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Investigation of a Selective Transglutaminase 2 Antagonist as a Novel Therapeutic Agent for Cystic Fibrosis
Full text linkCystic fibrosis (CF) is a genetic disorder, characterised by the presence of a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein. Currently approved therapeutic compounds all act by targeting CFTR directly. Yet, emerging evidence suggests that the proteostasis network within CF airway epithelial cells is severely disrupted, leading to fibrotic alterations of the extracellular matrix (ECM). TG2 is reported to be a key regulator of these pathogenic changes. With the advent of potent and selective inhibitors of TG2, a novel therapeutic avenue may now exist in CF. In this study, both immortalised and primary CF human bronchial epithelial cells (HBECs) were used to investigate the role of TG2, as regards the development of fibrosis in CF airway epithelia. It was shown that the deposition of TG2 and fibronectin is elevated in the ECM of both IB3 cells and CF primary HBECs. Notably, IB3 cells were found to undergo epithelial-mesenchymal transition (EMT)-derived myofibroblast transdifferentiation, with CF primary HBECs also exhibiting varying levels of EMT progression. Proof of concept experiments using CF primary HBECs revealed that the use of a CFTR corrector (VX-809) and TG2 specific inhibitor (1-155) in combination, could have a potentially additive therapeutic effect. A more in-depth investigation with IB3 cells, served to further validate these findings. It was demonstrated that the treatment of IB3 cells with VX-809 and 1-155, could completely reverse EMT-derived myofibroblast transdifferentiation and fully restore the barrier function of CF airway epithelium. Furthermore, the development of these pathogenic processes, was shown to be dependent on the interrelationship between extracellular TG2 and TGFβ1 signal transduction. The mechanism of cellular TG2 export in CF was also examined. Extracellular vesicles released by IB3 cells were determined to have increased TG2 expression and activity. IB3 cells were shown to secrete elevated levels of exosomes, which were found to be reduced after combination treatment with VX-809 and 1-155. The findings within this study confirm the importance of extracellular TG2 in the pathogenesis of CF and suggest that pharmacological inhibition of its aberrant activity, represents a viable therapeutic approach
The role of phytochemicals in modulating breast cancer resistance protein at the blood-brain barrier and the blood-tumour barrier
Full text linkThe blood-brain barrier (BBB) and the blood-tumour barrier (BTB) represent insidious obstacles for the delivery of anti-cancer agents to solid brain tumours, not only because of their morphological features, but also due to the presence of the drug efflux transporter breast cancer resistant protein (BCRP), localised at both the BBB and BTB. This efflux transporter restricts the permeation of anti-cancer agents across both barriers leading to suboptimal concentrations of drugs at the intended site of action. This work examined 13 naturally occurring phytochemicals, which were screened for their dual ability to modulate the efflux function of BCRP in addition to their anti-cancer properties in human LN229 glioblastoma cells, namely: (i) inhibition of cellular migration; (ii) activation of apoptosis; (iii) reactive oxygen species (ROS) production and (iv) activation of caspase pathways. Phytochemicals displayed minimal cytotoxicity, were able to modulate BCRP which led to enhancing the permeability of the fluorescent probe substrate H33342, in addition to inhibiting cellular migration. Hesperetin and baicalin displayed the optimal modulatory potential and demonstrated a similar ability to generate ROS and activate Caspase-3/7 when compared to the anti-cancer agents methotrexate and temozolomide. Subsequently, hesperetin was progressed as the optimal candidate, and its ability to permeate across the BBB was confirmed after conducting a permeability study using an in-vitro primary porcine brain microvascular endothelial cell (PBMEC) BBB model. We demonstrated that hesperetin was highly permeable across the BBB, can modulate the efflux function of BCRP and overall enhance the apparent permeability (Papp) of mitoxantrone and methotrexate. Thereafter, we assessed the impact of shear stress fashioned by laminal flow on the morphology of PBMEC using a Quasi Vivo 600® perfusion system. The results displayed a significant increase in Transepithelial Electrical Resistance (TEER) values, improved formation of zonula occludens-1 (ZO-1), and higher expression of efflux transporter proteins, suggesting the formation of a better in-vitro BBB model with hesperetin still being highly permeable across the barrier further confirming its ability to bypass the BBB and reach the BTB. This work highlights the anticancer and BCRP modulatory capabilities of phytochemicals as well as the ability of hesperetin to bypass the BBB
Engineering Bacteria for the Enhanced Production of High-value Chemicals
