2,065 research outputs found
Ruthenium metallotherapeutics: a targeted approach to combatting multidrug resistant pathogens
The discovery of antibiotics revolutionised healthcare practice. However due to overuse, inappropriate use, widespread prophylaxis therapy and the lack of new developments, the threat of antimicrobial resistance is now a major global threat to health. By 2050, it is estimated that mortality due to antimicrobial resistant infections will exceed 10 million people per annum, superseding cancer as the leading cause of global mortality. The use of drug repurposing to identify potential therapies which combat antimicrobial resistance is one potential solution. Metals have been used as antimicrobial agents throughout the history of medicine for a broad range of applications, including the use of Silver as an antimicrobial agent which dates back to antiquity. More recently, Ruthenium metallotherapeutic complexes have been shown to exhibit highly active antimicrobial properties by targeting a range of bacterial species, and in contrast to traditional antibiotics, these compounds are thought to elicit antibacterial activity at multiple sites within the bacterial cell, which may reduce the possibility of resistance evolution. This study aimed to evaluate the antimicrobial activity of a series of Ruthenium metallotherapeutic complexes against multidrug-resistant bacterial pathogens, with a focus on use within wound care applications.
Antimicrobial susceptibility assays identified two lead candidates, Hexaammineruthenium (III) chloride and [Chlorido(η6-p-cymene)(N-(4-chlorophenyl)pyridine-2-carbothioamide) ruthenium (II)] chloride which demonstrated activity against Pseudomonas aeruginosa and Staphylococcus aureus respectively with MIC values ranging between 4 μg mL-1 and 16 μg mL-1. Furthermore, Hexaammineruthenium (III) chloride demonstrated antibiofilm activity in both a time and concentration-dependent manner. Synergy studies combining lead complexes with antibiotics demonstrated the potential for use as resistance breakers. Subsequent in vitro infection modelling using scratch assays with skin cell lines, coupled with a 3D full thickness skin wound infection model was used to determine potential applied applications of Hexaammineruthenium (III) chloride for use as topical antimicrobial agent against P. aeruginosa infections.
Antimicrobial mechanistic studies demonstrated that Hexaammineruthenium (III) chloride targeted the bacterial cell ultrastructure of P. aeruginosa strain PAO1 as cell perturbations were observed when treated cells were analysed by scanning electron microscopy. Furthermore, exposure of P. aeruginosa PAO1 to Hexaammineruthenium (III) chloride also resulted in a concentration dependent membrane depolarisation, which further supported the antimicrobial mechanistic role.
Finally, global changes in gene expression following exposure of P. aeruginosa strain PAO1 to Hexaammineruthenium (III) chloride were explored by RNA sequencing. Genes involved in ribosome function, cofactor biosynthesis and membrane fusion were downregulated, which provided a further insight into the wider mechanisms of antibacterial activity.
The research conducted in the present study indicated the potential use of Hexaammineruthenium (III) chloride (and derivatives) as a potential treatment option for chronic wounds infected with P. aeruginosa, which could be applied as either a direct treatment or used within antimicrobial wound care applications
Analysis and monitoring of single HaCaT cells using volumetric Raman mapping and machine learning
No explorer reached a pole without a map, no chef served a meal without tasting, and no surgeon implants untested devices. Higher accuracy maps, more sensitive taste buds, and more rigorous tests increase confidence in positive outcomes. Biomedical manufacturing necessitates rigour, whether developing drugs or creating bioengineered tissues [1]–[4]. By designing a dynamic environment that supports mammalian cells during experiments within a Raman spectroscope, this project provides a platform that more closely replicates in vivo conditions. The platform also adds the opportunity to automate the adaptation of the cell culture environment, alongside spectral monitoring of cells with machine learning and three-dimensional Raman mapping, called volumetric Raman mapping (VRM). Previous research highlighted key areas for refinement, like a structured approach for shading Raman maps [5], [6], and the collection of VRM [7]. Refining VRM shading and collection was the initial focus, k-means directed shading for vibrational spectroscopy map shading was developed in Chapter 3 and exploration of depth distortion and VRM calibration (Chapter 4). “Cage” scaffolds, designed using the findings from Chapter 4 were then utilised to influence cell behaviour by varying the number of cage beams to change the scaffold porosity. Altering the porosity facilitated spectroscopy investigation into previously observed changes in cell biology alteration in response to porous scaffolds [8]. VRM visualised changed single human keratinocyte (HaCaT) cell morphology, providing a complementary technique for machine learning classification. Increased technical rigour justified progression onto in-situ flow chamber for Raman spectroscopy development in Chapter 6, using a Psoriasis (dithranol-HaCaT) model on unfixed cells. K-means-directed shading and principal component analysis (PCA) revealed HaCaT cell adaptations aligning with previous publications [5] and earlier thesis sections. The k-means-directed Raman maps and PCA score plots verified the drug-supplying capacity of the flow chamber, justifying future investigation into VRM and machine learning for monitoring single cells within the flow chamber
The development of liquid crystal lasers for application in fluorescence microscopy
Lasers can be found in many areas of optical medical imaging and their properties have enabled the rapid advancement of many imaging techniques and modalities. Their narrow linewidth, relative brightness and coherence are advantageous in obtaining high quality images of biological samples. This is particularly beneficial in fluorescence microscopy. However, commercial imaging systems depend on the combination of multiple independent laser sources or use tuneable sources, both of which are expensive and have large footprints. This thesis demonstrates the use of liquid crystal (LC) laser technology, a compact and portable alternative, as an exciting candidate to provide a tailorable light source for fluorescence microscopy.
