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A statistical analysis of spatially linked time series using data from the TOXBASE database to study Emergency Department and NHS phone line management of poisoned patients
Get PDFThe aim of this thesis is to examine the overall trends in poisoning, which will lead to an assessment of the relationship between poisons information database TOXBASE use and the management of poisoned patients within UK based emergency departments. Previous studies on the demographics of poisoning will be presented. This will cover important concepts in the study of toxicology before examining how specific demographic variables are linked to incidences of poisoning. A discussion of the agents used in cases of poisoning in recent history will be followed by a summary of legislation pertaining to poisoning and dangerous substances. The various services examined throughout the thesis will also be introduced. The first of these services, NHS 24, is described as an out of hours service for use by members of the public. The NHS 24 operator can advise on appropriate action given a description of the symptoms. These descriptions can be categorised as poisoning based on the presence of specific key words or phrases. Using generalised additive models, a consistent seasonal trend in poisoning calls to NHS 24 was found. The second service examined, TOXBASE, is a database provided by the National Poisons Information Service which provides information to clinical professionals on how to treat poisoning by a variety of substances. As in the NHS 24 analysis, generalised additive models have been used in order to assess the trends present in accesses made to the TOXBASE database by clinicians. The results from this analysis found that there was a consistent seasonal trend in TOXBASE accesses which peaked over summer and was similar to that seen in the NHS 24 call data. A third temporal analysis was carried out on data obtained from NHS information services pertaining to admissions and attendances due to poisoning, again showing similar results to the previous two analyses.;These analyses combined suggest an underlying trend in poisonings. Both the admissions data and TOXBASE access data were examined using funnel plots in order to determine whether there were any hospitals which were unusual in their admissions or their TOXBASE use. This analysis found some commonalities in those hospitals which are unusual in either their admission rates or TOXBASE use. The final step in this project was to link the TOXBASE access data with the attendances and admission data in order to examine whether there was any link between TOXBASE use and admission due to poisoning. The results of this indicate that there are associations between TOXBASE use and admissions, such that an increase in TOXBASE use indicates an increase in admissions due to drugs poisoning. However, it became clear that the data used were limited in their ability to show any direct impact of TOXBASE on admissions due to drug poisoning, and that more specific data, for example on toxicants involved and case severity, would potentially be useful in mitigating the obvious confounding present in these data. This thesis has provided new insight into patterns in cases of poisoning, as well as providing a strong basis for further analysis to establish whether there isa direct impact of TOXBASE use on patient management within UK emergency departments.The aim of this thesis is to examine the overall trends in poisoning, which will lead to an assessment of the relationship between poisons information database TOXBASE use and the management of poisoned patients within UK based emergency departments. Previous studies on the demographics of poisoning will be presented. This will cover important concepts in the study of toxicology before examining how specific demographic variables are linked to incidences of poisoning. A discussion of the agents used in cases of poisoning in recent history will be followed by a summary of legislation pertaining to poisoning and dangerous substances. The various services examined throughout the thesis will also be introduced. The first of these services, NHS 24, is described as an out of hours service for use by members of the public. The NHS 24 operator can advise on appropriate action given a description of the symptoms. These descriptions can be categorised as poisoning based on the presence of specific key words or phrases. Using generalised additive models, a consistent seasonal trend in poisoning calls to NHS 24 was found. The second service examined, TOXBASE, is a database provided by the National Poisons Information Service which provides information to clinical professionals on how to treat poisoning by a variety of substances. As in the NHS 24 analysis, generalised additive models have been used in order to assess the trends present in accesses made to the TOXBASE database by clinicians. The results from this analysis found that there was a consistent seasonal trend in TOXBASE accesses which peaked over summer and was similar to that seen in the NHS 24 call data. A third temporal analysis was carried out on data obtained from NHS information services pertaining to admissions and attendances due to poisoning, again showing similar results to the previous two analyses.;These analyses combined suggest an underlying trend in poisonings. Both the admissions data and TOXBASE access data were examined using funnel plots in order to determine whether there were any hospitals which were unusual in their admissions or their TOXBASE use. This analysis found some commonalities in those hospitals which are unusual in either their admission rates or TOXBASE use. The final step in this project was to link the TOXBASE access data with the attendances and admission data in order to examine whether there was any link between TOXBASE use and admission due to poisoning. The results of this indicate that there are associations between TOXBASE use and admissions, such that an increase in TOXBASE use indicates an increase in admissions due to drugs poisoning. However, it became clear that the data used were limited in their ability to show any direct impact of TOXBASE on admissions due to drug poisoning, and that more specific data, for example on toxicants involved and case severity, would potentially be useful in mitigating the obvious confounding present in these data. This thesis has provided new insight into patterns in cases of poisoning, as well as providing a strong basis for further analysis to establish whether there isa direct impact of TOXBASE use on patient management within UK emergency departments
