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Electrophysiology of Ion channels in miniaturised systems
Get PDFCell membranes form a natural protective boundary around cells and their organelles.Ion channels housed within these membranes comprise 1.5% of the human genomeand carry out essential cell signalling roles. As such, ion channels are importantpharmacological targets and a better understanding of their function would aid drugdiscovery as well as drug toxicity testing. Although there are methods of studying ionchannels, including patch clamping and artificial lipid bilayer system, a number ofdifficulties limit their experimental efficiency and practicality. In the case of artificialbilayer architectures, reconstitution of membrane proteins into bilayers is challenging. Here we demonstrate the design and development of a scalable dropletinterface bilayer system for single ion channel electrophysiology. A combination ofthis platform and the ion channel reconstitution method addresses the proteinintegration problem by improving the probability of channel incorporation to 29%.Single channel recordings of gramicidin, alamethicin and alpha hemolysin wereacquired for proof of concept work while eukaryotic ion channel BK electrophysiology provides evidence of the success of the project objectives. We anticipate that the suggested reconstitution method in conjunction with the platform developed in this research can be extended to study other pharmacologically relevant human ion channels.Cell membranes form a natural protective boundary around cells and their organelles.Ion channels housed within these membranes comprise 1.5% of the human genomeand carry out essential cell signalling roles. As such, ion channels are importantpharmacological targets and a better understanding of their function would aid drugdiscovery as well as drug toxicity testing. Although there are methods of studying ionchannels, including patch clamping and artificial lipid bilayer system, a number ofdifficulties limit their experimental efficiency and practicality. In the case of artificialbilayer architectures, reconstitution of membrane proteins into bilayers is challenging. Here we demonstrate the design and development of a scalable dropletinterface bilayer system for single ion channel electrophysiology. A combination ofthis platform and the ion channel reconstitution method addresses the proteinintegration problem by improving the probability of channel incorporation to 29%.Single channel recordings of gramicidin, alamethicin and alpha hemolysin wereacquired for proof of concept work while eukaryotic ion channel BK electrophysiology provides evidence of the success of the project objectives. We anticipate that the suggested reconstitution method in conjunction with the platform developed in this research can be extended to study other pharmacologically relevant human ion channels
Exploring Fresnel holography for optically guided atom interferometry with Bose-Einstein condensates
No full textPreviously held under moratorium from 26th October 2018 until 26th October 2022.The progress and practicality of quantum technologies, such as rotation sensing, are contingent on the portability of existing ultracold atom technologies and the exploration of new alternative techniques. In response to this, we integrate existing knowledge with new Fresnel zone plate (FZP) holograms to begin the development of a compact Bose-Einstein condensate (BEC) interferometry device. Utilising high precision microfabrication, FZPs are exciting candidates for the production of static trapping potentials useful to atomtronics, interferometry, and fundamental physics. They are particularly useful for quantum technologies due to their simplicity and the potential for low cost mass-production.We detail the build status of a new 87Rb BEC experiment, designed for prototyping new waveguide technologies for atom interferometry. The experiment is deliberately modular to enable smooth upgrades. This is complemented by brief reporting of results from an existing BEC experiment. Density fluctuations indicating underlying phase fluctuations were observed in an elongated cigar shaped BEC.The suitability of FZPs has been demonstrated with computational simulations comparing FZPs to spatial light modulators. Experimental imaging of various manufactured ring patterns gives an average RMS error in the brightest 10% of 3% with respect to trap depth. Typical optical profiles have residual limitations due to the imaging system, beam shape and alignment. The axial propagation of the potentials is presented experimentally and through numerical simulations; weak axial trapping is expected, though this is insufficient to support against gravity. A novel method by which the azimuthal optical potential at the FZP can be simply mapped into the imaged ring is also presented.The outlook for a second generation of kinoforms has been studied, with a view to allow for sub-wavelength spatial resolution and larger kinoforms. We detail a dark ring potential suitable for production with an FZP kinoform and map the parameter space possible for rings produced using FZPs.The progress and practicality of quantum technologies, such as rotation sensing, are contingent on the portability of existing ultracold atom technologies and the exploration of new alternative techniques. In response to this, we integrate existing knowledge with new Fresnel zone plate (FZP) holograms to begin the development of a compact Bose-Einstein condensate (BEC) interferometry device. Utilising high precision microfabrication, FZPs are exciting candidates for the production of static trapping potentials useful to atomtronics, interferometry, and fundamental physics. They are particularly useful for quantum technologies due to their simplicity and the potential for low cost mass-production.We detail the build status of a new 87Rb BEC experiment, designed for prototyping new waveguide technologies for atom interferometry. The experiment is deliberately modular to enable smooth upgrades. This is complemented by brief reporting of results from an existing BEC experiment. Density fluctuations indicating underlying phase fluctuations were observed in an elongated cigar shaped BEC.The suitability of FZPs has been demonstrated with computational simulations comparing FZPs to spatial light modulators. Experimental imaging of various manufactured ring patterns gives an average RMS error in the brightest 10% of 3% with respect to trap depth. Typical optical profiles have residual limitations due to the imaging system, beam shape and alignment. The axial propagation of the potentials is presented experimentally and through numerical simulations; weak axial trapping is expected, though this is insufficient to support against gravity. A novel method by which the azimuthal optical potential at the FZP can be simply mapped into the imaged ring is also presented.The outlook for a second generation of kinoforms has been studied, with a view to allow for sub-wavelength spatial resolution and larger kinoforms. We detail a dark ring potential suitable for production with an FZP kinoform and map the parameter space possible for rings produced using FZPs
