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The role of mitochondria in regulating smooth muscle cell proliferation and migration in pulmonary hypertension
Get PDFThis thesis was previously held under moratorium from 08/07/2020 to 08/07/2022Remodeling of the pulmonary arteries with proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is the hallmark of pulmonary hypertension, a proliferative disease with poor prognosis, which is exacerbated by hypoxic conditions. Hyperproliferation of PASMC underlies the failure of current therapy directed at vasodilation. Therefore, more direct anti-proliferative treatment, which targets the pulmonary vasculature is urgently sought. Signalling mechanisms that cause these cells to proliferate during hypoxia remain unclear. Recent studies highlight the role of mitochondrial alteration in PASMC of pulmonary hypertension, which is characterized by the glycolysis shift in metabolism and the increase in mitochondrial fragmentation that is also observed in cancer.The work presented in this thesis describes the influence of the mitochondria (particularly mitochondrial fission) on cell proliferation and migration of PASMCs during a short period of hypoxia and identifying the mitochondrial-dependent signalling in response to platelet-derived growth factor (PDGF) and hypoxia. Aims were first to determine the appropriate model of hypoxia-induced cell proliferation among different degrees of hypoxia. The second aim was to investigate the role of the mitochondria in regulating cell proliferation and migration using the established hypoxic cell model (3% O2).PASMCs were isolated from Sprague-Dawley rats. Cells were maintained under hypoxia for 24 h and compared with normoxic cultures. Cell proliferation was measured by [3H]-thymidine incorporation assay. The effect of mitochondrial inhibition was studied using mitochondrial dynamin-related protein-1 (DRP1) inhibitor Mdivi-1 (10 μM). The signalling proteins and genes were measured by Western blot and real-time qPCR techniques, respectively. Mitochondrial functions were assessed by measuring ATP level, reactive oxygen species (ROS; superoxide and H2O2) production, and cellular apoptosis. Mitochondrial morphology was examined using an epi-fluorescence microscope.During hypoxia, data show PDGF significantly enhances cell proliferation, HIF1α expression, and promotes mitochondrial dysfunction by decreasing mitochondrial fission, ATP, ROS (H2O2 release) and cellular apoptosis, which all exhibit the proliferative/apoptotic-resistant phenotype of PASMCs compared to PDGF stimulated cells in normoxia. In comparison to hypoxic background cells (cells quiesced with 0.1% FCS and maintained in 3% O2), PDGF caused a reduction in DRP1 expression and mitochondrial fragmentation, an increase in mTORC1 expression and a decrease in hypoxic genes (HIF1α and FIH-1).Interestingly, despite the reduced DRP1 expression and the presence of elongated mitochondria that were observed in the PDGF stimulated cells during hypoxia, inhibiting mitochondrial fission in these cells with Mdivi-1 slows cell proliferation and migration. It causes further reduction in HIF1α and DRP1 expression, promotes hyper-fused mitochondria, upregulates FIH-1 gene expression, inhibits mTORC1 expression and recovers the mitochondrial function possibly via increasing mitochondrial COX-II and ATPase 6 gene expression. Taken together, these data suggest PDGF under hypoxic conditions increases mTORC1 which could control mitochondrial fission and hypoxic genes (HIF1α and FIH-1) and significantly stimulates cell proliferation. However, DRP1 still seems essential in regulating cell proliferation.Therefore, the effect of mTORC1 knockdown in PDGF stimulated cells during hypoxia was studied to confirm the role of mTORC1 in mediating cell proliferation. Knockdown of the mTORC1 gene caused a significant decrease in cell proliferation associated with an increase in DRP1, HIF1α and FIH-1 gene expression. Finally, the inhibitory mechanism of Mdivi-1 following mTORC1 knockdown was studied. Mdivi-1 caused further reduction in cell proliferation, an increase in HIF1α gene and a decrease in FIH-1 expression in cells treated with siRNA against mTORC1. The results also showed Mdivi-1 enhanced HIF1α and FIH-1 expression and decreased mTORC1 expression in hypoxic background cells. Taken together, Mdivi-1 inhibits PDGF induced cell proliferation during hypoxia by increasing FIH-1, which is responsible for HIF1α degradation, via its direct inhibitory effect on mTORC1.Remodeling of the pulmonary arteries with proliferation and migration of pulmonary arterial smooth muscle cell (PASMC) is the hallmark of pulmonary hypertension, a proliferative disease with poor prognosis, which is exacerbated by hypoxic conditions. Hyperproliferation of PASMC underlies the failure of current therapy directed at vasodilation. Therefore, more direct anti-proliferative treatment, which targets the pulmonary vasculature is urgently sought. Signalling mechanisms that cause these cells to proliferate during hypoxia remain unclear. Recent studies highlight the role of mitochondrial alteration in PASMC of pulmonary hypertension, which is characterized by the glycolysis shift in metabolism and the increase in mitochondrial fragmentation that is also observed in cancer.The work presented in this thesis describes the influence of the mitochondria (particularly mitochondrial fission) on cell proliferation and migration of PASMCs during a short period of hypoxia and identifying the mitochondrial-dependent signalling in response to platelet-derived growth factor (PDGF) and hypoxia. Aims were first to determine the appropriate model of hypoxia-induced cell proliferation among different degrees of hypoxia. The second aim was to investigate the role of the mitochondria in regulating cell proliferation and migration using the established hypoxic cell model (3% O2).PASMCs were isolated from Sprague-Dawley rats. Cells were maintained under hypoxia for 24 h and compared with normoxic cultures. Cell proliferation was measured by [3H]-thymidine incorporation assay. The effect of mitochondrial inhibition was studied using mitochondrial dynamin-related protein-1 (DRP1) inhibitor Mdivi-1 (10 μM). The signalling proteins and genes were measured by Western blot and real-time qPCR techniques, respectively. Mitochondrial functions were assessed by measuring ATP level, reactive oxygen species (ROS; superoxide and H2O2) production, and cellular apoptosis. Mitochondrial morphology was examined using an epi-fluorescence