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The discrete coagulation-fragmentation system
Get PDFIn this thesis, we examine an infinite system of ordinary differential equations that models the evolution of fragmenting and coalescing discrete-sized particle clusters.We express this discrete coagulation-fragmentation system as an abstract Cauchy problem posed in an appropriate Banach space of sequences. The theory of operator semigroups is then used to establish the existence and uniqueness of solutions. We also investigate properties of the solutions, such as positivity, mass conservation and asymptotic behaviour. A main aim of this thesis is to relax the assumptions that have previously been required, when using a semigroupapproach, to obtain the existence and uniqueness of physically relevant solutions.Moreover, we consider the case when the infinite system is non-autonomous dueto time-dependent coagulation and fragmentation coefficients.In this thesis, we examine an infinite system of ordinary differential equations that models the evolution of fragmenting and coalescing discrete-sized particle clusters.We express this discrete coagulation-fragmentation system as an abstract Cauchy problem posed in an appropriate Banach space of sequences. The theory of operator semigroups is then used to establish the existence and uniqueness of solutions. We also investigate properties of the solutions, such as positivity, mass conservation and asymptotic behaviour. A main aim of this thesis is to relax the assumptions that have previously been required, when using a semigroupapproach, to obtain the existence and uniqueness of physically relevant solutions.Moreover, we consider the case when the infinite system is non-autonomous dueto time-dependent coagulation and fragmentation coefficients
Exploring the organisational, social and cultural factors influencing those employee attitudes and behaviours that impact the implementation of an information security culture within Omani organisations
Get PDFResearch has strongly established that the success of an information security program is heavily dependent upon the actions of the members of organisations that interact with the information security program. An appropriate information security culture is required to effectively influence and control the actions of the members within an organisation because of this interaction between people and the information security program.This thesis seeks to explore and study the current state of information security behaviour and discipline in public and private organisations in the context of Oman and investigates the challenges in developing an information security culture within these organisations. The key focus of the study is on an investigation and identification of the critical socio-cultural and organisational factors that affect the successful development and maintenance of a culture of information security within public organisations in the context of Oman. The study also aims to examine the difference between public andprivate organisations in Oman regarding information security practices.;Although many organisations in Oman have implemented technical solutions to protect information resources from adverse events, internal security breaches continue to occur.For this reason an emphasis on a culture of information security within organisations is required in order to make security an integral part of employees' daily work routines.Although, it is important in practice to address both technical and non-technical aspects when dealing with information security, the research described in this thesis concentrates upon non-technical approaches, and excludes consideration of the technological aspects.To achieve the study aim, the research reviewed and compared the roles of national culture; information security culture; organisational culture and employee behaviour within organisations, in order to determine the socio-cultural and organisational factors that potentially hinder an organisation in implementing, integrating, and maintaining a successful organisational information security culture. A review of related academic work was undertaken. In addition, the research used both quantitative and qualitative research methods to collect, analyse and integrate data from a survey questionnaire of 155 respondents semi-randomly selected from different Omani public and private organisations. The survey results formed the basis of hypotheses about the critical factors in developing effective information security practices in these organisations.;The IBM Statistical Package for the Social Sciences (SPSS version 22) with multiple regression was used to analyse the relationship between a dependent variable and several independent variables. To validate the identified critical factors further, thematic analysis was carried out using semi-structured open-ended interviews with specialist Information Technology (IT) and Information Security (IS) senior managers in fifteen selected public and private organisations.The data analysis indicates that security of information in Omani public organisations is not optimal. The findings show in general that these organisations have inadequate information security cultures. These organisations are facing several challenges. These include the remoteness of those in power from the issue and therefore a lack of senior management support and involvement. There is a lack of training and awareness. There is an absence of policies to develop a respect for collectivism, avoiding uncertainty and building a high level of trust, which would all help to support security of information.;The current study contributes in a number of ways to discussions and actions around these issues. Firstly, the findings can serve as a basis for Omani public organisations to reform their information security programs. The study identifies and investigates the most critical