Process physiology of antibody producing mammalian cell lines in batch and extended batch cultures
Get PDFThe increasing usage of monoclonal antibodies (mAbs), often expressed in Chinese Hamster Ovary (CHO) cells, for human therapy, has led to a focus on rational approaches to speed the development of cost-effective and highly productive cell lines. Understanding the process physiology of industrial CHO cell lines is important to making significant progress in cell line and culture development. In this study, which was underpinned by the supply of several industrial CHO cell lines by an industrial collaborator (Lonza Biologics) and newly developed industrial CHO media by another industry partner (Thermo Fisher Life Sciences), the effects of several process variables including different clones of cells, passage number, scale of culture, culture medium, feed supplements and culture modes (batch and fed-batch) on cell line physiology were investigated. GS-CHO 42 cell lines a high monoclonal antibody producer with low passage number (4) were observed to give better results compared to a less productive cell lines and higher passage numbers. In addition, three commercially available, chemically defined CHO cell culture media (CD-CHO, CD-OPTICHO and Dynamis) and two different types of feed supplements, CHO CD Efficient Feed A (EFA) and CHO CD Efficient Feed B (EFB) were evaluated in batch culture and fed-batch culture using the GS-CHO 42 cell line passage number 4. Cell culture in shake flasks and bioreactors showed clear effects of culture system on process physiology. In contrast, concentrated feed supplements did not help to increase the cell concentration and antibody titre. Amongst the three media tested, CD-CHO medium was found to be the best culture medium for GS-CHO 42 passage number 4 based upon the cell density, viability and Immunoglobulin (IgG) titre produced.A metabolomics study was carried out on samples from these cultures to observe the metabolic profiles under different culture conditions. Statistical analysis with Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) were performed using SIMCA version 14.0 to view the underlying global structure of the expression data. The results show that different metabolites were present under different culture conditions (different culture medium, scales and mode of culture) and were associated with different physiological behaviour of GS-CHO cell cultures. By using the results of this study, several bioprocessing strategiesincluding medium improvement, feeding strategy and downstream processing can be potentially implemented to achieve efficient CHO culture system. Nevertheless, more detailed studies are warranted to confirm and complement the existing information. The results in this work show how important process related information can be obtained with univariate and multivariate process analysis methods. Especially in cell culture process development, which is characterized by lengthy run times, a large number of influential and mutually interacting factors, as well as high-cost raw materials and process analytics, multivariate data analysis represents an attractive and versatile tools in process development.The increasing usage of monoclonal antibodies (mAbs), often expressed in Chinese Hamster Ovary (CHO) cells, for human therapy, has led to a focus on rational approaches to speed the development of cost-effective and highly productive cell lines. Understanding the process physiology of industrial CHO cell lines is important to making significant progress in cell line and culture development. In this study, which was underpinned by the supply of several industrial CHO cell lines by an industrial collaborator (Lonza Biologics) and newly developed industrial CHO media by another industry partner (Thermo Fisher Life Sciences), the effects of several process variables including different clones of cells, passage number, scale of culture, culture medium, feed supplements and culture modes (batch and fed-batch) on cell line physiology were investigated. GS-CHO 42 cell lines a high monoclonal antibody producer with low passage number (4) were observed to give better results compared to a less productive cell lines and higher passage numbers. In addition, three commercially available, chemically defined CHO cell culture media (CD-CHO, CD-OPTICHO and Dynamis) and two different types of feed supplements, CHO CD Efficient Feed A (EFA) and CHO CD Efficient Feed B (EFB) were evaluated in batch culture and fed-batch culture using the GS-CHO 42 cell line passage number 4. Cell culture in shake flasks and bioreactors showed clear effects of culture system on process physiology. In contrast, concentrated feed supplements did not help to increase the cell concentration and antibody titre. Amongst the three media tested, CD-CHO medium was found to be the best culture medium for GS-CHO 42 passage number 4 based upon the cell density, viability and Immunoglobulin (IgG) titre produced.A metabolomics study was carried out on samples from these cultures to observe the metabolic profiles under different culture conditions. Statistical analysis with Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) were performed using SIMCA version 14.0 to view the underlying global structure of the expression data. The results show that different metabolites were present under different culture conditions (different culture medium, scales and mode of culture) and were associated with different physiological behaviour of GS-CHO cell cultures. By using the results of this study, several bioprocessing strategiesincluding medium improvement, feeding strategy and downstream processing can be potentially implemented to achieve efficient CHO culture system. Nevertheless, more detailed studies are warranted to confirm and complement the existing information. The results in this work show how important process related information can be obtained with univariate and multivariate process analysis methods. Especially in cell culture process development, which is characterized by lengthy run times, a large number of influential and mutually interacting factors, as well as high-cost raw materials and process analytics, multivariate data analysis represents an attractive and versatile tools in process development