microscope.During hypoxia, data show PDGF significantly enhances cell proliferation, HIF1α expression, and promotes mitochondrial dysfunction by decreasing mitochondrial fission, ATP, ROS (H2O2 release) and cellular apoptosis, which all exhibit the proliferative/apoptotic-resistant phenotype of PASMCs compared to PDGF stimulated cells in normoxia. In comparison to hypoxic background cells (cells quiesced with 0.1% FCS and maintained in 3% O2), PDGF caused a reduction in DRP1 expression and mitochondrial fragmentation, an increase in mTORC1 expression and a decrease in hypoxic genes (HIF1α and FIH-1).Interestingly, despite the reduced DRP1 expression and the presence of elongated mitochondria that were observed in the PDGF stimulated cells during hypoxia, inhibiting mitochondrial fission in these cells with Mdivi-1 slows cell proliferation and migration. It causes further reduction in HIF1α and DRP1 expression, promotes hyper-fused mitochondria, upregulates FIH-1 gene expression, inhibits mTORC1 expression and recovers the mitochondrial function possibly via increasing mitochondrial COX-II and ATPase 6 gene expression. Taken together, these data suggest PDGF under hypoxic conditions increases mTORC1 which could control mitochondrial fission and hypoxic genes (HIF1α and FIH-1) and significantly stimulates cell proliferation. However, DRP1 still seems essential in regulating cell proliferation.Therefore, the effect of mTORC1 knockdown in PDGF stimulated cells during hypoxia was studied to confirm the role of mTORC1 in mediating cell proliferation. Knockdown of the mTORC1 gene caused a significant decrease in cell proliferation associated with an increase in DRP1, HIF1α and FIH-1 gene expression. Finally, the inhibitory mechanism of Mdivi-1 following mTORC1 knockdown was studied. Mdivi-1 caused further reduction in cell proliferation, an increase in HIF1α gene and a decrease in FIH-1 expression in cells treated with siRNA against mTORC1. The results also showed Mdivi-1 enhanced HIF1α and FIH-1 expression and decreased mTORC1 expression in hypoxic background cells. Taken together, Mdivi-1 inhibits PDGF induced cell proliferation during hypoxia by increasing FIH-1, which is responsible for HIF1α degradation, via its direct inhibitory effect on mTORC1
When engagement is for self-presentation : the role and impact of the ideal-self in social media engagement
Get PDFThis thesis was previously held under moratorium from 31st August 2021 until 31st August 2024.This thesis expands the concept of engagement beyond its representation of genuine behaviour. In the social media context, the thesis posits that individuals may hold a broader range of motivations and drivers including using engagement activity as part of a representation of their ideal self– rather than being driven by genuine behaviour as the extant literature generally expects. Via a two-phase, qualitative research design, this thesis explores how engagement can be affected by an individual’s ideal self and the effect on others. The first phase of the study conducted semi-structured interviews with 30 social media users to explore the role and impact of the ideal self in engagement. The results identified three types of engagement for self-presentation based on different relational and dispositional levels: staged engagement, contradictory engagement and faked engagement. These forms of engagement for self-presentation also had different drivers: self-differentiation, group belonging and self-enhancement. This revealed how engagement for self-presentation may affect other actors (i.e., individuals and focal firms). Hence, it was necessary to pose questions to organisations. The second phase of the study conducted semi-structured interviews with 7 organisations to understand the results of the first phase from organisational perspectives. The findings suggested that brand awareness, trust and control are key themes when attempting to understand how organisations view engagement for self-presentation. Overall, this thesis contributes to the literature on engagement by reinforcing the aspect of the self and enhancing the current understanding of how engagement may occur on social media and its impact on others. This research considers engagement as a form of self-presentation, reflecting the different relationships and dispositions of individuals to focal objects. This suggests self-presentation as an antecedent of engagement. The thesis also acknowledges the temporality of such engagement. Finally, the research contributes by showing how engagement for self-presentation may affect organisations.This thesis expands the concept of engagement beyond its representation of genuine behaviour. In the social media context, the thesis posits that individuals may hold a broader range of motivations and drivers including using engagement activity as part of a representation of their ideal self– rather than being driven by genuine behaviour as the extant literature generally expects. Via a two-phase, qualitative research design, this thesis explores how engagement can be affected by an individual’s ideal self and the effect on others. The first phase of the study conducted semi-structured interviews with 30 social media users to explore the role and impact of the ideal self in engagement. The results identified three types of engagement for self-presentation based on different relational and dispositional levels: staged engagement, contradictory engagement and faked engagement. These forms of engagement for self-presentation also had different drivers: self-differentiation, group belonging and self-enhancement. This revealed how engagement for self-presentation may affect other actors (i.e., individuals and focal firms). Hence, it was necessary to pose questions to organisations. The second phase of the study conducted semi-structured interviews with 7 organisations to understand the results of the first phase from organisational perspectives. The findings suggested that brand awareness, trust and control are key themes when attempting to understand how organisations view engagement for self-presentation. Overall, this thesis contributes to the literature on engagement by reinforcing the aspect of the self and enhancing the current understanding of how engagement may occur on social media and its impact on others. This research considers engagement as a form of self-presentation, reflecting the different relationships and dispositions of individuals to focal objects. This suggests self-presentation as an antecedent of engagement. The thesis also acknowledges the temporality of such engagement. Finally, the research contributes by showing how engagement for self-presentation may affect organisations