factors influencing the effectiveness of information security practices.There has been little research conducted to date that assists an understanding and management of the culture of information security within Omani public organisations.The researcher hope that this study will expand the body of knowledge in this area.Furthermore, this study is the first and only one to my knowledge that explores the influences of critical socio-cultural and organisational factors on employee behaviours and attitudes regarding the security of information in Omani organisations. However,further research is needed to improve our insight into information security in the context of Omani organisations. In addition, research which compares Omani organisations with other Gulf country organisations would provide further insight into information security in Oman.Research has strongly established that the success of an information security program is heavily dependent upon the actions of the members of organisations that interact with the information security program. An appropriate information security culture is required to effectively influence and control the actions of the members within an organisation because of this interaction between people and the information security program.This thesis seeks to explore and study the current state of information security behaviour and discipline in public and private organisations in the context of Oman and investigates the challenges in developing an information security culture within these organisations. The key focus of the study is on an investigation and identification of the critical socio-cultural and organisational factors that affect the successful development and maintenance of a culture of information security within public organisations in the context of Oman. The study also aims to examine the difference between public andprivate organisations in Oman regarding information security practices.;Although many organisations in Oman have implemented technical solutions to protect information resources from adverse events, internal security breaches continue to occur.For this reason an emphasis on a culture of information security within organisations is required in order to make security an integral part of employees' daily work routines.Although, it is important in practice to address both technical and non-technical aspects when dealing with information security, the research described in this thesis concentrates upon non-technical approaches, and excludes consideration of the technological aspects.To achieve the study aim, the research reviewed and compared the roles of national culture; information security culture; organisational culture and employee behaviour within organisations, in order to determine the socio-cultural and organisational factors that potentially hinder an organisation in implementing, integrating, and maintaining a successful organisational information security culture. A review of related academic work was undertaken. In addition, the research used both quantitative and qualitative research methods to collect, analyse and integrate data from a survey questionnaire of 155 respondents semi-randomly selected from different Omani public and private organisations. The survey results formed the basis of hypotheses about the critical factors in developing effective information security practices in these organisations.;The IBM Statistical Package for the Social Sciences (SPSS version 22) with multiple regression was used to analyse the relationship between a dependent variable and several independent variables. To validate the identified critical factors further, thematic analysis was carried out using semi-structured open-ended interviews with specialist Information Technology (IT) and Information Security (IS) senior managers in fifteen selected public and private organisations.The data analysis indicates that security of information in Omani public organisations is not optimal. The findings show in general that these organisations have inadequate information security cultures. These organisations are facing several challenges. These include the remoteness of those in power from the issue and therefore a lack of senior management support and involvement. There is a lack of training and awareness. There is an absence of policies to develop a respect for collectivism, avoiding uncertainty and building a high level of trust, which would all help to support security of information.;The current study contributes in a number of ways to discussions and actions around these issues. Firstly, the findings can serve as a basis for Omani public organisations to reform their information security programs. The study identifies and investigates the most critical factors influencing the effectiveness of information security practices.There has been little research conducted to date that assists an understanding and management of the culture of information security within Omani public organisations.The researcher hope that this study will expand the body of knowledge in this area.Furthermore, this study is the first and only one to my knowledge that explores the influences of critical socio-cultural and organisational factors on employee behaviours and attitudes regarding the security of information in Omani organisations. However,further research is needed to improve our insight into information security in the context of Omani organisations. In addition, research which compares Omani organisations with other Gulf country organisations would provide further insight into information security in Oman
Working with the enemy : relations between Italian partisans and the British forces (1943-1945)
