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The effect of weldable primer Paint thickness on weld quality and geometry
No full textThe purpose of this project was to provide data that could be used to support work by Cairns et al. [1] that aimed to optimise robotic fillet welding by identifying key contributing factors. The focus of this thesis was the effect of weld-through primers on the geometry and quality of a fillet weld. Steel samples were sent to a paint company, where the primer was applied top redetermined thickness ranges. The DFT of the sample plate and a randomly selected panel was then measured and systematically recorded for analysis. Test joints at three DFT ranges, were welded using five filler types and a mechanised welding rig at the University of Strathclyde. Varying test methods were used to identify any trends, that would suggest the coating was impacting the weld quality and/or geometry. Findings from the measurement of DFT casts doubt on data supplied by paint companies. This is a result of the effects of overspray, creating areas with a considerably higher DFT. Weld-through primer and HAZ area, showed a possible relationship. The samples where the paint was mechanically removed seemed in some cases to produce larger HAZ areas. It was concluded that the primer may limit the spread of heat, resulting in a smaller HAZ. The analysis of weld penetration data showed a possible relationship between weld-through primer and weld penetration. The sample joints, where the paint was mechanically removed, have produced welds with less penetration than those coated with primer. Discussion suggests, this could be a result of a surface tension effect from the coating and/or gases from welding, contaminating the gas shield and increasing the arc temperature. This work has also highlighted the potential for a model that would provide manufacturers using weld-through shop primers with a method of estimating internal weld metal porosity by measurement of DFT.The purpose of this project was to provide data that could be used to support work by Cairns et al. [1] that aimed to optimise robotic fillet welding by identifying key contributing factors. The focus of this thesis was the effect of weld-through primers on the geometry and quality of a fillet weld. Steel samples were sent to a paint company, where the primer was applied top redetermined thickness ranges. The DFT of the sample plate and a randomly selected panel was then measured and systematically recorded for analysis. Test joints at three DFT ranges, were welded using five filler types and a mechanised welding rig at the University of Strathclyde. Varying test methods were used to identify any trends, that would suggest the coating was impacting the weld quality and/or geometry. Findings from the measurement of DFT casts doubt on data supplied by paint companies. This is a result of the effects of overspray, creating areas with a considerably higher DFT. Weld-through primer and HAZ area, showed a possible relationship. The samples where the paint was mechanically removed seemed in some cases to produce larger HAZ areas. It was concluded that the primer may limit the spread of heat, resulting in a smaller HAZ. The analysis of weld penetration data showed a possible relationship between weld-through primer and weld penetration. The sample joints, where the paint was mechanically removed, have produced welds with less penetration than those coated with primer. Discussion suggests, this could be a result of a surface tension effect from the coating and/or gases from welding, contaminating the gas shield and increasing the arc temperature. This work has also highlighted the potential for a model that would provide manufacturers using weld-through shop primers with a method of estimating internal weld metal porosity by measurement of DFT
Phenotypic characterisation of wild type and mutant strains of adherent invasive Escherichia coli and Shigella, in vitro, ex vivo and in vivo models
Get PDFCrohn's disease (CD) is an inflammatory bowel disease (IBD) that is largely the result of aberrant immune responses, such as the increased production of proinflammatory cytokines including IL-1, TNF-ɑ, IFN- ɣ to microbial infections in genetically susceptible individuals.;Adherent-invasive Escherichia coli (AIEC) has been implicated in CD, as has Shigella sonnei due to the shared inflammatory characteristics and susceptible genetic background of Shigellosis and Crohn's disease patients, with both microorganisms showing resistance to antibiotics.;Resistance to antibiotics appears to be a factor in gastrointestinal diseases. Therefore, the present study sought to evaluate the key AIEC and Shigella sonnei virulence genes for their pathogenic roles and to address the problem of antibiotic resistance and also prevent the destruction of beneficial gut bacteria by targeting the dsbA gene (as one of the cardinal genes) using simple compounds such as the monoterpene, geraniol, which can inhibit the activity of bacteria rather than eradicate them.;This study employed a red lambda system to construct mutants of key virulence genes, such as dsbA, yadA, proQ, icsA and mxiD genes, in AIEC and Shigella. Murine cell line cultures in vitro (the RAW 264.7 macrophage cell line) and ex vivo (intestinal organoids derived from), were used together with in vivo (Galleria moth larvae) models to evaluate and ascertain the role of these genes in the pathogenesis of AIEC and Shigella.;The antimicrobial activities of geraniol were also evaluated by using the above models. Nitric oxide (NO) was estimated by measuring the production of nitrite using Griess reagent. Cell viability was assessed using MTT dye. The formation/growth rate of biofilms was also measured via a spectrophotometer to measure the optical density. Gentamicin protection assays were also used to determine the differences between wild type and mutated bacterial strains whereby RAW 264.7 cells were stimulated with cytokines in the presence of either geraniol or L-NAME.;The dsbA gene, shared by AIEC and Shigella sonnei, was one of the key genes found in both pathogens, so the deletion of this gene in the current study was found to have a major impact in terms of reducing bacterial virulence. However, although mutation of the yadA gene was not achieved in AIEC, the competitive assay found that yadA significantly reduced the death rate in moth larvae.;Moreover, Shigella sonnei genes for the Type III secretion system (TTSS), adherence, and RNA regulation also significantly reduced virulence. Geraniol was demonstrated to be effective in inhibiting bacterial infection and biofilm formation by AIEC and S. sonnei. Furthermore, it was found to reduce NO production caused by AIEC infection in RAW 264.7 cells, indicating control of inflammation characteristic of CD and shigellosis.;Differences in the reduction of adhesion were