Full text linkThere is a growing interest in the use of microbial cell factories to produce butanol, an industrial solvent and platform chemical. Biobutanol can also be used as a biofuel and represents a cleaner and more sustainable alternative to the use of conventional fossil fuels. Solventogenic Clostridia are the most popular microorganisms used due to the native expression of butanol synthesis pathways. A major drawback to the wide scale implementation and development of these technologies is the product toxicity of butanol. This study aims to develop a deeper understanding of butanol toxicity at the membrane. Using liposome membrane models and in vitro assays to investigate characteristics such as permeability, fluidity, and diameter, it was found that altering the composition of membranes can convey tolerance to butanol. The effect of butanol on membrane proteins was also investigated, with it causing unfolding of bacteriorhodopsin. The changes to the lipidome of Clostridium saccharoperbutylacetonicum N 1-4 in different butanol environments were investigated with thin layer chromatography and mass spectrometry. In higher butanol concentrations, levels of phosphatidylglycerol and oleic acid had increased significantly. Several metabolic targets were selected for the genetic engineering of Clostridium saccharoperbutylacetonicum N 1-4 (HMT) in an attempt to improve tolerance in butanol. The three targets investigated consisted of two membrane proteins and one enzyme. The first membrane protein, GlpF, a putative butanol channel which appeared to grant a butanol-independent advantage to growth when overexpressed. The second membrane protein, TtgB, is a transporter and overexpression increased cellular growth rate during fermentation. The final protein PssA is involved in lipid synthesis and showed no effect when overexpressed and knockouts appeared to inhibit growth in fermentation. Ultimately, this work highlights the detrimental impact of butanol-membrane interactions, how the cell responds and presents some novels strains, some of which have produced promising results
Aerogel Rafts Allowing for the Comparison of Novel Solvent-Based Biological Formulations During the Primary Drying Phase of Lyophilisation
Full text linkSilica aerogels (SA) are comprised of up to 90% air. As such, they are useful for many different techniques and delivery systems. In the present study, SA were successfully synthesised using the novel solvent tetraethyl butrane (TEB) as a reaction solvent via sol-gel formation and aging followed by a freezedrying (lyophilisation) process. Although the TEB solvent is expensive, it could still be a suitable alternative to traditional solvents for the synthesis of SA. The TEB solvent has a relatively short required gelation time that eliminates the need for the solvent exchange process, thereby allowing for a shorter synthesis time. Moreover, TEB solvent could be recycled. The lyophilisation process is one of the most common processes in pharmaceutical production; however, it is a relatively expensive technique due to the length of time it takes to complete. To reduce the cost of this process, the sublimation time should be minimised, as this is the longest of the three phases of lyophilisation. This is why it is very important to determine the end point of the sublimation phase. The second part of this study therefore involved producing SA combined with different substances. These were monitored by several sensors to determine the end point of their primary drying. Some successful formulations were synthesised when the SA was combined with agarose (AG), mannitol (MAT), polyvinyl alcohol (PVA) and ethyl cellulose (EC). SA/AG was the basic formula that monitored by thermocouple temperature, Corillonite V.2.3 and the microbalance sensors. The remaining formulations hardened the structure of the SA and significantly affected the sublimation time when measured by thermocouple sensor only. These findings suggest that SA produced by those substances (AG, MAT, PVA and EC) could decrease the end point of primary drying in the lyophilisation process and producing a better shape than pure SA
Synthesis, antimicrobial, and cytotoxicity evaluation of selective growth inhibitors of Clostridioides difficile
Full text linkThe World Health Organisation has estimated the cost of antimicrobial resistance (AMR) to the global economy to be $100,000,000,000,000 with AMR becoming the leading cause of death by 2050. Clostridioides difficile is a major healthcare-associated infection in the UK, Europe, the USA, and other developed countries. Strains of this microorganism that are less sensitive to the frontline therapies (metronidazole, vancomycin, and fidaxomicin) are emerging, causing great concern and negative prognosis for AMR. Developing new, potent, efficacious antimicrobial agents with a narrow spectrum of activity will provide alternative treatment options for C. difficile and AMR. Representative derivatives were prepared in good yield by Knoevenagel condensation of arylaldehydes with an active methylene substrate, in ethanol or water at reflux and without additional catalyst. Compounds were purified, characterised, and assayed against C. difficile NCTC 11204 to determine potency, and against Escherichia coli NCTC 35218 and Staphylococcus aureus NCTC 29213 to determine selectivity. The lead compound 5-[(5-Nitro-2-furyl)methylene]hexahydropyrimidine-2,4,6-trione (2.10) gave a minimum inhibitory concentration (MIC) value of 2 μg/mL against C. difficile NCTC 11204. Replacement of the nitro group by hydrogen 5-(2-furylmethylene)hexahydropyrimidine-2,4,6-trione (2.8) resulted in good selectivity. This inhibitory activity improved further, using isosteres. For example, the replacement of oxygen 2.10 by sulfur 5-[(5-Nitro-2-thienyl)methylene]hexahydropyrimidine-2,4,6-trione (2.16) gave a MIC value of 0.5 μg/mL against CD. Replacement of the nitro group in compound 2.16 by hydrogen 5-(thiophen-2-ylmethylene)pyrimidine-2,4,6(1H,3H,5H)-trione (2.20) resulted in the most potent compound in the series (MIC = 0.125 μg/mL) albeit with reduced selectivity. The active diene derivatives 5-[(E)-3-(2-Furyl)prop-2-enylidene]hexahydropyrimidine-2,4,6-trione (2.9), 5-[(E)-3-(5-Nitro-2-furyl)prop-2-enylidene]hexahydropyrimidine-2,4,6-trione (2.13), and 5-[(2-Methoxy-1-naphthyl)methylene]hexahydropyrimidine-2,4,6-trione (2.21) demonstrate scope for homologation in the inhibitors. Replacement of the five-membered aryl ring with benzene in compound 5-[(E)-3-(4-Methoxyphenyl) prop-2-enylidene]hexahydropyrimidine-2,4,6-trione (2.5) gave excellent selectivity but with reduced growth inhibitory against C. difficile. Isosteric modification provided derivatives with improved potency and selectivity against C. difficile compared with S. aureus and E. coli. Six representatives of the lead compounds that were tested for their cytotoxic activity against normal mice liver cells and colorectal cancer cells, where compound 2.8 showed the lowest cytotoxic activity against both cell lines. Of the compounds prepared and evaluated, a subset provided promising development candidates for testing against clinical isolates from patients presenting with C. difficile infection, to examine effectiveness against ribotypes that are less sensitive for frontline therapies