Get PDFThe Italo-British relationship during the Second World War has been the object of constant scrutiny and enquiry over the course of the years, especially as far as the relation between the Italian Resistance and British forces during the Italian campaign of 1943-1945 is concerned. Many have pointed out that the British actions were moved by political reasons, such as the fear of a possible Communist takeover after the war.According to this interpretation, this led to a general hostility in the British war machine towards Italians. Others have pointed out how this analysis is fundamentally flawed, as it focuses only on the political aspect of the relationship and does not account for the good relations that often Italian partisans and British forces were able to build in the field. However, both interpretations seem to exist independently of each other and the debate has reached a stale point over the course of the last few decades.Through the critical re-examination of three main topics (interactions on the field,propaganda and partisan disarmament) this thesis aims at filling the gulf between these two historiographical branches. The analysis is not only focused on British or Italian actions but also how these actions influenced the perception of each other. This point of view allows for a nuanced approach that considers representation and selfrepresentation as two key interpretative tools. Moreover, the elements unearthed in this way can be also evaluated against the historiography and public memory that followed the war, in order to frame elements of longue durée which may have influenced the representation of the Italo-British relationship in the future.The Italo-British relationship during the Second World War has been the object of constant scrutiny and enquiry over the course of the years, especially as far as the relation between the Italian Resistance and British forces during the Italian campaign of 1943-1945 is concerned. Many have pointed out that the British actions were moved by political reasons, such as the fear of a possible Communist takeover after the war.According to this interpretation, this led to a general hostility in the British war machine towards Italians. Others have pointed out how this analysis is fundamentally flawed, as it focuses only on the political aspect of the relationship and does not account for the good relations that often Italian partisans and British forces were able to build in the field. However, both interpretations seem to exist independently of each other and the debate has reached a stale point over the course of the last few decades.Through the critical re-examination of three main topics (interactions on the field,propaganda and partisan disarmament) this thesis aims at filling the gulf between these two historiographical branches. The analysis is not only focused on British or Italian actions but also how these actions influenced the perception of each other. This point of view allows for a nuanced approach that considers representation and selfrepresentation as two key interpretative tools. Moreover, the elements unearthed in this way can be also evaluated against the historiography and public memory that followed the war, in order to frame elements of longue durée which may have influenced the representation of the Italo-British relationship in the future
Nanoparticle-based assays for the detection of protein glycosylation in biopharmaceutical characterisation
Get PDFIn biopharmaceutical processing, the glycosylation of a therapeutic protein must be stringently monitored as this can affect the efficacy and safety of the final formulation. Current methods of analysis are time consuming, expensive, and require skilled personnel. Therefore, it would be advantageous to have rapid, cheap, and user-friendly assays to sensitively assess glycan composition. This research has made progress in the development of such assays.The first objective was to create sensing beacons which could be easily detected when binding to a target glycan occurred. Gold nanoparticles were investigated for this role due to their ease of functionalisation and optical properties. These were conjugated with a Raman-active molecule to act as a beacon, and a carbohydrate-binding protein via a heterobifunctional ligand. The conjugation procedure was shown to be successful and versatile, allowing for the attachment of various lectins using two different linking molecules.;Next, a solution-based assay was investigated to characterise the sensing capabilities of the functionalised gold nanoparticles. This could allow for the development of a glycosylation evaluation kit whereby a library of nanoparticle conjugates could be mixed with protein samples and analysed to assess glycan composition. Originally, the drive was to use surface enhanced resonance Raman scattering (SERRS) as a sensitive method of analysis. However, this was shown to be less useful in this setting, but localised surface plasmon resonance measurements proved to be a simple and rapid avenue for glycan characterisation. Detection of mannose was shown to have a limit of detection of 29.2 nM using the lectin from Galanthus nivalis. This technique was also shown to be versatile using various lectins for the detection of different glycan targets, and could potentially be more sensitive.A second route for glycan detection was investigated to provide a rapid and user-friendly method of analysis. Lateral flow assays were used to show the binding capabilities of the prepared nanoparticle conjugates, but had significantdisadvantages arising from the application of the target glycoprotein to the nitrocellulose surface as a spot rather than a line spanning the width of the strip. This led to an unsatisfactory assay sensitivity. To overcome this, a simpler and more sensitive format was investigated to create a high-throughput glycan analysis platform. For this to remain user-friendly, evaluation methods involved the use of a simple flatbed scanner to provide rapid colorimetric results, but this assay was also coupled with SERRS to investigate the capabilities of producing highly sensitive glycan detection. This