End-to-end optimization of lipid nanoparticle manufacturing for mRNA delivery

With the emergence of SARS-CoV-2 in 2020, mRNA vaccines have gained global attention. Currently, lipid nanoparticles (LNPs) are the most clinically advanced drug delivery system for the delivery of nucleic acids. Despite the extensive literature on LNPs in recent years, challenges persist regarding their development and production. In fact, most research papers focus on the therapeutic targets of LNPs, while less attention is given to understanding the challenges associated with their manufacturing, especially on an industrial-production scale, including scalability, reproducibility, encapsulation efficiency and long-term storage. This thesis focused on the end-to-end workflow of LNPs manufacturing, covering production, purification, and freeze-drying, while also addressing storage conditions. Beginning with LNP production, the effects of microfluidic parameters on LNP manufacturing were investigated while the preclinical scalable production of LNPs using various microfluidic devices was also evaluated. Moving on to purification, the second step of LNP manufacturing, the typical bottlenecks associated with this stage were assessed, with a focus on tangential flow filtration (TFF) as this method is commonly used on an industrial level. The effect of TFF speed and diafiltration volumes on LNPs characteristics were evaluated, along with the challenges related to scaling up the purification process. mRNA LNPs storage also represents a challenge due to the fragile nature of mRNA. With the aim of exploring lyophilisation as a technique for preserving mRNA LNPs, a series of freeze-drying cycles were conducted to identify the optimal parameters for producing mRNA LNPs with acceptable critical quality attributes (CQAs) and the in vitro and in vivo activity of the lyophilised product was evaluated to determine the effectiveness of the method. This thesis also explored the role of lipid selection in shaping the quality, stability, and performance of the final product. In particular, the contribution of PEGylated lipids having different alkyl chain lengths (DMG-PEG 2000 versus DSG-PEG 2000) to the physicochemical characteristics and performance of mRNA LNPs was investigated, as well as the impact in vitro and in vivo of the ionisable lipid (ALC-0315, DLin-MC3, and SM-102). The results presented demonstrate that all steps of LNP manufacturing influence the CQAs of the particles, from the choice of lipids, which can either limit or enhance their efficiency, to the selection of microfluidic parameters, buffers, purification methods, and lyophilisation conditions, highlighting the importance of carefully considering each individual step.With the emergence of SARS-CoV-2 in 2020, mRNA vaccines have gained global attention. Currently, lipid nanoparticles (LNPs) are the most clinically advanced drug delivery system for the delivery of nucleic acids. Despite the extensive literature on LNPs in recent years, challenges persist regarding their development and production. In fact, most research papers focus on the therapeutic targets of LNPs, while less attention is given to understanding the challenges associated with their manufacturing, especially on an industrial-production scale, including scalability, reproducibility, encapsulation efficiency and long-term storage. This thesis focused on the end-to-end workflow of LNPs manufacturing, covering production, purification, and freeze-drying, while also addressing storage conditions. Beginning with LNP production, the effects of microfluidic parameters on LNP manufacturing were investigated while the preclinical scalable production of LNPs using various microfluidic devices was also evaluated. Moving on to purification, the second step of LNP manufacturing, the typical bottlenecks associated with this stage were assessed, with a focus on tangential flow filtration (TFF) as this method is commonly used on an industrial level. The effect of TFF speed and diafiltration volumes on LNPs characteristics were evaluated, along with the challenges related to scaling up the purification process. mRNA LNPs storage also represents a challenge due to the fragile nature of mRNA. With the aim of exploring lyophilisation as a technique for preserving mRNA LNPs, a series of freeze-drying cycles were conducted to identify the optimal parameters for producing mRNA LNPs with acceptable critical quality attributes (CQAs) and the in vitro and in vivo activity of the lyophilised product was evaluated to determine the effectiveness of the method. This thesis also explored the role of lipid selection in shaping the quality, stability, and performance of the final product. In particular, the contribution of PEGylated lipids having different alkyl chain lengths (DMG-PEG 2000 versus DSG-PEG 2000) to the physicochemical characteristics and performance of mRNA LNPs was investigated, as well as the impact in vitro and in vivo of the ionisable lipid (ALC-0315, DLin-MC3, and SM-102). The results presented demonstrate that all steps of LNP manufacturing influence the CQAs of the particles, from the choice of lipids, which can either limit or enhance their efficiency, to the selection of microfluidic parameters, buffers, purification methods, and lyophilisation conditions, highlighting the importance of carefully considering each individual step