found between the dsbA mutant and wild-type strains for AIEC and Shigella in experiments using intestinal organoids directly infected. The co-culture of intestinal organoids with RAW 264.7 cells resulted in severe damage to the organoid cell structure and significant damage was also observed in response to LPS and cytokines such as TNF-ɑ and IFN-ɣ. This could have led to either the death of the organoids or protection of the organoid from bacterial induced damage when using geraniol treatment compared to L-NAME.;The data generated from these models and other experiments were consistent in that deletion of all the target genes were found to be important for virulence, with significant differences emerging between wild-type and mutated strains. This implies that these genes are important for bacterial virulence and pathogenesis in that the bacteria become harmless if they lose the genes by either mutation or inactivation.;Hence, targeting these genes can lead to the inactivation of the bacteria, which means that targeting the dsbA gene using geraniol, for example, in competitive inhibition with glutathione can lead to the inhibition of bacterial growth. This suggests that geraniol, which was found to have antimicrobial properties against the strains used in this study, may have a role to play in modulating the hyperinflammation feature of Crohn's disease and shigellosis.Crohn's disease (CD) is an inflammatory bowel disease (IBD) that is largely the result of aberrant immune responses, such as the increased production of proinflammatory cytokines including IL-1, TNF-ɑ, IFN- ɣ to microbial infections in genetically susceptible individuals.;Adherent-invasive Escherichia coli (AIEC) has been implicated in CD, as has Shigella sonnei due to the shared inflammatory characteristics and susceptible genetic background of Shigellosis and Crohn's disease patients, with both microorganisms showing resistance to antibiotics.;Resistance to antibiotics appears to be a factor in gastrointestinal diseases. Therefore, the present study sought to evaluate the key AIEC and Shigella sonnei virulence genes for their pathogenic roles and to address the problem of antibiotic resistance and also prevent the destruction of beneficial gut bacteria by targeting the dsbA gene (as one of the cardinal genes) using simple compounds such as the monoterpene, geraniol, which can inhibit the activity of bacteria rather than eradicate them.;This study employed a red lambda system to construct mutants of key virulence genes, such as dsbA, yadA, proQ, icsA and mxiD genes, in AIEC and Shigella. Murine cell line cultures in vitro (the RAW 264.7 macrophage cell line) and ex vivo (intestinal organoids derived from), were used together with in vivo (Galleria moth larvae) models to evaluate and ascertain the role of these genes in the pathogenesis of AIEC and Shigella.;The antimicrobial activities of geraniol were also evaluated by using the above models. Nitric oxide (NO) was estimated by measuring the production of nitrite using Griess reagent. Cell viability was assessed using MTT dye. The formation/growth rate of biofilms was also measured via a spectrophotometer to measure the optical density. Gentamicin protection assays were also used to determine the differences between wild type and mutated bacterial strains whereby RAW 264.7 cells were stimulated with cytokines in the presence of either geraniol or L-NAME.;The dsbA gene, shared by AIEC and Shigella sonnei, was one of the key genes found in both pathogens, so the deletion of this gene in the current study was found to have a major impact in terms of reducing bacterial virulence. However, although mutation of the yadA gene was not achieved in AIEC, the competitive assay found that yadA significantly reduced the death rate in moth larvae.;Moreover, Shigella sonnei genes for the Type III secretion system (TTSS), adherence, and RNA regulation also significantly reduced virulence. Geraniol was demonstrated to be effective in inhibiting bacterial infection and biofilm formation by AIEC and S. sonnei. Furthermore, it was found to reduce NO production caused by AIEC infection in RAW 264.7 cells, indicating control of inflammation characteristic of CD and shigellosis.;Differences in the reduction of adhesion were found between the dsbA mutant and wild-type strains for AIEC and Shigella in experiments using intestinal organoids directly infected. The co-culture of intestinal organoids with RAW 264.7 cells resulted in severe damage to the organoid cell structure and significant damage was also observed in response to LPS and cytokines such as TNF-ɑ and IFN-ɣ. This could have led to either the death of the organoids or protection of the organoid from bacterial induced damage when using geraniol treatment compared to L-NAME.;The data generated from these models and other experiments were consistent in that deletion of all the target genes were found to be important for virulence, with significant differences emerging between wild-type and mutated strains. This implies that these genes are important for bacterial virulence and pathogenesis in that the bacteria become harmless if they lose the genes by either mutation or inactivation.;Hence, targeting these genes can lead to the inactivation of the bacteria, which means that targeting the dsbA gene using geraniol, for example, in competitive inhibition with glutathione can lead to the inhibition of bacterial growth. This suggests that geraniol, which was found to have antimicrobial properties against the strains used in this study, may have a role to play in modulating the hyperinflammation feature of Crohn's disease and shigellosis
Beyond coverage as a metric for SDG 6 success in the decade of action (2020-2030), the sustainability burden of rural community handpumps in Malawi
Get PDFAccess to reliable and safe water has been recognised as a fundamental human right across the last 20 years of global goals. Handpumps have played a fundamental role in increasing the number of people with access to safe water for rural populations in low-income countries. However, monitoring indicators and investments based solely on increasing coverage alongside the challenge of maintaining handpumps across their intended life-cycle, may risk hiding low and inherently unsustainable services for rural communities in low-income regions.;This thesis addresses the need to move beyond coverage as a metric for success in the global goals. By investigating the sustainability burden on decentralised rural water supply in Malawi, through a comprehensive national monitoring dataset.;First, the impact a focus on drinking water coverage in global goals and Malawian rural water supply policy is addressed. The acceleration to meet targets, coupled with challenges of community based management, risks unsustainable infrastructure and a loss of the intended benefits. Second, the variation