paper-based analytical device still requires research, but has shown excellent potential for use as a valuable tool in biopharmaceutical glycan analysis.;Finally, the development of an enzyme-linked immunosorbent assay was investigated. This relied on the production of hydrogen peroxide by glucose oxidase when the target glycan was present. Hydrogen peroxide would then act as a reducing agent and induce the growth of gold nanoparticles in the presence of Au3+ cations. Although still in the early stages of development, this assay produced interesting results with many issues still to consider, but showed that it could have the capability of becoming a sensitive method of glycan detection. The common use of enzyme-linked immunosorbent assays in biochemical detection means that a glycosylation analysis kit based on this assay format could be an attractive alternative to current industrial analysis methods.In biopharmaceutical processing, the glycosylation of a therapeutic protein must be stringently monitored as this can affect the efficacy and safety of the final formulation. Current methods of analysis are time consuming, expensive, and require skilled personnel. Therefore, it would be advantageous to have rapid, cheap, and user-friendly assays to sensitively assess glycan composition. This research has made progress in the development of such assays.The first objective was to create sensing beacons which could be easily detected when binding to a target glycan occurred. Gold nanoparticles were investigated for this role due to their ease of functionalisation and optical properties. These were conjugated with a Raman-active molecule to act as a beacon, and a carbohydrate-binding protein via a heterobifunctional ligand. The conjugation procedure was shown to be successful and versatile, allowing for the attachment of various lectins using two different linking molecules.;Next, a solution-based assay was investigated to characterise the sensing capabilities of the functionalised gold nanoparticles. This could allow for the development of a glycosylation evaluation kit whereby a library of nanoparticle conjugates could be mixed with protein samples and analysed to assess glycan composition. Originally, the drive was to use surface enhanced resonance Raman scattering (SERRS) as a sensitive method of analysis. However, this was shown to be less useful in this setting, but localised surface plasmon resonance measurements proved to be a simple and rapid avenue for glycan characterisation. Detection of mannose was shown to have a limit of detection of 29.2 nM using the lectin from Galanthus nivalis. This technique was also shown to be versatile using various lectins for the detection of different glycan targets, and could potentially be more sensitive.A second route for glycan detection was investigated to provide a rapid and user-friendly method of analysis. Lateral flow assays were used to show the binding capabilities of the prepared nanoparticle conjugates, but had significantdisadvantages arising from the application of the target glycoprotein to the nitrocellulose surface as a spot rather than a line spanning the width of the strip. This led to an unsatisfactory assay sensitivity. To overcome this, a simpler and more sensitive format was investigated to create a high-throughput glycan analysis platform. For this to remain user-friendly, evaluation methods involved the use of a simple flatbed scanner to provide rapid colorimetric results, but this assay was also coupled with SERRS to investigate the capabilities of producing highly sensitive glycan detection. This paper-based analytical device still requires research, but has shown excellent potential for use as a valuable tool in biopharmaceutical glycan analysis.;Finally, the development of an enzyme-linked immunosorbent assay was investigated. This relied on the production of hydrogen peroxide by glucose oxidase when the target glycan was present. Hydrogen peroxide would then act as a reducing agent and induce the growth of gold nanoparticles in the presence of Au3+ cations. Although still in the early stages of development, this assay produced interesting results with many issues still to consider, but showed that it could have the capability of becoming a sensitive method of glycan detection. The common use of enzyme-linked immunosorbent assays in biochemical detection means that a glycosylation analysis kit based on this assay format could be an attractive alternative to current industrial analysis methods
Algorithmic enhancements to polynomial matrix factorisations
Get PDFIn broadband array processing applications, an extension of the eigenvalue decomposition (EVD) to parahermitian Laurent polynomial matrices - named the polynomial matrix EVD (PEVD) - has proven to be a useful tool for the decomposition of spacetime covariance matrices and their associated cross-spectral density matrices. Existing PEVD methods typically operate in the time domain and utilise iterative frameworks established by the second-order sequential best rotation (SBR2) or sequential matrix diagonalisation (SMD) algorithms.;However, motivated by recent discoveries that establish the existence of an analytic PEVD - which is rarely recovered by SBR2 or SMD - alternative algorithms that better meet analyticity by operating in the discrete Fourier transform (DFT)-domain have received increasing attention.;While offering promising results in applications including broadband MIMO and beamforming, the PEVD has seen limited deployment in hardware due to its high computational complexity. If the PEVD is to be fully utilised, overcoming this bottleneck is paramount. This thesis therefore seeks to reduce the computational cost of iterative PEVD algorithms - with particular emphasis on SMD - through the development of several novel algorithmic improvements.;While these are effective, the complexity of the optimised algorithms still grows