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Identification of sphingosine 1-phosphate receptors in exosomes released from breast cancer cells
Get PDFSphingosine-1-phosphate (S1P) is a bioactive lipid that promotes cell survival, proliferation, migration, angiogenesis and immune response, which are critical processes in cancer progression. Although some essential roles of intracellular S1P have recently been revealed, the majority of its biological effects are recognised to be mediated through activation of its cognate S1P1-5 receptors. This work demonstrates for the first time that the sphingosine 1-phosphate receptor 2 (S1P2, Mr = 42kDa) is shed in Hsp70 and CD63 containing exosomes from MDA-MB-231 breast cancer cells.;These exosomes can be taken up by fibroblasts where exosomal S1P2 receptor is proteolysed to a constitutively active shorter form of S1P2 (S1P2, Mr=38 kDa) which activates ERK-1/2 and stimulates DNA synthesis in fibroblasts. Therefore, conditioned medium and an exosomal preparation containing shed S1P2 and isolated from MDA-MB-231 cells was able to stimulate the ERK-1/2 pathway and DNA synthesis in fibroblasts.;Moreover, S1P2 siRNA knockdown in MDA-MB-231 cells reduced the ability of conditioned medium and exosomal preparations containing S1P2 to induce ERK-1/2 activation in fibroblasts. This finding links the constitutively active shorter form of S1P2 released from cancer cells with the proliferation of fibroblasts required for metastatic spread. In addition, it was demonstrated that S1P4 is released from MDA-MB-453 cells breast cancer cells.;However, conditioned medium containing S1P4 failed to activate ERK-1/2 in fibroblasts, suggesting that ERK-1/2 signalling is specifically regulated by the short form S1P2. Future work requires in vivo study of the truncated version of S1P2 receptor in an orthotopic graft mouse model to examine its impact on cancer metastasis.;In addition, S1P and inhibitors of sphingosine kinase (SK) increase the release of S1P4 from MDA-MB-453 cells. These findings, suggest that exogenous S1P might enhance S1P4 internalisation and its subsequent release into CM. SK inhibitors might increase intracellular ceramide, which has been demonstrated to be enriched in lipid raft microdomains and regulate MVBs formation and cargo sorting into ILVs (exosomes).;S1P and SK inhibitors had no effect on S1P2 release, suggesting that the effect on S1P4 is cell type-specific. In addition, it was demonstrated that the combination of the ceramide kinase (CERK) inhibitor, NVP-231 and the SK1 inhibitor, PF-543 produced less than additive effect on DNA synthesis, suggesting a functional interaction or overlapping regulation of CERK and SK1 in MDA-MB-453 and MDA-MB-231 breast cancer cells survival.;Therefore, modulating the sphingolipid rheostat using CERK and SK1 inhibitor failed to sensitise breast cancer cells to cell death. Further investigation is required to examine the effect of CERK inhibitor alone and in combination with SK1 inhibitors on S1P4 release.Taken together; the findings of this study, suggest a novel regulation of cancer progression by the S1P signalling pathway.Sphingosine-1-phosphate (S1P) is a bioactive lipid that promotes cell survival, proliferation, migration, angiogenesis and immune response, which are critical processes in cancer progression. Although some essential roles of intracellular S1P have recently been revealed, the majority of its biological effects are recognised to be mediated through activation of its cognate S1P1-5 receptors. This work demonstrates for the first time that the sphingosine 1-phosphate receptor 2 (S1P2, Mr = 42kDa) is shed in Hsp70 and CD63 containing exosomes from MDA-MB-231 breast cancer cells.;These exosomes can be taken up by fibroblasts where exosomal S1P2 receptor is proteolysed to a constitutively active shorter form of S1P2 (S1P2, Mr=38 kDa) which activates ERK-1/2 and stimulates DNA synthesis in fibroblasts. Therefore, conditioned medium and an exosomal preparation containing shed S1P2 and isolated from MDA-MB-231 cells was able to stimulate the ERK-1/2 pathway and DNA synthesis in fibroblasts.;Moreover, S1P2 siRNA knockdown in MDA-MB-231 cells reduced the ability of conditioned medium and exosomal preparations containing S1P2 to induce ERK-1/2 activation in fibroblasts. This finding links the constitutively active shorter form of S1P2 released from cancer cells with the proliferation of fibroblasts required for metastatic spread. In addition, it was demonstrated that S1P4 is released from MDA-MB-453 cells breast cancer cells.;However, conditioned medium containing S1P4 failed to activate ERK-1/2 in fibroblasts, suggesting that ERK-1/2 signalling is specifically regulated by the short form S1P2. Future work requires in vivo study of the truncated version of S1P2 receptor in an orthotopic graft mouse model to examine its impact on cancer metastasis.;In addition, S1P and inhibitors of sphingosine kinase (SK) increase the release of S1P4 from MDA-MB-453 cells. These findings, suggest that exogenous S1P might enhance S1P4 internalisation and its subsequent release into CM. SK inhibitors might increase intracellular ceramide, which has been demonstrated to be enriched in lipid raft microdomains and regulate MVBs formation and cargo sorting into ILVs (exosomes).;S1P and SK inhibitors had no effect on S1P2 release, suggesting that the effect on S1P4 is cell type-specific. In addition, it was demonstrated that the combination of the ceramide kinase (CERK) inhibitor, NVP-231 and the SK1 inhibitor, PF-543 produced less than additive effect on DNA synthesis, suggesting a functional interaction or overlapping regulation of CERK and SK1 in MDA-MB-453 and MDA-MB-231 breast cancer cells survival.;Therefore, modulating the sphingolipid rheostat using CERK and SK1 inhibitor failed to sensitise breast cancer cells to cell death. Further investigation is required to examine the effect of CERK inhibitor alone and in combination with SK1 inhibitors on S1P4 release.Taken together; the findings of this study, suggest a novel regulation of cancer progression by the S1P signalling pathway