Contextual comparing and constraining : a grounded theory study of undergraduate engineering group design projects

Engineering design projects are significant components of engineering undergraduate students’ programmes and can be transformative in developing important professional competencies such as communication and teamwork skills. This study contributes to the limited knowledge in qualitative research into undergraduate engineering design projects by exploring the questions: what are the prominent concerns of engineering students undertaking such design projects and how do they manage these? The study involved an exploration of undergraduate Chemical Engineering students undertaking a substantial design project at the University of Strathclyde. Student groups were observed in supervisory meetings to sensitise the researcher and accompany the primary data collection method of intensive interviews. Informing the approach to the study was Grounded Theory (GT) methods for analysis, these involved techniques such as initial (line-by-line) coding; memo-writing; focused coding and theoretical sampling. Following extensive coding and categorisation of concepts, data from other studies in engineering design projects conducted by Goncher (2012) and Morgan (2017) were introduced and integrated into the emerging understanding. This research demonstrated that students’ beliefs around the significance of the design projects; their socialisation with others and their expectations of the educational setting impacted their approach to design significantly. Students engaged in social processes related to comparing and constraining to manage a range of contextual factors. Comparing involved evaluating various design ideas and solutions to make sense of open-ended problems; while constraining refers to the methods used to limit the design space and context to reduce ambiguity. These processes also relate to both social dynamics (such as collaborative efforts and information sharing) and technical factors (like minimising risks and relying on models). Students use such strategies to refine their designs iteratively and align with project requirements to constrain their emergent designs. Supervisory feedback also plays a crucial role in guiding these processes, with effective feedback helping to constrain creative exploration within feasible boundaries.Engineering design projects are significant components of engineering undergraduate students’ programmes and can be transformative in developing important professional competencies such as communication and teamwork skills. This study contributes to the limited knowledge in qualitative research into undergraduate engineering design projects by exploring the questions: what are the prominent concerns of engineering students undertaking such design projects and how do they manage these? The study involved an exploration of undergraduate Chemical Engineering students undertaking a substantial design project at the University of Strathclyde. Student groups were observed in supervisory meetings to sensitise the researcher and accompany the primary data collection method of intensive interviews. Informing the approach to the study was Grounded Theory (GT) methods for analysis, these involved techniques such as initial (line-by-line) coding; memo-writing; focused coding and theoretical sampling. Following extensive coding and categorisation of concepts, data from other studies in engineering design projects conducted by Goncher (2012) and Morgan (2017) were introduced and integrated into the emerging understanding. This research demonstrated that students’ beliefs around the significance of the design projects; their socialisation with others and their expectations of the educational setting impacted their approach to design significantly. Students engaged in social processes related to comparing and constraining to manage a range of contextual factors. Comparing involved evaluating various design ideas and solutions to make sense of open-ended problems; while constraining refers to the methods used to limit the design space and context to reduce ambiguity. These processes also relate to both social dynamics (such as collaborative efforts and information sharing) and technical factors (like minimising risks and relying on models). Students use such strategies to refine their designs iteratively and align with project requirements to constrain their emergent designs. Supervisory feedback also plays a crucial role in guiding these processes, with effective feedback helping to constrain creative exploration within feasible boundaries

The political determinants of public infrastructure investment in Middle Income Countries : the visibility and targetability of white elephants