rapidly with the spatial dimensions of the decomposition. Steps are therefore taken to convert the sequential form of SMD to a novel reduced dimensionality and partially parallelisable divide-and-conquer architecture. The resulting algorithms are shown to converge an order of magnitude faster than existing methods for large spatial dimensions, and are well-suited to application scenarios with many sensors.;Further in this thesis, an investigation into DFT-based algorithms highlights their potential to offer compact, analytic solutions to the PEVD. Subsequently, two novel DFT-based algorithms improve upon an existing method by reducing decomposition error and eliminating a priori knowledge requirements. Finally, an innovative strategy is shown to be capable of extracting a minimum-order solution to the PEVD.In broadband array processing applications, an extension of the eigenvalue decomposition (EVD) to parahermitian Laurent polynomial matrices - named the polynomial matrix EVD (PEVD) - has proven to be a useful tool for the decomposition of spacetime covariance matrices and their associated cross-spectral density matrices. Existing PEVD methods typically operate in the time domain and utilise iterative frameworks established by the second-order sequential best rotation (SBR2) or sequential matrix diagonalisation (SMD) algorithms.;However, motivated by recent discoveries that establish the existence of an analytic PEVD - which is rarely recovered by SBR2 or SMD - alternative algorithms that better meet analyticity by operating in the discrete Fourier transform (DFT)-domain have received increasing attention.;While offering promising results in applications including broadband MIMO and beamforming, the PEVD has seen limited deployment in hardware due to its high computational complexity. If the PEVD is to be fully utilised, overcoming this bottleneck is paramount. This thesis therefore seeks to reduce the computational cost of iterative PEVD algorithms - with particular emphasis on SMD - through the development of several novel algorithmic improvements.;While these are effective, the complexity of the optimised algorithms still grows rapidly with the spatial dimensions of the decomposition. Steps are therefore taken to convert the sequential form of SMD to a novel reduced dimensionality and partially parallelisable divide-and-conquer architecture. The resulting algorithms are shown to converge an order of magnitude faster than existing methods for large spatial dimensions, and are well-suited to application scenarios with many sensors.;Further in this thesis, an investigation into DFT-based algorithms highlights their potential to offer compact, analytic solutions to the PEVD. Subsequently, two novel DFT-based algorithms improve upon an existing method by reducing decomposition error and eliminating a priori knowledge requirements. Finally, an innovative strategy is shown to be capable of extracting a minimum-order solution to the PEVD
Framing, explaining and forming the National Improvement Framework
Get PDFThe National Improvement Framework (NIF) is intended to bring together the Scottish Government's priorities for education into one source. It promotes educational improvement through two main aims: achieving excellence and equity. It was published in January 2016 and is updated annually.;Adams' (2016) original approach to policy analysis is used to examine how the NIF has been formed through discourse (for example, conversations) at the micro level (policy-forming). However, these do not occur outside the social, cultural, historical or economic sphere in which they take place (Gee, 2012). Therefore, it is necessary to consider how policy ideas are framed through wider Discourse (policy-framing), and subsequently presented and explained in policy pronouncements (policy-explaining).;Fairclough's Critical Discourse Analysis was used to examine policy-framing and policy-explaining. The literature was used as data to highlight wider Discourse that framed the NIF. The NIF is framed within Discourse that espouses a Scottish identity through education, education in a globalised society and as a means to achieve equity. The internal relations, organisation and structure of the NIF policy text was examined to understand how the NIF was explained. Words and images are used that position education as a top priority with children placed centre staged. There is a focus on improvement and numbers that has positioned data as a symbolic form of capital.;Semi-structured interviews were undertaken with seventeen primary school teachers and analysed thematically to show how the NIF was formed. Since the NIF was published, there has been an increase in formal assessment and data tracking. Teachers are engaging in discussions about data, and children are being referred to by their 'data double' when achievement is reduced to colour coding on a spreadsheet. The distribution of pupil equity funding (PEF) is sometimes being prioritised for children in P1-P3. These practices are conceptualised as gaming by schools, although often resisted by teachers, which is not always commensurate with the aims of the Scottish curriculum.;It is recommended that the purpose of the NIF and its relationship to CfE is made clear to teachers. Schools need to examine the practices that have formed around tracking and the distribution of PEF. Local authorities and the Scottish Government need to examine the practices that have formed, and the increased sense of accountability felt by teachers, due to 'reaching into' schools' data.The National Improvement Framework (NIF) is intended to bring together the Scottish Government's priorities for education into one source. It promotes educational improvement through two main aims: achieving excellence and equity. It was published in January 2016 and is updated annually.;Adams' (2016) original approach to policy analysis is used to examine how the NIF has