Novel methods and processes for the chiral resolution of fine chemicals
Get PDFPreviously held under moratorium from 1st March 2021 until 1st March 2023Chiral molecules occur in left- and right-handed configurations that can be considered asmirror images (enantiomers), and that, like hands, cannot be superimposed onto each other.Despite their similarity, the biological activity of enantiomers inside living organisms can becompletely different. While one enantiomer may have a desirable therapeutic effect, the othermay induce no response or even be harmful. Most pharmaceuticals and agrochemicals arechiral. The search of this specificity at the molecular level led to the development of manyasymmetric syntheses. However, the enantioselective synthetic approach is not alwayspossible and the mixture of equal amounts of both enantiomers (racemate) is obtained instead.Then, the separation of these enantiomers, known as chiral resolution, becomes the alternativepathway. Chiral resolution techniques have already been developed, but they still presentsome limitations. For that, the aim of this thesis is to develop novel methodologies andprocesses that integrate membrane filtration, crystallisation, and extraction technologiesfor the chiral resolution of fine chemical racemates.In chapter 3, the effects of pH and temperature on the solubility and phase diagrams ofthree racemic compound-forming systems are described. This preliminary study of effectspermits the development of a method to accurately estimate the eutectic points and phasediagrams of these systems. Additionally, the method also gives the possibility to determinethe class of crystal lattice (conglomerate, racemic compound, or solid solution) of any chiralcompound. The ultimate objective of this chapter is to provide a workflow process todetermine fast and accurately the phase diagrams of racemic compounds.The chiral resolution of conglomerates has been successful by means of crystallisationbased techniques such as preferential crystallisation. Nevertheless, this type of chiralcompounds alongside solid solutions are rare with racemic compounds being the mostabundant. The latter class requires an initial enantiomeric enrichment which must go beyondthe eutectic composition to after being able to selectively crystallise an enantiopure product.The main objective of chapter 4 is to assess if the combination of membrane filtration andcrystallisation technologies enables the continuous chiral resolution of racemic compounds.This has been carried out through computerised modelling and optimisation of the integratedVIIprocess and has shown potential to further explore and test the process with other chiralsystems.The separation of enantiomers can also be attempted by leveraging the potential distinctiveenantiospecific interactions between enantiomers and chiral solvents. In that way, chapter 5evaluates the chiral recognition capacity of the relatively new chiral solvent cyrene and findsits potential for chiral resolution process applications founded on both enantioselective liquidliquid extraction and crystallisation.The work in this thesis has achieved its aim in developing novel methodologies andprocesses for the chiral resolution of fine chemical racemates by providing a less laboriousworkflow process for the fast and accurate determination of ternary phase diagrams ofracemic compounds, which has served to propose a novel chiral resolution process for thisclass of chiral compounds that integrates both membrane filtration and crystallisationtechnologies. In addition, the enantiomeric discrimination power found in the chiralsolvent cyrene has made possible to propose this solvent for its use in the separation ofenantiomers by means of enantioselective liquid-liquid extraction and crystallisation. Thefindings described in this text will then facilitate the study, understanding, andcharacterisation of chiral resolution processes based on enantioselective interactions inseparation techniques, such as membrane filtration, crystallisation, and extraction. Thenew methodologies and processes developed in this thesis have the potential to addresscurrent and future challenges in the separation of enantiomers, which is of high interestfor the fine chemical industry.Chiral molecules occur in left- and right-handed configurations that can be considered asmirror images (enantiomers), and that, like hands, cannot be superimposed onto each other.Despite their similarity, the biological activity of enantiomers inside living organisms can becompletely different. While one enantiomer may have a desirable therapeutic effect, the othermay induce no response or even be harmful. Most pharmaceuticals and agrochemicals arechiral. The search of this specificity at the molecular level led to the development of manyasymmetric syntheses. However, the enantioselective synthetic approach is not alwayspossible and the mixture of equal amounts of both enantiomers (racemate) is obtained instead.Then, the separation of these enantiomers, known as chiral resolution, becomes the alternativepathway. Chiral resolution techniques have already been developed, but they still presentsome limitations. For that, the aim of this thesis is to develop novel methodologies andprocesses that integrate membrane filtration, crystallisation, and extraction technologiesfor the chiral resolution of fine chemical racemates.In chapter 3, the effects of pH and temperature on the solubility and phase diagrams ofthree racemic compound-forming systems are described. This preliminary study of effectspermits the development of a method to accurately estimate the eutectic points and phasediagrams of these systems. Additionally, the method also gives the possibility to determinethe class of crystal lattice (conglomerate, racemic compound, or solid solution) of any chiralcompound. The ultimate objective of this chapter is to provide a workflow process todetermine fast and accurately the phase diagrams of racemic compounds.The chiral resolution of conglomerates has been successful by means of crystallisationbased techniques such as preferential crystallisation. Nevertheless, this type of chiralcompounds alongside solid solutions are rare with racemic compounds being the mostabundant. The latter class requires an initial enantiomeric enrichment which must go beyondthe eutectic composition to after being able to selectively