Middle Income Countries are often stuck in a trap, due to the political distortions that drive their allocation of investment. This dissertation explores these political determinants of public infrastructure investment in Middle-Income Countries [MICs], focusing on how political competition drives the allocation of investment. I argue that insecure incumbents in MICs, prioritize investments in economic infrastructure over social infrastructure due to their higher visibility and targetability, which make them politically valuable. Theoretically, when accountability is low, this misallocation can lead to inefficient, wasteful investments and "White Elephants" that hinder development. By adopting a political economy approach, the dissertation disaggregates investment categories by function to examine the visibility and targetability mechanisms and their distortionary effects on resource allocation. Using empirical analysis of MICs and a detailed case study, this dissertation shows how low horizontal and vertical accountability mean that politics can distort the allocation of investment, resulting in overinvestment in potentially wasteful-albeit politically valuable- economic infrastructure at the expense of -often-more necessary social investments. The findings contribute to understanding, not only why MICs overinvest in economic infrastructure, but also why investment in MICs often fails to translate into growth, providing critical insights into the political dynamics behind resource misallocation and its implications for escaping the MIT.Middle Income Countries are often stuck in a trap, due to the political distortions that drive their allocation of investment. This dissertation explores these political determinants of public infrastructure investment in Middle-Income Countries [MICs], focusing on how political competition drives the allocation of investment. I argue that insecure incumbents in MICs, prioritize investments in economic infrastructure over social infrastructure due to their higher visibility and targetability, which make them politically valuable. Theoretically, when accountability is low, this misallocation can lead to inefficient, wasteful investments and "White Elephants" that hinder development. By adopting a political economy approach, the dissertation disaggregates investment categories by function to examine the visibility and targetability mechanisms and their distortionary effects on resource allocation. Using empirical analysis of MICs and a detailed case study, this dissertation shows how low horizontal and vertical accountability mean that politics can distort the allocation of investment, resulting in overinvestment in potentially wasteful-albeit politically valuable- economic infrastructure at the expense of -often-more necessary social investments. The findings contribute to understanding, not only why MICs overinvest in economic infrastructure, but also why investment in MICs often fails to translate into growth, providing critical insights into the political dynamics behind resource misallocation and its implications for escaping the MIT

Modelling of pulsed electric field treatment on microorganisms : transient electric field and forces acting on cell membrane, local thermal effects

Irreversible or reversible pores could be generated in the cell membrane of microorganisms by pulsed electric field (PEF) treatment, which is generally called electroporation. Such process could be used for inactivation of microorganisms or bio-medical extraction. Both reversible and irreversible pores can be generated in bio-membranes this changes the permeabilization of the cell membrane, with the former allowing for transfection (DNA, RNA, etc.) and the latter for cell inactivation and bio fuel extraction. However, the PEF treatment is generally considered as ‘nonthermal’ due to the lesser significance of the thermal effect among the treatment samples. Although PEF is reported to be a non-thermal method, local heating effects which were not reported before does occur among biological cells during PEF treatment, different level of thermal excitation will be investigated in this study. Besides, the exact mechanism between the pulsed electric field and microorganisms were not fully understood. This study aimed to investigate the interaction between pulsed electric field and microorganisms, with thermal effects (local heating effects) also taken into account. Three different novel analytical models were developed in this study: a linear model, a QuickField model and a COMSOL model. ‘Hot spots’ (due to local heating effects) were observed in the models and the characteristics of local heating effects were also investigated. The contribution of induced electric field strength in cell membrane and local heating effects were evaluated for electroporation process during PEF treatment. The results suggest that the significant induced electric field strength in cell membrane made the main contribution to electroporation. However, local heating effects could be significant when the treatment samples were highly conductive. The thermal force and electromagnetic force on the cell membrane were also investigated. Finally, the situation of penetrated membrane (pore was included in the cell membrane) was also modelled and it was found that the local heating effects in the penetrated membrane were significant and could enhance the expansion of pores. The cell nucleus was also included in the novel QuickField and COMSOL models, which were used to investigate the interactions between microorganism and external electric field, both electric field strength in membranes (cell membrane and nuclear membrane) and thermal effects were investigated. It was observed that, with nano-second PEF treatment, the induced electric field strength in the cell nucleus was strong enough to cause electroporation. Thermal effects could also be generated in cytoplasm. The experimental works were performed using a self-built HV Blumlein generator. Different test cells were used to investigate the inactivation process of PEF treatment with different number of impulses. An alternative plasma treatment was also implemented to compare the inactivation effects between PEF treatment and Plasma treatment with the same Blumlein generator. It was found that the plasma treatment in metallic dish test cell could achieve stronger inactivation compared with PEF treatment with the same number of impulses.Irreversible or reversible pores could be generated in the cell membrane of microorganisms by pulsed electric field (PEF) treatment, which is generally called electroporation. Such process could be used for inactivation of microorganisms or bio-medical extraction. Both reversible and irreversible pores can be generated in bio-membranes this changes the permeabilization of the cell membrane, with the former allowing for transfection (DNA, RNA, etc.) and the latter for cell inactivation and bio fuel extraction. However, the PEF treatment is generally considered as ‘nonthermal’ due to the lesser significance of the thermal effect among the treatment samples. Although PEF is reported to be a non-thermal method, local heating effects which were not reported before does occur among biological cells during PEF treatment, different level of thermal excitation will be investigated in this study. Besides, the exact mechanism between the pulsed electric field and microorganisms were not fully understood. This study aimed to investigate the interaction between pulsed electric field and microorganisms, with thermal effects (local heating effects) also taken into account. Three different novel analytical models were developed in this study: a linear model, a QuickField model and a COMSOL model. ‘Hot spots’ (due to local heating effects) were observed in the models and the characteristics of local heating effects were also investigated. The contribution of induced electric field strength in cell membrane and local heating effects were evaluated for electroporation process during PEF treatment. The results suggest that the significant induced electric field strength in cell membrane made the main contribution to electroporation. However, local heating effects could be significant when the treatment samples were highly conductive. The thermal force and electromagnetic force on the cell membrane were also investigated. Finally, the situation of penetrated membrane (pore was included in the cell membrane) was also modelled and it was found that the local heating effects in the penetrated membrane were significant and could enhance the expansion of pores. The cell nucleus was also included in the novel QuickField and COMSOL models, which were used to investigate the interactions between microorganism and external electric field, both electric field strength in membranes (cell membrane and nuclear membrane) and thermal effects were investigated. It was observed that, with nano-second PEF treatment, the induced electric field strength in the cell nucleus was strong enough to cause electroporation. Thermal effects could also be generated in cytoplasm. The experimental works were performed using a self-built HV Blumlein generator. Different test cells were used to investigate the inactivation process of PEF treatment with different number of impulses. An alternative plasma treatment was also implemented to compare the inactivation effects between PEF treatment and Plasma treatment with the same Blumlein generator. It was found that the plasma treatment in metallic dish test cell could achieve stronger inactivation compared with PEF treatment with the same number of impulses