of tariffs to maintain water supply infrastructure are investigated. Significant explanatory variables associated with considering affordability and operations and maintenance costs are identified through regression analysis. Finally, the principles of life-cycle costing are adopted to determine the capacity of rural service providers sustaining infrastructure across their intended life-cycle.;Findings show low costing repairs are prioritised while high costing repairs are left until the complete failure of the asset. Regression analysis further identifies significant variables that increase the likelihood of handpump breakdown.;As the global goals move into the decade of action (2020-2030), increased efforts towards capacity building, localising the goals, significant explanatory factors and identifying risks relating to sustaining services are required. True representation of rural service provision may be misrepresented if the lessons of the global goals to date are not fed back into monitoring strategies and investment appraisal.Access to reliable and safe water has been recognised as a fundamental human right across the last 20 years of global goals. Handpumps have played a fundamental role in increasing the number of people with access to safe water for rural populations in low-income countries. However, monitoring indicators and investments based solely on increasing coverage alongside the challenge of maintaining handpumps across their intended life-cycle, may risk hiding low and inherently unsustainable services for rural communities in low-income regions.;This thesis addresses the need to move beyond coverage as a metric for success in the global goals. By investigating the sustainability burden on decentralised rural water supply in Malawi, through a comprehensive national monitoring dataset.;First, the impact a focus on drinking water coverage in global goals and Malawian rural water supply policy is addressed. The acceleration to meet targets, coupled with challenges of community based management, risks unsustainable infrastructure and a loss of the intended benefits. Second, the variation of tariffs to maintain water supply infrastructure are investigated. Significant explanatory variables associated with considering affordability and operations and maintenance costs are identified through regression analysis. Finally, the principles of life-cycle costing are adopted to determine the capacity of rural service providers sustaining infrastructure across their intended life-cycle.;Findings show low costing repairs are prioritised while high costing repairs are left until the complete failure of the asset. Regression analysis further identifies significant variables that increase the likelihood of handpump breakdown.;As the global goals move into the decade of action (2020-2030), increased efforts towards capacity building, localising the goals, significant explanatory factors and identifying risks relating to sustaining services are required. True representation of rural service provision may be misrepresented if the lessons of the global goals to date are not fed back into monitoring strategies and investment appraisal
Characterising the biosynthesis and mechanism of action of Aurodox from Streptomyces goldiniensis
Get PDFAurodox, a specialised metabolite from the soil bacterium Streptomyces goldiniensis was discovered in 1973 and was originally investigated for its antibacterial properties. However, aurodox has been recently identified from large-scale compound screens as an inhibitor of the Enteropathogenic Escherichia coli (EPEC) Type III Secretion System (T3SS).;Therefore, to gain an understanding of its mechanism of action and to assess the suitability of this molecule for repurposing as an anti-virulence compound, a multidisciplinary approach to understanding aurodox was used. The biosynthesis of aurodox by S. goldiniensis was investigated through sequencing the whole genome of S. goldiniensis to enable the identification of the putative aurodox biosynthetic gene cluster (BGC).;This BGC was then cloned and expressed in multiple heterologous hosts including Streptomyces coelicolor M1152, confirming that this BGC is responsible for aurodox production. In-depth analysis of the BGC supports a model of a polyketide synthase pathway involving a combination of both cis and transacyltransferases which synthesise the aurodox polyketide backbone before tailoring enzymes are recruited to form the final aurodox precursor, kirromycin, which is also produced by S. collinus.;These studies suggest that the SAM-dependent Omethyltransferase AurM* is responsible for catalysing the conversion of kirromycin to aurodox in S. goldiniensis through the methylation of the pyridone moiety. Furthermore, multiple aurodox resistance genes at distinct loci have been identified and their role in aurodox resistance has been explored. The mechanism of action of aurodox has also been investigated.;Whole transcriptome analysis, cell infection and GFP-reporter assays were used to demonstrate that aurodox transcriptionally downregulates the expression of the Locus of Enterocyte Effacement (LEE) pathogenicity island- which encodes for the T3SS, acting via its master regulator, Ler. We have also observed these effects across other enteric pathogens carrying a homologous T3SS such as Enterohemorrhagic Escherichia coli (EHEC). Significantly, unlike traditional antibiotics, aurodox does not induce the production of shiga toxin.Aurodox, a specialised metabolite from the soil bacterium Streptomyces goldiniensis was discovered in 1973 and was originally investigated for its antibacterial properties. However, aurodox has been recently identified from large-scale compound screens as an inhibitor of the Enteropathogenic Escherichia coli (EPEC) Type III Secretion System (T3SS).;Therefore, to gain an understanding of its mechanism of action and to assess the suitability of this molecule for repurposing as an anti-virulence compound, a multidisciplinary approach to understanding aurodox was used. The biosynthesis of aurodox by S. goldiniensis was investigated through sequencing the whole genome of S. goldiniensis to enable the identification of the putative aurodox biosynthetic gene cluster (BGC).;This BGC was then cloned and expressed in multiple heterologous hosts including Streptomyces coelicolor M1152, confirming that this BGC is responsible for aurodox production. In-depth analysis of the BGC supports a model of a polyketide synthase pathway involving a combination of both cis and transacyltransferases which synthesise the aurodox polyketide backbone before tailoring enzymes are recruited to form the final aurodox precursor, kirromycin, which is also produced by S. collinus.;These studies suggest that the SAM-dependent Omethyltransferase AurM* is responsible for catalysing the conversion of kirromycin to aurodox in S. goldiniensis through the methylation of the pyridone moiety. Furthermore, multiple aurodox resistance genes at distinct loci have been identified and their role in aurodox resistance has been explored. The mechanism of action of aurodox has also been investigated.;Whole transcriptome analysis, cell infection and GFP-reporter assays were used to demonstrate that aurodox transcriptionally downregulates the expression of the Locus of Enterocyte Effacement (LEE) pathogenicity island- which encodes for the T3SS, acting via its master regulator, Ler. We have also observed these effects across other enteric pathogens carrying a homologous T3SS such as Enterohemorrhagic Escherichia coli (EHEC). Significantly, unlike traditional antibiotics, aurodox does not induce the production of shiga toxin
