5,137 research outputs found

    Exploring transition metal catalysis in water for <i>in vivo </i>applications

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    Transition metal catalysis proves a powerful tool to achieve otherwise synthetically challenging, or even impossible, transformations with (high) selectivity and is therefore employed in various areas of chemistry. Recently, transition metal-catalysed reactions have been successfully performed in cells (in vitro) and living systems (in vivo). The achievements made thus far reveal the potential of transition metal catalysis and its applications in such biological settings. Interestingly, the scope is limited compared to the breadth of transition metal-catalysed reactions that have been unlocked for synthetic applications. Translating transition metal-catalysed reactions from flasks to cells is non-trivial as the conditions in cells are fairly different compared to the highly controlled and adaptable conditions achieved in a flask. The development of catalytic systems for future applications in vivo therefore proceeds through many steps, starting with evaluating their reactivity, selectivity, and stability in water and under biologically relevant and biomimetic conditions. By exploring transition metal-catalysed reactions in water for in vivo applications, this dissertation has contributed to the subfield of bioorthogonal chemistry devoted to complementing Nature’s repertoire of reactions. Our studies have revealed the challenges associated with the performance of transition metal catalysis in aqueous media and how a detailed understanding of a catalytic system can address them. Apart from these fundamental studies, we have performed explorative studies under biologically relevant and biomimetic conditions in the context of intracellular drug synthesis. Moreover, we have developed a new and compatible protocol that enables detailed kinetic studies in complex reaction media, comparable to the cellular environment, to facilitate the translation of transition metal catalysis from flasks to cells

    Application of disposable chiral plasmonics for biosensing and Raman spectroscopy

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    This thesis explores the capabilities of disposable chiral plasmonic metafilm assays, termed Disposable Plasmonic Assays, as a promising platform for biosensing and surface-enhanced Raman spectroscopy. The sensing and Raman properties of these metafilms arise from the excitation of surface plasmons when exposed to incident light. These plasmonic properties strongly depend on the geometric characteristics of the constituent nanostructures found in the metafilms. Specifically, the primary nanostructure employed throughout this research is the chiral 'shuriken' star, which generates chiral electromagnetic fields exhibiting greater chiral asymmetry than circularly polarized light. Monitoring changes in the resonance positions of the characteristic optical rotatory dispersion spectra produced by the Disposable Plasmonic Assays allows for the observation of surface binding events. By measuring resonance shift data and through the utilisation of various gold film functionalisation techniques, these assays are demonstrated as versatile, label-free biosensing platforms capable of specifically detecting a wide range of target proteins and virus particles from complex solutions. Furthermore, the multiplexing performance of these assays is showcased, enabling the detection of multiple different antigens and virions in a single experiment. These results highlight the potential of plasmonic metafilms as rapid and disposable point-of-care immunoassays for diagnostic applications. In addition to biosensing, the chiral geometry of Disposable Plasmonic Assays is exploited for the chiral discrimination of metal nanoparticles and small molecules using Surface Enhanced Raman Spectroscopy (SERS). By linking helicoid shaped gold nanoparticles to the metafilm surface via a dithiol linker, the chiral properties of both nanoparticles and metafilms combine, resulting in the creation of differential electromagnetic 'hotspot' regions based on their symmetry combinations. The electromagnetic intensity in these regions corresponds to the SERS signal obtained from the achiral dithiol linker molecule, facilitating a deeper understanding of the chirally dependent SERS phenomenon. These findings serve to validate and explain the differential SERS data obtained enantiomers of biomolecules and drug molecules from silver modified Disposable Plasmonic Assays

    Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies

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    Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies

    Pathophysiological role and therapeutic potential of extracellular vesicles in cancer