I, victim : the creation of the ‘victim’ in Scots criminal law

This thesis sets out to answer the question: what is the victim as a legal construct in Scots law? This thesis demonstrates that the victim as a legal concept is protean and has changed over time, shaped and reshaped by external factors, to meet the needs of the legal system and times into which it is introduced. This thesis proposes that a new concept of the victim has been created in Scots law and will examine the socio-political factors which gave rise to this new legal phenomenon. It will then examine how international legal mechanisms enabled this new concept of the victim to permeate the centre of the Scottish criminal justice system. Finally, it will propose how this new legal construct of the victim might be understood as compatible with the framework of the current legal system, and without having to abandon the fundamental pillars of that system or creating a paradox within the system itself. Through exploring its central question, this thesis addresses three key themes: First, the relationship between the victim and the state, and specifically the impact the changing relationship between these two parties has on our understanding of the victim and the criminal justice system. Second, the impact of victim creation on our criminal justice discourse, specifically how differing attitudes of the legal function of the victim, can lead to incoherent discourse and conceptual paradox within the system itself. Thirdly, the role of a rights-based model in conceptualising the victim in Scots law, specifically the rights of recognition, access and participation and how this might offer the solution to a unifying construct of the victim.This thesis sets out to answer the question: what is the victim as a legal construct in Scots law? This thesis demonstrates that the victim as a legal concept is protean and has changed over time, shaped and reshaped by external factors, to meet the needs of the legal system and times into which it is introduced. This thesis proposes that a new concept of the victim has been created in Scots law and will examine the socio-political factors which gave rise to this new legal phenomenon. It will then examine how international legal mechanisms enabled this new concept of the victim to permeate the centre of the Scottish criminal justice system. Finally, it will propose how this new legal construct of the victim might be understood as compatible with the framework of the current legal system, and without having to abandon the fundamental pillars of that system or creating a paradox within the system itself. Through exploring its central question, this thesis addresses three key themes: First, the relationship between the victim and the state, and specifically the impact the changing relationship between these two parties has on our understanding of the victim and the criminal justice system. Second, the impact of victim creation on our criminal justice discourse, specifically how differing attitudes of the legal function of the victim, can lead to incoherent discourse and conceptual paradox within the system itself. Thirdly, the role of a rights-based model in conceptualising the victim in Scots law, specifically the rights of recognition, access and participation and how this might offer the solution to a unifying construct of the victim