Analysis of CSPα mutants that cause adult-onset neuronal ceroid lipofuscinosis
No full textCysteine-string Protein (CSPα) has been linked to neurodegeneration and mutations in CSPα are known to cause Adult-onset Ceroid Lipofuscinosis (ANCL). CSPα’s normal function relies on its targeting to synaptic vesicles by palmitoylation – the attachment offatty acids to cysteine residues. Palmitoylation is mediated by several Palmitoyl-Transferase (PAT) enzymes, such as zDHHC3, zDHHC7 and zDHHC17. zDHHC17 bindsto all its known substrates such as, CSPα through its ankyrin repeat domain by recognising CSPα’s zDHHC ankyrin-repeat binding motif (zDABM), which contains the consensus sequence ΨβXXQP, where proline-175 is the central residue for CSPα’s association. Despite the known requirement of the zDABM for CSPα binding to zDHHC17,the importance of the zDABM for palmitoylation has not yet been demonstrated.Therefore, this investigation determined how disruption of the zDABM affected palmitoylation of CSPα. Furthermore, PAT co-expression with CSPα has been shown to increase aggregation in ANCL mutants in vitro; therefore, we also aimed to determine the importance of the zDABM in the aggregation of ANCL mutant CSPα. in-silico methods were also used to predict changes in post-translational modifications (PTMs), and changes in the monomeric and homodimeric structure of CSPα caused by ANCL mutations. It was found that disruption of the zDABM sequence led to a zDHHC17-dependent increase in the levels of both palmitoylated and non-palmitoylated CSPα.Moreover, disruption of the zDABM sequence had no influence on the aggregation of ANCL mutants. A method for solubilisation of the CSPα aggregates using Triton-X100lysis and Sodium Dodecyl Sulfate (SDS) incubation was identified; however, further investigation of the solubilised aggregates was not possible due to restrictions on laboratory work. Structural predictions of CSPα identified that proline-175 in the zDABM sequence directly interacts with L115. Furthermore, this interaction, in addition to the structure and localisation of the zDABM sequence is considerably compromised in theANCL mutants. Structural homodimer predictions also found altered dimer formation fo rthe L115R mutant that could provide a mechanism of aggregation that is independent of palmitoylation. Collectively, these analyses have uncovered potential novel functions of proline-175 in the zDABM of CSPα, which may be relevant to both the expression of the protein and its aggregation in ANCL.Cysteine-string Protein (CSPα) has been linked to neurodegeneration and mutations in CSPα are known to cause Adult-onset Ceroid Lipofuscinosis (ANCL). CSPα’s normal function relies on its targeting to synaptic vesicles by palmitoylation – the attachment offatty acids to cysteine residues. Palmitoylation is mediated by several Palmitoyl-Transferase (PAT) enzymes, such as zDHHC3, zDHHC7 and zDHHC17. zDHHC17 bindsto all its known substrates such as, CSPα through its ankyrin repeat domain by recognising CSPα’s zDHHC ankyrin-repeat binding motif (zDABM), which contains the consensus sequence ΨβXXQP, where proline-175 is the central residue for CSPα’s association. Despite the known requirement of the zDABM for CSPα binding to zDHHC17,the importance of the zDABM for palmitoylation has not yet been demonstrated.Therefore, this investigation determined how disruption of the zDABM affected palmitoylation of CSPα. Furthermore, PAT co-expression with CSPα has been shown to increase aggregation in ANCL mutants in vitro; therefore, we also aimed to determine the importance of the zDABM in the aggregation of ANCL mutant CSPα. in-silico methods were also used to predict changes in post-translational modifications (PTMs), and changes in the monomeric and homodimeric structure of CSPα caused by ANCL mutations. It was found that disruption of the zDABM sequence led to a zDHHC17-dependent increase in the levels of both palmitoylated and non-palmitoylated CSPα.Moreover, disruption of the zDABM sequence had no influence on the aggregation of ANCL mutants. A method for solubilisation of the CSPα aggregates using Triton-X100lysis and Sodium Dodecyl Sulfate (SDS) incubation was identified; however, further investigation of the solubilised aggregates was not possible due to restrictions on laboratory work. Structural predictions of CSPα identified that proline-175 in the zDABM sequence directly interacts with L115. Furthermore, this interaction, in addition to the structure and localisation of the zDABM sequence is considerably compromised in theANCL mutants. Structural homodimer predictions also found altered dimer formation fo rthe L115R mutant that could provide a mechanism of aggregation that is independent of palmitoylation. Collectively, these analyses have uncovered potential novel functions of proline-175 in the zDABM of CSPα, which may be relevant to both the expression of the protein and its aggregation in ANCL
Spatiotemporal and temporal forecasting of ambient air pollution levels through data-intensive hybrid artificial neural network models
Get PDFOutdoor air pollution (AP) is a serious public threat which has been linked to severe respiratory and cardiovascular illnesses, and premature deaths especially among those residing in highly urbanised cities. As such, there is a need to develop early-warning and risk management tools to alleviate its effects. The main objective of this research is to develop AP forecasting models based on Artificial Neural Networks (ANNs) according to an identified model-building protocol from existing related works. Plain, hybrid and ensemble ANN model architectures were developed to estimate the temporal and spatiotemporal variability of hourly NO2 levels in several locations in the Greater London area. Wavelet decomposition was integrated with Multilayer Perceptron (MLP) and Long Short-term Memory (LSTM) models to address the issue of high variability of AP data and improve the estimation of peak AP levels. Block-splitting and crossvalidation procedures have been adapted to validate the models based on Root Mean Squared Error (RMSE), Mean