Use of alkali activated fly ash binder for soil stablisation : a multi-scale approach
No full textThis study addresses the use of alkali activated fly ash-based binder to improve engineering characteristics of soils, and as a substitute to usual high-carbon footprint stabilisers such as lime and Ordinary Portland Cement. In particular, it examines the use of a calcium-rich fly ash from coal combustion binder activated by a sodium-based alkaline solution for kaolin treatment. The global aim is to explore the feasibility of using this binder as a potential soil stabiliser. To do so, a multi-scale analysis that conjointly explores the physicochemical evolution of the system, its microstructure and mechanical performances was carried out.At a particle level, calcium-rich particles from fly ash constitute the reactive part of the mix. Their dissolution releases calcium that subsequently combines with silicon and potentially aluminium to form chains whose structure resembles the one of Calcium Silicate Hydrate encountered in Portland Cement and responsible of a mechanical improvement. At a microstructural level, a denser material is consequently formed overtime because of the filling of pores by the new compounds. Different heterogenous matrices of various porosity and arrangement are however observed across the material and owed to the high heterogeneity of fly ash whose particles locally react differently.Finally, at a macroscopic level, those changes lead to an improvement of the treated soil resistance to compression and shear forces. The maximum stability is achieved after 28 days for 10 % of added binder, and with an effectiveness suitable for field applications. This study hence confirms a positive feasibility potential of using calciumrichfly ash-based alkali activated binder for kaolin soil stabilisation.This study addresses the use of alkali activated fly ash-based binder to improve engineering characteristics of soils, and as a substitute to usual high-carbon footprint stabilisers such as lime and Ordinary Portland Cement. In particular, it examines the use of a calcium-rich fly ash from coal combustion binder activated by a sodium-based alkaline solution for kaolin treatment. The global aim is to explore the feasibility of using this binder as a potential soil stabiliser. To do so, a multi-scale analysis that conjointly explores the physicochemical evolution of the system, its microstructure and mechanical performances was carried out.At a particle level, calcium-rich particles from fly ash constitute the reactive part of the mix. Their dissolution releases calcium that subsequently combines with silicon and potentially aluminium to form chains whose structure resembles the one of Calcium Silicate Hydrate encountered in Portland Cement and responsible of a mechanical improvement. At a microstructural level, a denser material is consequently formed overtime because of the filling of pores by the new compounds. Different heterogenous matrices of various porosity and arrangement are however observed across the material and owed to the high heterogeneity of fly ash whose particles locally react differently.Finally, at a macroscopic level, those changes lead to an improvement of the treated soil resistance to compression and shear forces. The maximum stability is achieved after 28 days for 10 % of added binder, and with an effectiveness suitable for field applications. This study hence confirms a positive feasibility potential of using calciumrichfly ash-based alkali activated binder for kaolin soil stabilisation
A classical view of the quantum vacuum
Get PDFIn the coming years, a previously unexplored regime of quantum electrodynamics will be opened up to experimental study for the first time: the strong-field regime. Under the influence of strong electromagnetic fields, virtual particles in the quantum vacuum become polarised, and wave propagation in regions of strong field becomes nonlinear.This Thesis explores this regime using nonlinear vacuum electrodynamics.The nonlinear nature of the vacuum imbues a region of strong field with an effective refractive index, such that wave propagation becomes analogous to propagation in a medium. This permits a novel view of an old problem concerning the energy-momentum tensor of light. In the context of light interacting with a medium two rival forms exist of the energy-momentum exist, each supposedly supported by theoretical and experimental evidence. By translating the problem to nonlinear electrodynamics, where the medium is replaced by a strong electromagnetic field, it is found that a much more precise statement can be made about which formulation should be adopted. Maxwellian electrodynamics is known to be invariant under the conformal group, an extension of the usual Poincaré symmetry group. In general, nonlinear electrodynamics is invariant under Poincaré symmetries, and not the extended conformal group.;The conformal group has been exploited in a wide range of areas of physics to simplify difficult problems. The possibility of using a conformally invariant, nonlinear theory of electrodynamics to describe strong-field physics is investigated. An entire class of conformally invariant nonlinear theories of electrodynamics is found, and their structure analysed. The role such theories may have in strong-field physics is then assessed,and it is found that in (3 + 1) spacetime dimensions, the only physically meaningful conformally invariant theory of electrodynamics is Maxwell's theory.A charged particle moving through a medium emits Cherenkov radiation when its velocity exceeds the phase velocity of light in that medium. Under the influence of a strong electromagnetic field the nonlinear nature of the vacuum allows for the possibility of high-energy particles to radiate via the Cherenkov process. The properties of this vacuum Cherenkov radiation are analysed from first principles, and applied to two physically relevant examples. It is found that this radiation process may be relevant to the excess signals of high-energy photons in astrophysical observations.In the coming years, a previously unexplored regime of quantum electrodynamics will be opened up to experimental study for the first time: the strong-field regime. Under the influence of strong electromagnetic fields, virtual particles in the quantum vacuum become polarised, and wave propagation in regions of strong field becomes nonlinear.This Thesis explores this regime using nonlinear vacuum electrodynamics.The nonlinear nature of the vacuum imbues a region of strong field with an effective refractive index, such that wave propagation becomes analogous to propagation