crystallise an enantiopure product.The main objective of chapter 4 is to assess if the combination of membrane filtration andcrystallisation technologies enables the continuous chiral resolution of racemic compounds.This has been carried out through computerised modelling and optimisation of the integratedVIIprocess and has shown potential to further explore and test the process with other chiralsystems.The separation of enantiomers can also be attempted by leveraging the potential distinctiveenantiospecific interactions between enantiomers and chiral solvents. In that way, chapter 5evaluates the chiral recognition capacity of the relatively new chiral solvent cyrene and findsits potential for chiral resolution process applications founded on both enantioselective liquidliquid extraction and crystallisation.The work in this thesis has achieved its aim in developing novel methodologies andprocesses for the chiral resolution of fine chemical racemates by providing a less laboriousworkflow process for the fast and accurate determination of ternary phase diagrams ofracemic compounds, which has served to propose a novel chiral resolution process for thisclass of chiral compounds that integrates both membrane filtration and crystallisationtechnologies. In addition, the enantiomeric discrimination power found in the chiralsolvent cyrene has made possible to propose this solvent for its use in the separation ofenantiomers by means of enantioselective liquid-liquid extraction and crystallisation. Thefindings described in this text will then facilitate the study, understanding, andcharacterisation of chiral resolution processes based on enantioselective interactions inseparation techniques, such as membrane filtration, crystallisation, and extraction. Thenew methodologies and processes developed in this thesis have the potential to addresscurrent and future challenges in the separation of enantiomers, which is of high interestfor the fine chemical industry
Real world outcomes with systemic anti-cancer treatments for advanced melanoma : experience from the West of Scotland 2010-2018
Get PDFBackground:Ipilimumab was the first systemic anti-cancer therapy (SACT) to show survival benefit in clinical trials for advanced (unresectable or metastatic) melanoma. (Hodi et al. 2010) Vemurafenib, dabrafenib with trametinib, pembrolizumab and nivolumab followed but it is recognised that clinical trial findings are not always representative of real world practice (Donia et al. 2017). It was proposed that electronic record linkage (ERL) of routinely captured healthcare data with SACT prescriptions would generate real world data (RWD) to supplement clinical trial results for treatment decision making.Methods:Patients starting SACT for advanced melanoma between 1.11.10 and 31.12.17 in the West of Scotland were identified and followed up until 31.3.18. Prescribing records from the Chemotherapy Electronic Prescribing and Administration System (CEPAS) were linked with routinely captured healthcare data including the Scottish Cancer Registry, anonymised then accessed in the NHSGGC Safe Haven. Multivariable Cox regression models estimated impact of patient baseline characteristics and SACT on OS. ERL methodology was validated using results from individual patient case notes (IPLR).Results:Median OS varied from 18.5 months (95%CI 14.4-not estimable) for ipilimumab with nivolumab to 5.6 months (4.5-7.3) with dabrafenib. Dabrafenib with trametinib (HR 0.42, p=0.0014) and ipilimumab with nivolumab (HR 0.50, p=0.0352) had a positive impact on OS compared to ipilimumab monotherapy. Baseline characteristics including LDH levels above the upper limit of normal; ECOG performance status ≥2 and mucosal melanomas had a negative impact on OS.Data availability differences between ERL and IPLR (some laboratory results and disease characteristics) did not have a statistically significant impact on the hazard ratios for each SACT, except dabrafenib, in the multivariable Cox model.Conclusion:Our RWD showed reduced median OS compared to the pivotal clinical trials but it included patients often excluded from clinical trials. ERL is a valid method for obtaining real world outcomes.Background:Ipilimumab was the first systemic anti-cancer therapy (SACT) to show survival benefit in clinical trials for advanced (unresectable or metastatic) melanoma. (Hodi et al. 2010) Vemurafenib, dabrafenib with trametinib, pembrolizumab and nivolumab followed but it is recognised that clinical trial findings are not always representative of real world practice (Donia et al. 2017). It was proposed that electronic record linkage (ERL) of routinely captured healthcare data with SACT prescriptions would generate real world data (RWD) to supplement clinical trial results for treatment decision making.Methods:Patients starting SACT for advanced melanoma between 1.11.10 and 31.12.17 in the West of Scotland were identified and followed up until 31.3.18. Prescribing records from the Chemotherapy Electronic Prescribing and Administration System (CEPAS) were linked with routinely captured healthcare data including the Scottish Cancer Registry, anonymised then accessed in the NHSGGC Safe Haven. Multivariable Cox regression models estimated impact of patient baseline characteristics and SACT on OS. ERL methodology was validated using results from individual patient case notes (IPLR).Results:Median OS varied from 18.5 months (95%CI 14.4-not estimable) for ipilimumab with nivolumab to 5.6 months (4.5-7.3) with dabrafenib. Dabrafenib with trametinib (HR 0.42, p=0.0014) and ipilimumab with nivolumab (HR 0.50, p=0.0352) had a positive impact on OS compared to ipilimumab monotherapy. Baseline characteristics including LDH levels above the upper limit of normal; ECOG performance status ≥2 and mucosal melanomas had a negative impact on OS.Data availability differences between ERL and IPLR (some laboratory results and disease characteristics) did not have a statistically significant impact on the hazard ratios for each SACT, except dabrafenib, in the multivariable Cox model.Conclusion:Our RWD showed reduced median OS compared to the pivotal clinical trials but it included patients often excluded from clinical trials. ERL is a valid method for obtaining real world outcomes
Gold nanorod based nanoprobes for biomedical applications
