low-fidelity prototype was constructed by turning the frameworks’ components into software widgets that resembled widgets on a software application’s toolbar. Eight experienced criminal intelligence analysts from West Midlands Police and Belgium’s Local and Federal Police forces took part in the evaluation study. Participants had to construct their rationale using the available components as part of a simulated robbery crime scenario, which used real anonymised crime data from West Midlands Police force. The evaluation study made use of a Likert scale questionnaire to capture the participant’s views on how the frameworks’ components aided participants with; understanding what was going on in the analysis, lines-of-enquiry and; the changes in their level of confidence pertaining to their rationale. A non-parametric, one sample z-test was used for reporting the statistical results. The significance is at 5% (α=0.05) against a median of 3 for the z-test, where μ =3 represents neutral. The participants reported a positive experience with the framework’s components and results show that the framework’s components aided them with formulating their rationale and understanding how confident they were during different phases of constructing their rationale

    Enabling high-throughput image analysis with deep learning-based tools

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    Microscopes are a valuable tool in biological research, facilitating information gathering with different magnification scales, samples and markers in single-cell and whole-population studies. However, image acquisition and analysis are very time-consuming, so efficient solutions are needed for the required speed-up to allow high-throughput microscopy. Throughout the work presented in this thesis, I developed new computational methods and software packages to facilitate high-throughput microscopy. My work comprised not only the development of these methods themselves but also their integration into the workflow of the lab, starting from automating the microscopy acquisition to deploying scalable analysis services and providing user-friendly local user interfaces. The main focus of my thesis was YeastMate, a tool for automatic detection and segmentation of yeast cells and sub-type classification of their life-cycle transitions. Development of YeastMate was mainly driven by research on quality control mechanisms of the mitochondrial genome in S. cerevisiae, where yeast cells are imaged during their sexual and asexual reproduction life-cycle stages. YeastMate can automatically detect both single cells and life-cycle transitions, perform segmentation and enable pedigree analysis by determining origin and offspring cells. I developed a novel adaptation of the Mask R-CNN object detection model to integrate the classification of inter-cell connections into the usual detection and segmentation analysis pipelines. Another part of my work focused on the automation of microscopes themselves using deep learning models to detect wings of D. melanogaster. A microscope was programmed to acquire large overview images and then to acquire detailed images at higher magnification on the detected coordinates of each wing. The implementation of this workflow replaced the process of manually imaging slides, usually taking hours to do so, with a fully automated, end-to-end solution
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