Username Construction and Identity Performance in Dark Web Child Sexual Abuse Communications
Full text linkChild sex abusers use the dark web to exchange illicit imagery and share advice. As part of their identity work, users can make use of a number of resources – linguistic , social, cultural, situational – to form their online identity and create a unique username. Employing corpus-assisted discourse analysis with a focus on social actor representation, this thesis investigates child sex abusers naming practices and uncovers the resources child sex abusers draw on when crafting their usernames and how those relate to users’ discursively performed identities. The data analysed stems from a corpus of eight dark web discussion fora related to child sexual abuse consisting of 295,799 forum posts written by 32,410 unique users. In study 1, the usernames are manually segmented and are then classified thematically into categories. 20 username categories emerged which are typical for users’ naming choices in the dark web CSA context. The analysis shows that users draw on a number of resources to create their usernames and specific considerations regarding anonymity impact those choices. Interestingly, users in this high risk/low trust environment still opt for proper names as parts of their usernames. For study 2, forum posts for the most frequent posters in the data (n=1837) are analysed through a corpus-assisted approach examining the usernames and forum contributions for a subset of 5 username categories. Findings suggest that users who identify in a certain way will also show linguistic behaviours related to that self-assigned identity and engage in discussions of topics which can be inferred from the username choice. Study 3 dives deeper into the 5 selected subcorpora and applies a thematic as well as a detailed concordance analysis for top keywords identified against the full corpus of most frequent posters. The results show that while similarities between the subgroups are present, differences between the subgroups show clear distinctive features by type of user. Users engage in discussions of topics directly or indirectly linked to their self-assigned identities and different types of users are shown to be more or less concerned with topics such as age of consent, security measures or the exchange of CSAM. In addition to adding to the linguistic literature on identity performance, child sexual abuse and offender communities, this research opens up possibilities for law enforcement policing such online environments: findings can aid agencies in identifying targets and aid undercover officers in choosing appropriate usernames when engaging in infiltration tasks
Neuromorphic Computing and Machine Learning for Nonlinearity Mitigation in Optical Communication Systems
Full text linkNonlinear fibre propagation and transceiver impairments limit the performance of coherent optical communication systems. Established digital signal processing techniques such as digital back-propagation and Volterra equalisation can mitigate Kerr-induced nonlinear distortions, but their computational complexity and energy consumption hinder real-time deployment. This thesis develops machine-learning and neuromorphic approaches to optical channel equalisation, focusingon performance–complexity trade-offs and practical hardware feasibility. A unified training and benchmarking framework is introduced for coherent transmission scenarios, employing Q-factor and bit-error-rate alongside implementation measures including operation counts, memory footprint, and inference latency. Within this framework, a broad class of equalisers is analysed, including multi-layer perceptrons, convolutional and recurrent neural networks, and complex-valued neural networks that process in-phase and quadrature components. Model compression is studied: pruning, quantisation, and weight clustering are jointly optimised using Bayesian optimisation to identify Pareto-efficient configurations that preserve equalisation performance while reducing computational load, memory usage, and latency. Experimental evaluations on edge-device platforms demonstrate feasibility under realistic receiver constraints. The thesis also explores hardware–software co-design. Optical phase conjugation is integrated with neural equalisers to offload part of the nonlinearity compensation to the optical domain, enabling smaller models and lower digital complexity. To address analogue noise in photonic neuromorphic computing, robustness is assessed under additive and signal-dependent noise; noise-aware training, stochastic-resonance neurons, and ensemble-based “crowd equalisation” are proposed to enhance resilience. Finally, a neuromorphic equaliser combining spiking neural networks with a streaming RWKV time-mixing module is introduced. By exploiting event-driven sparsity and constant-memory sequential processing, this architecture achieves competitive equalisation performance with reduced computational and energy requirements compared to conventional deep learning models
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