On the role of focal spot size in ultra-intense laser-solid interaction physics
Get PDFThis thesis reports on experimental investigations examining the role of laser-pulse focal spot size on key aspects of laser-solid interactions, namely laser-driven proton acceleration and laser-energy absorption, at the current state-of-the-art peak laser intensities (1020-1021 W/cm2). This includes the development of new optical tools and diagnostics to achieve these intensities and explore the resultant physics.;The interaction of intense laser pulses (>1018 W/cm2) with solid foils has received considerable attention over the last few decades, motivated by their ability to generate high energy particles (electrons and ions), photons (x-rays, γ-rays and THz emission) and gigagauss magnetic fields.The development of these novel sources of high energy particles and radiation requires understanding of the underpinning physics on parameters such as the laser focal spot size. The work reported here is structured into three main studies.;The first study presents the development of an ellipsoidal F/1 focusing plasma mirror (FPM) capable of increasing the peak intensity achievable on a petawatt level laser system, through focal spot size reduction. A factor of 2.5 reduction in spot size (from 4.0 μm to 1.6 μm [FWHM]) is achieved when compared to F/3.1 focusing with a conventional (solid state) optic. This corresponded to a factor of 3.6 enhancement in peak intensity, taking into account changes in plasma mirror reflectivity and focal spot quality.;The sensitivity of FPM operation to misalignment is also investigated, this is vital for its successful development. An example use of a FPM, in an investigation of laser-driven proton acceleration, is demonstrated. The intensity increase (3x1020W/cm2 to 1021 W/cm2) results in a factor of 2 increase in the maximum energy (from 27 MeV to 53 MeV) of sheath-accelerated protons from a foil target. This study helps to move the concept of focusing plasma mirrors beyond demonstration, towards routinely used tools in laser-plasma research, and enables a window into future research through the intensity enhancement achieved.;The developed F/1 FPM is employed in the second study to investigate the inuence of using tightly focused (near-wavelength sized) focal spot geometry on the properties of beams of accelerated protons generated by the target normal sheath acceleration (TNSA) mechanism. When comparing beam measurements for tight focusing to relativity larger (x2.5 larger) focal spot irradiation, significant variations are found.;These include, a slower maximum proton energy scaling with laser intensity (from I0:6 to I0:2), a x 2.5 enhancement in laser-to-proton energy conversion efficiency and a significant degreeo f target edge emitted protons with tight focusing compared to the larger focal spot measurements. The findings are explained in terms of changes to the evolution dynamics of the rear surface electron spatial distribution, with enhanced lateral electron spreading and subsequent recirculation, under tight focusing compared to the larger spot.;Through 2D particle-in-cell simulations this effect is suggested to derive from the front surface interaction dynamics, with a wider angular distribution of fast electrons throughout the target observed with tight focusing. This study is timely considering the employment of low F/# optics is a pulse focusing scheme under consideration by several existing, and future laser facilities as a route to higher intensities.;The final study reports on an investigation of laser-energy absorption into dense plasma. Using a suite of diagnostics, the total reected laser energy as a function of intensity is measured, distinguishing between the inuence of laser energy and focal spot size on energy absorption. Good agreement is found with previously published data on the scaling of absorption with intensity, by variation of pulse energy.;However,when the intensity is controlled by variation of the focal spot size, higher absorption values are measured (45% with a relatively large [270 μm FWHM] focal spot, compared to 22% with a tight focus [7 μm FWHM], both at an intensity of ~5 x1017 W/cm2) and a slower absorption scaling is observed, relative to the pulse energy variation case.;Through 2D particle-in-cell simulations this difference is shown to arise from additional energy gained by the population of electrons recirculating within the target due to multiple interactions with the laser pulse, a process dependent upon; the pulse duration, target thickness, focal spot size, and the energy spectrum and divergence of the fast electrons. A simple geometric electron recirculation model is presented to explore this absorption concept. This investigation has important consequences for fundamental understanding, application development and, most immediately, for experimental methodology.This thesis reports on experimental investigations examining the role of laser-pulse focal spot size on key aspects of laser-solid interactions, namely laser-driven proton acceleration and laser-energy absorption, at the current state-of-the-art peak laser intensities (1020-1021 W/cm2). This includes the development of new optical tools and diagnostics to achieve these intensities and explore the resultant physics.;The interaction of intense laser pulses (>1018 W/cm2) with solid foils has received considerable attention over the last few decades, motivated by their ability to generate high energy particles (electrons and ions), photons (x-rays, γ-rays and THz emission) and gigagauss magnetic fields.The development of these novel sources of high energy particles and radiation requires understanding of the underpinning physics on parameters such as the laser focal spot size. The work reported here is structured into three main studies.;The first study presents the development of an ellipsoidal F/1 focusing plasma mirror (FPM) capable of increasing the peak intensity achievable on a petawatt level laser system, through focal spot size reduction. A factor of 2.5 reduction in spot size (from 4.0 μm to 1.6 μm [FWHM]) is achieved when compared to F/3.1 focusing with a conventional (solid state) optic. This corresponded to a factor of 3.6 enhancement in peak intensity, taking into account changes in plasma mirror reflectivity and focal spot quality.;The sensitivity of FPM operation to misalignment is also investigated, this is vital for its successful development. An example use of a FPM, in an investigation of laser-driven proton acceleration, is demonstrated. The intensity increase (3x1020W/cm2 to 1021 