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    Extracellular vesicles (EVs) are nanosized lipid bilayer vesicles that are endogenously generated through various biogenesis pathways within most cellular entities. Subsequently, they are released into the extracellular milieu to facilitate intercellular communication. They are composed of diverse bioactive molecules with important roles in physiological and pathological states. Over the past few decades, the therapeutic potential of EVs has garnered significant interest in the drug delivery field. However, deepened understanding of EV biology and further technological advances are needed to bridge the gap between research and clinical translation. In this thesis, we address these challenges and investigate EVs as novel biomedical agents. EVs are crucial components of physiological processes and disease development. Sensitive visualisation techniques are needed to better understand their function as therapeutic agents. In paper I, a bioluminescent labelling system was developed to track EVs in vitro and in vivo. The system uses genetic modifications to enable the encapsulation of sensitive luciferase-variants in EVs. The system was used in vivo to enable highly sensitive detection of EV distribution pattern. Exogenously administered EVs were found to rapidly distribute within different organs, with a preference for the spleen, lung, and liver. In addition to endogenously engineered EVs for in vivo tracking, exogenously engineered EVs can be utilised as promising drug delivery platforms. However, cargo loading is often insufficient, requiring improved EV loading approaches. In paper II, we developed an optimised cargo loading method using electroporation. An optimised protocol was designed to load EVs with doxorubicin, which increased cargo loading, EV recovery, and drug potency by 190-fold over free doxorubicin. Owing to their potential to cross biological barriers, transport bioactive cargo, and targetability, EVs can be exploited as delivery vehicles for targeting of therapeutics. EVs were used as delivery vectors in paper III by coating their surfaces with an Fc domain-specific antibodybinding moiety. These Fc-EVs were then decorated with various IgG antibodies and targeted to cells of interest. In vitro and in vivo antibody targeting studies showed the broad potential of this technology for cancer therapy. The platform efficiently targeted EVs to cancer cells, including HER2 and PD-L1 positive cells. As proof of concept, Fc-EVs with PD-L1 antibody accumulate in tumour tissue and, when loaded with doxorubicin, reduce tumour burden, and increase survival in melanoma-bearing mice. Despite significant EV engineering advances, we have a limited understanding of the biology of tumour-derived extracellular vesicles (tEVs). In paper IV, we investigated the role of in vitrogenerated melanoma-derived EVs as indirect communicators in tumour-induced haematopoiesis dysregulation. The tEVs, which contain high levels of angiogenic factors like VEGF, osteopontin, and tissue factor, were found to cause splenomegaly, extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and anaemia in syngeneic mice. These findings suggest that tEVs dysregulate haematopoiesis during the immune escape phase of cancer immunoediting, making them potential targets for overcoming immune evasion and restoring normal haematopoiesis. To summarise, the tools generated in this thesis, including the ability to detect EVs in vivo, effective cargo loading, display antibody binding moieties on EV surfaces for targeting, and understanding the pathophysiological role of tEVs, contribute to the advancement of EVs for biomedical purposes, and clinical translation down the line

    Design and Optimisation of a Modular Industrial Air Scrubber Utilising Surfactant Absorption Technology

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    A novel air scrubbing technology using a misting application and water additives has successfully demonstrated the capability of scrubbing H2S at a water treatment plant. The air scrubber design utilises a solution of potable water and a water additive that exploits chemical surfactant technology as the scrubbing agent. The air scrubber equipment is modular in design, so that its performance can be tailored to the application in the testing environment. The modularity aspect allows for customisation to benefit the technology in high and low pollutant concentrations and with a range of air volume flow rates. The design allows for retrofit in existing systems for air scrubbing, and can be used in series or parallel for increased flow rate and removal efficiency. The technology of surfactant induced absorption with water is able to be studied by the ability to vary the water flow rate to a rotary atomiser, and alter the exposure time of water droplets to polluted air streams over a range of air flow rates, of 5 seconds exposure and above. The testing site proved to be a challenging area to utilise for the research, with the fluctuating properties of the pollutant gas streams, the multiple gas species and by-products present, and changing environmental factors. The first test results of air scrubbing was observed at an air flow rate of 1000 m3 per hour, with 200 litres per hour of water and scrubbing additive at a concentration of 1.25%. The obtained absorption efficiency was 21.9%, where the polluted gas at the inlet had a concentration of H2S in excess of 2000 PPM. Further tests of the air scrubber resulted in removal efficiency performances of 55% where the inlet H2S concentration was at 500 PPM. The scrubbing additive was reduced to a 0.5% concentration into 200 litres per hour of water, and an air flow rate of 1000 m3 per hour. Despite the low H2S removal efficiency calculated in the testing of the design, considerable efficiency gains can be made by optimising several features. These would include improvements in sealing joints between parts in the modular design, improving the atomisation method and reaction chamber to further prolong the water droplet lifespan increasing absorption capacity, and research and testing to the effects on surfactant additives and concentrations for increased absorption efficiency. This project has shown promising results, highlighted areas for further analysis, and testing should be performed to further validate the efficacy of the technology and the design

