1,095 research outputs found

    R-Pyocin Regulation, Release, and Susceptibility in Pseudomonas aeruginosa

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    Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a major determinant of declining lung function in individuals with cystic fibrosis (CF). P. aeruginosa possesses many intrinsic antibiotic resistance mechanisms and isolates from chronic CF lung infections develop increasing resistance to multiple antibiotics over time. Chronic infection with P. aeruginosa remains one of the main causes of mortality and morbidity in CF patients, thus new therapeutic interventions are necessary. R-type pyocins are narrow spectrum, phage tail-like bacteriocins, specifically produced by P. aeruginosa to kill other strains of P. aeruginosa. Due to their specific anti-pseudomonal activity and similarity to bacteriophage, R-pyocins have potential as additional therapeutics for P. aeruginosa, either in isolation, in combination with antibiotics, or as an alternative to phage therapy. There are five subtypes of R-pyocin (types R1-R5), and it is thought that each P. aeruginosa strain uniquely produces only one of these, suggesting a degree of strain-specificity. P. aeruginosa from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. It is known P. aeruginosa populations in CF chronic lung infection become phenotypically and genotypically diverse over time, however, little is known of the efficacy of R-pyocins against heterogeneous populations. Even less is known regarding the timing and regulation of R-pyocins in CF lung infections, or if P. aeruginosa utilizes R-pyocin production during infection for competition or otherwise – which may influence pressure towards R-pyocin resistance. In this work, I evaluated R-pyocin type and susceptibility among P. aeruginosa isolates sourced from CF infections and found that (i) R1-pyocins are the most prevalent R-type among respiratory infection and CF strains; (ii) a large proportion of P. aeruginosa strains lack R-pyocin genes entirely; (iii) isolates from P. aeruginosa populations collected from the same patient at a single time point have the same R-pyocin type; (iv) there is heterogeneity in susceptibility to R-pyocins within P. aeruginosa populations and (v) susceptibility is likely driven by diversity of LPS phenotypes within clinical populations. These findings suggest that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage, which is important for consideration of antimicrobials as therapeutics. To investigate the prevalence of R2-pyocin susceptible strains in CF, I then utilized 110 isolates of P. aeruginosa collected from five individuals with CF to test for R2-pyocin susceptibility and identify LPS phenotypes. From our collection we i) estimated that approximately 83% of sputum samples contain heterogenous P. aeruginosa populations without R2-pyocin resistant isolates and all sputum samples contained susceptible isolates; ii) we found that there is no correlation between R2-pyocin susceptibility and LPS phenotypes, and iii) we estimate that approximately 76% of isolates sampled from sputum lack O-specific antigen, 42% lack common antigen, and 27% exhibit altered LPS cores. This finding highlights that perhaps LPS packing density may play a more influential role in mediating R-pyocin susceptibility in infection. Finding the majority of our sampled P. aeruginosa populations to be R2-pyocin susceptible further supports the potential of these narrow-spectrum antimicrobials despite facing heterogenous susceptibility among diverse populations. In order to evaluate how R-pyocins may influence strain competition and growth in CF lung infection, I assessed R-pyocin activity in an infection-relevant environment (Synthetic Cystic Fibrosis Sputum Medium; SCFM2) and found that (i) R-pyocins genes are transcribed more in the CF nutrient environment than in rich laboratory medium and (ii) in a structured, CF-like environment, R-pyocin induction is costly to producing strains in competition rather than beneficial. Our work suggests that R-pyocins may not be essential in CF lung infection and can be costly to producing cells in the presence of stress response-inducing stimuli, such as those commonly found in infection. In this thesis I have studied R-pyocin susceptibility, regulation and release utilizing a biobank of whole populations of P. aeruginosa collected from 11 individuals with CF, as well as the CF infection model (SCFM) to understand the mechanisms of R-pyocin activity in an infection-relevant context and the role R-pyocins play in shaping P. aeruginosa populations during infection. The findings of this work have illuminated the impact of P. aeruginosa heterogeneity on R-pyocin susceptibility, furthered our understanding of R-pyocins as potential therapeutics, and built upon our knowledge of bacteriocin-mediated interactions.Ph.D

    Exploring communication and collective behaviour between spatially organised inorganic protocell communities