Investigating the expression and function of IL-16 in central nervous system under normal and inflammatory conditions
Get PDFMultiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). MS pathophysiology is complex and CD4+ T cells are one of the key mediators involved in the disease initiation and progression. Interestingly CD4 is also the receptor for interleukin-16 (IL-16), a pro-inflammatory cytokine. Previous re-search has shown that IL-16 is involved in the development of several autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Graves' disease (GD) and MS, but the exact role of IL-16 during MS initiation and progression remains unclear. Therefore, the aim of this study was to understand the role of IL-16 in CNS inflammation by: (i) examining the expression and distribution of IL-16 in the CNS and investigating whether expression levels correlate with the se-verity of neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS; and (ii) by further determining the function of IL-16 in the CNS under normal physiological condition. We immunised C57BL/6j mice with myelin oligodendrocyte glycoprotein (MOG35-55) to induce EAE and utilised immunohistochemistry to study the expression of IL-16 and its receptor CD4 in the lymphoid and CNS tissues at different stages of EAE. While CD4 was expressed in lymphoid tissues of both EAE and control mice, CD4 expression in CNS tissues was only observed during EAE. For IL-16, its levels in the lymphoid organs and CNS tissues of EAE mice were significantly increased com-pared to tissues of naïve and PBS controls, as confirmed by the quantification of the percentage of IL-16+ cells in tissues. Surprisingly, soluble IL-16 produced by spleno-cytes was similar between groups of naïve, and PBS or MOG35-55 immunised mice, and there was no difference with or without MOG35-55 stimulation in the culture.;To identify the cell source of IL-16 in the lymphoid and CNS tissues we next utilised double immunofluorescence staining and our data showed co-localisation of IL-16 with some immune cells during EAE, with CD11b+ cells (macrophages and microglia) expressing the highest percentage of IL-16 in both types of tissues. However, in CNS, IL-16 was also co-localised with CNS resident cells including neurons in both EAE and control mice and astrocytes only during EAE. However further study is required to fully understand the exact underlying action mechanisms of IL-16 in EAE. Following that we investigated the role of IL-16 in CNS function by examining whether IL-16 modulates neuronal excitability and synaptic activity in mouse primary hippo-campal cultures. We observed application of recombinant IL-16 (rIL-16) protein impaired sEPSC frequency and amplitude in a CD4-independent manner. We examined the mechanisms underlying these effects with rIL-16 reducing GluA1 S831 phosphorylation and inhibiting Na+ channel function. Taken together, our data suggest that IL-16 expression levels in both lymphoid organs and CNS tissues correlate with CNS inflammation, and CD11b+ cells are the main source of IL-16 in both tissues during CNS inflammation in our EAE disease model. Under normal physiological condition, IL-16 reduces neuronal excitability and synap-tic activity via multiple mechanisms and it is likely that its function is not solely dependent on the presence of CD4. Clearly IL-16 have a role both under normal and pathophysiological state but whether it is detrimental or beneficial is yet to be identified and requires further investigation to identify the exact role and potential of IL-16 in treatment of MS.Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). MS pathophysiology is complex and CD4+ T cells are one of the key mediators involved in the disease initiation and progression. Interestingly CD4 is also the receptor for interleukin-16 (IL-16), a pro-inflammatory cytokine. Previous re-search has shown that IL-16 is involved in the development of several autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Graves' disease (GD) and MS, but the exact role of IL-16 during MS initiation and progression remains unclear. Therefore, the aim of this study was to understand the role of IL-16 in CNS inflammation by: (i) examining the expression and distribution of IL-16 in the CNS and investigating whether expression levels correlate with the se-verity of neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS; and (ii) by further determining the function of IL-16 in the CNS under normal physiological condition. We immunised C57BL/6j mice with myelin oligodendrocyte glycoprotein (MOG35-55) to induce EAE and utilised immunohistochemistry to study the expression of IL-16 and its receptor CD4 in the lymphoid and CNS tissues at different stages of EAE. While CD4 was expressed in lymphoid tissues of both EAE and control mice, CD4 expression in CNS tissues was only observed during EAE. For IL-16, its levels in the lymphoid organs and CNS tissues of EAE mice were significantly increased com-pared to tissues of naïve and PBS controls, as confirmed by the quantification of the percentage of IL-16+ cells in tissues. Surprisingly, soluble IL-16 produced by spleno-cytes was similar between groups of naïve, and PBS or MOG35-55 immunised mice, and there was no difference with or without MOG35-55 stimulation in the culture.;To identify the cell source of IL-16 in the lymphoid and CNS tissues we next utilised double immunofluorescence staining and our data showed co-localisation of IL-16 with some immune cells during EAE, with CD11b+ cells (macrophages and microglia) expressing the highest percentage of IL-16 in both types of tissues. However, in CNS, IL-16 was also co-localised with CNS resident cells including neurons in both EAE and control mice and astrocytes only during EAE. However further study is required