Deep learning : assisted visualisation of cellular structure in microscopy

Within a few years, deep learning (DL) has become an important staple in image processing in microscopy, often showing superior performance in comparison to classical methods. Specifically, the ability of an artificial neural network (ANN) to learn a complex mapping function between image domains has found wide application. Its utilisation in fields such as image restoration, image segmentation, and artificial labelling allows cell biologists to increase knowledge output from their microscopy data. However, the performance of an ANN for such tasks is limited by the quality of the data that is used for the training. In practice, high quality ground truth data can be difficult to acquire with microscopy due to the multitude of limitations of imaging setups as well as labelling protocols. Alternatively, target data for the training can be generated through manual annotation but this is often unfeasible as it is extremely time- and labour-intensive, and requires a high level of expert knowledge. In this thesis, methods are introduced for image-to-image translation that were developed while taking those limitations into account, with the main aim of enhancing the microscopic visualisation of routinely studied cellular structures in cell biology such as the actin cytoskeleton and the microtubule network. Two methods are presented. The first, Label2Label (L2L), is a new restoration method in fluorescence microscopy that enhances the image contrast of cellular structure. L2L does not require clean target data for the training. Instead, clear differences in image quality observable between non-identical fluorescent labels are capitalised on to systematically train a network to increase structural contrast. The idea to dual-label a cell to acquire training data makes L2L relatively straightforward to implement in practice. Moreover, results show that a multi-scale structural similarity loss function can enhance the performance in L2L when implemented carefully. Furthermore, the use of artificial labelling in interference reflection microscopy (IRM) is explored. In artificial labelling, an ANN is trained to translate between images acquired with the label-free IRM technique and fluorescence images. Both images differ in their specificity; contrast in label-free images stems from all cell components, whereas fluorescence images are ideally highly specific to the cellular target that was labelled. Consequently, with artificial labelling, specific cellular structures can be selectively visualised in label-free images. For the translation between IRM images and fluorescence images of a focal adhesion-marker, different cases were explored where target data exhibited either a low or a high contrast of the focal adhesions, and were paired or unpaired to the IRM inputs. Results presented in this work show that a network can be successfully trained for artificial labelling in IRM. Moreover, high quality paired target images are not necessarily required for that task. Here, a newly developed framework, the so-called 2LGAN, that is a variant of a generative adversarial network, performed highest for such a case. Notably, L2L and artificial labelling in IRM out-perform classical methods as pre-processing step for downstream quantitative image analysis. Both methods could have important impact in the future for the visualisation of other cellular structures not explored in this work. One such structure could be the plasma membrane which is difficult to visualise with microscopy but is of high interest in cell biology.Within a few years, deep learning (DL) has become an important staple in image processing in microscopy, often showing superior performance in comparison to classical methods. Specifically, the ability of an artificial neural network (ANN) to learn a complex mapping function between image domains has found wide application. Its utilisation in fields such as image restoration, image segmentation, and artificial labelling allows cell biologists to increase knowledge output from their microscopy data. However, the performance of an ANN for such tasks is limited by the quality of the data that is used for the training. In practice, high quality ground truth data can be difficult to acquire with microscopy due to the multitude of limitations of imaging setups as well as labelling protocols. Alternatively, target data for the training can be generated through manual annotation but this is often unfeasible as it is extremely time- and labour-intensive, and requires a high level of expert knowledge. In this thesis, methods are introduced for image-to-image translation that were developed while taking those limitations into account, with the main aim of enhancing the microscopic visualisation of routinely studied cellular structures in cell biology such as the actin cytoskeleton and the microtubule network. Two methods are presented. The first, Label2Label (L2L), is a new restoration method in fluorescence microscopy that enhances the image contrast of cellular structure. L2L does not require clean target data for the training. Instead, clear differences in image quality observable between non-identical fluorescent labels are capitalised on to systematically train a network to increase structural contrast. The idea to dual-label a cell to acquire training data makes L2L relatively straightforward to implement in practice. Moreover, results show that a multi-scale structural similarity loss function can enhance the performance in L2L when implemented carefully. Furthermore, the use of artificial labelling in interference reflection microscopy (IRM) is explored. In artificial labelling, an ANN is trained to translate between images acquired with the label-free IRM technique and fluorescence images. Both images differ in their specificity; contrast in label-free images stems from all cell components, whereas fluorescence images are ideally highly specific to the cellular target that was labelled. Consequently, with artificial labelling, specific cellular structures can be selectively visualised in label-free images. For the translation between IRM images and fluorescence images of a focal adhesion-marker, different cases were explored where target data exhibited either a low or a high contrast of the focal adhesions, and were paired or unpaired to the IRM inputs. Results presented in this work show that a network can be successfully trained for artificial labelling in IRM. Moreover, high quality paired target images are not necessarily required for that task. Here, a newly developed framework, the so-called 2LGAN, that is a variant of a generative adversarial network, performed highest for such a case. Notably, L2L and artificial labelling in IRM out-perform classical methods as pre-processing step for downstream quantitative image analysis. Both methods could have important impact in the future for the visualisation of other cellular structures not explored in this work. One such structure could be the plasma membrane which is difficult to visualise with microscopy but is of high interest in cell biology