W/cm2) results in a factor of 2 increase in the maximum energy (from 27 MeV to 53 MeV) of sheath-accelerated protons from a foil target. This study helps to move the concept of focusing plasma mirrors beyond demonstration, towards routinely used tools in laser-plasma research, and enables a window into future research through the intensity enhancement achieved.;The developed F/1 FPM is employed in the second study to investigate the inuence of using tightly focused (near-wavelength sized) focal spot geometry on the properties of beams of accelerated protons generated by the target normal sheath acceleration (TNSA) mechanism. When comparing beam measurements for tight focusing to relativity larger (x2.5 larger) focal spot irradiation, significant variations are found.;These include, a slower maximum proton energy scaling with laser intensity (from I0:6 to I0:2), a x 2.5 enhancement in laser-to-proton energy conversion efficiency and a significant degreeo f target edge emitted protons with tight focusing compared to the larger focal spot measurements. The findings are explained in terms of changes to the evolution dynamics of the rear surface electron spatial distribution, with enhanced lateral electron spreading and subsequent recirculation, under tight focusing compared to the larger spot.;Through 2D particle-in-cell simulations this effect is suggested to derive from the front surface interaction dynamics, with a wider angular distribution of fast electrons throughout the target observed with tight focusing. This study is timely considering the employment of low F/# optics is a pulse focusing scheme under consideration by several existing, and future laser facilities as a route to higher intensities.;The final study reports on an investigation of laser-energy absorption into dense plasma. Using a suite of diagnostics, the total reected laser energy as a function of intensity is measured, distinguishing between the inuence of laser energy and focal spot size on energy absorption. Good agreement is found with previously published data on the scaling of absorption with intensity, by variation of pulse energy.;However,when the intensity is controlled by variation of the focal spot size, higher absorption values are measured (45% with a relatively large [270 μm FWHM] focal spot, compared to 22% with a tight focus [7 μm FWHM], both at an intensity of ~5 x1017 W/cm2) and a slower absorption scaling is observed, relative to the pulse energy variation case.;Through 2D particle-in-cell simulations this difference is shown to arise from additional energy gained by the population of electrons recirculating within the target due to multiple interactions with the laser pulse, a process dependent upon; the pulse duration, target thickness, focal spot size, and the energy spectrum and divergence of the fast electrons. A simple geometric electron recirculation model is presented to explore this absorption concept. This investigation has important consequences for fundamental understanding, application development and, most immediately, for experimental methodology
The management of foreign direct investment risk by three Norwegian firms in the 1960s and 1970s
Get PDFThis thesis investigates the internationalisation and risk management strategies of three Norwegian manufacturing firms that first commenced Foreign Direct Investment (FDI) in the late 1960s and early 1970s. The three firms are: Dyno Industrier; Norcem; and Elkem-Spigerverket. Dyno invested in West Germany, England, Singapore, Denmark and Finland; Norcem invested in Ghana, Liberia, the Philippines, and Ras al-Khaimah; and Elkem-Spigerverket invested in the United Kingdom. The three firms were relatively early to invest abroad in comparison to the majority of Norwegian firms. For all three firms, risk and risk management was an important factor in their investment decisions. This research examines why the three firms decided to invest abroad, the context of their investment decision, how they viewed the risk involved with the investments, and which strategies they implemented to manage the risks. A comparison between the three firms is drawn in order to identify similarities and differences in their risk-management strategies and investment decisions. The thesis also investigates the extent to which modern risk management was practised by the three firms in the 1960s and the 1970s. The research was carried out using historical methods, primarily based on company archives, company magazines, government archives, and newspaper articles, which are used to highlight the firms' investments, the contexts to those, and their risk management strategies. Oral history interviews were conducted with four former senior managers and decision-makers in the three selected firms. The investments made by the three firms are described and discussed in individual chapters, followed by a comparison and discussion of the three firms' risk management strategies. The research finds that several risk management strategies were used by the three firms when they invested abroad. State guarantees, shared ownership, and networking/relationships with local governments were particularly important as risk management strategies.This thesis investigates the internationalisation and risk management strategies of three Norwegian manufacturing firms that first commenced Foreign Direct Investment (FDI) in the late 1960s and early 1970s. The three firms are: Dyno Industrier; Norcem; and Elkem-Spigerverket. Dyno invested in West Germany, England, Singapore, Denmark and Finland; Norcem invested in Ghana, Liberia, the Philippines, and Ras al-Khaimah; and Elkem-Spigerverket invested in the United Kingdom. The three firms were relatively early to invest abroad in comparison to the majority of Norwegian firms. For all three firms, risk and risk management was an important factor in their investment decisions. This research examines why the three firms decided to invest abroad, the context of their investment decision, how they viewed the risk involved with the investments, and which strategies they implemented to manage the risks. A comparison between the three firms is drawn in order to identify similarities and differences in their risk-management strategies and investment decisions. The thesis also investigates the extent to which modern risk management was practised by the three firms in the 1960s and the 1970s. The research was carried out using historical methods, primarily based on company archives, company magazines, government archives, and newspaper articles, which are used to highlight the firms' investments, the contexts to those, and their risk management strategies. Oral history interviews were conducted with four former senior managers and decision-makers in the three selected firms. The investments made by the three firms are described and discussed in individual chapters, followed by a comparison and discussion of the three firms' risk management strategies. The research finds that several risk management strategies were used by the three firms when they invested abroad. State guarantees, shared ownership, and networking/relationships with local governments were particularly important as risk management strategies
