Dissertations / Theses on the topic 'Biophysical characterisation'
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1
Sarkar, Supti. "Biophysical characterisation of plasmid formulation." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1446862/.
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DNA vaccination and gene therapy offer significant advantages in the treatment of many intractable diseases but many technical challenges must be overcome before the potential of these techniques are realised. A major challenge for non-viral vector particles is their physical instability in physiological conditions. The delivery vector investigated was a Lipid-Peptide-DNA (LPD) complex. The initial system was composed of lipofectin, peptide 6 and a 6.9 kb plasmid DNA. The aim of this work was to study parameters with a view of optimising the biophysical characteristics of the system, namely with regard to storage and transportation. The effects of ionic strength and pH on colloidal stability and structure of the gene delivery vector were investigated using dynamic and static light scattering. Results showed increased levels of aggregation at physiological concentrations (150mM NaCl), although a more stable system was observed in distilled water and at extreme sah conditions of IM. The rates of aggregation were found to be related to the zeta potential of the system and could be predicted using Monte Carlo simulations. Fractal dimension of complexes (where higher values correspond to more compact structures) showed values of 1.6 for 150 mM NaCl and 2.4 for IM NaCl. Lipids, peptides and plasmid DNA were investigated to see the effect of chemistry and physical structure on the aggregation properties of the system. Lipid studies assessing the release of fluorescent calcein from a variety of lipids, over a range of pH found Cl4 Unsaturated + DOPE had the greatest level of release when observed at pH 5.0. The effect of increasing plasmid DNA size (number of base pairs 5.7-72) was also investigated, larger plasmid sizes showed greater stability although further investigation is required to confirm this.
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Damianoglou, Angeliki. "Biophysical characterisation of peptides and proteins." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/3664/.
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Venkatesan, Meenakshi. "Biophysical characterisation of PAK/Cdc42 interaction." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620265.
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Tsytlonok, Maksym. "Biophysical characterisation of linear repeat proteins." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610764.
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Herbert, Andrew Peter. "Biophysical characterisation of measles virus receptors." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/15013.
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To date two human proteins have been identified that can serve as the receptor for measles virus, namely membrane cofactor protein (MCP) and signalling lymphocyte activation molecule (SLAM). It is thought that a more through understanding of the structure and behaviour of the interacting regions of these receptors, will facilitate a more detailed understanding of the pathogenesis of MV. This thesis describes efforts to clone, express and characterise the relevant fragments of these receptors. MCP carries a functionally critical N-glycan on its second module (MCP2) and the crystal structure of MCP12 (expressed in mammalian cells) implies a structural role for this carbohydrate moiety. MCP12 and MCP2 were successfully cloned and expressed as secreted proteins in Pichia pastoris at levels sufficient for biophysical characterisation. Proton and 15N Nuclear magnetic resonance spectroscopy (NMR) studies, differential scanning calorimetry and mass spectrometry were employed to investigate the recombinant proteins. Both the natural sequences of MCP12 and MCP2 fragments expressed in P. pastoris were hyperglycosylated and therefore these recombinant fragments were investigated in both glycosylated and deglycolylated forms in order to study the effect of the glycans on module 2 of MCP, and with a view to detailed structural and dynamic studies using NMR. The data obtained with the P. pastoris-expressed fragments are not inconsistent with the hypothesis that the N-glycan on module 2 of the wild-type MCP is required for this molecule to be properly folded. After extensive investigations, it was concluded that the inability of P. pastoris to attach the natural glycan is a barrier to more detailed structural studies. In an effort to produce MCP2 with a better defined glycosylation profile, efforts were made to express, this fragment in insect cells which are known to produce a ‘more natural’ glycosylation profile on proteins. Moreover, the MCP2 fragment was also expressed in E. coli, in order to produce a form of MCP2 that had never been glycosylated.
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Tully, Mark David. "Biophysical characterisation of SAP97 and AKAP79." Thesis, University of Liverpool, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548812.
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Clewes, Oliver. "Recombinant proneurotrophins : production, purification and biophysical characterisation." Thesis, University of Bristol, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443699.
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Philippe, Didier. "Biophysical characterisation of 'Staphylococcus aureus' defence proteins." Thesis, University of Bath, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426285.