Get PDFPreviously held under moratorium from 31st March 2021 until 31st March 2023Small gold nanorods (SGNRs) are nanorods that are ~13-50 nm in length and less than 10 nm in diameter. The SGNRs manifest excellent optical properties arising from localised surface plasmon resonance including strong optical absorption and scattering tunable from the visible to the infrared region of electromagnetic spectrum. The SGNRs have higher absorption to scattering ratio compared to the large gold nanorods (LGNRs), making the SGNRs good photothermal agents. Moreover, the SGNRs have ease of subcellular accessibility, high internalization rate and large surface area to volume ratio for binding of analytes. These attributes make the SGNRs good candidates in various biomedical applications such as biosensing, imaging and delivery of drugs. This study is aimed at developing gold nanorod based nanoprobes for detecting molecular biomarkers at the single cell level, and characterization of the optical properties and the photothermal effects of gold nanorods for photothermal therapy of cancer. A systematic study on the growth conditions was carried out and a reliable method has been developed for the synthesis of stable SGNRs with good control over size and shape, and high yield of rods. The SGNRs were successfully functionalized with hairpin DNA (hpDNA) for targeting messenger RNA (mRNA). Moreover, this work investigated the influence of gold nanorods size and media on their photothermal effect. Theoretical calculation revealed that the SGNRs have higher photothermal efficiency than the LGNRs in solution. However, in solution the SGNRs generated slightly more heat at off-resonance illumination while the LGNRs generated more heat than SGNRs at plasmon resonance excitation. Nevertheless, the experimental study revealed that the SGNRs generated more heat than the LGNRs when both are in gel media that is close to cell enviroment. Furthermore, aptamer functionalized SGNRs nanoprobes were developed for targeting cancer cells. The SGNRs based nanoprobes were found to have higher photothermal effect in cancer cells compared to the LGNRs nanoprobes. In addition, the SGNR based nanoprobes were found to be more sensitive than the LGNR based nanoprobes in detecting RNA cancer biomarkers in cells and exosomes. iv This work demonstrates the capability of the gold nanorod based nanoprobes in detecting the cancer biomarker RNA in a blood serum.Small gold nanorods (SGNRs) are nanorods that are ~13-50 nm in length and less than 10 nm in diameter. The SGNRs manifest excellent optical properties arising from localised surface plasmon resonance including strong optical absorption and scattering tunable from the visible to the infrared region of electromagnetic spectrum. The SGNRs have higher absorption to scattering ratio compared to the large gold nanorods (LGNRs), making the SGNRs good photothermal agents. Moreover, the SGNRs have ease of subcellular accessibility, high internalization rate and large surface area to volume ratio for binding of analytes. These attributes make the SGNRs good candidates in various biomedical applications such as biosensing, imaging and delivery of drugs. This study is aimed at developing gold nanorod based nanoprobes for detecting molecular biomarkers at the single cell level, and characterization of the optical properties and the photothermal effects of gold nanorods for photothermal therapy of cancer. A systematic study on the growth conditions was carried out and a reliable method has been developed for the synthesis of stable SGNRs with good control over size and shape, and high yield of rods. The SGNRs were successfully functionalized with hairpin DNA (hpDNA) for targeting messenger RNA (mRNA). Moreover, this work investigated the influence of gold nanorods size and media on their photothermal effect. Theoretical calculation revealed that the SGNRs have higher photothermal efficiency than the LGNRs in solution. However, in solution the SGNRs generated slightly more heat at off-resonance illumination while the LGNRs generated more heat than SGNRs at plasmon resonance excitation. Nevertheless, the experimental study revealed that the SGNRs generated more heat than the LGNRs when both are in gel media that is close to cell enviroment. Furthermore, aptamer functionalized SGNRs nanoprobes were developed for targeting cancer cells. The SGNRs based nanoprobes were found to have higher photothermal effect in cancer cells compared to the LGNRs nanoprobes. In addition, the SGNR based nanoprobes were found to be more sensitive than the LGNR based nanoprobes in detecting RNA cancer biomarkers in cells and exosomes. iv This work demonstrates the capability of the gold nanorod based nanoprobes in detecting the cancer biomarker RNA in a blood serum
Design and development of an electrochemical and infrared spectroscopic medical device for serum-based cancer diagnostics
Get PDFPreviously held under moratorium from 17 June 2020 until 22 June 2022Development of an integrated electrochemical and spectroscopic serum diagnosticdevice would have significant potential as a triage tool for brain cancer and Hodgkin’slymphoma in primary care settings to better inform clinical decisions and facilitateprompt referral to secondary care, whilst reducing financial and practical constraintsplaced on current diagnostic modalities. Additionally, the ability to detect IDH1molecular status from serum samples would have significant clinical value to patientsand clinicians alike, ultimately allowing earlier diagnosis and improved planning ofsurgery and treatment therapeutics. To this extent, the medical device hasdemonstrated the ability to electrochemically detect the biomarker CCL17/TARC at387-50,000pg/ml concentrations with R2 = 0.979 and limit of detection of 387pg/mlin spiked buffer samples. Thereafter, the medical device demonstrated successfuldiagnosis of Hodgkin’s lymphoma in all 11 tested clinical patient samples.Additionally, ATR-FTIR discriminated between Hodgkin’s lymphoma and healthycontrols in 200 patient samples with sensitivity of 83.2 ± 6.6% and specificity of 85.3± 8.1% with Random Forest classification, highlighting differences in proteinsecondary structures within clinical serum samples. The integrated diagnostic platformfurther demonstrated the ability to electrochemically detect IDH1-R132H proteins inspiked buffer samples at 0.05-10,000ng/ml concentrations with R2 = 0.958. However,it was not subsequently possible to detect IDH1 mutant proteins in clinical serumsamples. Nevertheless, ATR-FTIR demonstrated the ability to discriminate betweenIDH1 molecular status in 104 glioma patients through consideration of the globalmolecular signatures ofserum samples with sensitivity of 89.0 ± 11.3% and specificityof 88.2 ± 10.1% with