The effects of 7β-OH Epiandrosterone on Cytokine production by human immune cells
Get PDFDuring inflammatory responses, tumour necrosis factor alpha (TNF-α) and interleukin-1 (IL-1b) are the major pro-inflammatory cytokines that can stimulate the production of secondary mediators such as prostaglandins (PGs) including PGE2 which are responsible for the symptoms of inflammation. 7β-hydroxy epiandrosterone (7β OHEpiA) is a naturally occurring steroid that has recently been reported to have cytoprotective actions and can prevent ischaemia-reperfusion induced cell death. This action appears to be related to the production of PGD2 and its spontaneous metabolite15-deoxy PGJ2 (15-d-PGJ2). However, previous studies have provided little information about the actions of this steroid on immune/inflammatory responses and whether any of its actions may be mediated via glucocorticoid receptors. Therefore, this study investigated the effects of 7β OH-EpiA on TNF-α and IL-1β in LPS stimulated whole human blood, isolated monocytes and the human monocytic cell line THP-1 and also its effects on the production of anti-inflammatory cytokines such as IL-4 and IL-10. Whole human blood was used directly, and responses were compared to monocytesisolated from whole human blood and to THP-1 cells which were cultured continuously. Levels of cytokines (TNF-α, IL-1β, IL-4 and IL-10) and prostaglandins (PGE2, PGD2 and 15d-PGJ2), in plasma or cell supernatants, were measured by ELISA and enzyme immunoassay (EIA) respectively. In addition to this, the effect of 7β OHEpiA on the expression of various genes (including TNF-α, TLR-4, PGDS and PTGDR2) in THP-1 cells involved in inflammatory responses was evaluated by quantitative real-time PCR.7β OH-EpiA reduced TNF-α and IL-1β production at low concentrations (< 0.1 μM) in blood monocytes and THP-1 cells. In addition, the glucocorticoid analogue dexamethasone also significantly reduced the LPS-induced production of both cytokines but at higher concentrations (≥ 50 μM). 7β OH-EpiA did not affect the level of the anti-inflammatory cytokines IL-4 or IL10 whereas dexamethasone significantly increased the quantity of IL-4 at concentrations ≥ 1 μM. The glucocorticoid and progesterone receptor antagonist mifepristone did not alter the inhibitory action of 7βOH-EpiA on LPS-induced TNF-α production but reversed the dexamethasone-induced suppression of TNF-α. The non-steroidal cyclooxygenase inhibitor, ketoprofen,reversed the 7β OH-EpiA-induced suppression of LPS-stimulated increases in the concentration of TNF-a. With respect to prostaglandin production, LPS increased concentrations of PGE2, PGD2 and 15d-PGJ2 in both whole human blood and isolated monocytes. 7β OH-EpiA alone selectively increased concentrations of PGD2 and 15d-PGJ2 in contrast to decreasing the level of PGE2. Dexamethasone reduced the LPS stimulated increase in production of PGE2, PGD2 and 15d-PGJ2. There were no effects of mifepristone on either the 7β OH-EpiA-induced increase in PGD2 and 15d-PGJ2 production or the decrease in PGE2 production. Ketoprofen was confirmed to directly inhibit the production of all PGs in both blood and monocytes. With respect to the actions of 7β OH-EpiA on gene expression, alone it did not affect expression of the TLR-4 gene in THP-1 cells, a small decrease in TLR4 expression was observed with both LPS and 7β OH-EpiA versus LPS alone. 7β OH-EpiA did, however, decrease the LPS-stimulated increase in TNF-a gene expression in THP-1cells. With respect to PGD2 processes, 7β OH-EpiA increased the quantity of mRNAfor the PGDS (PGD synthase) gene and increased the concentration of PTGDR2 (DP2receptor) expression.The data indicates that the actions of 7β OH-EpiA on TNF-α production are highly unlikely to be mediated via a glucocorticoid or progesterone steroid receptor. They could be mediated via the selective upregulation of prostaglandin production,specifically PGD2 or 15d-PGJ2 which were increased by 7β OH-EpiA. Coupled with the observations that the cyclooxygenase inhibitor, ketoprofen reversed the suppressive actions of 7β OH-EpiA on TNF-α production and ketoprofen was directly confirmed to inhibit the production of all PGs, this implies that 7β OH-EpiA acts via the induction of PG biosynthesis. The identity of the PG is not certain but both PGD2 or 15d-PGJ2 are possible candidates as both were able to suppress LPS-stimulated TNF-αproduction. Nevertheless, at present the receptor for 7β OH-EpiA that initiates the increase in PGD2 and 15d-PGJ2 levels remains unknown.During inflammatory responses, tumour necrosis factor alpha (TNF-α) and interleukin-1 (IL-1b) are the major pro-inflammatory cytokines that can stimulate the production of secondary mediators such as prostaglandins (PGs) including PGE2 which are responsible for the symptoms of inflammation. 7β-hydroxy epiandrosterone (7β OHEpiA) is a naturally occurring steroid that has recently been reported to have cytoprotective actions and can prevent ischaemia-reperfusion induced cell death. This action appears to be related to the production of PGD2 and its spontaneous metabolite15-deoxy PGJ2 (15-d-PGJ2). However, previous studies have provided little information about the actions of this steroid on immune/inflammatory responses and whether any of its actions may be mediated via glucocorticoid receptors. Therefore, this study investigated the effects of 7β OH-EpiA on TNF-α and IL-1β in LPS stimulated whole human blood, isolated monocytes and the human monocytic cell line THP-1 and also its effects on the production of anti-inflammatory cytokines such as IL-4 and IL-10. Whole human blood was used directly, and responses were compared to monocytesisolated from whole human blood and to THP-1 cells which were cultured continuously. Levels of cytokines (TNF-α, IL-1β, IL-4 and IL-10) and prostaglandins (PGE2, PGD2 and 15d-PGJ2), in plasma or cell supernatants, were measured by ELISA and enzyme immunoassay (EIA) respectively. In addition to this, the effect of 7β OHEpiA on the expression of various genes (including TNF-α, TLR-4, PGDS and PTGDR2) in THP-1 cells involved in inflammatory responses was evaluated by quantitative real-time PCR.7β OH-EpiA reduced TNF-α and IL-1β production at low concentrations (< 0.1 μM) in blood monocytes and THP-1 cells. In addition, the glucocorticoid analogue dexamethasone also significantly reduced the LPS-induced production of both cytokines but at higher concentrations (≥ 50 μM). 