Absolute Error (MAE), and Willmott’s index of agreement (IA). The results of the proposed models present better performance than those from the benchmark models. For instance, the proposed wavelet-based hybrid approach provided 39.15% and 28.58% reductions in RMSE and MAE indices, respectively, on the performance of the benchmark MLP model results for the temporal forecasting of NO2 levels. The same approach reduced the RMSE and MAE indices of the benchmark LSTM model results by 12.45% and 20.08%, respectively, for the spatiotemporal estimation of NO2 levels in one site at Central London. The proposed hybrid deep learning approach offers great potential to be operational in providing air pollution forecasts in areas without a reliable database. The model-building protocol adapted in this thesis can also be applied to studies using measurements from other sites.Outdoor air pollution (AP) is a serious public threat which has been linked to severe respiratory and cardiovascular illnesses, and premature deaths especially among those residing in highly urbanised cities. As such, there is a need to develop early-warning and risk management tools to alleviate its effects. The main objective of this research is to develop AP forecasting models based on Artificial Neural Networks (ANNs) according to an identified model-building protocol from existing related works. Plain, hybrid and ensemble ANN model architectures were developed to estimate the temporal and spatiotemporal variability of hourly NO2 levels in several locations in the Greater London area. Wavelet decomposition was integrated with Multilayer Perceptron (MLP) and Long Short-term Memory (LSTM) models to address the issue of high variability of AP data and improve the estimation of peak AP levels. Block-splitting and crossvalidation procedures have been adapted to validate the models based on Root Mean Squared Error (RMSE), Mean Absolute Error (MAE), and Willmott’s index of agreement (IA). The results of the proposed models present better performance than those from the benchmark models. For instance, the proposed wavelet-based hybrid approach provided 39.15% and 28.58% reductions in RMSE and MAE indices, respectively, on the performance of the benchmark MLP model results for the temporal forecasting of NO2 levels. The same approach reduced the RMSE and MAE indices of the benchmark LSTM model results by 12.45% and 20.08%, respectively, for the spatiotemporal estimation of NO2 levels in one site at Central London. The proposed hybrid deep learning approach offers great potential to be operational in providing air pollution forecasts in areas without a reliable database. The model-building protocol adapted in this thesis can also be applied to studies using measurements from other sites
Performance management in Thai R&D organizations : exploring the interplay between R&D institutions and R&D contexts
Get PDFMeasuring research and development (R&D) performance has become a fundamental concern for R&D organisations. However, the complexity of measurement problems in R&D organisations has resulted in a situation where there is an excess of literature around the areas of R&D measurement, and yet a scarcity of generally accepted measurement approaches (Brown & Gobeli, 1992). This might be because the design of performance measurement (PM) for an R&D organisation combines several interrelated contexts that make each R&D measurement unique. This thesis, therefore, reviews several major R&D distinctions which should be taken into account when the R&D PM design is considered. These considerations are R&D measurement levels and perspectives, R&D key measures, R&D key drivers, and types of R&D institutions.;Taking a quality-based approach, the thesis combines several techniques, i.e. in-depth interviews, cognitive mapping interviews, document analysis, multiple case studies, and cross-case analysis. The interviews involve 30 respondents who are all experienced in R&D management in four different Thai R&D institutes, under the Ministry of Science and Technology.;The results indicate issues in three areas of investigation. First, the four cases studied measure R&D performance at different levels, for different purposes, and applying different measures and techniques. At a corporate level, instead of emphasising financial areas, the output measurement seems to be significant, as well as deliberative to quantitative methods. Meanwhile, at a team level, the measures highlight both quantitative and qualitative measures, for the purpose of monitoring the process and progress of research.;Second, the output mixes, stage of R&D, and sources of research questions could lead to the identification of three major types of R&D organisations: discipline-based, profession-based, and domain-based. The R&D measures that a firm applies seem to be interrelated with the type of R&D institution that firm represents.;Finally, the main key driver in this study is R&D collaboration. However, collaboration functions differ according to the different types of R&D organisations. A discipline-based organisation tends to use collaboration as a tool to explore new knowledge and to strengthen the firm's competency, whereas a profession-based organisation tends to use collaboration to gain market information and increase its ability to utilise R&D.;The study developed implications of both theoretical and managerial importance, identifying patterns of interrelationship between R&D institutions and key performance measures, and between R&D institutions and their collaboration mechanisms. Additionally, the main managerial implication could benefit R&D management practitioners, R&D managers, and R&D policymakers. Overall, the study's results demonstrate the importance of understanding the constraints of each R&D measurement context, i.e. levels of measurement, areas of measurement, and stages of R&D, for the performance measurement system.;Also, this study shows that each type of R&D institute may significantly be interrelated with other features, i.e. with key measures, and with key driver's mechanisms. Conducive to measuring and managing R&D performance efficiently, managers may realise the unique role of each type of R&D organisation (as well as its key measures and performance drivers) and design their performance measurement accordingly. Therefore, the benefits of this study may be seen as practical knowledge which could be employed to design R&D PM and, ultimately, to complement a strategic formulation to improve a firm's R&D performance.Measuring research and development (R&D) performance has become a fundamental concern for R&D organisations. However, the complexity of measurement problems in R&D organisations has resulted in a situation where there is an excess of literature around the areas of R&D measurement, and yet a scarcity of generally accepted measurement approaches (Brown & Gobeli, 