Formulation and manufacture of lipid nanoparticles using the micropore AXF crossflow systems

Since its discovery in 1961, the therapeutic utilisation of messenger RNA (mRNA) to combat viral infection and genetic predispositions became a strong focal point for the field of molecular biology. Unlike DNA, mRNA’s transient single strand structure alleviated concerns associated with long-circulation times and consequential offtarget toxicity whilst simultaneously harbouring a reduced risk of genetic recombination. Although promising, challenges facing effective mRNA delivery to the cell cytosol hindered its therapeutic potential. Both exogenous mRNA’s intrinsic immunostimulatory activity and susceptibility to host endonucleases inhibited mRNA’s translational activity and potency. The result led to the exploration of two avenues to mitigate these pitfalls: nucleoside modification of mRNA to protect against innate immune clearance mechanisms and optimisation of lipid nanoparticles (LNPs) to enable successful delivery and uptake to the cell cytosol. Expedited by the SARSCoV2 response, both played critical roles in the rapid development and success of Pfizer-BioNTech’s Comirnaty and Moderna’s Spikevax response. Despite the success of this LNP- based vaccine roll out, bottlenecks in the manufacture of LNPs highlighted the necessity for a scale-independent, uniform design, capable of formulating LNPs at benchtop to manufacturing throughputs. As such, varying microfluidic-based synthesis methods are being explored, one of which is the AXF crossflow technology developed by Micropore Technologies. This project aims to develop a thorough understanding of the effect of the process parameters and fluid mixing dynamics within the range of Micropore’s crossflow devices to support the fast and easy scale-up of LNP production processes and to enable local vaccine manufacture within developing countries. This will be achieved by evaluating the impact of operating speed, lipid concentration, choice of ionizable lipid on LNP critical quality attributes (CQAs) using Micropore AXF-Mini crossflow device. Succeeding this, LNP efficacy in vitro and methods scaling to an in vivo dosage concentration will be evaluated.Since its discovery in 1961, the therapeutic utilisation of messenger RNA (mRNA) to combat viral infection and genetic predispositions became a strong focal point for the field of molecular biology. Unlike DNA, mRNA’s transient single strand structure alleviated concerns associated with long-circulation times and consequential offtarget toxicity whilst simultaneously harbouring a reduced risk of genetic recombination. Although promising, challenges facing effective mRNA delivery to the cell cytosol hindered its therapeutic potential. Both exogenous mRNA’s intrinsic immunostimulatory activity and susceptibility to host endonucleases inhibited mRNA’s translational activity and potency. The result led to the exploration of two avenues to mitigate these pitfalls: nucleoside modification of mRNA to protect against innate immune clearance mechanisms and optimisation of lipid nanoparticles (LNPs) to enable successful delivery and uptake to the cell cytosol. Expedited by the SARSCoV2 response, both played critical roles in the rapid development and success of Pfizer-BioNTech’s Comirnaty and Moderna’s Spikevax response. Despite the success of this LNP- based vaccine roll out, bottlenecks in the manufacture of LNPs highlighted the necessity for a scale-independent, uniform design, capable of formulating LNPs at benchtop to manufacturing throughputs. As such, varying microfluidic-based synthesis methods are being explored, one of which is the AXF crossflow technology developed by Micropore Technologies. This project aims to develop a thorough understanding of the effect of the process parameters and fluid mixing dynamics within the range of Micropore’s crossflow devices to support the fast and easy scale-up of LNP production processes and to enable local vaccine manufacture within developing countries. This will be achieved by evaluating the impact of operating speed, lipid concentration, choice of ionizable lipid on LNP critical quality attributes (CQAs) using Micropore AXF-Mini crossflow device. Succeeding this, LNP efficacy in vitro and methods scaling to an in vivo dosage concentration will be evaluated