PLS classification.Development of an integrated electrochemical and spectroscopic serum diagnosticdevice would have significant potential as a triage tool for brain cancer and Hodgkin’slymphoma in primary care settings to better inform clinical decisions and facilitateprompt referral to secondary care, whilst reducing financial and practical constraintsplaced on current diagnostic modalities. Additionally, the ability to detect IDH1molecular status from serum samples would have significant clinical value to patientsand clinicians alike, ultimately allowing earlier diagnosis and improved planning ofsurgery and treatment therapeutics. To this extent, the medical device hasdemonstrated the ability to electrochemically detect the biomarker CCL17/TARC at387-50,000pg/ml concentrations with R2 = 0.979 and limit of detection of 387pg/mlin spiked buffer samples. Thereafter, the medical device demonstrated successfuldiagnosis of Hodgkin’s lymphoma in all 11 tested clinical patient samples.Additionally, ATR-FTIR discriminated between Hodgkin’s lymphoma and healthycontrols in 200 patient samples with sensitivity of 83.2 ± 6.6% and specificity of 85.3± 8.1% with Random Forest classification, highlighting differences in proteinsecondary structures within clinical serum samples. The integrated diagnostic platformfurther demonstrated the ability to electrochemically detect IDH1-R132H proteins inspiked buffer samples at 0.05-10,000ng/ml concentrations with R2 = 0.958. However,it was not subsequently possible to detect IDH1 mutant proteins in clinical serumsamples. Nevertheless, ATR-FTIR demonstrated the ability to discriminate betweenIDH1 molecular status in 104 glioma patients through consideration of the globalmolecular signatures ofserum samples with sensitivity of 89.0 ± 11.3% and specificityof 88.2 ± 10.1% with PLS classification
Dynamic analysis of a spar-type offshore floating wind turbine and its mooring system
Get PDFThe demand for developing sustainable and renewable energy is increasing due to the greenhouse gas effect and the reducing availability of fossil fuels. Wind energy is one of the cleanest and most sustainable renewable resources. People mainly use two types of wind turbines - onshore and offshore, to harvest the wind energy. Compared with onshore wind, offshore wind resources allow more reliable generation of electricity. In shallow water, bottom-fixed turbines may be used. However, as water depths increase it becomes increasingly difficult to build suitable fixed foundations, and floating offshore wind turbines become attractive. The OC3-Hywind is an offshore floating wind turbine (OFWT) supported by a spar platform and held in place with three mooring lines. It is important to study the dynamic responses of the floating platform and the mooring system behaviour so that the OFWT can be designed and constructed safely and economically and generate electricity with reliability. However, there are not many studies which have validated the numerical simulation results for floating offshore wind turbines with physical experiments while published studies rarely show the details of the mooring line motion behaviour for OFWTs. This thesis investigates the dynamic behaviour of a spar-type OFWT including its mooring systems, analysed under various environmental loads both by using state-of-the-art numerical software and conducting an experiment campaign at the Kelvin Hydrodynamics Laboratory. The free decay test has been carried out first to get the spar platform's motions natural frequencies and damping characteristics. A range of regular and irregular waves has been applied to the platform (both with and without realistic mooring lines) to obtain the platform motion RAO (response amplitude operator) - which can be used to predict the platform dynamic responses under other wave conditions - and to examine the reliability of the numerical predictions under realistic sea conditions. For the mooring line tensions and motion, the software shows its limitations in the calculation. A non-linear snatching phenomenon has been observed at the tank for some wave frequencies, which has rarely been discussed in published research. It is very important to study this non-linear behaviour as it is shown that snatching leads to the high instantaneous mooring line loads and platform accelerations, which could cause the failure of the mooring lines in the real structure.The demand for developing sustainable and renewable energy is increasing due to the greenhouse gas effect and the reducing availability of fossil fuels. Wind energy is one of the cleanest and most sustainable renewable resources. People mainly use two types of wind turbines - onshore and offshore, to harvest the wind energy. Compared with onshore wind, offshore wind resources allow more reliable generation of electricity. In shallow water, bottom-fixed turbines may be used. However, as water depths increase it becomes increasingly difficult to build suitable fixed foundations, and floating offshore wind turbines become attractive. The OC3-Hywind is an offshore floating wind turbine (OFWT) supported by a spar platform and held in place with three mooring lines. It is important to study the dynamic responses of the floating platform and the mooring system behaviour so that the OFWT can be designed and constructed safely and economically and generate electricity with reliability. However, there are not many studies which have validated the numerical simulation results for floating offshore wind turbines with physical experiments while published studies rarely show the details of the mooring line motion behaviour for OFWTs. This thesis investigates the dynamic behaviour of a spar-type OFWT including its mooring systems, analysed under various environmental loads both by using state-of-the-art numerical software and conducting an experiment campaign at the Kelvin Hydrodynamics Laboratory. The free decay test has been carried out first to get the spar platform's motions natural frequencies and damping characteristics. A range of regular and irregular waves has been applied to the platform (both with and without realistic mooring lines) to obtain the platform motion RAO (response amplitude operator) - which can be used to predict the platform dynamic responses under other wave conditions - and to examine the reliability of the numerical predictions under realistic sea conditions. For the mooring line tensions and motion, the software shows its limitations in the calculation. A non-linear snatching phenomenon has been observed at the tank for some wave frequencies, which has rarely been discussed in published research. It is very important to study this non-linear behaviour as it is shown that snatching leads to the high instantaneous mooring line loads and platform accelerations, which could cause the failure of the mooring lines in the real structure