1992). This might be because the design of performance measurement (PM) for an R&D organisation combines several interrelated contexts that make each R&D measurement unique. This thesis, therefore, reviews several major R&D distinctions which should be taken into account when the R&D PM design is considered. These considerations are R&D measurement levels and perspectives, R&D key measures, R&D key drivers, and types of R&D institutions.;Taking a quality-based approach, the thesis combines several techniques, i.e. in-depth interviews, cognitive mapping interviews, document analysis, multiple case studies, and cross-case analysis. The interviews involve 30 respondents who are all experienced in R&D management in four different Thai R&D institutes, under the Ministry of Science and Technology.;The results indicate issues in three areas of investigation. First, the four cases studied measure R&D performance at different levels, for different purposes, and applying different measures and techniques. At a corporate level, instead of emphasising financial areas, the output measurement seems to be significant, as well as deliberative to quantitative methods. Meanwhile, at a team level, the measures highlight both quantitative and qualitative measures, for the purpose of monitoring the process and progress of research.;Second, the output mixes, stage of R&D, and sources of research questions could lead to the identification of three major types of R&D organisations: discipline-based, profession-based, and domain-based. The R&D measures that a firm applies seem to be interrelated with the type of R&D institution that firm represents.;Finally, the main key driver in this study is R&D collaboration. However, collaboration functions differ according to the different types of R&D organisations. A discipline-based organisation tends to use collaboration as a tool to explore new knowledge and to strengthen the firm's competency, whereas a profession-based organisation tends to use collaboration to gain market information and increase its ability to utilise R&D.;The study developed implications of both theoretical and managerial importance, identifying patterns of interrelationship between R&D institutions and key performance measures, and between R&D institutions and their collaboration mechanisms. Additionally, the main managerial implication could benefit R&D management practitioners, R&D managers, and R&D policymakers. Overall, the study's results demonstrate the importance of understanding the constraints of each R&D measurement context, i.e. levels of measurement, areas of measurement, and stages of R&D, for the performance measurement system.;Also, this study shows that each type of R&D institute may significantly be interrelated with other features, i.e. with key measures, and with key driver's mechanisms. Conducive to measuring and managing R&D performance efficiently, managers may realise the unique role of each type of R&D organisation (as well as its key measures and performance drivers) and design their performance measurement accordingly. Therefore, the benefits of this study may be seen as practical knowledge which could be employed to design R&D PM and, ultimately, to complement a strategic formulation to improve a firm's R&D performance
Design of novel delivery systems to probe alternative routes of administration for a self-amplifying RNA rabies vaccine
Get PDFCurrent vaccine design aims to develop safer vaccines based on one or few selected antigens. RNA-based vaccines can be engineered to encode any antigen of interest and have the potential for rapid, inexpensive and scalable manufacturing and have an acceptable safety profile. Moreover, they enable in situ antigen expression, mimicking a real viral infection hence eliciting robust humoral and cellular-mediated immune responses. RNA vaccines therefore represent a versatile tool to fight infectious diseases and emerging pathogens effectively and rapidly. Furthermore, the antigen can be designed in a self-amplifying RNA (SAM) to enhance the immunogenicity and to reduce the therapeutic dose compared to conventional non-amplifying mRNA vaccines.;RNAs can be encapsulated in delivery systems to protect them against degradation upon injection and to facilitate their delivery in host cells. Among them, lipid-based delivery systems and, more specifically, lipid nanoparticles (LNPs) are efficient non-viral delivery systems for RNA and SAM vaccines. Within this thesis, a panel of cationic LNPs (cLNPs), based on existing cationic lipids (e.g. DOTAP and DDA), was designed to deliver a SAM vaccine. The rabies virus was used as a model, as there is an established correlate of protection (neutralising antibodies) and there exist efficacious vaccines in the market (e.g. Rabipur) to be used as comparators. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG), the only target for neutralising antibodies, was used;Microfluidics-based methods for producing cLNPs of desired physicochemical properties were developed and optimal operating parameters (e.g. total flow rate and flow rate ratio) were established. Most promising SAM-cLNP candidates were chosen according to their physicochemical attributes, their ability to protect SAM from enzymatic degradation and their capacity to associate with cells and to induce antigen expression. These formulations were well retained at the injection site when administered intramuscularly or intradermally, while they were rapidly cleared following intranasal administration.;On the other hand, SAM-cLNPs induced protective levels of anti-RVG antibodies following intramuscular injection in mice and RVG-specific polyfunctional T cell responses even with a dose as low as 0.15 μg RVG-SAM. Remarkably, the immune responses elicited by SAM-cLNPs were comparable to Rabipur, a commercial vaccine based on an inactivated rabies virus, and a cationic nanoemulsion, a safe and well-established SAM delivery system which is currently being investigated in a phase I clinical trial in humans (as of September 2019). Intradermal administration of SAM-cLNPs resulted in similar humoral and cell-mediated immune responses, while significantly weaker immune responses were achieved when administered intranasally.Current vaccine design aims to develop safer vaccines based on one or few selected antigens. RNA-based vaccines can be engineered to encode any antigen of interest and have the potential for rapid, inexpensive and scalable manufacturing and have an acceptable safety profile. Moreover, they enable in situ antigen expression, mimicking a real viral infection hence eliciting robust humoral and cellular-mediated immune responses. RNA vaccines therefore represent a versatile tool to fight infectious diseases and emerging pathogens effectively and rapidly. Furthermore, the antigen can be designed in a self-amplifying RNA (SAM) to enhance the immunogenicity