to fully understand the exact underlying action mechanisms of IL-16 in EAE. Following that we investigated the role of IL-16 in CNS function by examining whether IL-16 modulates neuronal excitability and synaptic activity in mouse primary hippo-campal cultures. We observed application of recombinant IL-16 (rIL-16) protein impaired sEPSC frequency and amplitude in a CD4-independent manner. We examined the mechanisms underlying these effects with rIL-16 reducing GluA1 S831 phosphorylation and inhibiting Na+ channel function. Taken together, our data suggest that IL-16 expression levels in both lymphoid organs and CNS tissues correlate with CNS inflammation, and CD11b+ cells are the main source of IL-16 in both tissues during CNS inflammation in our EAE disease model. Under normal physiological condition, IL-16 reduces neuronal excitability and synap-tic activity via multiple mechanisms and it is likely that its function is not solely dependent on the presence of CD4. Clearly IL-16 have a role both under normal and pathophysiological state but whether it is detrimental or beneficial is yet to be identified and requires further investigation to identify the exact role and potential of IL-16 in treatment of MS
Molecularly imprinted polymers for applications in proteomics and diagnostics
Get PDFAmongst enumerated types of biomarkers are Pro-Gastrin-Releasing Peptide,Aβ-amyloid and α-Synuclein peptides. ProGRP is reported as a highly specific new biomarker for Small Cell Lung Cancer (SCLC). Aggregates of Aβ and α-Synuclein peptides are in the group of biomarkers with importance in the diagnosis of Alzheimer's Disease (AD) and Parkinson's Disease (PD), respectively. Therapy and/or drugs work most efficiently in the early stage of AD, PD and SCLC, however in most cases diagnoses are made too late and only after a time when significant progress of pathological change is observed. Therefore, there is a significant demand in the market for cheap and fast diagnostic tools that could allow for the reliable detection of AD, PD and SCLC in their early stages. Molecularly imprinted polymers (MIPs) are synthetic materials with outstanding affinity and selectivity for a given target molecule. Their low production cost, reusability and high reproducibility make them attractive candidates for the next generation of separation materials, with potential applications to AD, PD and SCLC.;The purpose of this study was to design, synthesise, characterise and exploit the next generation of MIPs that can enable the selective extractions of targets from native blood or cerebrospinal fluid samples. In this Thesis, a generic synthetic protocol for the synthesis of protein-imprinted polymers was developed. The method allows for the rapid production of peptide imprinted polymers in a convenient microsphere format. In fact, four different formats of MIPs for the peptide-based biomarkers were developed and described. Selected materials were supplied to the PEPMIP partners for the determination of their molecular recognition character and use in proteomics and diagnostics applications. A new, sensitive on-line assay for SCLC has been developed using these materials and good progress made towards a magnetic capture protocol.Amongst enumerated types of biomarkers are Pro-Gastrin-Releasing Peptide,Aβ-amyloid and α-Synuclein peptides. ProGRP is reported as a highly specific new biomarker for Small Cell Lung Cancer (SCLC). Aggregates of Aβ and α-Synuclein peptides are in the group of biomarkers with importance in the diagnosis of Alzheimer's Disease (AD) and Parkinson's Disease (PD), respectively. Therapy and/or drugs work most efficiently in the early stage of AD, PD and SCLC, however in most cases diagnoses are made too late and only after a time when significant progress of pathological change is observed. Therefore, there is a significant demand in the market for cheap and fast diagnostic tools that could allow for the reliable detection of AD, PD and SCLC in their early stages. Molecularly imprinted polymers (MIPs) are synthetic materials with outstanding affinity and selectivity for a given target molecule. Their low production cost, reusability and high reproducibility make them attractive candidates for the next generation of separation materials, with potential applications to AD, PD and SCLC.;The purpose of this study was to design, synthesise, characterise and exploit the next generation of MIPs that can enable the selective extractions of targets from native blood or cerebrospinal fluid samples. In this Thesis, a generic synthetic protocol for the synthesis of protein-imprinted polymers was developed. The method allows for the rapid production of peptide imprinted polymers in a convenient microsphere format. In fact, four different formats of MIPs for the peptide-based biomarkers were developed and described. Selected materials were supplied to the PEPMIP partners for the determination of their molecular recognition character and use in proteomics and diagnostics applications. A new, sensitive on-line assay for SCLC has been developed using these materials and good progress made towards a magnetic capture protocol
Subject relativizers in early modern drama : a study based on the visualizing English print project parsed corpus of early modern drama