in a medium. This permits a novel view of an old problem concerning the energy-momentum tensor of light. In the context of light interacting with a medium two rival forms exist of the energy-momentum exist, each supposedly supported by theoretical and experimental evidence. By translating the problem to nonlinear electrodynamics, where the medium is replaced by a strong electromagnetic field, it is found that a much more precise statement can be made about which formulation should be adopted. Maxwellian electrodynamics is known to be invariant under the conformal group, an extension of the usual Poincaré symmetry group. In general, nonlinear electrodynamics is invariant under Poincaré symmetries, and not the extended conformal group.;The conformal group has been exploited in a wide range of areas of physics to simplify difficult problems. The possibility of using a conformally invariant, nonlinear theory of electrodynamics to describe strong-field physics is investigated. An entire class of conformally invariant nonlinear theories of electrodynamics is found, and their structure analysed. The role such theories may have in strong-field physics is then assessed,and it is found that in (3 + 1) spacetime dimensions, the only physically meaningful conformally invariant theory of electrodynamics is Maxwell's theory.A charged particle moving through a medium emits Cherenkov radiation when its velocity exceeds the phase velocity of light in that medium. Under the influence of a strong electromagnetic field the nonlinear nature of the vacuum allows for the possibility of high-energy particles to radiate via the Cherenkov process. The properties of this vacuum Cherenkov radiation are analysed from first principles, and applied to two physically relevant examples. It is found that this radiation process may be relevant to the excess signals of high-energy photons in astrophysical observations
Characterisation of disuse-related osteoporosis in an animal model of spinal cord injury
Get PDFInjury to the spinal cord can result in paralysis below the level of injury. A secondary complication of the removal of muscle-driven bone stimulation is the development of rapid osteoporosis in the bones of the paralysed limbs. The severe deterioration of both bone quantity and quality means that spinal cord injury (SCI) patients are at a significantly higher risk of fragility fractures in the lower extremities than the able-bodied population.;These fractures occur most commonly around the knee (distal femur and proximal tibia). This thesis presents a characterisation of the time-course effects a complete SCI has on the fracture-prone distal femur in a rat model. The aims are to characterise the quality and distribution of bone and to provide a uniquely detailed description of its response to SCI at various time points post-injury.;Bone quality is assessed using i) ex vivo micro-Computed Tomography (μCT) for global and site-specific analysis of both trabecular and cortical bone morphometry and densitometry, and ii) three-point bending and torsional mechanical testing to provide whole-bone structural and material level properties.;Evidence is presented that SCI-induced osteoporosis is site-specific within the same appendicular bone. A rapid and severe deterioration of metaphyseal trabecular bone was observed, after just 2 weeks trabecular volume fraction (BV/TV) had decreased by 59% compared to age-matched sham-operated controls. This resulted in a compromised structure composed of on average 53% fewer and 15% thinner trabeculae compared to control.;At later time points post-SCI there were no further significant reductions in metaphyseal BV/TV, although significant microstructural changes did occur. On the other hand, the more distally located epiphyseal trabecular bone was structurally more resistant to SCI-induced osteoporosis. There was a 23% decrease in BV/TV at 2 weeks post-SCI compared to control, characterised by a 15% decrease in trabecular thickness, thus unlike metaphyseal trabecular structures, the epiphyseal structure's connectivity was maintained. At later time points post-SCI there was a growth-related increase in epiphyseal BV/TV.;Rapid changes to cortical bone were also seen, with distal-metaphyseal regions experiencing the most severe decrease in cortical area at 2 weeks post-SCI compared to control. The varying degrees of change in the amount of both trabecular and cortical bone appears concomitant with each region's bone surface to volume ratio. Analysis of more chronic time points post-SCI (6, 10 and 16 weeks) highlights that caution must be exercised when interpreting results from rodent studies.;The analysis performed here indicates that SCI-induced bone changes are a combination of bone loss and suppressed bone growth. No difference in cortical tissue mineral density was observed between SCI and control groups at any time-points assessed, indicating that the decreases in whole-bone mechanical properties observed due to SCI were primarily a result of changes to the spatial distribution of bone.;Cumulatively, this thesis illustrates that SCI-induced osteoporosis has detrimentally affected the spatial distribution of both trabecular and cortical bone in site-specific ways, but the bone material itself does not appear affected.Injury to the spinal cord can result in paralysis below the level of injury. A secondary complication of the removal of muscle-driven bone stimulation is the development of rapid osteoporosis in the bones of the paralysed limbs. The severe deterioration of both bone quantity and quality means that spinal cord injury (SCI) patients are at a significantly higher risk of fragility fractures in the lower extremities than the able-bodied population.;These fractures occur most commonly around the knee (distal femur and proximal tibia). This thesis presents a characterisation of the time-course effects a complete SCI has on the fracture-prone distal femur in a rat model. The aims are to characterise the quality and distribution of bone and to provide a uniquely detailed description of its response to SCI at various time points post-injury.;Bone quality is assessed using i) ex vivo micro-Computed Tomography (μCT) for global and site-specific analysis of both trabecular and cortical bone morphometry and densitometry, and ii) three-point bending and torsional mechanical testing to provide whole-bone structural and material level properties.;Evidence is presented that SCI-induced osteoporosis is site-specific within the same appendicular bone. A rapid and severe deterioration of metaphyseal trabecular bone was observed, after just 2 weeks trabecular volume fraction (BV/TV) had