and to reduce the therapeutic dose compared to conventional non-amplifying mRNA vaccines.;RNAs can be encapsulated in delivery systems to protect them against degradation upon injection and to facilitate their delivery in host cells. Among them, lipid-based delivery systems and, more specifically, lipid nanoparticles (LNPs) are efficient non-viral delivery systems for RNA and SAM vaccines. Within this thesis, a panel of cationic LNPs (cLNPs), based on existing cationic lipids (e.g. DOTAP and DDA), was designed to deliver a SAM vaccine. The rabies virus was used as a model, as there is an established correlate of protection (neutralising antibodies) and there exist efficacious vaccines in the market (e.g. Rabipur) to be used as comparators. To this end, a SAM vaccine encoding the rabies virus glycoprotein (RVG), the only target for neutralising antibodies, was used;Microfluidics-based methods for producing cLNPs of desired physicochemical properties were developed and optimal operating parameters (e.g. total flow rate and flow rate ratio) were established. Most promising SAM-cLNP candidates were chosen according to their physicochemical attributes, their ability to protect SAM from enzymatic degradation and their capacity to associate with cells and to induce antigen expression. These formulations were well retained at the injection site when administered intramuscularly or intradermally, while they were rapidly cleared following intranasal administration.;On the other hand, SAM-cLNPs induced protective levels of anti-RVG antibodies following intramuscular injection in mice and RVG-specific polyfunctional T cell responses even with a dose as low as 0.15 μg RVG-SAM. Remarkably, the immune responses elicited by SAM-cLNPs were comparable to Rabipur, a commercial vaccine based on an inactivated rabies virus, and a cationic nanoemulsion, a safe and well-established SAM delivery system which is currently being investigated in a phase I clinical trial in humans (as of September 2019). Intradermal administration of SAM-cLNPs resulted in similar humoral and cell-mediated immune responses, while significantly weaker immune responses were achieved when administered intranasally
Analysis of Leishmania GSK3 beta signalling pathways
No full textAccording to the World Health Organisation (WHO), leishmaniasis belongs to the twenty-one neglected tropical diseases affecting over 1 million people each year in 98 countries worldwide. The mortality rate is estimated to be 70,000 annual deaths indicating the need of a treatment (Torres-Guerrero et al., 2017). Protein kinases are potential antileishmanial drug targets. GSK3β is involved in important cellular processes such as cell proliferation and apoptosis. LmxGSK3β has been proven essential for the survival of L. mexicana in the promastigote insect stage and amastigote mammalian stage of the parasite suggesting that this is a protein worth investigating further (Munro, 2013). To serve this scope, the present project aimed to confirm potential substrates of this protein. From a list with putative substrates of LmxGSK3β, which was already generated using BioID in our lab, LmxDynamin and LmxKIN13-1 were used for further analyses. Moreover, a peptide derived from LmxUBH5 was found to interact with LxmGSK3β in a kinase assay, thus this protein was included as a putative substrate in this investigation.;To address the hypothesis that LmxDynamin, LmxKIN13-1 and LmxUBH5 are bona fide LmxGSK3β substrates CRISPR/Cas9 technology and bioinformatics tools were utilised. Attempts to tag LmxDynamin with mNeonGreen (mNG) using CRISPR/Cas9 were unsuccessful. Likewise, a knockout of LmxUBH5 could not be obtained. It might be that LmxUBH5 is essential for the parasite and hence its deletion is not possible. CRISPR/Cas9 allowed C- and N- terminal mNG-fusion of LmxKIN13-1 and LmxUBH5, respectively. Diagnostic PCRs detected the correct integration of donor DNA fragments into the L. mexicana target gene loci. Immunoblot analysis confirmed expression of the full-length fusion proteins. LmxUBH5 was found in the cytoplasm of promastigotes in all stages of the cell cycle. Some of the transfected cells appeared with normal morphology, while some cells were affected by the tagging and showed an aberrant cell shape and their cytokinesis started from the posterior end of the cell, opposite from the flagellum. LmxKIN13-1 localised in the nucleus. It was only present during mitosis. Its homologue in Trypanosoma brucei, TbKIN13-1, showed similar results (Wickstead et al., 2010). A three-dimensional model of the LmxKIN13-1 motor domain was generated based on the structure of the Plasmodium falciparum kinesin pKinI (PDB 1ry6). The motor domain is at the centre of the protein classifying LmxKIN13-1 as an M-kinesin. It contains the conserved ATP-binding and microtubule interaction sites.;The ATP-binding site is comprised of the N1/P loop (GQTGSGKT), the N2/Switch I (NxxSSRSH), the N3/Switch II (DLAGxER) and N4 (RxRP). The microtubule interaction site consists of the α4-helix (KECIR), loop 2 (KVD), loop 8 (LRxLEDxKxxVxxGL) and loop 12 (QNKxHTPVSK), which are highly conserved among proteins of the kinesin-13 family in different Leishmania spp and in T. brucei. The central location of the motor domain is a general feature of kinesin13-1 family members. This study presents evidence that LmxKIN13-1 is involved in mitosis. Therefore, as a potential interaction partner of LmxGSK3β, LmxKIN13-1 activity might be regulated by this kinase allowing the development of a mitotic inhibitor. This could be then used to design a drug against leishmaniasis. Further analysis utilising Miniturbo-2C BioID and kinase assays with recombinantly expressed proteins were planned to investigate this aspect but could not be performed due to the COVID-19 pandemic.According to the World Health Organisation (WHO), leishmaniasis belongs to the twenty-one neglected tropical diseases affecting over 1 million people each year in 98 countries worldwide. The mortality rate is estimated to be 70,000 annual deaths indicating the need of a treatment (Torres-Guerrero et al., 2017). Protein kinases are potential antileishmanial drug targets. GSK3β is involved in important cellular processes such as cell proliferation and apoptosis. LmxGSK3β has been proven essential for the survival of L. mexicana in the promastigote insect stage and amastigote mammalian stage of the parasite suggesting that this is a protein worth investigating further (Munro, 2013). To serve this scope, the present project aimed to confirm potential substrates of this protein. From a list with putative substrates of LmxGSK3β, which was already generated using BioID in our lab, LmxDynamin and LmxKIN13-1 were used for further analyses. Moreover, a peptide derived from LmxUBH5 was found to interact with LxmGSK3β in a kinase assay, thus this protein was included as a putative substrate