    Raman Spectroscopy Techniques for the Detection and Management of Breast Cancer

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    Breast cancer has recently become the most common cancer worldwide, and with increased incidence, there is increased pressure on health services to diagnose and treat many more patients. Mortality and survival rates for this particular disease are better than other cancer types, and part of this is due to the facilitation of early diagnosis provided by screening programmes, including the National Health Service breast screening programme in the UK. Despite the benefits of the programme, some patients undergo negative experiences in the form of false negative mammograms, overdiagnosis and subsequent overtreatment, and even a small number of cancers are induced by the use of ionising radiation. In addition to this, false positive mammograms cause a large number of unnecessary biopsies, which means significant costs, both financially and in terms of clinicians' time, and discourages patients from attending further screening. Improvement in areas of the treatment pathway is also needed. Surgery is usually the first line of treatment for early breast cancer, with breast conserving surgery being the preferred option compared to mastectomy. This type of operation achieves the same outcome as mastectomy - removal of the tumour - while allowing the patient to retain the majority of their normal breast tissue for improved aesthetic and psychological results. Yet, re-excision operations are often required when clear margins are not achieved, i.e. not all of the tumour is removed. This again has implications on cost and time, and increases the risk to the patient through additional surgery. Currently lacking in both the screening and surgical contexts is the ability to discern specific chemicals present in the breast tissue being assessed/removed. Specifically relevant to mammography is the presence of calcifications, the chemistry of which holds information indicative of pathology that cannot be accessed through x-rays. In addition, the chemical composition of breast tumour tissue has been shown to be different to normal tissue in a variety of ways, with one particular difference being a significant increase in water content. Raman spectroscopy is a rapid, non-ionising, non-destructive technique based on light scattering. It has been proven to discern between chemical types of calcification and subtleties within their spectra that indicate the malignancy status of the surrounding tissue, and differentiate between cancerous and normal breast tissue based on the relative water contents. Furthermore, this thesis presents work aimed at exploring deep Raman techniques to probe breast calcifications at depth within tissue, and using a high wavenumber Raman probe to discriminate tumour from normal tissue predominantly via changes in tissue water content. The ability of transmission Raman spectroscopy to detect different masses and distributions of calcified powder inclusions within tissue phantoms was tested, as well as elucidating a signal profile of a similar inclusion through a tissue phantom of clinically relevant thickness. The technique was then applied to the measurement of clinically active samples of bulk breast tissue from informed and consented patients to try to measure calcifications. Ex vivo specimens were also measured with a high wavenumber Raman probe, which found significant differences between tumour and normal tissue, largely due to water content, resulting in a classification model that achieved 77.1% sensitivity and 90.8% specificity. While calcifications were harder to detect in the ex vivo specimens, promising results were still achieved, potentially indicating a much more widespread influence of calcification in breast tissue, and to obtain useful signal from bulk human tissue is encouraging in itself. Consequently, this work demonstrates the potential value of both deep Raman techniques and high wavenumber Raman for future breast screening and tumour margin assessment methods

    Non-invasive and non-intrusive diagnostic techniques for gas-solid fluidized beds – A review