Information seeking behaviour of UK retail banking customers towards online purchase adoption: an integrated UTAUT2 model

Digitization has continued to transform the way we live and work. It is steadily changing the UK retail banking sector, where the rapid adoption of digital business models has led to the closure of traditional bank branches, a trend expected to persist. A smooth transition to a digital service model requires a deeper understanding of consumer behaviour. This study investigates consumer behaviour by integrating the Unified Theory of Acceptance and Use of Technology 2 (UTAUT2) with the Everyday Life Information Seeking (ELIS) model as its theoretical framework. A deductive approach was employed, using an online survey distributed nationwide via convenience sampling. A total of 438 responses were received, of which 377 were valid. Findings from five research questions and 18 hypotheses, tested through PLS-SEM and PLS-MGA, highlight challenges in understanding online information available on bank websites, a lack of well-trained advisors, fear, poor user interfaces and complex purchasing processes as key barriers; and the need for information on security, charges, and requirements for tutorials and glossary of terms. Many respondents noted that visiting bank branches is the most preferred information source, suggesting consumers lack the knowledge needed to make online purchasing decisions, and quick access and reliability of information are key determinants of channel choice. Younger consumers are more influenced by perceived information, while older consumers rely more heavily on perceived trust. By integrating UTAUT2 with ELIS, this research advances interdisciplinary collaboration. The proposed integrated model incorporates trust into UTAUT2, a prerequisite for online transactions, and further explores digital information-seeking behaviour through ELIS. Addressing information and trust concerns can enhance technology adoption in financial markets. The qualitative themes identified could inform interview questions for future mixed-method research. Additionally, larger datasets could support multi-group analysis with three classifications, providing deeper insights than a two-group approach.Digitization has continued to transform the way we live and work. It is steadily changing the UK retail banking sector, where the rapid adoption of digital business models has led to the closure of traditional bank branches, a trend expected to persist. A smooth transition to a digital service model requires a deeper understanding of consumer behaviour. This study investigates consumer behaviour by integrating the Unified Theory of Acceptance and Use of Technology 2 (UTAUT2) with the Everyday Life Information Seeking (ELIS) model as its theoretical framework. A deductive approach was employed, using an online survey distributed nationwide via convenience sampling. A total of 438 responses were received, of which 377 were valid. Findings from five research questions and 18 hypotheses, tested through PLS-SEM and PLS-MGA, highlight challenges in understanding online information available on bank websites, a lack of well-trained advisors, fear, poor user interfaces and complex purchasing processes as key barriers; and the need for information on security, charges, and requirements for tutorials and glossary of terms. Many respondents noted that visiting bank branches is the most preferred information source, suggesting consumers lack the knowledge needed to make online purchasing decisions, and quick access and reliability of information are key determinants of channel choice. Younger consumers are more influenced by perceived information, while older consumers rely more heavily on perceived trust. By integrating UTAUT2 with ELIS, this research advances interdisciplinary collaboration. The proposed integrated model incorporates trust into UTAUT2, a prerequisite for online transactions, and further explores digital information-seeking behaviour through ELIS. Addressing information and trust concerns can enhance technology adoption in financial markets. The qualitative themes identified could inform interview questions for future mixed-method research. Additionally, larger datasets could support multi-group analysis with three classifications, providing deeper insights than a two-group approach

Decipher, disarm and disengage : understanding the biosynthesis and self-resistance mechanisms of kirromycin-like, elfamycin producing, streptomyces.