in this investigation.;To address the hypothesis that LmxDynamin, LmxKIN13-1 and LmxUBH5 are bona fide LmxGSK3β substrates CRISPR/Cas9 technology and bioinformatics tools were utilised. Attempts to tag LmxDynamin with mNeonGreen (mNG) using CRISPR/Cas9 were unsuccessful. Likewise, a knockout of LmxUBH5 could not be obtained. It might be that LmxUBH5 is essential for the parasite and hence its deletion is not possible. CRISPR/Cas9 allowed C- and N- terminal mNG-fusion of LmxKIN13-1 and LmxUBH5, respectively. Diagnostic PCRs detected the correct integration of donor DNA fragments into the L. mexicana target gene loci. Immunoblot analysis confirmed expression of the full-length fusion proteins. LmxUBH5 was found in the cytoplasm of promastigotes in all stages of the cell cycle. Some of the transfected cells appeared with normal morphology, while some cells were affected by the tagging and showed an aberrant cell shape and their cytokinesis started from the posterior end of the cell, opposite from the flagellum. LmxKIN13-1 localised in the nucleus. It was only present during mitosis. Its homologue in Trypanosoma brucei, TbKIN13-1, showed similar results (Wickstead et al., 2010). A three-dimensional model of the LmxKIN13-1 motor domain was generated based on the structure of the Plasmodium falciparum kinesin pKinI (PDB 1ry6). The motor domain is at the centre of the protein classifying LmxKIN13-1 as an M-kinesin. It contains the conserved ATP-binding and microtubule interaction sites.;The ATP-binding site is comprised of the N1/P loop (GQTGSGKT), the N2/Switch I (NxxSSRSH), the N3/Switch II (DLAGxER) and N4 (RxRP). The microtubule interaction site consists of the α4-helix (KECIR), loop 2 (KVD), loop 8 (LRxLEDxKxxVxxGL) and loop 12 (QNKxHTPVSK), which are highly conserved among proteins of the kinesin-13 family in different Leishmania spp and in T. brucei. The central location of the motor domain is a general feature of kinesin13-1 family members. This study presents evidence that LmxKIN13-1 is involved in mitosis. Therefore, as a potential interaction partner of LmxGSK3β, LmxKIN13-1 activity might be regulated by this kinase allowing the development of a mitotic inhibitor. This could be then used to design a drug against leishmaniasis. Further analysis utilising Miniturbo-2C BioID and kinase assays with recombinantly expressed proteins were planned to investigate this aspect but could not be performed due to the COVID-19 pandemic
Removal of polycyclic aromatic hydrocarbons and other contaminants from hydrocarbon fluids
Get PDFThis thesis was previously held under moratorium from 30/06/2020 to 30/06/2022Polycyclic aromatic hydrocarbons (PAHs), compounds that are produced during the combustion of fuels, is one example of contaminants found in automotive lubricants. PAHs can aggregate and form solid deposits. The deposits can be dispersed by the inclusion of oil-soluble additives into the lubricant. However, these additives can have negative effects on the lubricant performance. Unfortunately, current technologies do not remove contaminants; rather, they serve to minimise any damage caused by them. It is proposed that supplementing current technologies with suitable solid-phase, polymeric additives may be of benefit. It is hypothesised that contaminants can be removed from hydrocarbon fluids using polymer-supported chemistries in the solid-phase.Precipitation polymerisation (PP) and non-aqueous dispersion (NAD) polymerisation were investigated to synthesise a library of aromatic-based, crosslinked polymer particles. Hypercrosslinking chemistry was also utilised to impart high specific surface areas into some materials. To test the applicability of removing PAHs from a hydrocarbon fluid, each polymer was contacted with a solution of PAHs in heptane.These investigations proved fruitful, showing that upwards of 90% of certain PAHs could be removed from a heptane solution using crosslinked polymers.The synthesis of polymer resins by suspension polymerisation was investigated, with an aim of producing polymer particles that were of an appropriate size for containment within an engine lubricant system. A library of resins was successfully designed, synthesised and characterised. Gel-type, macroreticular and hypercrosslinked materials were synthesised to give a breadth of specific surface areas bearing varied functionality.Finally, contamination removal from an engine lubricant in situ, using some of the synthesised materials, was undertaken. It was concluded that using solid phase-polymers as contaminant removal devices was effective.Some of the work presented in this thesis forms the basis for the patent application W02017178593A2 (Removal of aromatic compounds from a hydrocarbon fluid).Polycyclic aromatic hydrocarbons (PAHs), compounds that are produced during the combustion of fuels, is one example of contaminants found in automotive lubricants. PAHs can aggregate and form solid deposits. The deposits can be dispersed by the inclusion of oil-soluble additives into the lubricant. However, these additives can have negative effects on the lubricant performance. Unfortunately, current technologies do not remove contaminants; rather, they serve to minimise any damage caused by them. It is proposed that supplementing current technologies with suitable solid-phase, polymeric additives may be of benefit. It is hypothesised that contaminants can be removed from hydrocarbon fluids using polymer-supported chemistries in the solid-phase.Precipitation polymerisation (PP) and non-aqueous dispersion (NAD) polymerisation were investigated to synthesise a library of aromatic-based, crosslinked polymer particles. Hypercrosslinking chemistry was also utilised to impart high specific surface areas into some materials. To test the applicability of removing PAHs from a hydrocarbon fluid, each polymer was contacted with a solution of PAHs in heptane.These investigations proved fruitful, showing that upwards of 90% of certain PAHs could be removed from a heptane solution using crosslinked polymers.The synthesis of polymer resins by suspension polymerisation was investigated, with an aim of producing polymer particles that were of an appropriate size for containment within an engine lubricant system. A library of resins was successfully designed, synthesised and characterised. Gel-type, macroreticular and hypercrosslinked materials were synthesised to give a breadth of specific surface areas bearing varied functionality.Finally, contamination removal from an engine lubricant in situ, using some of the synthesised materials, was undertaken. It was concluded that using solid phase-polymers as contaminant removal devices was effective.Some of the work presented in this thesis forms the basis for the patent application W02017178593A2 (Removal of aromatic compounds from a hydrocarbon fluid)
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