7β OH-EpiA did not affect the level of the anti-inflammatory cytokines IL-4 or IL10 whereas dexamethasone significantly increased the quantity of IL-4 at concentrations ≥ 1 μM. The glucocorticoid and progesterone receptor antagonist mifepristone did not alter the inhibitory action of 7βOH-EpiA on LPS-induced TNF-α production but reversed the dexamethasone-induced suppression of TNF-α. The non-steroidal cyclooxygenase inhibitor, ketoprofen,reversed the 7β OH-EpiA-induced suppression of LPS-stimulated increases in the concentration of TNF-a. With respect to prostaglandin production, LPS increased concentrations of PGE2, PGD2 and 15d-PGJ2 in both whole human blood and isolated monocytes. 7β OH-EpiA alone selectively increased concentrations of PGD2 and 15d-PGJ2 in contrast to decreasing the level of PGE2. Dexamethasone reduced the LPS stimulated increase in production of PGE2, PGD2 and 15d-PGJ2. There were no effects of mifepristone on either the 7β OH-EpiA-induced increase in PGD2 and 15d-PGJ2 production or the decrease in PGE2 production. Ketoprofen was confirmed to directly inhibit the production of all PGs in both blood and monocytes. With respect to the actions of 7β OH-EpiA on gene expression, alone it did not affect expression of the TLR-4 gene in THP-1 cells, a small decrease in TLR4 expression was observed with both LPS and 7β OH-EpiA versus LPS alone. 7β OH-EpiA did, however, decrease the LPS-stimulated increase in TNF-a gene expression in THP-1cells. With respect to PGD2 processes, 7β OH-EpiA increased the quantity of mRNAfor the PGDS (PGD synthase) gene and increased the concentration of PTGDR2 (DP2receptor) expression.The data indicates that the actions of 7β OH-EpiA on TNF-α production are highly unlikely to be mediated via a glucocorticoid or progesterone steroid receptor. They could be mediated via the selective upregulation of prostaglandin production,specifically PGD2 or 15d-PGJ2 which were increased by 7β OH-EpiA. Coupled with the observations that the cyclooxygenase inhibitor, ketoprofen reversed the suppressive actions of 7β OH-EpiA on TNF-α production and ketoprofen was directly confirmed to inhibit the production of all PGs, this implies that 7β OH-EpiA acts via the induction of PG biosynthesis. The identity of the PG is not certain but both PGD2 or 15d-PGJ2 are possible candidates as both were able to suppress LPS-stimulated TNF-αproduction. Nevertheless, at present the receptor for 7β OH-EpiA that initiates the increase in PGD2 and 15d-PGJ2 levels remains unknown
Continuous crystallization of multicomponent materials
Get PDFThe challenges of developing continuous crystallization processes of multicomponent crystals are addressed within this thesis. Multicomponent crystals such as co-crystals and solid solutions, can be used to modify physical properties of active pharmaceuticals, agrochemicals and other materials. These can result in enhanced product properties such as higher solubility, faster dissolution, better stability or improved manufacturability in downstream processing through desirable morphology and better powder flowability. Continuous manufacturing is routinely used in many industries but is a new trend in the manufacture of pharmaceuticals driven by the potential to reduce plant footprint and intermediate inventory, improve yields, reduce lead time, implement real time monitoring and automation and make processes safer.Compared to crystallization of single component crystals, additional component and solid phases introduce additional complexity in the phase diagram. Co-crystal phase diagram measurement in a series of solvents can be very time consuming compared to a solubility curve of a single component. A semi-empirical approach of modeling phase diagrams as well as new methods of measuring phase diagrams of multicomponent materials are presented to accelerate the time to obtain a phase diagram compared to traditional approaches. Transitions from small scale batch crystallization to continuous crystallization is also demonstrated here for co-crystals and solid solutions with high selectivity and reproducibility with respect to the solid phase produced.The challenges of developing continuous crystallization processes of multicomponent crystals are addressed within this thesis. Multicomponent crystals such as co-crystals and solid solutions, can be used to modify physical properties of active pharmaceuticals, agrochemicals and other materials. These can result in enhanced product properties such as higher solubility, faster dissolution, better stability or improved manufacturability in downstream processing through desirable morphology and better powder flowability. Continuous manufacturing is routinely used in many industries but is a new trend in the manufacture of pharmaceuticals driven by the potential to reduce plant footprint and intermediate inventory, improve yields, reduce lead time, implement real time monitoring and automation and make processes safer.Compared to crystallization of single component crystals, additional component and solid phases introduce additional complexity in the phase diagram. Co-crystal phase diagram measurement in a series of solvents can be very time consuming compared to a solubility curve of a single component. A semi-empirical approach of modeling phase diagrams as well as new methods of measuring phase diagrams of multicomponent materials are presented to accelerate the time to obtain a phase diagram compared to traditional approaches. Transitions from small scale batch crystallization to continuous crystallization is also demonstrated here for co-crystals and solid solutions with high selectivity and reproducibility with respect to the solid phase produced
Performance enhancement for filter bank multicarrier methods in multi-antenna wireless communication systems