No full textThis dissertation is an attempt to make the most of recent developments in natural language processing and vast resources of texts of early modern drama recently released by Early English Books Online - Text Creation Partnership (EEBO-TCP). Specifically, compiling a syntactically parsed corpus of early modern drama consisting of 1,244 plays, this dissertation seeks to improve the performance of a syntactic parser on EEBO-TCP texts to conduct large-scale research on early modern relativization. Special emphasis will be placed on retrieval of subject zero relativizers, which, being invisible in texts, are often assumed to be impossible to collect automatically.;It will be demonstrated that subject zero relativizers can indeed be retrieved automatically if we run texts through a syntactic parser; that in order to maximize the capability of the parser to locate relativizers, EEBO-TCP texts have to undergo several layers of text processing that affects historical spelling, contracted forms and punctuation; that the parsed corpus enables us to see the distribution of relativizers across genres and from a diachronic perspective as well as from a perspective of linguistic contexts in which they are employed.This dissertation is an attempt to make the most of recent developments in natural language processing and vast resources of texts of early modern drama recently released by Early English Books Online - Text Creation Partnership (EEBO-TCP). Specifically, compiling a syntactically parsed corpus of early modern drama consisting of 1,244 plays, this dissertation seeks to improve the performance of a syntactic parser on EEBO-TCP texts to conduct large-scale research on early modern relativization. Special emphasis will be placed on retrieval of subject zero relativizers, which, being invisible in texts, are often assumed to be impossible to collect automatically.;It will be demonstrated that subject zero relativizers can indeed be retrieved automatically if we run texts through a syntactic parser; that in order to maximize the capability of the parser to locate relativizers, EEBO-TCP texts have to undergo several layers of text processing that affects historical spelling, contracted forms and punctuation; that the parsed corpus enables us to see the distribution of relativizers across genres and from a diachronic perspective as well as from a perspective of linguistic contexts in which they are employed
Real options and strategic interactions in imperfectly competitive markets
Get PDFAs Smit and Trigeorgis (2004), Smit and Ankum (1993), and others have observed, an options-based approach to strategic investment needs to be considered from the perspective of competitive market structures. The models in this thesis show the synergies obtainable when real options ideas are embedded in industrial organisation-type frameworks. The results are insights that neither field, thus far, has captured independently.Firstly, we present a real options model in an investment game of incomplete information in a duopoly where product market competition influences the value of the investment and entry times are endogenously determined. We show that type-asymmetry or the level of initial demand, independently, or together, as in extant models, are insufficient criteria upon which endogenous roles under uncertainty may be determined when firms have private information over their types. Rather, ex post market structures are determined by threshold functions whose images lie in the type-space of the firms. These results are discussed in detail along with numerical examples.Secondly, whilst the advertising literature has particular focus on either the informative or persuasive effects of advertising efforts and views advertising investments as intertemporal expenditures, this thesis addresses the brand loyalty aspects of advertising in a new market which, asides from routine expenditure, requires a lumpy initial investment outlay in the development of a viable competitive advertising campaign. We view this as a real options investment with a differential game played at the advertising-efforts level.Lastly, empirical analyses on the impact of celebrity endorsements have largely been inconclusive. In this thesis, we model investments in brand equity using celebrity endorsers with embedded options over the investment opportunity. The aim is to determine optimal strategies when the arrival of the investment itself follows a random process and when firms can update their beliefs about the risk profile of the celebrity.As Smit and Trigeorgis (2004), Smit and Ankum (1993), and others have observed, an options-based approach to strategic investment needs to be considered from the perspective of competitive market structures. The models in this thesis show the synergies obtainable when real options ideas are embedded in industrial organisation-type frameworks. The results are insights that neither field, thus far, has captured independently.Firstly, we present a real options model in an investment game of incomplete information in a duopoly where product market competition influences the value of the investment and entry times are endogenously determined. We show that type-asymmetry or the level of initial demand, independently, or together, as in extant models, are insufficient criteria upon which endogenous roles under uncertainty may be determined when firms have private information over their types. Rather, ex post market structures are determined by threshold functions whose images lie in the type-space of the firms. These results are discussed in detail along with numerical examples.Secondly, whilst the advertising literature has particular focus on either the informative or persuasive effects of advertising efforts and views advertising investments as intertemporal expenditures, this thesis addresses the brand loyalty aspects of advertising in a new market which, asides from routine expenditure, requires a lumpy initial investment outlay in the development of a viable competitive advertising campaign. We view this as a real options investment with a differential game played at the advertising-efforts level.Lastly, empirical analyses on the impact of celebrity endorsements have largely been inconclusive. In this thesis, we model investments in brand equity using celebrity endorsers with embedded options over the investment opportunity. The aim is to determine optimal strategies when the arrival of the investment itself follows a random process and when firms can update their beliefs about the risk profile of the celebrity
Domain decomposition methods for time-harmonic elastic waves