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    A living system profoundly relies on mass, information and energy interactions through cell-cell and cell-environment networks. As a step towards understanding such interactions, it is beneficial to design and create bottom-up artificial living systems from non-living components, with a specific focus on synergistic interactivity between artificial cells (protocells) and their local environment. Although there are several routes for fabricating protocellular systems, we recognise key challenges associated with a) developing protocellular models with high levels of organisational tunability, b) achieving cell-environment bilateral communication, and c) realising autonomous self-assembly and regulation of protocell systems. The aim of this thesis is thus to review some matrix-based and matrix-free methods of inorganic protocell (colloidosome) 3D-spatial organisation, as judicious system designs capable of cell-cell and cell-environment communication, collective behaviours, and dynamic self-assembly, in close relation with local environments.The first experimental chapter details assembly of colloidosomes within hydrogel or coacervate-based matrices. A droplet microfluidic technique is employed as a novel method for encapsulating segregated colloidosome colonies within alginate hydrogel microspheres. The technique exploits high tunability for customisable size, ratio, microscale geometry, and 3D-patterning parameters. Benefiting from the versatility associated with such matrix-based systems, the second experimental chapter develops 3D-organised colloidosomes for collective signalling and emergent behaviours. Notably, spatially segregated colonies show proximity-mediated chemical communication with increased kinetics compared to analogous homogenous arrangements. This proximity-enhanced colloidosome signalling is exploited, alongside segregated ionic/covalent crosslinking transitions in the environment, to obtain simultaneous structural degradation and resilience of hydrogel hemispheres as a programmable mechanism for protocell ejection. Colloidosomes are also employed as simple signalling hotspots within coacervate-matrix systems. The final experimental chapter aims to re-imagine colloidosome organisation into a matrix-free system, capable of dynamic self-assembly and self-sorting via electrostatically-active membrane appendages. Alginate-coated and chitosan-coated colloidosomes are either co-assembled or self-sorted, in response to varied pH environments. Again, these systems are highly coordinated with their environment and as such, can be spatially pattered according to temporal pH changes through endogenous enzyme catalysis. Furthermore, a spatiotemporal effect on the rate of colloidosome communication in the presence of a hostile guest molecule is demonstrated. <br/

    (b2023 to 2014) The UNBELIEVABLE similarities between the ideas of some people (2006-2016) and my ideas (2002-2008) in physics (quantum mechanics, cosmology), cognitive neuroscience, philosophy of mind, and philosophy (this manuscript would require a REVOLUTION in international academy environment!)

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    (b2023 to 2014) The UNBELIEVABLE similarities between the ideas of some people (2006-2016) and my ideas (2002-2008) in physics (quantum mechanics, cosmology), cognitive neuroscience, philosophy of mind, and philosophy (this manuscript would require a REVOLUTION in international academy environment!

    A War of Words: The Forms and Functions of Voice-Over in the American World War II Film — An Interdisciplinary Analysis

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    Aside from being American World War II films, what else do the following films have in common? The Big Red One; Hacksaw Ridge; Harts War; Mister Roberts; Stalag 17; and The Thin Red Line — all have voice-over in them. These, and hundreds of other war films have voice-overs that are sometimes the thoughts of a fearful soldier; the wry observations of a participant-observer; or the declarations of all-knowing authoritative figures. There are voice-overs blasted out through a ships PA system; as the reading of a heart-breaking letter; or as the words of a dead comrade, heard again in the mind of a haunted soldier. This thesis questions why is voice-over such a recurring phenomenon in these films? Why is it conveyed in so many different forms? What are the terms for those different forms? What are their narrative functions? A core component of this thesis is a new taxonomy of the six distinct forms of voice-over: acousmatic, audioemic, epistolary, objective, omniscient, and subjective. However, the project is more than a structuralist taxonomy that merely serves to identify, and define those forms. It is also a close examination of their narrative functions beyond the unimaginative trope that voice-over in war films is simply a convenient storytelling device. Through interdisciplinarity — combined with a realist framework — I probe the correlations between: the conditions, codification, and suppression of speech within the U.S. military, and the manifestations of that experience through the cinematic device, and genre convention of voice-over. In addition, I present a radically new interpretation of the voice-overs in The Thin Red Line (Terrence Malick, 1998) as being both a choric meta-memorial to James Jones; and a Greek tragedy — with its replication of the stagecraft of Aeschylus, in its use of the cosmic frame, and the inclusion of a collective character, which I have named ‘The Chorus of Unknown Soldiers’. The overall result is a more logical, and nuanced explanation of the forms, functions, and prevalent use of voice-over in the American World War II film

    Structural optimization in steel structures, algorithms and applications

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    L'abstract è presente nell'allegato / the abstract is in the attachmen

    Developing a novel and versatile approach to study populations of microbes on surfaces

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    Spatial structure, for example regarding antibiotic gradients, is an important topic of investigation in microbial ecology and evolution. Experiments investigating pop- ulation dynamics in spatially-structured environments are often performed on agar plates. Whilst inexpensive and straightforward, these provide only rudimentary temporal and spatial control of environmental conditions. In chemostats and microfluidic devices, for well-mixed and micrometre-scale environments, respectively, regulating media inflow and outflow enables environ- mental control. We combine proven use of agar surfaces with such flow-enabled control in a novel, low-cost fluidic device; the device comprises an elastomer supporting base with a thin agar sheet on top on which microbes grow. Indented channels in the base allow flow of media/antibiotics below the agar surface. A Raspberry-Pi-operated camera allows for time-lapse imaging suitable for quantita- tive image analysis. As a proof of principle, we used our device for extended and robust growth of non-motile E. coli and motile P. aeruginosa maintaining the initial speed with which colonies propagate over three days, whilst a continual speed decrease occurred on agar plates. Guided by simulations of flow and diffusion, we then used the device to create stable antibiotic gradients within the agar. Along these gradients, we found P. aeruginosa exhibit unique microbial growth patterns with local adaptations. Because flow below the agar surface can be controlled spatially and temporally, the device promises a range of applications for studying microbial ecology and evolution in spatially continuous environments at a substrate-air interface.Engineering and Physical Sciences Research Council (EPSRC