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Chen, Ho Ann. "Biophysical characterisation of rat CD2 domain 1." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392332.
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Harvey, Richard Douglas. "Biophysical characterisation of vesicles containing nonionic surfactants." Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396320.
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Sklepari, Meropi. "Stability and biophysical characterisation of protein therapeutics." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/98558/.
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For the past two decades, the development of protein therapeutics has significantly expanded with numerous biopharmaceutical and biosimilar products entering the medicine market every year, and even more queuing in the pipeline globally. Biologics are very complex molecules and therefore extremely sensitive to minor changes in the manufacturing process, which can result in heterogeneity and affect the stability, potency and immunogenicity of the final product. Public health organisations, such as EMA (European Medicines Agency), require that biological products should be extensively tested for their similarity to the original drug (in the case of a biosimilar) as well as to products from different batches (batch-to-batch comparison). The issued guidelines focus, among other tests, on physicochemical characterisation of these molecules. The suggested analytical techniques, however, are only vaguely named in the specifications, leaving the final decision to the manufacturers. The present work focuses on the use of different combinations of analytical techniques with an aim to demonstrate similarity or dissimilarity between two or more samples. The selected instrumental techniques are characterised by their simplicity and are able to detect structural differences and microheterogeneity of the active ingredient in different samples, aggregation, degradation and post-translational modifications (PTMs). Seven studies were completed in total, each one to a different extent, and these included protein therapeutics such as insulin and monoclonal antibodies. The applied techniques served for primary (MS),* secondary (far-UV CD, FT-IR) and tertiary structure (near-UV CD, fluorescence) comparison of the examined samples. Particle size comparability and detection of aggregation was achieved with DLS, and higher-order structure comparison with 1D 1 H-NMR. Coupling of the techniques with temperature-dependent measurements enabled further comparison on the thermal stability of the samples and provided confidence in the observed (at room temperature) results. The acquired empirical experience pointed out the advantages and disadvantages of each technique compared to the rest of the techniques, possible solutions to the encountered challenges, and the cases that one technique can be used instead of another or as complementary to it. Finally, a potential SOP is suggested, advising on which biophysical techniques should be used depending on the structure of the protein that is examined and its formulation.
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Kearney, Alice. "Biophysical characterisation of interactions involving CD2 family members." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437361.
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Brown, Peter N. "Biophysical and structural characterisation of protein-peptide interactions." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/3982.
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Proliferating cell nuclear antigen (PCNA) is an essential protein in the cell. It is involved in transcription and many types of DNA repair and replication. Homologues of this protein are found in all orders of life. The high level of conservation and essential nature of PCNA infers that it may be a potential drug target for anti-caner drugs in humans and also a potential anti-parasitic target. X-ray structures of PCNA from Homo sapiens (Hs), Schizosaccharomyces pombe (Sp) and Leishmania major (Lm) are now available and can be used as a template for structure based drug design. In this work PCNA from these three species have been prepared in milligram quantities for biochemical and biophysical studies. The previously unknown structure of LmPCNA has been solved in an uncomplexed form and also complexed with a dodecapeptide to a resolution of 3.0Å. A comparison of PCNA structures and their peptide complexes for the three species identifies structural differences which may be relevant in analysing thermodynamic contributions of binding. All eukaryotic PCNA molecules exist as ring shaped trimers which form around DNA. In this work the oligomeric state of LmPCNA has been determined to be hexameric both in solution and in the crystal. It has also been hypothesised that HsPCNA is hexameric however these would seem to form hexamers in which the trimeric rings associate “back-to-back” while LmPCNA trimers would seem to associate “face-to-face”. The binding affinities for these three PCNAs have been determined with a selection of peptides derived from the Hs p21 protein. This work has shown, using a selection of different techniques including Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) and Dynamic Scanning Fluorimetry (DSF); that HsPCNA and SpPCNA have similar affinities for a 12mer peptide (Kd of ~1μM) however LmPCNA shows significantly weaker interactions (Kd of ~10μM). This is most likely due to divergence in the sequence and structure of LmPCNA. A systematic investigation by SPR on the effect of peptide linker length on binding has been carried out using a series of synthesised peptides with different lengths of chemical spacer. The series of streptavidin immobilised peptides show that longer spacers are required for the recovery of the PCNA peptide binding affinity. The results presented in this work indicate that a linker length of at least 20Å is required for measurable protein binding activity. This interaction is improved with longer peptide spacers.