been formed through discourse (for example, conversations) at the micro level (policy-forming). However, these do not occur outside the social, cultural, historical or economic sphere in which they take place (Gee, 2012). Therefore, it is necessary to consider how policy ideas are framed through wider Discourse (policy-framing), and subsequently presented and explained in policy pronouncements (policy-explaining).;Fairclough's Critical Discourse Analysis was used to examine policy-framing and policy-explaining. The literature was used as data to highlight wider Discourse that framed the NIF. The NIF is framed within Discourse that espouses a Scottish identity through education, education in a globalised society and as a means to achieve equity. The internal relations, organisation and structure of the NIF policy text was examined to understand how the NIF was explained. Words and images are used that position education as a top priority with children placed centre staged. There is a focus on improvement and numbers that has positioned data as a symbolic form of capital.;Semi-structured interviews were undertaken with seventeen primary school teachers and analysed thematically to show how the NIF was formed. Since the NIF was published, there has been an increase in formal assessment and data tracking. Teachers are engaging in discussions about data, and children are being referred to by their 'data double' when achievement is reduced to colour coding on a spreadsheet. The distribution of pupil equity funding (PEF) is sometimes being prioritised for children in P1-P3. These practices are conceptualised as gaming by schools, although often resisted by teachers, which is not always commensurate with the aims of the Scottish curriculum.;It is recommended that the purpose of the NIF and its relationship to CfE is made clear to teachers. Schools need to examine the practices that have formed around tracking and the distribution of PEF. Local authorities and the Scottish Government need to examine the practices that have formed, and the increased sense of accountability felt by teachers, due to 'reaching into' schools' data
Risk assessment due to load demand and electricity price forecast uncertainty
Get PDFThis thesis introduces methodologies for load demand forecasting and electricity price forecasting. The autoregressive integrated moving average (ARIMA) models, seasonal autoregressive integrated moving average (SARIMA) models and artificial neural network (ANN) techniques are introduced to forecast load demand. And the forecasting process of load demand includes monthly, seasonal, annual and multi-step-ahead. Similarly, the same forecasting methods are used for electricity price forecasting. In terms of forecast error analysis method, the root mean square percentage error (RMSPE) and the mean absolute percentage error (MAPE) are used to observe the accuracy of forecasting results. After obtaining the forecasting results, this thesis proposes a risk index method to observe the forecast error more intuitively. The risk indexes are presented based on the load demand forecast errors and electricity price forecast errors respectively. In addition, this thesis investigates the financial risk by combining the errors made by load demand forecasting and electricity price forecasting. The Value-at-Risk (VaR) and Expected Shortfall (ES) methods in economic theory are used to analyse the financial risks. Moreover, to present the actual risk that the market participants have to bear, the daily, monthly, seasonal and annual total financial risks under three different preconditions are compared.This thesis introduces methodologies for load demand forecasting and electricity price forecasting. The autoregressive integrated moving average (ARIMA) models, seasonal autoregressive integrated moving average (SARIMA) models and artificial neural network (ANN) techniques are introduced to forecast load demand. And the forecasting process of load demand includes monthly, seasonal, annual and multi-step-ahead. Similarly, the same forecasting methods are used for electricity price forecasting. In terms of forecast error analysis method, the root mean square percentage error (RMSPE) and the mean absolute percentage error (MAPE) are used to observe the accuracy of forecasting results. After obtaining the forecasting results, this thesis proposes a risk index method to observe the forecast error more intuitively. The risk indexes are presented based on the load demand forecast errors and electricity price forecast errors respectively. In addition, this thesis investigates the financial risk by combining the errors made by load demand forecasting and electricity price forecasting. The Value-at-Risk (VaR) and Expected Shortfall (ES) methods in economic theory are used to analyse the financial risks. Moreover, to present the actual risk that the market participants have to bear, the daily, monthly, seasonal and annual total financial risks under three different preconditions are compared
A combined perfectly matching layer and infinite element formulation for unbounded wave problems
Get PDFNo abstract available.No abstract available
Development of a cardiac patch for the regeneration of infarcted hearts