decreased by 59% compared to age-matched sham-operated controls. This resulted in a compromised structure composed of on average 53% fewer and 15% thinner trabeculae compared to control.;At later time points post-SCI there were no further significant reductions in metaphyseal BV/TV, although significant microstructural changes did occur. On the other hand, the more distally located epiphyseal trabecular bone was structurally more resistant to SCI-induced osteoporosis. There was a 23% decrease in BV/TV at 2 weeks post-SCI compared to control, characterised by a 15% decrease in trabecular thickness, thus unlike metaphyseal trabecular structures, the epiphyseal structure's connectivity was maintained. At later time points post-SCI there was a growth-related increase in epiphyseal BV/TV.;Rapid changes to cortical bone were also seen, with distal-metaphyseal regions experiencing the most severe decrease in cortical area at 2 weeks post-SCI compared to control. The varying degrees of change in the amount of both trabecular and cortical bone appears concomitant with each region's bone surface to volume ratio. Analysis of more chronic time points post-SCI (6, 10 and 16 weeks) highlights that caution must be exercised when interpreting results from rodent studies.;The analysis performed here indicates that SCI-induced bone changes are a combination of bone loss and suppressed bone growth. No difference in cortical tissue mineral density was observed between SCI and control groups at any time-points assessed, indicating that the decreases in whole-bone mechanical properties observed due to SCI were primarily a result of changes to the spatial distribution of bone.;Cumulatively, this thesis illustrates that SCI-induced osteoporosis has detrimentally affected the spatial distribution of both trabecular and cortical bone in site-specific ways, but the bone material itself does not appear affected
A numerical and analytical study of size effects in free vibration of heterogeneous materials
Get PDFIn this thesis, the influence of the periodic microstructure on the dynamic mechanical behaviour of geometrically similar heterogeneous samples, namely 2D beams and 3D plates, with different dimensions and boundary textures but constant aspect ratio has been numerically investigated. Beam samples of a representative material comprised of 2D unit-cells were created using the conventional finite element analysis (FEA) to identify and quantify size effects existing in flexural modal frequencies when the scale of microstructure becomes comparable to the macroscopic dimensions. The unit cells were created so as to keep the overall properties of the material at the macroscopic scale constant despite variations in the void or inclusions volume fraction. The finite element numerical results were then compared against the analytical results obtained from the enhanced nonlocal Timoshenko beam which incorporates the Eringen small length scale coefficients, but the values obtained for the coefficient exhibited size dependency. Accordingly, 2D analysis using a novel finite element method (MPFEM) or, alternatively, the control volume based finite element method (CVFEM) was carried out by incorporating micropolar constitutive behaviour into their formulation. The numerical predictions using either MPFEM or CVFEM were then matched with the FEA results to obtain additional constitutive parameters featuring in planar micropolar elasticity theory. The 2D models were then extruded to form square 3D plates as a straightforward progression. These samples demonstrated a moderate degree of anisotropy, which increased with volume fraction. Nevertheless, the 3D-MPFEM models which assume isotropy agreed with the dynamic behaviour of FEA nonhomogeneous models with low volume fractions, which were mildly anisotropic. Subsequently, to reduce the anisotropy, 3D square plate samples with a square-pyramidal geometry, or a body-centred cubic, arrangement of spherical voids and inclusions were modelled which demonstrated approximately isotropic characteristics for which the 3D-MPFEM results agreed with the finite element results at lower mode numbers.In this thesis, the influence of the periodic microstructure on the dynamic mechanical behaviour of geometrically similar heterogeneous samples, namely 2D beams and 3D plates, with different dimensions and boundary textures but constant aspect ratio has been numerically investigated. Beam samples of a representative material comprised of 2D unit-cells were created using the conventional finite element analysis (FEA) to identify and quantify size effects existing in flexural modal frequencies when the scale of microstructure becomes comparable to the macroscopic dimensions. The unit cells were created so as to keep the overall properties of the material at the macroscopic scale constant despite variations in the void or inclusions volume fraction. The finite element numerical results were then compared against the analytical results obtained from the enhanced nonlocal Timoshenko beam which incorporates the Eringen small length scale coefficients, but the values obtained for the coefficient exhibited size dependency. Accordingly, 2D analysis using a novel finite element method (MPFEM) or, alternatively, the control volume based finite element method (CVFEM) was carried out by incorporating micropolar constitutive behaviour into their formulation. The numerical predictions using either MPFEM or CVFEM were then matched with the FEA results to obtain additional constitutive parameters featuring in planar micropolar elasticity theory. The 2D models were then extruded to form square 3D plates as a straightforward progression. These samples demonstrated a moderate degree of anisotropy, which increased with volume fraction. Nevertheless, the 3D-MPFEM models which assume isotropy agreed with the dynamic behaviour of FEA nonhomogeneous models with low volume fractions, which were mildly anisotropic. Subsequently, to reduce the anisotropy, 3D square plate samples with a square-pyramidal geometry, or a body-centred cubic, arrangement of spherical voids and inclusions were modelled which demonstrated approximately isotropic characteristics for which the 3D-MPFEM results agreed with the finite element results at lower mode numbers
SERS & efficacy assessment of anti-cancer drug-nanoparticle conjugates