Get PDFThe construction of optimal solvers for high frequency Helmholtz-type equations is highly problematic. After discretisation of the previous equations by a finite element method,the underlying linear systems are usually large and difficult to solve both by direct and iterative methods. Domain decomposition methods are hybrid methods in the sense that they use an iterative coupling of smaller problems that are solved by direct methods,and rely on the splitting the global problem into local problems on smaller subdomains. These methods can be used as iterative solvers but also as preconditioners in a Krylov type method. That is the reason why transmission conditions between subdomains are very important.In this manuscript, we start by an overview of main domain decomposition methods and focus first on their use as preconditioners. Then we consider these methods from an iterative point of view and perform a convergence study of non-overlapping and overlapping Schwarz methods with Dirichlet and Robin interface conditions, by analysing their behaviour and conclude on their convergence properties which prove to be very poor when used as solvers. The theoretical findings are illustrated by numerical results.Then we present more sophisticated methods, namely the optimised Schwarz algorithms,which use more effective transmission conditions depending on some parameters which are solutions of min-max problems.The Schwarz preconditioners defined previously were one-level, meaning only the information from the neighbouring domains is used. This has the undesired consequence that the number of iterations needed to reach convergence increases with the number of subdomains. For this reason we have tested numerically two-level preconditioners,based on a coarse grid correction, this very simple idea giving promising results.The construction of optimal solvers for high frequency Helmholtz-type equations is highly problematic. After discretisation of the previous equations by a finite element method,the underlying linear systems are usually large and difficult to solve both by direct and iterative methods. Domain decomposition methods are hybrid methods in the sense that they use an iterative coupling of smaller problems that are solved by direct methods,and rely on the splitting the global problem into local problems on smaller subdomains. These methods can be used as iterative solvers but also as preconditioners in a Krylov type method. That is the reason why transmission conditions between subdomains are very important.In this manuscript, we start by an overview of main domain decomposition methods and focus first on their use as preconditioners. Then we consider these methods from an iterative point of view and perform a convergence study of non-overlapping and overlapping Schwarz methods with Dirichlet and Robin interface conditions, by analysing their behaviour and conclude on their convergence properties which prove to be very poor when used as solvers. The theoretical findings are illustrated by numerical results.Then we present more sophisticated methods, namely the optimised Schwarz algorithms,which use more effective transmission conditions depending on some parameters which are solutions of min-max problems.The Schwarz preconditioners defined previously were one-level, meaning only the information from the neighbouring domains is used. This has the undesired consequence that the number of iterations needed to reach convergence increases with the number of subdomains. For this reason we have tested numerically two-level preconditioners,based on a coarse grid correction, this very simple idea giving promising results
The effect of nanofibers for improving the dynamic and mechanical properties of composite laminates and their application as self-powered sensors
Get PDFOne of the main objectives of this thesis is to investigate the effect of the inclusion of nanofibers on the global dynamical and mechanical properties of composite laminates. For this aim, the natural frequencies, damping, interlaminar strength and impact response of pristine and nanomodified composites are determined experimentally and numerically using a finite element model. Experimental and numerical results showed a significant effect of the nanofibers on the mechanical and dynamic properties of the composites. Nanomodified composites demonstrated a consistent increase in the damping, interlaminar strength and impact resistance. However, the variation in the natural frequencies was very small. This study expands the knowledge about the macro mechanical and dynamical properties of composites reinforced with nanofibers. Furthermore, it proposes finite element models to simulate with high accuracy the mechanical and vibratory behaviour of pristine and nanomodified composites. These findings are of great interest for the research community and for industry as composite laminates reinforced with nanofibers could be potentially used to improve the properties of the composite structures used in aircrafts, wind turbines and other civil structures.The other major objective of the thesis is to investigate the potential applications of nanofibers as self-powered triboelectric sensors. The first triboelectric sensor was invented in 2012 and it has become one of the most important innovations in the field of self-powered sensing technologies. As compared to traditional sensors, triboelectric sensors are self-powered and do not require and external power supply or battery to sense the mechanical stimulus. As a result, they are maintenance free and energy independent, which results in important cost-savings. In this work, the potential of two new classes of triboelectric sensors for monitoring of pressures and impacts is demonstrated. To carry out this investigation, the developed triboelectric sensors are subjected to controlled pressures and impacts using the techniques of dynamic mechanical analysis and drop weight