Get PDFPreviously held under moratorium from 13 June 2019 until 28 July 2021The inflammation that occurs after cardiac ischemia/reperfusion leads to high levels of oxidative stress, which produces deleterious effects that ultimately limit regeneration of the myocardium. This stress response can be prolonged, thereby affecting various stages of cardiac repair remodelling. Efforts to control such remodelling and stimulate cardiac tissue regeneration have therefore included the use of antioxidant or anti-inflammatory drug strategies. To date, such approaches have involved delivery of the drugs orally or by injection. The use of a scaffold that can be placed onto the heart immediately following surgery would allow sustained delivery of high concentrations of drug in situ, directly targeting the affected area. Moreover, implantation of such a material has the potential to encourage cell infiltration into the matrix, thereby promoting regeneration. The ultimate aim of this project was to develop a cardiac scaffold loaded with a drug with antioxidant and anti-inflammatory activity. Pyruvate is a well characterised anti-inflammatory and antioxidant drug. It has been added to cardioplegia by some surgical groups for its potential cardioprotective effects, although the therapeutic value of sustained delivery of this drug into the myocardium following cardiac surgery has not been investigated. Ethyl Pyruvate (EP) is a more stable form of this drug and was therefore the drug selected for investigation in the present study. Various alginate scaffolds were developed as drug delivery vehicles, with EP release characterised in vitro. Alginate gels prepared with (1-2%) low viscosity high guluronate alginate and crosslinked with 1:1 (0.6-1%) calcium chloride solution provided sustained release of about 2,000 – 3,000 µM of EP over 28-days period, characterised by an initial burst of about 85% of EP released in the first week, and the remaining EP was released over the following weeks.Since it is likely that an optimal scaffold for cardiac regeneration will have a porous structure with interconnected pores to allow cell infiltration and proliferation, a series of macro-porous alginate scaffolds were developed. Different methods were used to prepare the scaffolds and thus different EP release profiles were obtained. Overall, the scaffolds prepared with 1% low viscosity high guluronate alginate and double crosslinked with 1:1 (0.4-1%) calcium gluconate solution and 0.2 M calcium chloride bath and prepared with one cycle of free-drying (method 6) released about 5,000- 5,500 µg over 28 days (with 88-98% drug loading efficiency), the highest compared to the other formulations. The potential therapeutic benefits of such EP release were then investigated in vitro. When primary rat cardiac fibroblast cells were exposed to hydrogen peroxide (150 μM) in the presence of EP (1,000 – 20,000 μM), EP improved cell viability as measured by alamarBlue assay. Moreover, at (1,000 - 10,000 μM), EP significantly increased cell viability compared to the control. In contrast, EP had no protective effect on the cells that had been previously exposed to H2O2 (150 μM) for 24 hours. Alginate macro-porous scaffolds (prepared using method 6), which showed high porosity, the best EP release profiles and the highest EP loading efficiency, were then tested in a cardiac fibroblasts culture and cell viability was measured by Neutral Red assay after 5 days. In order to improve cell attachment in the scaffolds prepared in this study, Arginine-Glycine-Aspartic acid modified RGD alginate was also used to prepared the scaffolds for the cardiac fibroblast study. Cells seeded onto the RGD- Alginate + EP scaffolds presented higher cell viability compared to the scaffolds without EP, demonstrating that the cells benefit from the structure of the scaffold, as well as from the presence of EP. This study has demonstrated for the first time that alginate represents a suitable delivery system for providing sustained release of EP. The protective effective of this drug on cardiac fibroblasts shown here, combined with the promising cell viability observed within the delivery scaffold, mean that this approach has significant potential for future development towards clinical evaluation.The inflammation that occurs after cardiac ischemia/reperfusion leads to high levels of oxidative stress, which produces deleterious effects that ultimately limit regeneration of the myocardium. This stress response can be prolonged, thereby affecting various stages of cardiac repair remodelling. Efforts to control such remodelling and stimulate cardiac tissue regeneration have therefore included the use of antioxidant or anti-inflammatory drug strategies. To date, such approaches have involved delivery of the drugs orally or by injection. The use of a scaffold that can be placed onto the heart immediately following surgery would allow sustained delivery of high concentrations of drug in situ, directly targeting the affected area. Moreover, implantation of such a material has the potential to encourage cell infiltration into the matrix, thereby promoting regeneration. The ultimate aim of this project was to develop a cardiac scaffold loaded with a drug with antioxidant and anti-inflammatory activity. Pyruvate is a well characterised anti-inflammatory and antioxidant drug. It has been added to cardioplegia by some surgical groups for its potential cardioprotective effects, although the therapeutic value of sustained delivery of this drug into the myocardium following cardiac surgery has not been investigated. Ethyl Pyruvate (EP) is a more stable form of this drug and was therefore the drug selected for investigation in the present study. Various alginate scaffolds were developed as drug delivery vehicles, with EP release characterised in vitro. Alginate gels prepared with (1-2%) low viscosity high guluronate alginate and crosslinked with 1:1 (0.6-1%) calcium chloride solution provided sustained release of about 2,000 – 3,000 µM of EP over 28-days period, characterised by an initial burst of about 85% of EP released in the first week, and the remaining EP was released over the following weeks.Since it is likely that an optimal scaffold for cardiac regeneration will have a porous structure with interconnected pores to allow cell infiltration and proliferation, a series of macro-porous alginate scaffolds were developed. Different methods were used to prepare the scaffolds and thus different EP release profiles were obtained. Overall, the scaffolds prepared with 1% low viscosity high guluronate alginate and double crosslinked with 