Get PDFThis thesis investigates filter bank based multicarrier modulation using offset quadrature amplitude modulation (FBMC/OQAM), which is characterised by a critically sampled FBMC system that achieves full spectral efficiency in the sense of being free of redundancy. As a starting point, a performance comparison between FBMC/OQAM and oversampled (OS) FBMC systems is made in terms of per-subband fractionally spaced equalisation in order to compensate for the transmission distortions caused by dispersive channels. Simulation results show the reduced performance in equalising FBMC/OQAM compared to OS-FBMC,where the advantage for the latter stems from the use of guard bands. Alternatively,the inferior performance of FBMC/OQAM can be assigned to the inability of a per-subband equaliser to address the problem of potential intercarrier interference(ICI) in this system.The FBMC/OQAM system is analysed by representing the equivalent transmultiplexed channel including the filter banks as a polynomial matrix. The formulated polynomial matrix is demonstrated as a tri-diagonal matrix plus two corner elements which indicates that the induced ICI is limited to the direct adjacent spectrally overlapped subchannels. Based on polynomial matrix algebra, an equaliser is proposed which considers the cross terms between subchannels rather than performing a per-subband equalisation. The proposed equaliser is obtained through the inversion of the channel polynomial matrix; due to its reduced-rank nature, this inversion requires the extension of pseudo-inversion principles to the domain of polynomial matrices, and the inclusion of a regularisation term for enhanced stability and system performance. Some numerical examples demonstrate the ability of the proposed equaliser to suppress both ISI and ICI. Furthermore, this thesis combines FBMC/OQAM with multi-antenna architectures.In this scenario, the FBMC/OQAM system will not only suffer from ISI and ICI but also from spatial or inter-antenna interference (IAI). The multiple input multiple-output (MIMO) channel including the filter bank system is formulated as a polynomial matrix. A polynomial matrix pseudo-inverse of the equivalent channel polynomial matrix is proposed to approximately eliminate ISI, ICI, and IAI. Examples and simulation results are presented to underpin the performance of the proposed architecture.This thesis investigates filter bank based multicarrier modulation using offset quadrature amplitude modulation (FBMC/OQAM), which is characterised by a critically sampled FBMC system that achieves full spectral efficiency in the sense of being free of redundancy. As a starting point, a performance comparison between FBMC/OQAM and oversampled (OS) FBMC systems is made in terms of per-subband fractionally spaced equalisation in order to compensate for the transmission distortions caused by dispersive channels. Simulation results show the reduced performance in equalising FBMC/OQAM compared to OS-FBMC,where the advantage for the latter stems from the use of guard bands. Alternatively,the inferior performance of FBMC/OQAM can be assigned to the inability of a per-subband equaliser to address the problem of potential intercarrier interference(ICI) in this system.The FBMC/OQAM system is analysed by representing the equivalent transmultiplexed channel including the filter banks as a polynomial matrix. The formulated polynomial matrix is demonstrated as a tri-diagonal matrix plus two corner elements which indicates that the induced ICI is limited to the direct adjacent spectrally overlapped subchannels. Based on polynomial matrix algebra, an equaliser is proposed which considers the cross terms between subchannels rather than performing a per-subband equalisation. The proposed equaliser is obtained through the inversion of the channel polynomial matrix; due to its reduced-rank nature, this inversion requires the extension of pseudo-inversion principles to the domain of polynomial matrices, and the inclusion of a regularisation term for enhanced stability and system performance. Some numerical examples demonstrate the ability of the proposed equaliser to suppress both ISI and ICI. Furthermore, this thesis combines FBMC/OQAM with multi-antenna architectures.In this scenario, the FBMC/OQAM system will not only suffer from ISI and ICI but also from spatial or inter-antenna interference (IAI). The multiple input multiple-output (MIMO) channel including the filter bank system is formulated as a polynomial matrix. A polynomial matrix pseudo-inverse of the equivalent channel polynomial matrix is proposed to approximately eliminate ISI, ICI, and IAI. Examples and simulation results are presented to underpin the performance of the proposed architecture
Fast blind adaptive equalisation for multiuser CDMA systems
Get PDFIn order to improve communication over a dispersive channel in a CDMA system, we have to re-establish the orthogonally of codes which are used when combining input signals from many users onto a single communication path, as otherwise the performance of such system is limited significantly by inter-symbol interference (ISI) and multiuser access interference (MAI). In order to achieve this, adaptive filters are employed. A variety of adaptive schemes to remove ISI and MAI have been reported in the literature, some of which rely on training sequences, such as the Least Mean Squares (LMS) and Recursive Least Squares (RLS) algorithms, or on blind adaptation, such as the Constant Modulus Algorithm (CMA) or the Decision Directed algorithm (DD), which has similar convergence properties as the LMS in the absence of decision errors, the CMA is relatively slow compared to the DD algorithm but more robust in converging to a suitable solution. This thesis is concerned with developing a new robust and low-complexity blind multiuser equalisation over frequency selective channels. A robust pilot-assisted equalisation strategy is developed for the partially loaded time division duplex (TDD) component of the universal mobile telecommunications system (UMTS). In addition to training-based equalisation performed using the midamble of a data packet, some of the unused spreading codes are exploited to upload pilots in order to perform anadditional semi-blind adaptation over the payload of a packet. The latter ensures continuious adaptation and better tracking performance. The affine projection concept along with the concurrent constant modulus algorithm (CMA) and decision-directed (DD) mode are implemented to update the equaliser weights. Computer simulations are used to assess the performance of the proposed adaptation strategy over various UMTS TDD time bursts. A new low complexity adaptive technique is derived for blind multiuser equalisation basedon fitting the probability density function (PDF) of the equalizer output to the desired PDF of the corresponding symbol alphabet, i.e. matched-PDF. The cost function of the proposed technique can be measured by a stochastic gradient descent approach. The performance of the proposed adaptation strategy is assessed by a number of simulations, and benchmarked against FIRMER-CMA under QPSK modulation. The matched-PDF algorithm is used for the equalisation of Space-Time Block Coding (STBC) and Time-Reversal Space Time Block Coding (TR-STBC) signals transmitted over dispersive MIMO channels. The