Get PDFThis thesis was previously held under moratorium from 8 November 2019 to 8 November 2021.Non-small cell lung cancer (NSCLC) is the result of tumour development from genetically-mutated epithelial cells lining the surface of the lung. Clinical response to treatments is generally gauged by comparing tumour sizes pre- and posttreatment on computerised tomography. If no response is observed, it is difficult to understand why the patient is not responding to treatement. Measuring the success of the drug at the intracellular level, for example: drug entry, accumulation and binding to its active site, can be difficult to observe directly when the drug is not labelled. Being able to determine the intracellular accumulation (concentration), biological effect and fate (metabolism or degradation) of the drug could inform preclinical development of future generations of drugs. The purpose of this study was to functionalise a nanoparticle (NP) with an anticancer drug to be intracellularly imaged without the use of a dye. The aim was to track the uptake, distribution and release kinetics of the small molecule drug from the NP conjugate within the cell at clinically relevant levels. Here, the inherent chemical signature of the small molecule tyrosine kinase inhibitor (SM-TKI) erlotinib (EL) that targets a mutant epidermal growth factor receptor (EGFR), was selected as a proof of concept for optical imaging in cancerous cells. The alkyne bond within EL was detected with the vibrational spectroscopy technique, Raman microspectroscopy. Raman scattered signals can be enhanced when the analyte is associated with the surface of a NP (surface enhanced Raman spectroscopy (SERS)). Two metals were compared for signal detection, namely gold and silver NPs. Once EL conjugate imaging was achieved, the anti-proliferative efficacy and cytotoxicity of the NP conjugated erlotinib to the free drug was assessed. EL-NP conjugates were characterised in solution and mapped in fixed cells by SERS at 300 nM EL. This was a high resolution time-lapsed intracellular localisation study. The alkyne signal from silver conjugates were found to occupy more of the cell volume over time 32.6% versus gold 0.2% of the cell volume at 24 hours. The dynamic release of EL from the silver NP was observed from 4 hours by tracking a Raman shift of the alkyne vibration from bound to free-EL. The intracellular uptake was confirmed via correlative dark-field (DF) microscopy, single particle inductively coupled plasma- mass spectrometry (spICP-MS) and TEM. Conjugates were present at 1-10,000 particles per cell and a final concentration of 39 nM EL was delivered. The fate of gold NPs were illustrated by TEM to be different when compared to the silver, as they tend to accumulate more in membrane bound organelles than the silver NPs. This is likely to have consequences for the efficacy of drug responses. A SERS & Efficacy Assessment of Anti-Cancer Drug-Nanoparticle Conjugates v microfluidics device was also employed to demonstrate the potential for 3D SERS imaging at physiological conditions to monitor conjugated EL in real-time. This is the first example of a dye-free SERS approach for drug detection within cells using 3D mapping and a microfluidic device for live cell imaging. Cell viability was assessed during a fluorescent image time-course study to evaluate the cytotoxicity and efficacy of the nano-delivery method employed versus parent (free) drug formulation. It was found that the silver NP conjugate was almost as effective as the free drug. In contrast, the gold NP conjugate was completely ineffective on EL-sensitive cells. It was shown that that NP toxicity on cells must be determined empirically per NP and per cell type, as the EL-insensitive cell line was far more susceptible to silver NP toxicity than the EL-sensitive cell line. Examining the inhibition of EGFR phosphorylation elucidated reduced expression levels in EL-silver NP conjugate treated cells, the gold counterpart had no effect on inhibiting activation of EGFR. This was demonstrated by immunofluorescent imaging for localisation and Western immunoblots for quantitation (of phosphorylated/total EGFR (p/tEGFR) protein). Functional proteomics in the form of reverse phase protein array (RPPA) analysis was carried out to assess the downstream effects of the conjugates compared with unconjugated (free) EL on drug-sensitive cells. Hierarchical cluster analysis (HCA) and interactive network maps of RPPA for downstream signal transduction phosphorylation studies confirmed the earlier efficacy results from cell viability studies. EGFR and associated signalling events were similarly disrupted in free-EL and silver conjugate but were unaffected in the gold conjugate samples. Interestingly, RPPA highlighted another key difference in that free-EL induced more of a DNA damage response than in the conjugate-treated EL-sensitive cells. In conclusion, it was determined that nanoparticles could be useful as the next generation of dye-label-free theranostics for multiple purposes, for example: as diagnostic, treatment and prognostic markers. To enable this, the NPs must be rendered safe enough for use in humans, specifically targeted to the cancerous cells and loaded with a drug moiety that can be released near the active site. Although these studies have revealed potential utility in drug imaging, there are clearly a number of key hurdles to be overcome if NP-drug conjugates are to be used in a clinical setting for cancer treatment. However, their potential to aid in the tracking the intracellular fate of the drug could help in nanoformulation drug discovery pipelines.Non-small cell lung cancer (NSCLC) is the result of tumour development from genetically-mutated epithelial cells lining the surface of the lung. Clinical response to treatments is generally gauged by comparing tumour sizes pre- and posttreatment on computerised tomography. If no response is observed, it is difficult to understand why the patient is not responding to treatement. Measuring the success of the drug at the intracellular level, for example: drug entry, accumulation and binding to its active site, can be difficult to observe directly when the drug is not labelled. Being able to determine the intracellular accumulation (concentration), biological effect and fate (metabolism or degradation) of the drug could inform preclinical development of future generations of drugs. The purpose of this study was to functionalise a nanoparticle (NP) with an anticancer drug to be intracellularly imaged without the use of a dye. The aim was to track the uptake, distribution and release kinetics of the small molecule drug from the NP conjugate within the cell at clinically relevant levels. Here, the inherent chemical signature of the small molecule tyrosine kinase inhibitor (SM-TKI) erlotinib (EL) that targets a mutant epidermal growth factor receptor (EGFR), was selected as a proof of concept for optical imaging in cancerous