Firstly, to improve the laser performance parameters such that high power and high specification lasers could be realised; device fabrication improvements were presented. Studies exploring the effect of alignment layer rubbing depth and the device cell gap spacing on laser performance were conducted. The results were the first of their kind and produced advances in fabrication that were critical to repeatedly realising stable, single-mode LC laser outputs with sufficient power to conduct microscopy. These investigations also aided with the realisation of laser diode pumping of LC lasers. Secondly, the identification of optimum dye concentrations for single and multi-dye systems were used to optimise the LC laser mixtures for optimal performance. These investigations resulted in novel results relating to the gain media in LC laser systems. Collectively, these advancements yielded lasers of extremely low threshold, comparable to the lowest reported thresholds in the literature.
A portable LC laser system was integrated into a microscope and used to perform fluorescence microscopy. Successful two-colour imaging and multi-wavelength switching ability of LC lasers were exhibited for the first time. The wavelength selectivity of LC lasers was shown to allow lower incident average powers to be used for comparable image quality. Lastly, wavelength selectivity enabled the LC laser fluorescence microscope to achieve high enough sensitivity to conduct quantitative fluorescence measurements. The development of LC lasers and their suitability to fluorescence microscopy demonstrated in this thesis is hoped to push towards the realisation of commercialisation and application for the technology
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Policy options for food system transformation in Africa and the role of science, technology and innovation
As recognized by the Science, Technology and Innovation Strategy for Africa – 2024 (STISA-2024), science, technology and innovation (STI) offer many opportunities for addressing the main constraints to embracing transformation in Africa, while important lessons can be learned from successful interventions, including policy and institutional innovations, from those African countries that have already made significant progress towards food system transformation. This chapter identifies opportunities for African countries and the region to take proactive steps to harness the potential of the food and agriculture sector so as to ensure future food and nutrition security by applying STI solutions and by drawing on transformational policy and institutional innovations across the continent. Potential game-changing solutions and innovations for food system transformation serving people and ecology apply to (a) raising production efficiency and restoring and sustainably managing degraded resources; (b) finding innovation in the storage, processing and packaging of foods; (c) improving human nutrition and health; (d) addressing equity and vulnerability at the community and ecosystem levels; and (e) establishing preparedness and accountability systems. To be effective in these areas will require institutional coordination; clear, food safety and health-conscious regulatory environments; greater and timely access to information; and transparent monitoring and accountability systems
Beam scanning by liquid-crystal biasing in a modified SIW structure
A fixed-frequency beam-scanning 1D antenna based on Liquid Crystals (LCs) is designed for application in 2D scanning with lateral alignment. The 2D array environment imposes full decoupling of adjacent 1D antennas, which often conflicts with the LC requirement of DC biasing: the proposed design accommodates both. The LC medium is placed inside a Substrate Integrated Waveguide (SIW) modified to work as a Groove Gap Waveguide, with radiating slots etched on the upper broad wall, that radiates as a Leaky-Wave Antenna (LWA). This allows effective application of the DC bias voltage needed for tuning the LCs. At the same time, the RF field remains laterally confined, enabling the possibility to lay several antennas in parallel and achieve 2D beam scanning. The design is validated by simulation employing the actual properties of a commercial LC medium
Inmovilización de enzimas en MOFs: diseño y aplicaciones
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de Lectura: 24-02-202
Single-molecule detection and characterisation of alpha-synuclein aggregates
Aberrant protein aggregation is a predominant feature of many neurodegenerative disorders.