performance is demonstrated in a number of simulations and benchmarked against other blind schemes such as: CMA, Newton's method, and the Conjugate Gradient method. A thorough evaluation is carried out taking into consideration the complexity of each implementation in terms of multiply-accumulate (MAC) operations required per iteration. Finally, some variations of matched-PDF algorithm are proposed to improve the equaliser performance, including concurrent matched-PDF and decision directed, matched-PDF with affine projection algorithm, as well as pilot assisted equalisation based on matched-PDF algorithm for partially loaded systems.In order to improve communication over a dispersive channel in a CDMA system, we have to re-establish the orthogonally of codes which are used when combining input signals from many users onto a single communication path, as otherwise the performance of such system is limited significantly by inter-symbol interference (ISI) and multiuser access interference (MAI). In order to achieve this, adaptive filters are employed. A variety of adaptive schemes to remove ISI and MAI have been reported in the literature, some of which rely on training sequences, such as the Least Mean Squares (LMS) and Recursive Least Squares (RLS) algorithms, or on blind adaptation, such as the Constant Modulus Algorithm (CMA) or the Decision Directed algorithm (DD), which has similar convergence properties as the LMS in the absence of decision errors, the CMA is relatively slow compared to the DD algorithm but more robust in converging to a suitable solution. This thesis is concerned with developing a new robust and low-complexity blind multiuser equalisation over frequency selective channels. A robust pilot-assisted equalisation strategy is developed for the partially loaded time division duplex (TDD) component of the universal mobile telecommunications system (UMTS). In addition to training-based equalisation performed using the midamble of a data packet, some of the unused spreading codes are exploited to upload pilots in order to perform anadditional semi-blind adaptation over the payload of a packet. The latter ensures continuious adaptation and better tracking performance. The affine projection concept along with the concurrent constant modulus algorithm (CMA) and decision-directed (DD) mode are implemented to update the equaliser weights. Computer simulations are used to assess the performance of the proposed adaptation strategy over various UMTS TDD time bursts. A new low complexity adaptive technique is derived for blind multiuser equalisation basedon fitting the probability density function (PDF) of the equalizer output to the desired PDF of the corresponding symbol alphabet, i.e. matched-PDF. The cost function of the proposed technique can be measured by a stochastic gradient descent approach. The performance of the proposed adaptation strategy is assessed by a number of simulations, and benchmarked against FIRMER-CMA under QPSK modulation. The matched-PDF algorithm is used for the equalisation of Space-Time Block Coding (STBC) and Time-Reversal Space Time Block Coding (TR-STBC) signals transmitted over dispersive MIMO channels. The performance is demonstrated in a number of simulations and benchmarked against other blind schemes such as: CMA, Newton's method, and the Conjugate Gradient method. A thorough evaluation is carried out taking into consideration the complexity of each implementation in terms of multiply-accumulate (MAC) operations required per iteration. Finally, some variations of matched-PDF algorithm are proposed to improve the equaliser performance, including concurrent matched-PDF and decision directed, matched-PDF with affine projection algorithm, as well as pilot assisted equalisation based on matched-PDF algorithm for partially loaded systems
Aromatic ynamines : a new bio-orthogonal reactive group for step-efficient, sequential bioconjugation
Get PDFThe Cu-catalysed alkyne-azide cycloaddition (CuAAC) or 'click' reaction is a powerful and robust bio-orthogonal reaction that exclusively produces 1,4-substituted triazoles. Despite its extensive utility in chemical biology, the ability to differentiate alkyne subtypes has received little attention as a tool for the construction of discrete bioconjugates. This work highlights the utility of aromatic ynamines as a new click reagent for sequential bioconjugation. Aromatic ynamines are superior click reagents with enhanced chemical reactivity relative to conventional alkynes. This unique and orthogonal reactivity profile circumvents the need for conventional protecting group strategies.;This project will also highlight the biocompatibility of these reagents as a new tool for protecting-group free sequential CuAAC bioconjugation of oligonucleotides in the presence of more accessible competing alkyne substrate (Scheme 1). This strategy allows the formation of a new platform for specific labelling using fluorescent and PET probes as much as specific targeting and drug delivery. Importantly, higher reactivity of aromatic ynamines allows lower copper loading, thereby decreases toxicity and side reactions on biomolecules.;[Graphic with the following title:] Scheme 1. Chemoslective, sequential CuAAC bioconjugation using enhanced reactivity of aromatic ynaminesThe Cu-catalysed alkyne-azide cycloaddition (CuAAC) or 'click' reaction is a powerful and robust bio-orthogonal reaction that exclusively produces 1,4-substituted triazoles. Despite its extensive utility in chemical biology, the ability to differentiate alkyne subtypes has received little attention as a tool for the construction of discrete bioconjugates. This work highlights the utility of aromatic ynamines as a new click reagent for sequential bioconjugation. Aromatic ynamines are superior click reagents with enhanced chemical reactivity relative to conventional alkynes. This unique and orthogonal reactivity profile circumvents the need for conventional protecting group strategies.;This project will also highlight the biocompatibility of these reagents as a new tool for protecting-group free sequential CuAAC bioconjugation of oligonucleotides in the presence of more accessible competing alkyne substrate (Scheme 1). This strategy allows the formation of a new platform for specific labelling using fluorescent and PET probes as much as specific targeting and drug delivery. Importantly, higher reactivity of aromatic ynamines allows lower copper loading, thereby decreases toxicity and side reactions on biomolecules.;[Graphic with the following title:] Scheme 1. Chemoslective, sequential CuAAC bioconjugation using enhanced reactivity of aromatic ynamine
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