cells. The alkyne bond within EL was detected with the vibrational spectroscopy technique, Raman microspectroscopy. Raman scattered signals can be enhanced when the analyte is associated with the surface of a NP (surface enhanced Raman spectroscopy (SERS)). Two metals were compared for signal detection, namely gold and silver NPs. Once EL conjugate imaging was achieved, the anti-proliferative efficacy and cytotoxicity of the NP conjugated erlotinib to the free drug was assessed. EL-NP conjugates were characterised in solution and mapped in fixed cells by SERS at 300 nM EL. This was a high resolution time-lapsed intracellular localisation study. The alkyne signal from silver conjugates were found to occupy more of the cell volume over time 32.6% versus gold 0.2% of the cell volume at 24 hours. The dynamic release of EL from the silver NP was observed from 4 hours by tracking a Raman shift of the alkyne vibration from bound to free-EL. The intracellular uptake was confirmed via correlative dark-field (DF) microscopy, single particle inductively coupled plasma- mass spectrometry (spICP-MS) and TEM. Conjugates were present at 1-10,000 particles per cell and a final concentration of 39 nM EL was delivered. The fate of gold NPs were illustrated by TEM to be different when compared to the silver, as they tend to accumulate more in membrane bound organelles than the silver NPs. This is likely to have consequences for the efficacy of drug responses. A SERS & Efficacy Assessment of Anti-Cancer Drug-Nanoparticle Conjugates v microfluidics device was also employed to demonstrate the potential for 3D SERS imaging at physiological conditions to monitor conjugated EL in real-time. This is the first example of a dye-free SERS approach for drug detection within cells using 3D mapping and a microfluidic device for live cell imaging. Cell viability was assessed during a fluorescent image time-course study to evaluate the cytotoxicity and efficacy of the nano-delivery method employed versus parent (free) drug formulation. It was found that the silver NP conjugate was almost as effective as the free drug. In contrast, the gold NP conjugate was completely ineffective on EL-sensitive cells. It was shown that that NP toxicity on cells must be determined empirically per NP and per cell type, as the EL-insensitive cell line was far more susceptible to silver NP toxicity than the EL-sensitive cell line. Examining the inhibition of EGFR phosphorylation elucidated reduced expression levels in EL-silver NP conjugate treated cells, the gold counterpart had no effect on inhibiting activation of EGFR. This was demonstrated by immunofluorescent imaging for localisation and Western immunoblots for quantitation (of phosphorylated/total EGFR (p/tEGFR) protein). Functional proteomics in the form of reverse phase protein array (RPPA) analysis was carried out to assess the downstream effects of the conjugates compared with unconjugated (free) EL on drug-sensitive cells. Hierarchical cluster analysis (HCA) and interactive network maps of RPPA for downstream signal transduction phosphorylation studies confirmed the earlier efficacy results from cell viability studies. EGFR and associated signalling events were similarly disrupted in free-EL and silver conjugate but were unaffected in the gold conjugate samples. Interestingly, RPPA highlighted another key difference in that free-EL induced more of a DNA damage response than in the conjugate-treated EL-sensitive cells. In conclusion, it was determined that nanoparticles could be useful as the next generation of dye-label-free theranostics for multiple purposes, for example: as diagnostic, treatment and prognostic markers. To enable this, the NPs must be rendered safe enough for use in humans, specifically targeted to the cancerous cells and loaded with a drug moiety that can be released near the active site. Although these studies have revealed potential utility in drug imaging, there are clearly a number of key hurdles to be overcome if NP-drug conjugates are to be used in a clinical setting for cancer treatment. However, their potential to aid in the tracking the intracellular fate of the drug could help in nanoformulation drug discovery pipelines
Design of vaccine nanotechnology-based delivery systems, the effect of CpGODN TLR9 agonist-protein antigen conjugates anchored to liposomes
Get PDFThe efficiency of CpG oligonucleotides as Toll like receptor (TLR) 9 agonist has been well established along the last few years. Although CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity have generated concern for the use of CpGODN. In an effort to increase stability, localise action and reduce dosage, different strategies have been approached, such as conjugation of CpGODN with immunogenic agents or encapsulation/adsorption of CpGODN into/onto liposomes resulting in enhanced immunopotency compared to coadministration of free CpGODN and antigen. Despite the advances in the field, the effect of conjugation of TLR9 to antigen in combination with liposomes on the immunogenicity of protein-based vaccines has not been explored yet.In this present study, thiol-maleimide chemistry was utilised for the covalent ligation between protein antigen and CpGODN TLR9 agonist, which did not alter protein's ability to be recognised by specific antibodies or activation of receptor by TLR9 agonist. Thanks to its negative charge, protein conjugate was electrostatically bound to cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The designed system GBS67-CpGODN+L shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following immunisation through the intramuscular (i.m.) route, cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) formed a vaccine depot at the injection site, which translated into notable increase of functional immune responses compared to the simple coadministration of GBS67, CpGODN and liposomes (GBS67+CpGODN+L). This effect seems due to increased total IgG level and specifically of IgG2a subtype, although no specific Th1/Th2-driven response was found.This work demonstrates that the conjugation of TLR9 agonist to GBS67 in conjunction with adsorption on cationic liposomes, can promote codelivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. The findings of this study highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms achieving therapeutic effect at lower doses.The efficiency of CpG oligonucleotides as Toll like receptor (TLR) 9 agonist has been well established along the last few years. Although CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity have generated concern for the use of CpGODN. In an effort to increase stability, localise action and reduce