    Realisms and the Body after War: Document, Truth, and Critique in Postrevolutionary Mexico and Weimar Germany

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    This thesis examines realisms and representations of the body in postrevolutionary Mexico, using the critical framework surrounding Neue Sachlichkeit and post-World War I Weimar society as a comparative model. Through imagery of soldiers, women, and class disparity, artists utilized realisms to relay record, suggest truth, and create criticism

    Animals in Dutch travel writing, 1800-present

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    Apart from humans, animals play a pivotal role in travel literature. However, the way they are represented in texts can vary from living companions to metaphorical entities. Existing studies mainly focus on the representation of conventional or unconventional roles that are assigned to animals from around the Napoleonic age until now, roles that have been subject to change and that tell us a lot about human reflections on encounters with non-human creatures and the position of man in this rapidly changing world. In this edited volume, scholars from the Netherlands and abroad analyse the roles that animals play in Dutch travel literature from 1800 to the present. In this way, we aim to provide new insights into the relationships between man and animals, in textual expressions and real life, and to add the ‘Dutch case’ to the flourishing international field of travel writing studies

    Biophysical modelling of bacterial colonisation of urinary catheters

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    I developed the first mathematical model for the infection dynamics of a urinary catheter. Urinary catheters are thin tubes which are inserted into the urethra to drain the bladder. They are commonly used for patients undergoing surgery under anaesthesia, or for elderly patients in long term care facilities. Unfortunately, urinary catheters develop bacterial infections at rates up to 5% per catheter day, with potential complications including catheter blockage, bladder damage, and kidney infections. Attempts to prevent or mitigate infections have been largely ineffective, and through mathematical modelling I seek to understand why. The primary aim of my PhD was to uncover routes by which techniques and approaches from physics can contribute to tackling catheter-associated urinary tract infections (CAUTI). I have provided new clinical insight by developing a novel population dynamics model and applying it to reveal the key factors determining how, why, and when catheters get infected. CAUTI are complex phenomena, involving the host physiology in the urethra and bladder, multiple catheter surfaces, urine flow dynamics within the catheter lumen (the inner channel), and bacterial growth and behaviour. Guided by discussions with clinical collaborators, I considered this system (of catheter, host, and colonising bacteria) as a set of smaller subsystems. I divided the model into 4 subsystems, in which bacteria first grow and colonise the extraluminal surface (the outside of the catheter), before spreading into the bladder. The bacteria grow in the urine in the bladder, and then are swept down through the catheter lumen, where finally some adhere on the intraluminal surface. I then identified suitable mathematical descriptions for the change in the bacterial populations. These descriptions take the form of Fisher, logistic growth, and convection-diffusion equations, which, using insight from clinicians, I coupled together. I implemented this model computationally by developing code (in C++ and Python) to numerically solve the coupled equations, applying this to explore the effects of varying the properties of the catheter, host, and bacteria. I found the rate of urine production by the kidneys to be critical in determining the outcome of bacterial infection, as it governs a transition between a high bacterial density state in the bladder and a ‘washed-out’ state whereby bacteria may grow on the catheter surfaces, but there is no bacteriuria (no bacterial growth in the urine). These results are highly significant for the prevention and mitigation of CAUTI, as they imply that increasing fluid intake may reduce the likelihood of bacteriuria. I also discovered that the urethral length determines the timescale over which infection may occur, giving important insight into observed gendered differences in infection rates in short-term catheterisation. Finally, my model suggests an avenue for future work to investigate the origin of infections, by studying bacterial distributions across the catheter surface, and comparing with modelled distributions to determine the initial conditions. I interpreted the model predictions in the context of clinical data, finding new perspectives on both in vitro and in vivo previous studies. By considering how clinical interventions correspond to changes in model parameters, I classified clinical interventions as postponement, mitigation, or prevention, and discussed the contexts in which those interventions might be effective. I applied the model to predict the outcome of a clinical trial of catheter interventions, showing that the model provides quantitatively better fits to clinical data than previous fits applied in the literature, and successfully qualitatively predicted trial outcomes, including identifying an outcome (reduced incidence of bacteriuria associated with the use of silver-alloy catheters in males) that was present in the study dataset, but not discussed in the original study. My work suggests physical mechanisms that explain clinical observations, demonstrating how from basic assumptions many complex phenomena emerge. Through the development of a population dynamics model with direct clinical implications, I applied physics to make an important contribution to CAUTI research
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