1:1 (0.4-1%) calcium gluconate solution and 0.2 M calcium chloride bath and prepared with one cycle of free-drying (method 6) released about 5,000- 5,500 µg over 28 days (with 88-98% drug loading efficiency), the highest compared to the other formulations. The potential therapeutic benefits of such EP release were then investigated in vitro. When primary rat cardiac fibroblast cells were exposed to hydrogen peroxide (150 μM) in the presence of EP (1,000 – 20,000 μM), EP improved cell viability as measured by alamarBlue assay. Moreover, at (1,000 - 10,000 μM), EP significantly increased cell viability compared to the control. In contrast, EP had no protective effect on the cells that had been previously exposed to H2O2 (150 μM) for 24 hours. Alginate macro-porous scaffolds (prepared using method 6), which showed high porosity, the best EP release profiles and the highest EP loading efficiency, were then tested in a cardiac fibroblasts culture and cell viability was measured by Neutral Red assay after 5 days. In order to improve cell attachment in the scaffolds prepared in this study, Arginine-Glycine-Aspartic acid modified RGD alginate was also used to prepared the scaffolds for the cardiac fibroblast study. Cells seeded onto the RGD- Alginate + EP scaffolds presented higher cell viability compared to the scaffolds without EP, demonstrating that the cells benefit from the structure of the scaffold, as well as from the presence of EP. This study has demonstrated for the first time that alginate represents a suitable delivery system for providing sustained release of EP. The protective effective of this drug on cardiac fibroblasts shown here, combined with the promising cell viability observed within the delivery scaffold, mean that this approach has significant potential for future development towards clinical evaluation
Molecular analysis of MAP kinase kinase signaling in Leishmania
Get PDFMitogen-activated protein kinase patways play important roles in L. mexicana cell biology. This research characterised two yet unstudied MAP2Ks (LmxPK3 and LmxPK6) in Leishmania and studied putative signal transduction between MAP2Ks and MAPKs involved in regulating flagellum length. LmxPK6 is closely related to the STE7 kinase family, and LmxPK3 is related to CAMK. Recombinant GST-LmxPK6 could not be obtained, but GST-LmxPK3 could be purified in sufficient amounts to prove kinase activity by phosphorylation of the generic substrate MBP.;Only single allele deletion mutants could be generated for LmxPK6. Multiple attempts to obtain a null mutant were unsuccessful. This might suggest that LmxPK6 is an essential kinase of L. mexicana. However, an LmxPK3 null mutant was successfully generated, relying on the LmxMPK12 flanking regions to guarantee sufficient neomycin phosphotransferase resistance marker gene expression. GFP fused to LmxPK3 at either the C-terminus or the N-terminus showed that LmxPK3 localised in the cytosol and flagellum.;A null mutant of LmxPK3 showed similar lesion development in BALB/c mice as wild type L. mexicana, and the lesion-derived amastigotes differentiated back to promastigotes and grew in culture suggesting that LmxPK3 does not play a role in Leishmania differentiation. Hence, LmxPK3 is not a drug target against leishmaniasis. Interactions between LmxPK4 and LmxMPK3 were investigated in vitro by co-expression of the two kinases in Escherichia coli followed by purification and kinase assays.;MS/MS analysis showed that LmxPK4 phosphorylates LmxMPK3 at SER183, THR194 and TYR196 of the TDY motif. Using split-GFP for the first time in Leishmania promastigotes showed an interaction between LmxPK4 and LmxMPK3 in vivo by fluorescence in distinct areas of the cytosol and formation of normal length flagella when expressed in the LmxMPK3 null mutant. A hypothesis of how LmxPK4 and LmxMKK can jointly regulate intraflagellar transport was generated.Mitogen-activated protein kinase patways play important roles in L. mexicana cell biology. This research characterised two yet unstudied MAP2Ks (LmxPK3 and LmxPK6) in Leishmania and studied putative signal transduction between MAP2Ks and MAPKs involved in regulating flagellum length. LmxPK6 is closely related to the STE7 kinase family, and LmxPK3 is related to CAMK. Recombinant GST-LmxPK6 could not be obtained, but GST-LmxPK3 could be purified in sufficient amounts to prove kinase activity by phosphorylation of the generic substrate MBP.;Only single allele deletion mutants could be generated for LmxPK6. Multiple attempts to obtain a null mutant were unsuccessful. This might suggest that LmxPK6 is an essential kinase of L. mexicana. However, an LmxPK3 null mutant was successfully generated, relying on the LmxMPK12 flanking regions to guarantee sufficient neomycin phosphotransferase resistance marker gene expression. GFP fused to LmxPK3 at either the C-terminus or the N-terminus showed that LmxPK3 localised in the cytosol and flagellum.;A null mutant of LmxPK3 showed similar lesion development in BALB/c mice as wild type L. mexicana, and the lesion-derived amastigotes differentiated back to promastigotes and grew in culture suggesting that LmxPK3 does not play a role in Leishmania differentiation. Hence, LmxPK3 is not a drug target against leishmaniasis. Interactions between LmxPK4 and LmxMPK3 were investigated in vitro by co-expression of the two kinases in Escherichia coli followed by purification and kinase assays.;MS/MS analysis showed that LmxPK4 phosphorylates LmxMPK3 at SER183, THR194 and TYR196 of the TDY motif. Using split-GFP for the first time in Leishmania promastigotes showed an interaction between LmxPK4 and LmxMPK3 in vivo by fluorescence in distinct areas of the cytosol and formation of normal length flagella when expressed in the LmxMPK3 null mutant. A hypothesis of how LmxPK4 and LmxMKK can jointly regulate intraflagellar transport was generated
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