dosage, different strategies have been approached, such as conjugation of CpGODN with immunogenic agents or encapsulation/adsorption of CpGODN into/onto liposomes resulting in enhanced immunopotency compared to coadministration of free CpGODN and antigen. Despite the advances in the field, the effect of conjugation of TLR9 to antigen in combination with liposomes on the immunogenicity of protein-based vaccines has not been explored yet.In this present study, thiol-maleimide chemistry was utilised for the covalent ligation between protein antigen and CpGODN TLR9 agonist, which did not alter protein's ability to be recognised by specific antibodies or activation of receptor by TLR9 agonist. Thanks to its negative charge, protein conjugate was electrostatically bound to cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The designed system GBS67-CpGODN+L shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following immunisation through the intramuscular (i.m.) route, cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) formed a vaccine depot at the injection site, which translated into notable increase of functional immune responses compared to the simple coadministration of GBS67, CpGODN and liposomes (GBS67+CpGODN+L). This effect seems due to increased total IgG level and specifically of IgG2a subtype, although no specific Th1/Th2-driven response was found.This work demonstrates that the conjugation of TLR9 agonist to GBS67 in conjunction with adsorption on cationic liposomes, can promote codelivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. The findings of this study highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms achieving therapeutic effect at lower doses
Microfluidics for assessing the behaviour of deformable biological objects
Get PDFBiological fluids, composed of polymeric solutions or suspensions of deformable particles, commonly present complex rheological behaviour. It is well known that particle-fluid interactions at the microscale dictate the macroscopic flow behaviour of these fluids, however the exact link in numerous situations is still missing. Recently, microfluidic techniques have been widely employed to study the dynamics of microscopic particles under flow.;Even though such techniques present a range of advantages, including the precise control of the flow conditions, as well as the consumption of a small amount of sample, the design of the microfluidic geometries still mostly relies on a trial-and-error approach. In this thesis, we experimentally test a set of microfluidic geometries, the design of which was previously optimised based on theoretical considerations or by means of numerical simulations in order to achieve specific flow conditions.;In addition, we have used complex observation techniques to study the dynamics of solutions and suspensions under flow, identifying microscopic dynamics as well as the major limitations of the microfluidic devices. Biological fluids such as solutions of DNA molecules and red blood cells suspensions were investigated in shear-dominated and extension-dominated flows and the performance of the optimised flow geometries for the study of such biological fluids was demonstrated.Biological fluids, composed of polymeric solutions or suspensions of deformable particles, commonly present complex rheological behaviour. It is well known that particle-fluid interactions at the microscale dictate the macroscopic flow behaviour of these fluids, however the exact link in numerous situations is still missing. Recently, microfluidic techniques have been widely employed to study the dynamics of microscopic particles under flow.;Even though such techniques present a range of advantages, including the precise control of the flow conditions, as well as the consumption of a small amount of sample, the design of the microfluidic geometries still mostly relies on a trial-and-error approach. In this thesis, we experimentally test a set of microfluidic geometries, the design of which was previously optimised based on theoretical considerations or by means of numerical simulations in order to achieve specific flow conditions.;In addition, we have used complex observation techniques to study the dynamics of solutions and suspensions under flow, identifying microscopic dynamics as well as the major limitations of the microfluidic devices. Biological fluids such as solutions of DNA molecules and red blood cells suspensions were investigated in shear-dominated and extension-dominated flows and the performance of the optimised flow geometries for the study of such biological fluids was demonstrated
Experimental demonstration of high-fidelity entanglement via Rydberg blockade
Get PDFThe strong dipole interactions of Rydberg atoms are ideal candidates to facilitate interactions between neutral atoms to generate entanglement for quantum information processing. This offers the potential to scale to large atom arrays through well established techniques for neutral atoms, overcoming limitations of other architectures for quantum information processing. This thesis presents the design and development of an experiment for quantum information processing using Rydberg atoms, concluding with the deterministic preparation of two caesium atomic qubits in a maximumly entangled Bell state.The experiment presented achieves low error readout of two single atoms held in optical tweezers using new imaging technology as an alternative to what is typically used in the field, offering a cost effective solution whilst maintaining high shot to shot retention as is necessary for qubit operations. Qubit manipulations are demonstrated with fast two-photon rotations between the hyperfine ground states and the 69S1/2 Rydberg state. Due to the cold single atom temperatures achieved, T ≈ 10 μK, the ground-Rydberg dephasing times measured through Ramsey spectroscopy find coherence times around twice that of previously reported experiments, over an order of magnitude greater than the gate time.;Demonstration of Rydberg blockade between two atoms with a separation of 6 μm is shown with an almost compete suppression to the doubly excited state and observation of a √2-enhancement of coupling to an entangled symmetric |W〉 state. Finally the |W〉 state is mapped to the ground state qubit levels to create a maximally entangled Bell state achieving a loss-corrected fidelity of Ƒpairs = 0:81 ± 0:05. This result represents the highest corrected ground state neutral atom entanglement fidelity via Rydberg blockade and is equal to that achieved via Rydberg dressing . The limitation of this Bell state preparation is primarily due to laser phase noise as found in other