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Fairweather, Victoria S. S. "Biophysical characterisation of DNA-binding proteins and oxidoreductases." Thesis, University of Bristol, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.508074.
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Ang, H. C. "Biophysical characterisation and rescue of p53 cancer mutants." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596120.
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The aim of this thesis was to use biophysical methods to characterise the stabilities and DNA binding properties of monomeric and tetrameric p53 cancer mutants, and to study various approaches aimed at rescuing structural mutants of p53. A detailed study of the destabilising effects of p53 mutations was performed using differential scanning calorimetry and urea denaturation, while equilibrium binding of p53 mutants to a specific promoter sequence, gadd45, was studied using fluorescence anisotropy and analytical ultracentrifugation. This thesis will also discuss how p53 structural mutants may be rescued by suppressor mutations that either increase the overall protein stability of compensate specifically for oncogenically induced loss of interactions. Stability and DNA-binding measurements showed that the destabilising effects of mutations H168R and R249S were not additive, and that these mutations in combination restored DNA binding. Similar biophysical techniques were used in analysing a series of p53 core domain mutants in which the residue Ser-116 in the middle of flexible loop L1 was mutated. One mutant, S1116C, was found to be more stable than previously predicted. The crystal structure showed how the mutation had led to formation of a new hydrogen-bonding network. Altogether, these protein-engineering studies provided useful insights into possible ways to rescue p53 function. A small set of compounds was selected based on the nature of proteins and peptides that were known to interact with p53. NMR spectroscopy was used to screen for compounds binding to the protein target and to probe for atomic detail of binding interactions. It was shown that 15N-1H HSQC could be used for screening and deconvoluting mixtures of compounds, in the presence of 5% v/v d6-DMSO, at relatively high throughput.
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Baldwin, Andrew Keith. "Biophysical characterisation of a liposomal gene delivery system." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405147.
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Hanlon, Michael Richard. "Molecular modelling and biophysical characterisation of three proteins." Thesis, Birkbeck (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326154.
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Hussain, F. "The biochemical and biophysical characterisation of p47phox activation." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444240/.
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The multi-component enzyme NADPH oxidase plays a central role in host defence against microbial infection, due to its ability to support the production of reactive oxygen species. The enzyme is dormant in resting cells where its six hetero-subunits are separated into cytoplasmic and membrane compartments. Activation and assembly of the NADPH oxidase is a complex process that is regulated, in large part, by changes in protein-protein and protein-lipid interactions. An important step in the activation process is the phosphorylation-induced translocation of the cytoplasmic P40phox-p67phox-p47phox complex to the membrane-bound heterodimeric P22phox-gp91phox complex (flavocytochrome b.sss). This interaction is prevented in the resting state due to an intramolecular interaction in p47phox that maintains the protein in an auto-inhibited conformation. Phosphorylation of eight serine residues in p47phox is essential for enzyme activation, of which five are located in the polybasic region and are known to relieve the auto-inhibition. Glutamate mutants were made to mimic phosphoserine residues, and various biochemical and biophysical techniques were used to demonstrate that phosphorylation of the remaining three serine residues, located in the C-terminal region, do not contribute to the release of auto-inhibition, and hence do not promote binding to p22phox. Instead, they weaken the interaction with p67phox. Based on these data, a new model for phosphorylation-induced changes in NADPH oxidase assembly is proposed. Binding of the polybasic region and p22phox to the tandem SH3 domains of p47phox occurs with high affinity. This affinity is mediated through the formation of a 'superSH3 domain', which is largely dependent on two structural features: the covalent SH3-SH3 linker and a 'GWW' motif located in the n-Src loops of either SH3 domain. Mutant proteins were made and quantitative binding assays were performed to demonstrate that the chemical nature of the linker is more important for the intramolecular interaction with the polybasic region in the auto-inhibited state, than for an intermolecular interaction such as that with p22phox in the active state. Furthermore, it was shown that the tryptophan residues of the GWW motif play different roles in the stabilisation of the superSH3 domain conformation. Their precise role depends on whether additional interactions between the superSH3 domain and its target can occur outside of the conserved ligand binding site, as observed in the auto-inhibited state. These data suggest that the sequence requirements for the formation of a superSH3 domain are relatively flexible, making it more likely that other proteins containing multiple SH3 domains may also form a superSH3 domain.