impact tests, respectively. The experimental results proved that the sensors generated electric responses are affected by the magnitude of the mechanical stimulus and their amplitude increases linearly under stronger pressures/impacts. Furthermore, the sensor electric responses show a large detection range, high sensitivity, good reproducibility, and fast response time, which is essential for the practical applications of the sensor. The main contributions of this study are the development of two novel triboelectric nanogenerators and active sensors based on polyvinyl fluoride nanofibers and their investigation for its potential use for monitoring of pressures and impacts. The results of this work successfully demonstrated that the developed triboelectric sensor measure dynamic pressures and impacts in real time, which has important applications in monitoring systems, vehicle safety, and touch screens. These findings are the utmost importance as the new developed sensors could be utilized as sustainable and maintenance free sensors with the advantages of easy fabrication and low-cost processing technology.In conclusion, this thesis contributes to the knowledge about nanofibers in terms of their applications to improve material and structural properties of composite laminates and their use as two novel nanostructured sensors for detection and measurement of pressures and impacts.One of the main objectives of this thesis is to investigate the effect of the inclusion of nanofibers on the global dynamical and mechanical properties of composite laminates. For this aim, the natural frequencies, damping, interlaminar strength and impact response of pristine and nanomodified composites are determined experimentally and numerically using a finite element model. Experimental and numerical results showed a significant effect of the nanofibers on the mechanical and dynamic properties of the composites. Nanomodified composites demonstrated a consistent increase in the damping, interlaminar strength and impact resistance. However, the variation in the natural frequencies was very small. This study expands the knowledge about the macro mechanical and dynamical properties of composites reinforced with nanofibers. Furthermore, it proposes finite element models to simulate with high accuracy the mechanical and vibratory behaviour of pristine and nanomodified composites. These findings are of great interest for the research community and for industry as composite laminates reinforced with nanofibers could be potentially used to improve the properties of the composite structures used in aircrafts, wind turbines and other civil structures.The other major objective of the thesis is to investigate the potential applications of nanofibers as self-powered triboelectric sensors. The first triboelectric sensor was invented in 2012 and it has become one of the most important innovations in the field of self-powered sensing technologies. As compared to traditional sensors, triboelectric sensors are self-powered and do not require and external power supply or battery to sense the mechanical stimulus. As a result, they are maintenance free and energy independent, which results in important cost-savings. In this work, the potential of two new classes of triboelectric sensors for monitoring of pressures and impacts is demonstrated. To carry out this investigation, the developed triboelectric sensors are subjected to controlled pressures and impacts using the techniques of dynamic mechanical analysis and drop weight impact tests, respectively. The experimental results proved that the sensors generated electric responses are affected by the magnitude of the mechanical stimulus and their amplitude increases linearly under stronger pressures/impacts. Furthermore, the sensor electric responses show a large detection range, high sensitivity, good reproducibility, and fast response time, which is essential for the practical applications of the sensor. The main contributions of this study are the development of two novel triboelectric nanogenerators and active sensors based on polyvinyl fluoride nanofibers and their investigation for its potential use for monitoring of pressures and impacts. The results of this work successfully demonstrated that the developed triboelectric sensor measure dynamic pressures and impacts in real time, which has important applications in monitoring systems, vehicle safety, and touch screens. These findings are the utmost importance as the new developed sensors could be utilized as sustainable and maintenance free sensors with the advantages of easy fabrication and low-cost processing technology.In conclusion, this thesis contributes to the knowledge about nanofibers in terms of their applications to improve material and structural properties of composite laminates and their use as two novel nanostructured sensors for detection and measurement of pressures and impacts
Xenophagy in response to gram-positive staphylococcus aureus and gram-negative salmonella enterica sv. typhimurium
No full textAutophagy is an important process for cell survival in the human body, which plays a critical role in fighting infections. Some infections exploit the autophagic system and are often promoted by autophagy. Recent evidence has suggested that Staphylococcus aureus has specialised mechanisms to evade xenophagy, thus allowing bacterial survival and replication within autophagosomes, leading to eventual cell death. ULK1 is a serine/threonine kinase that plays an essential role during the early steps of autophagosome biogenesis, but its roles during xenophagy following Staphylococcus aureus infection have been unclear. ULK1 represents an excellent candidate for drug targeting to control autophagy under various settings.This study aimed to investigate the role of autophagy in defence against two disease causing bacteria that are known for their ability to damage cells: Salmonella entericasv. Typhimurium and Staphylococcus