It has long been recognised that aggregates of alpha-synuclein (α-syn) drive pathogenesis in
Parkinson’s Disease (PD), and it is widely accepted that small α-syn oligomers are the key
cytotoxic species in PD. Notably, however, these oligomeric species are difficult to characterise
using traditional biochemical ensemble methods due to their high level of heterogeneity
and low abundance. Single-molecule fluorescence microscopy techniques have emerged
as a suitable approach to circumventing this problem, enabling the detection of individual
aggregates amongst monomeric protein and thus facilitating the identification, quantification,
and characterisation of rare oligomeric species. However, cellular mechanisms of α-syn aggregation
are poorly understood. Furthermore, there remains some limitations to the singlemolecule
techniques currently available. This thesis describes the work completed to address
some of these issues.
Chapter 1 provides the contextual background for the work presented in this thesis, detailing
the biological aspects of α-syn, its aggregation, and its implications in PD, as well as outlining
the single-molecule techniques used to investigate aggregate species. Chapter 2 describes
the methodologies undertaken in this thesis, and chapters 3 to 5 describe the findings made
using the single-molecule techniques which were utilised and developed in this work.
One primary approach for studying species in single-molecule experiments involves directly
labelling biomolecules of interest with a suitable fluorophore. Early steps in α-syn aggregation
have previously been identified using fluorescently tagged α-syn and single-molecule Förster
resonance energy transfer (smFRET) in vitro; however, the characterisation of early aggregate
formation in cells has thus far been difficult to achieve. Chapter 3 describes the use of duallabelled
α-syn to detect and characterise aggregates formed both intracellularly and in vitro
via smFRET, using both single-molecule confocal microscopy coupled with microfluidics and
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total internal reflection fluorescence microscopy (TIRFM) to determine both the sizes and
structures of the oligomers formed. This work reveals the presence of distinct oligomeric
species in vitro and in neurons resulting from structural conversion during early aggregate
formation.
The approach taken in Chapter 3 is highly suitable for investigating aggregate formation
resulting from the addition of exogenous α-syn to samples of interest. However, such an
approach is not ideal for the detection and characterisation of endogenous aggregates due to
issues with the covalent labelling of cellular protein. Extrinsic amyloid dyes are typically used
as an alternative approach to labelled protein; however, such dyes are non-protein-specific
and bind to the common amyloid beta-sheet motif. As an alternative, the work presented in
Chapter 4 describes a novel single-molecule method to specifically detect and characterise
α-syn aggregates with high sensitivity, making use of a high-affinity antibody labelled with
orthogonal fluorophores which is combined with fast-flow microfluidics and single-molecule
confocal microscopy. This enables the quantification and size approximation of α-syn aggregates
at picomolar concentrations, both in vitro and in biological samples.
Although the kinetics of α-syn aggregation have been studied extensively, much of our current
knowledge stems from ensemble averaging techniques which are associated with high levels
of variability and are not conducive to detecting the earliest steps in aggregate formation.
In addition, there remains uncertainty surrounding the effect of familial variants and posttranslational
modifications (PTM) on aggregation. Chapter 5 encompasses the study of the effects
of the ubiquitous N-terminal acetylation PTM, in addition to the familial, rapid-onset G51D
mutation, on α-syn aggregation, using the novel detection method developed in Chapter 4.
This is used in conjunction with single-molecule detection with thioflavin-T (ThT) to reveal new
insights into the aggregation of α-syn variants.
Overall, the work presented here provides new insights into the aggregation of α-syn via the
use and development of single-molecule techniques. The advancements made have added
to the current understanding of the molecular mechanisms of α-syn aggregation, both in
vitro and in neurons, and have also been used to develop a novel single-molecule detection
method for α-syn aggregates. The work presented in this thesis has resulted in two published
papers, ’Pathological structural conversion of alpha-synuclein at the mitochondria induces
neuronal toxicity’ in Nature Neuroscience, and ’Single-molecule two-color coincidence detection
of unlabeled alpha-synuclein aggregates’ in Angewandte Chemie International Edition.
Furthermore, the novel detection method presented here holds promise for measuring α-syn
oligomeric load in clinical samples due to its high sensitivity and specificity for α-syn aggregates.
This may therefore be used in future studies for identifying, detecting, and studying
potential biomarkers in PD, with potential use in disease diagnosis. It is therefore expected
that the work from this thesis will be used to aid researchers towards better understanding the
mechanisms of α-syn aggregation, both in vitro and in clinical samples
Science and Innovations for Food Systems Transformation
This Open Access book compiles the findings of the Scientific Group of the United Nations Food Systems Summit 2021 and its research partners. The Scientific Group was an independent group of 28 food systems scientists from all over the world with a mandate from the Deputy Secretary-General of the United Nations. The chapters provide science- and research-based, state-of-the-art, solution-oriented knowledge and evidence to inform the transformation of contemporary food systems in order to achieve more sustainable, equitable and resilient systems
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