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    Gas-solid fluidized-bed systems offer great advantages in terms of chemical reaction efficiency and temperature control where other chemical reactor designs fall short. For this reason, they have been widely employed in a range of industrial application where these properties are essential. Nonetheless, the knowledge of such systems and the corresponding design choices, in most cases, rely on a heuristic expertise gained over the years rather than on a deep physical understanding of the phenomena taking place in fluidized beds. This is a huge limiting factor when it comes to the design, the scale-up and the optimization of such complex units. Fortunately, a wide array of diagnostic techniques has enabled researchers to strive in this direction, and, among these, non-invasive and non-intrusive diagnostic techniques stand out thanks to their innate feature of not affecting the flow field, while also avoiding direct contact with the medium under study. This work offers an overview of the non-invasive and non-intrusive diagnostic techniques most commonly applied to fluidized-bed systems, highlighting their capabilities in terms of the quantities they can measure, as well as advantages and limitations of each of them. The latest developments and the likely future trends are also presented. Neither of these methodologies represents a best option on all fronts. The goal of this work is rather to highlight what each technique has to offer and what application are they better suited for

    Long-Molecule Assessment of Ribosomal DNA and RNA

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    The genes encoding ribosomal RNA and their transcriptional products are essential for life, however, remain poorly understood. Even with the advent of long-range sequencing methodologies, rDNA loci are difficult to study and remain obscure, prompting the consideration of alternative methods to probing this critical region of the genome. The research outlined in this thesis utilises molecular combing, a fibre stretching technique, to isolate DNA molecules measuring more than 5 Mbp in length. The capture of DNA molecules of this size should assist in exploring the architecture of entire rDNA clusters at the single-molecule level. Combining molecular combing with SNP targeting probes, this study aims to distinguish and assess the arrangement of rDNA promoter variants which have been shown to exhibit dramatically different environmental sensitivity. Additionally, through the application of Oxford Nanopore Technologies direct RNA sequencing, the work here has demonstrated the capture of near full-length rRNA primary transcripts, which will allow for assessing post-transcriptional modification across the length of multiple coding subunits within a single molecule, for the first time. Furthermore, an exploration of RNA modification profiles across sample types representative of different developmental stages has been conducted. This study predicts many sites to be differentially modified across these different developmental conditions, several of which are known to be important for, if not crucial in ribosome biogenesis and function. The work outlined in this thesis provides a framework for future studies to conduct long-molecule, genetic, and epitranscriptome profiling of this vital region of the genome, and its dynamic response to a changing environment

    Functional Nanomaterials and Polymer Nanocomposites: Current Uses and Potential Applications

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    This book covers a broad range of subjects, from smart nanoparticles and polymer nanocomposite synthesis and the study of their fundamental properties to the fabrication and characterization of devices and emerging technologies with smart nanoparticles and polymer integration

    Physics of drying complex fluid drop: flow field, pattern formation, and desiccation cracks

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    Drying complex fluids is a common phenomenon where a liquid phase transforms into a dense or porous solid. This transformation involves several physical processes, such as the diffusion of liquid molecules into the surrounding atmosphere and the movement of dispersed phases through evaporation-driven flow. As a result, the solute forming a dried deposit exhibits unique patterns and often displays structural defects like desiccation cracks, buckling, or wrinkling. Various drying configurations have been utilized to study the drying of colloids, the process of their consolidation, and fluid-flow dynamics. This review focuses on the drying of colloids and the related phenomena, specifically the drying-induced effects observed during sessile drop drying. We first present a theoretical overview of the physics of drying pure and binary liquid droplets, followed by drying colloidal droplets. Then, we explain the phenomena of pattern formation and desiccation cracks. Additionally, the article briefly describes the impact of evaporation-driven flows on the accumulation of particles and various physical parameters that influence deposit patterns and cracks.Comment: 20 pages, 28 figures, Accepted in Physics of Fluids. arXiv admin note: substantial text overlap with arXiv:2011.1402
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