The role of sphingolipids in autosomal dominant polycystic kidney disease (ADPKD)
No full textAutosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of renal cysts and eventual renal failure. There is currently no cure for ADPKD, but various treatments are available to alleviate the symptoms. In ADPKD, cystic renal epithelia are prevalent by the upregulation of various growth regulating signalling pathways, such as ERK-1/2 and PI3K/Akt/mTOR, which leads to elevated kidney epithelial cell proliferation. Dysregulation of their primary cilia (evident by decreased levels of acetylated α-tubulin) is also a crucial factor in defective renal cystic epithelia. Sphingolipids contribute to regulating these cellular processes. Two isoforms of sphingosine kinase (SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine-1-phosphate (S1P) from sphingosine.;We sought to determine the effects of modulating these enzymes, either separately or together, on the signalling pathways implicated in ADPKD. Five novel SK inhibitors (SK2-selective,SK1-selective or dual SK1/SK2 inhibitor) were evaluated. Comparison was made with a glucosylceramide synthase inhibitor (Genz-123346) as blocking the synthesis of glucosylceramide is emerging as a possible therapeutic for ADPKD. The current study shows that the SK2-selective inhibitors, ST-066, ST-081 and HWG-35D, and the dualSK1/SK2 inhibitor, PLR-11, decreased DNA synthesis, PCNA expression, Aktphosphorylation and ERK-1/2 phosphorylation, but had no major effect on apoptosis, autophagy, α-tubulin acetylation or primary cilia in Madin-Darby Canine Kidney(MDCK) cells. Collectively, these data suggest that ST-066, ST-081, HWG-35D andPLR-11 affect signalling pathways of relevance to cyst growth; however, the potential application of SK2 inhibitors in ADPKD therapeutics remains to be determined.Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of renal cysts and eventual renal failure. There is currently no cure for ADPKD, but various treatments are available to alleviate the symptoms. In ADPKD, cystic renal epithelia are prevalent by the upregulation of various growth regulating signalling pathways, such as ERK-1/2 and PI3K/Akt/mTOR, which leads to elevated kidney epithelial cell proliferation. Dysregulation of their primary cilia (evident by decreased levels of acetylated α-tubulin) is also a crucial factor in defective renal cystic epithelia. Sphingolipids contribute to regulating these cellular processes. Two isoforms of sphingosine kinase (SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine-1-phosphate (S1P) from sphingosine.;We sought to determine the effects of modulating these enzymes, either separately or together, on the signalling pathways implicated in ADPKD. Five novel SK inhibitors (SK2-selective,SK1-selective or dual SK1/SK2 inhibitor) were evaluated. Comparison was made with a glucosylceramide synthase inhibitor (Genz-123346) as blocking the synthesis of glucosylceramide is emerging as a possible therapeutic for ADPKD. The current study shows that the SK2-selective inhibitors, ST-066, ST-081 and HWG-35D, and the dualSK1/SK2 inhibitor, PLR-11, decreased DNA synthesis, PCNA expression, Aktphosphorylation and ERK-1/2 phosphorylation, but had no major effect on apoptosis, autophagy, α-tubulin acetylation or primary cilia in Madin-Darby Canine Kidney(MDCK) cells. Collectively, these data suggest that ST-066, ST-081, HWG-35D andPLR-11 affect signalling pathways of relevance to cyst growth; however, the potential application of SK2 inhibitors in ADPKD therapeutics remains to be determined
Broadly tuneable crystalline Raman lasers
No full textHere is presented the first demonstration of a continuous-wave third-Stokes crystalline Raman laser. This was achieved via intracavity Raman conversion of a vertical external cavity surface emitting laser (VECSEL), also known as a semiconductor disk laser (SDL). The VECSEL-pumped Raman laser achieved Watt-level output and broad tuneability within the water transmission window at 1.7 µm, offering potential applications in remote sensing and medical imaging. In this thesis the design and characterization of such a cascaded Raman laser are described.;The attractive performance of VECSELs based on InGaAs for emission around 1 µm, now being implemented in commercial cw laser systems, has spurred the VECSEL research community to widen the fundamental spectral coverage beyond 1.2 µm via more sophisticated and at the same time more complex growth and fabrication processes. These devices nonetheless lend themselves very well to nonlinear frequency conversion, primarily owing to the high-intensity fields generated intracavity, thus bypassing diffcult epitaxy and processing to access long wavelengths with high power and low output coupling.;Raman media, as x(3)-type materials, are powerful tools as wavelength converters thanks to the simplified optics required to achieve stimulated Raman scattering -which is the basis of the Raman laser- when compared to most common x(2)-based systems.;This thesis reports 1.1 W continuous-wave Raman laser emission centred at 1.73 µm from third-Stokes generation in a KGW crystal within an InGaAs-based VECSEL designed for fundamental emission at 1.18 µm. This system also demonstrated broad tuneability from 1696-1761 nm when the VECSEL was tuned from 1163-1193.2 nm, exceptional beam quality (M2 ≈ 1) and instrument-limited narrow linewidth of 48 pm FWHM.;In addition, simultaneous output coupling at the first and second Stokes was also attained, with maximum output power of 1.1 and 0.8 W at 1.5 µm and 1.32 µm,respectively tuneable from 1473-1522 nm and 1300-1338 nm. Having originally intended to reach 1.7 µm with a synthetic crystal of diamond and two Stokes shifts (the Raman shift is larger than in KGW), in this work three Stokes shifts in KGW were utilized since the deposition of robust anti-reflection coatings was more feasible on this crystal for such a broad wavelength range (1.1-1.7 µm).;Nevertheless, the first Stokes shift in diamond, being AR-coated for 1.1-1.4 µm operation and pumped by the same VECSEL structure, resulted in Raman laser emission at an important water absorption region around 1.4 µm, with maximum 2.3 W output power and tuneable