aureus. A further aim was to study the role of the ULK1 complex in xenophagy following infection by Staphylococcus aureus and furthermore to test ULK1 inhibitors as a novel therapy to restrict MRSA infection in cells. In addition, in this project, CRISPR genetic selection approaches were developed, aimed to find new host cell genes required for Staphylococcus aureus and Salmonella enterica sv. Typhimurium infection. The results indicated two different roles of autophagy: 1) to provide a protective niche for MRSA, and 2) to provide a mechanism to fight infection by Salmonella enterica sv.Typhimurium. Importantly, treatment of cells with a ULK1/2 small molecule inhibitor strongly inhibited cell killing following infection by MRSA. However, ULK1/2 inhibition made cells more sensitive to cell death following infection by Salmonella enterica sv.Typhimurium. Thus, ULK1 inhibitors may be a novel therapeutic method for fighting infection by MRSA.Also, in this project, we found that screening with a freely available CRISPR-Cas9 library successfully identified the host genes essential for the toxicity of cells by MRSA(NCTC8325) or Salmonella enterica sv. Typhimurium. This was confirmed byfunctional validation and may open the door for novel putative therapeutic targets in future.Autophagy is an important process for cell survival in the human body, which plays a critical role in fighting infections. Some infections exploit the autophagic system and are often promoted by autophagy. Recent evidence has suggested that Staphylococcus aureus has specialised mechanisms to evade xenophagy, thus allowing bacterial survival and replication within autophagosomes, leading to eventual cell death. ULK1 is a serine/threonine kinase that plays an essential role during the early steps of autophagosome biogenesis, but its roles during xenophagy following Staphylococcus aureus infection have been unclear. ULK1 represents an excellent candidate for drug targeting to control autophagy under various settings.This study aimed to investigate the role of autophagy in defence against two disease causing bacteria that are known for their ability to damage cells: Salmonella entericasv. Typhimurium and Staphylococcus aureus. A further aim was to study the role of the ULK1 complex in xenophagy following infection by Staphylococcus aureus and furthermore to test ULK1 inhibitors as a novel therapy to restrict MRSA infection in cells. In addition, in this project, CRISPR genetic selection approaches were developed, aimed to find new host cell genes required for Staphylococcus aureus and Salmonella enterica sv. Typhimurium infection. The results indicated two different roles of autophagy: 1) to provide a protective niche for MRSA, and 2) to provide a mechanism to fight infection by Salmonella enterica sv.Typhimurium. Importantly, treatment of cells with a ULK1/2 small molecule inhibitor strongly inhibited cell killing following infection by MRSA. However, ULK1/2 inhibition made cells more sensitive to cell death following infection by Salmonella enterica sv.Typhimurium. Thus, ULK1 inhibitors may be a novel therapeutic method for fighting infection by MRSA.Also, in this project, we found that screening with a freely available CRISPR-Cas9 library successfully identified the host genes essential for the toxicity of cells by MRSA(NCTC8325) or Salmonella enterica sv. Typhimurium. This was confirmed byfunctional validation and may open the door for novel putative therapeutic targets in future
Essays on private investments in public equity
Get PDFThis PhD thesis, comprised of 3 essays, assesses the activity of Private Investments in Public Equity (PIPEs) and provides new insights on PIPEs behaviour. In the second chapter, I document PIPEs performance around the world and assess whether cross-country regulatory and institutional differences can explain the variation in PIPEs valuation. I document a significant decline in the market valuation around the announcement of PIPEs, especially from 2004 to 2015 and find that firms participating in the PIPE market during these years have worse fundamentals in terms of size, profitability and operating performance. I further find that PIPE issuers are followed by a significant long-term underperformance globally. Finally, consistent with the Law and Finance theory, I show that country governance quality matters, as issuing firms operating in countries with better regulatory quality and higher law enforcement outperform others. In the third chapter, I examine the stock returns and volume prior to PIPE announcements, to document whether there is a price run ahead of the public announcement of the issues. Focusing in the UK and the US, that both have high levels of PIPE activity but differ in the PIPE regulatory environment and insider trading treatment, I assess whether the price run patterns are affected by regulatory differences. In addition, assessing the contemporaneous relationship between abnormal returns and volume, I examine whether the price runs can be explained by leaked information. I find abnormal returns and abnormal volume prior to the announcement of PIPEs in both markets. I further find support of the information leakage hypothesis for US issuers. In the fourth chapter, I assess registered insider trades around PIPEs in the UK. I examine whether insiders adjust their trading strategies before the PIPE issue. I find that insiders significantly increase their net sales in the pre-announcement PIPE period, with the results being robust after controlling for time effects and comparing with a matched sample of control firms.This PhD thesis, comprised of 3 essays, assesses the activity of Private Investments in Public Equity (PIPEs) and provides new insights on PIPEs behaviour. In the second chapter, I document PIPEs performance around the world and assess whether cross-country regulatory and institutional differences can explain the variation in PIPEs valuation. I document a