experiments and is verified through the long coherence times measured in this thesis. Generation of entanglement in the magnetically insensitive hyperfine states of caesium allows long coherence times to be achieved with Ramsey spectroscopy used to measured transverse dephasing times of T*₂ = 10± 1 ms and T'2 = 150 ± 20 ms, offering an excellent platform for quantum computation.The strong dipole interactions of Rydberg atoms are ideal candidates to facilitate interactions between neutral atoms to generate entanglement for quantum information processing. This offers the potential to scale to large atom arrays through well established techniques for neutral atoms, overcoming limitations of other architectures for quantum information processing. This thesis presents the design and development of an experiment for quantum information processing using Rydberg atoms, concluding with the deterministic preparation of two caesium atomic qubits in a maximumly entangled Bell state.The experiment presented achieves low error readout of two single atoms held in optical tweezers using new imaging technology as an alternative to what is typically used in the field, offering a cost effective solution whilst maintaining high shot to shot retention as is necessary for qubit operations. Qubit manipulations are demonstrated with fast two-photon rotations between the hyperfine ground states and the 69S1/2 Rydberg state. Due to the cold single atom temperatures achieved, T ≈ 10 μK, the ground-Rydberg dephasing times measured through Ramsey spectroscopy find coherence times around twice that of previously reported experiments, over an order of magnitude greater than the gate time.;Demonstration of Rydberg blockade between two atoms with a separation of 6 μm is shown with an almost compete suppression to the doubly excited state and observation of a √2-enhancement of coupling to an entangled symmetric |W〉 state. Finally the |W〉 state is mapped to the ground state qubit levels to create a maximally entangled Bell state achieving a loss-corrected fidelity of Ƒpairs = 0:81 ± 0:05. This result represents the highest corrected ground state neutral atom entanglement fidelity via Rydberg blockade and is equal to that achieved via Rydberg dressing . The limitation of this Bell state preparation is primarily due to laser phase noise as found in other experiments and is verified through the long coherence times measured in this thesis. Generation of entanglement in the magnetically insensitive hyperfine states of caesium allows long coherence times to be achieved with Ramsey spectroscopy used to measured transverse dephasing times of T*₂ = 10± 1 ms and T'2 = 150 ± 20 ms, offering an excellent platform for quantum computation
Control of the stability of pulsed 2 μm lasers
Get PDFPulse-to-pulse uctuations in the output of Q-switched 2 μm lasers are a major obstacle to commercialisation. This jitter is present in all pulse parameters: build-up time, pulse duration, peak power and energy. The severity of the jitter is dependent on how far above threshold the laser can be pumped; lower gain coupled with reabsorption losses contribute to the level of jitter observed in 2 μm lasers. This thesis presents a detailed investigation into the jitter in a Q-switched Tm:YAP laser and tests methods of reducing the jitter. Target jitter levels of <1 ns for the build-up time and <3% for the energy were identified based on similar commercial Nd-based systems.;A full characterisation of the level of jitter in all pulse parameters was undertaken and the results were used as a benchmark to gauge the efficacy of subsequent improvements. The use of etalons to limit the laser mode content increased the build-up time jitter by an order of magnitude and the energy jitter by a factor of three. This is thought to be due to increased mode competition.;The cavity was transferred to a ruggedised housing with the minimum degrees of freedom required for alignment. This was done to reduce the variation of losses due to mechanical vibrations and was successful in reducing the jitter. This housing also allowed the purging of the cavity with dry air to reduce atmospheric water absorption. Through this combination, the build-up time jitter was reduced to 1.9 ns and pulse energy jitter to 1.8 %, representing the best performance achieved in this work.;The pre-lase technique was employed to enforce laser oscillation on a single longitudinal mode. Pre-lase is a two-step process that allows the build-up of a weak single-frequency pulse that seeds the main output. This led to increased jitter in the build-up time (to 19 ns) and energy (to 16 %) due to the inherent levels of jitter in the high-threshold seed pulse.Pulse-to-pulse uctuations in the output of Q-switched 2 μm lasers are a major obstacle to commercialisation. This jitter is present in all pulse parameters: build-up time, pulse duration, peak power and energy. The severity of the jitter is dependent on how far above threshold the laser can be pumped; lower gain coupled with reabsorption losses contribute to the level of jitter observed in 2 μm lasers. This thesis presents a detailed investigation into the jitter in a Q-switched Tm:YAP laser and tests methods of reducing the jitter. Target jitter levels of <1 ns for the build-up time and <3% for the energy were identified based on similar commercial Nd-based systems.;A full characterisation of the level of jitter in all pulse parameters was undertaken and the results were used as a benchmark to gauge the efficacy of subsequent improvements. The use of etalons to limit the laser mode content increased the build-up time jitter by an order of magnitude and the energy jitter by a factor of three. This is thought to be due to increased mode competition.;The cavity was transferred to a ruggedised housing with the minimum degrees of freedom required for alignment. This was done to reduce the variation of losses due to mechanical vibrations and was successful in reducing the jitter. This housing also allowed the purging of the cavity with dry air to reduce atmospheric water absorption. Through this combination, the build-up time jitter was reduced to 1.9 ns and pulse energy jitter to 1.8 %, representing the best performance achieved in this work.;The pre-lase technique was employed to enforce laser oscillation on a single longitudinal mode. Pre-lase is a two-step process that allows the build-up of a weak single-frequency pulse that seeds the main output. This led to increased jitter in the build-up time (to 19 ns) and energy (to 16 %) due to the inherent levels of jitter in the high-threshold seed pulse
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