over a range of 42 nm.Here is presented the first demonstration of a continuous-wave third-Stokes crystalline Raman laser. This was achieved via intracavity Raman conversion of a vertical external cavity surface emitting laser (VECSEL), also known as a semiconductor disk laser (SDL). The VECSEL-pumped Raman laser achieved Watt-level output and broad tuneability within the water transmission window at 1.7 µm, offering potential applications in remote sensing and medical imaging. In this thesis the design and characterization of such a cascaded Raman laser are described.;The attractive performance of VECSELs based on InGaAs for emission around 1 µm, now being implemented in commercial cw laser systems, has spurred the VECSEL research community to widen the fundamental spectral coverage beyond 1.2 µm via more sophisticated and at the same time more complex growth and fabrication processes. These devices nonetheless lend themselves very well to nonlinear frequency conversion, primarily owing to the high-intensity fields generated intracavity, thus bypassing diffcult epitaxy and processing to access long wavelengths with high power and low output coupling.;Raman media, as x(3)-type materials, are powerful tools as wavelength converters thanks to the simplified optics required to achieve stimulated Raman scattering -which is the basis of the Raman laser- when compared to most common x(2)-based systems.;This thesis reports 1.1 W continuous-wave Raman laser emission centred at 1.73 µm from third-Stokes generation in a KGW crystal within an InGaAs-based VECSEL designed for fundamental emission at 1.18 µm. This system also demonstrated broad tuneability from 1696-1761 nm when the VECSEL was tuned from 1163-1193.2 nm, exceptional beam quality (M2 ≈ 1) and instrument-limited narrow linewidth of 48 pm FWHM.;In addition, simultaneous output coupling at the first and second Stokes was also attained, with maximum output power of 1.1 and 0.8 W at 1.5 µm and 1.32 µm,respectively tuneable from 1473-1522 nm and 1300-1338 nm. Having originally intended to reach 1.7 µm with a synthetic crystal of diamond and two Stokes shifts (the Raman shift is larger than in KGW), in this work three Stokes shifts in KGW were utilized since the deposition of robust anti-reflection coatings was more feasible on this crystal for such a broad wavelength range (1.1-1.7 µm).;Nevertheless, the first Stokes shift in diamond, being AR-coated for 1.1-1.4 µm operation and pumped by the same VECSEL structure, resulted in Raman laser emission at an important water absorption region around 1.4 µm, with maximum 2.3 W output power and tuneable over a range of 42 nm
Dynamics of impurities and trapped Bose-Einstein Condensates
Get PDFThe high degree of control that exists in experiments with cold atoms, and Bose-Einstein condensates, in particular, allows us to design systems with chosen geometries. These provide us with simple model systems that are well understood from first principles microscopically, and with parameters controlled by external fields, as tools to explore new frontiers in the context of non-equilibrium dynamics. These possibilities can be used for a wide variety of applications, ranging from quantum simulations to quantum metrology. In this thesis, we investigate designing the confinement of tightly trapped impurities in a BEC reservoir to study their dissipative dynamics, and to study non-equilibrium dynamics of BECs confined in a tilted ring trap (both with ultracold atoms and polaritons).;In the first part we explore tightly confined impurities, immersed in a weakly trapped BEC and initially excited in the strongly confined direction, to study cooling of the impurities to low-temperature states under realistic experimental conditions. This scheme, combined with dissipative state engineering, sets the basis for adaption of laser cooling techniques for the production of low-entropy states in quantum simulators. We can also use this system to access non-Markovian dynamics by changing the ratios of relevant timescales using control over the trapping of the impurities and the reservoir.;In the second part we study the dynamics of a BEC confined in a 1D tilted ring trap, both with ultracold atoms and long-lifetime polaritons. We study the collective oscillations of a BEC in a tilted ring trap to characterise the effects generated by the interplay between non-linearities due to anharmonicity and non-linearities due to interactions. In the case of polaritons, we make comparison with experimentally observed features, and gain an understanding of the thermalisation process in such systems.The high degree of control that exists in experiments with cold atoms, and Bose-Einstein condensates, in particular, allows us to design systems with chosen geometries. These provide us with simple model systems that are well understood from first principles microscopically, and with parameters controlled by external fields, as tools to explore new frontiers in the context of non-equilibrium dynamics. These possibilities can be used for a wide variety of applications, ranging from quantum simulations to quantum metrology. In this thesis, we investigate designing the confinement of tightly trapped impurities in a BEC reservoir to study their dissipative dynamics, and to study non-equilibrium dynamics of BECs confined in a tilted ring trap (both with ultracold atoms and polaritons).;In the first part we explore tightly confined impurities, immersed in a weakly trapped BEC and initially excited in the strongly confined direction, to study cooling of the impurities to low-temperature states under realistic experimental conditions. This scheme, combined with dissipative state engineering, sets the basis for adaption of laser cooling techniques for the production of low-entropy states in quantum simulators. We can also use this system to access non-Markovian dynamics by changing the ratios of relevant timescales using control over the trapping of the impurities and the reservoir.;In the second part we study the dynamics of a BEC confined in a 1D tilted ring trap, both with ultracold atoms and long-lifetime polaritons. We study the collective oscillations of a BEC in a tilted ring trap to characterise the effects generated by the interplay between non-linearities due to anharmonicity and non-linearities due to interactions. In the case of polaritons, we make comparison with experimentally observed features, and gain an understanding of the thermalisation process in such systems
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