significant decline in the market valuation around the announcement of PIPEs, especially from 2004 to 2015 and find that firms participating in the PIPE market during these years have worse fundamentals in terms of size, profitability and operating performance. I further find that PIPE issuers are followed by a significant long-term underperformance globally. Finally, consistent with the Law and Finance theory, I show that country governance quality matters, as issuing firms operating in countries with better regulatory quality and higher law enforcement outperform others. In the third chapter, I examine the stock returns and volume prior to PIPE announcements, to document whether there is a price run ahead of the public announcement of the issues. Focusing in the UK and the US, that both have high levels of PIPE activity but differ in the PIPE regulatory environment and insider trading treatment, I assess whether the price run patterns are affected by regulatory differences. In addition, assessing the contemporaneous relationship between abnormal returns and volume, I examine whether the price runs can be explained by leaked information. I find abnormal returns and abnormal volume prior to the announcement of PIPEs in both markets. I further find support of the information leakage hypothesis for US issuers. In the fourth chapter, I assess registered insider trades around PIPEs in the UK. I examine whether insiders adjust their trading strategies before the PIPE issue. I find that insiders significantly increase their net sales in the pre-announcement PIPE period, with the results being robust after controlling for time effects and comparing with a matched sample of control firms
Developing spectroscopic biofluid diagnostics : monitoring and therapeutic profiling of melanoma patients
Get PDFThe ability to diagnose melanoma prior to metastasis could revolutionise the clinical environment. This would allow improved patient care via monitoring, rapid follow up of high-risk cases and improve patient mortality and morbidity. Biomedical spectroscopy can diagnose a wide range of pathologies however no study has made it from the laboratory into a clinical setting as a regulated spectroscopic test. To facilitate translation this thesis describes: • Development of the optimal methodology for the quantification of protein biomarkers. It was demonstrated that concentrations as low as 0.66 ± 0.05 mg mL-1, with a linearity of 0.992, can be achieved within patient samples. • Analysis of liquid serum samples led to discrimination of cancer vs. non-cancer with a sensitivity of 95.4 % and a specificity of 81.8 %, compared to the air-dried data set, which achieved 92.4 % and 84.4 %, respectively. Analysis of liquid samples removes the rate determining air drying step. Digitally drying the liquid spectrum was investigated to determine if an improvement could be achieved. The optimal result was achieved through the use of an extended multiplicative scatter correction algorithm, providing a sensitivity of 91.2 % and a specificity of 77.3 %. • Discussion and investigation of a longitudinal melanoma biobank containing 311 samples, from 110 patients. The use of recently developed, novel, clinical attenuated total reflectance-Fourier transform infrared (ATR-FTIR) technology was explored and achieved the ability to determine BRAF status in melanoma patients with a sensitivity and specificity of 77.7 % and 75.0 %, respectively. Finally, developments towards spectroscopic precision medicine and categorising of melanoma patients, based on analysis of their individual disease and treatment journeys, was completed. This thesis showcases the development of ATR-FTIR spectroscopy to allow for clinical translation and enable detection and monitoring of melanoma, for close monitoring of high-risk patients and the progression of therapeutic methods.The ability to diagnose melanoma prior to metastasis could revolutionise the clinical environment. This would allow improved patient care via monitoring, rapid follow up of high-risk cases and improve patient mortality and morbidity. Biomedical spectroscopy can diagnose a wide range of pathologies however no study has made it from the laboratory into a clinical setting as a regulated spectroscopic test. To facilitate translation this thesis describes: • Development of the optimal methodology for the quantification of protein biomarkers. It was demonstrated that concentrations as low as 0.66 ± 0.05 mg mL-1, with a linearity of 0.992, can be achieved within patient samples. • Analysis of liquid serum samples led to discrimination of cancer vs. non-cancer with a sensitivity of 95.4 % and a specificity of 81.8 %, compared to the air-dried data set, which achieved 92.4 % and 84.4 %, respectively. Analysis of liquid samples removes the rate determining air drying step. Digitally drying the liquid spectrum was investigated to determine if an improvement could be achieved. The optimal result was achieved through the use of an extended multiplicative scatter correction algorithm, providing a sensitivity of 91.2 % and a specificity of 77.3 %. • Discussion and investigation of a longitudinal melanoma biobank containing 311 samples, from 110 patients. The use of recently developed, novel, clinical attenuated total reflectance-Fourier transform infrared (ATR-FTIR) technology was explored and achieved the ability to determine BRAF status in melanoma patients with a sensitivity and specificity of 77.7 % and 75.0 %, respectively. Finally, developments towards spectroscopic precision medicine and categorising of melanoma patients, based on analysis of their individual disease and treatment journeys, was completed. This thesis showcases the development of ATR-FTIR spectroscopy to allow for clinical translation and enable detection and monitoring of melanoma, for close monitoring of high-risk patients and the progression of therapeutic methods
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