Elfamycin antibiotics exhibit activity against Gram-positive bacteria by inhibiting translation via elongation factor EF-Tu. Elfamycins are characterised by mode of action rather than chemical structure, though minor structural modifications in these antibiotics can significantly alter their biological activity. Aurodox, a kirromycin-like elfamycin, was shown to inhibit the Type III Secretion System (T3SS) of Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli along with effective anti-virulence treatment. This study aimed to investigate the diversity of kirromycin-like elfamycin biosynthetic gene clusters (BGCs), their evolution, resistance mechanisms, and how biosynthesis may be manipulated to create modified elfamycin variants which many be active against the T3SS of EHEC. Genome sequencing of the kirromycin producer Streptomyces ramocissimus was performed, and the BGC responsible for kirromycin production through comparison to the BGC found in Streptomyces collinus. Comparative analyses between kirromycin-like BGCs revealed the potential for natural variation in producing identical compounds via different genetic pathways. Additionally, a kirromycin-like BGC was identified in Streptomyces ISL094, which contained an additional methyltransferase on the BGC, similar to the aurodox BGC of S. goldiniensis, suggesting it could be an aurodox producer. Moreover, aurodox and kirromycin were found to downregulate the T3SS of EHEC, a trait previously thought to be unique to aurodox. Key genes within the aurodox BGC were manipulated and the BGC heterologously expressed to infer function of their roles in aurodox biosynthesis, where their derivatives showed similar effects on the T3SS. The protein, AurM*, thought to methylate the kirromycin molecule creating aurodox was assayed for methyltransferase activity in vitro, but activity was not demonstrated on kirromycin as a substrate. Finally, a potential "moonlighting" role for EF-Tu proteins is suggested, indicating that they might have additional functions related to T3SS regulation, where the knockdown phenotype of aurodox on the T3SS of EHEC was reversed when EF-Tu was expressed in EHEC.Elfamycin antibiotics exhibit activity against Gram-positive bacteria by inhibiting translation via elongation factor EF-Tu. Elfamycins are characterised by mode of action rather than chemical structure, though minor structural modifications in these antibiotics can significantly alter their biological activity. Aurodox, a kirromycin-like elfamycin, was shown to inhibit the Type III Secretion System (T3SS) of Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli along with effective anti-virulence treatment. This study aimed to investigate the diversity of kirromycin-like elfamycin biosynthetic gene clusters (BGCs), their evolution, resistance mechanisms, and how biosynthesis may be manipulated to create modified elfamycin variants which many be active against the T3SS of EHEC. Genome sequencing of the kirromycin producer Streptomyces ramocissimus was performed, and the BGC responsible for kirromycin production through comparison to the BGC found in Streptomyces collinus. Comparative analyses between kirromycin-like BGCs revealed the potential for natural variation in producing identical compounds via different genetic pathways. Additionally, a kirromycin-like BGC was identified in Streptomyces ISL094, which contained an additional methyltransferase on the BGC, similar to the aurodox BGC of S. goldiniensis, suggesting it could be an aurodox producer. Moreover, aurodox and kirromycin were found to downregulate the T3SS of EHEC, a trait previously thought to be unique to aurodox. Key genes within the aurodox BGC were manipulated and the BGC heterologously expressed to infer function of their roles in aurodox biosynthesis, where their derivatives showed similar effects on the T3SS. The protein, AurM*, thought to methylate the kirromycin molecule creating aurodox was assayed for methyltransferase activity in vitro, but activity was not demonstrated on kirromycin as a substrate. Finally, a potential "moonlighting" role for EF-Tu proteins is suggested, indicating that they might have additional functions related to T3SS regulation, where the knockdown phenotype of aurodox on the T3SS of EHEC was reversed when EF-Tu was expressed in EHEC