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Dobson, John Andrew. "Biophysical characterisation of biopharmaceuticals under defined flow fields." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/20779/.
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Flow induced aggregation occurs during the manufacturing process of biopharmaceuticals. An understanding of the effects of extensional flow is required as a means of predicting the response of experimental protein molecules to flow conditions during the down-stream manufacturing operations. Adequate prediction methods allow for elimination of proteins susceptible to flow induced aggregation, reducing development costs. An experimental device is designed and implemented to subject the proteins BSA, β2-microglobulin (β2m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs) to a defined and quantified flow field dominated by extensional flow. CFD analysis is used to accurately characterise the flow field throughout the geometry. Through simulation and bespoke post-processing it is possible to determine the magnitude of extensional strain which the fluid is subjected to. The device is then modified to allow for a comparison to be made between the strain and shear which is inherent in any flow device. The work shows that the device induces protein aggregation after exposure to an extensional flow field for 0.36 – 1.8 ms, at concentrations as low as 0.5 mg ml−1. Correlation is drawn between the extent of aggregation and the applied strain rate, as well as protein concentration, structural properties, and sequence of the protein. A method of equating the forces present within the flow to those experienced by a single molecule within the fluid continuum are also presented.
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Mortuza, Gulnahar B. "Cytochrome bâ‚… : biophysical characterisation of a haem binding protein." Thesis, Queen Mary, University of London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312882.
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Mohammed, Diar. "Biophysical characterisation of the skin at a molecular level." Thesis, University College London (University of London), 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.579673.
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This thesis investigates the skin barrier properties at a molecular level and possible methods to enhance model drug (niacinamide) permeation across model membranes and human skin. These can serve to improve the skin barrier function, understanding biophysical properties of the skin and more importantly developing a healthier and better appearance of the skin. In normal skin, stratum corneum desquamation and maturation are a carefully balanced process by the underlying rate of proliferation. The corneodesmosomes are degraded by serine proteases such as kallikrein 5 and kallikrein 7. This progressive process is precisely controlled to maintain skin integrity and TransEpidermal Water Loss (TEWL). As a result, the primary object is to develop and validate minimally invasive techniques to characterise and examine the skin barrier function. The skin's barrier properties is investigated at a molecular level as a function of depth, anatomical site, age, gender and ethnicity using relatively non-invasive techniques in vivo. Further experimental work involved understanding the influence of the UK's most prescribed and cheapest cream 'Aqueous Cream B.P.' on the SC. The techniques indicate that application of Aqueous Cream B.P. as a leave-on moisturiser can be damaging to the skin by reducing the corneocyte surface area, decreasing cell maturities, increasing selected desquamatory and inflammatory serine protease activities and TEWL. Using the techniques developed as well as confocal Raman spectrometry, the SC was investigated after application of a cosmetic and pharmaceutical active niacinamide in a range of formulations. The results recommend application of moisturiser containing niacinamide can improve the skin's barrier function. Finally, the permeation of niacinamide in vitro through model synthetic membrane and human skin under infinite and clinical doses were investigated. The results were then compared with the in vivo permeation of niacinamide. A range of formulations together with commercially available products that contain similar amount of the model drug were examined. There were no correlation between the flux of niacinamide through synthetic membranes and human skin. These techniques can effectively be applied in vivo to examine the skin barrier function at a molecular level and investigate the role of various formulations on the skin's barrier integrity. These assist the development of 'smarter' and more cost-effective pharmaceutical or cosmetic dermal formulations.
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Spencer, Kelly-Anne. "Biophysical characterisation of the infectious bronchitis virus nucleocapsid protein." Thesis, University of Leeds, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436017.
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Saul, Louise. "Biophysical characterisation of the Cannabinoid Receptor interacting protein CRIP1a." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/biophysical-characterisation-of-the-cannabinoid-receptor-interacting-protein-crip1a(45acbe47-1468-45f3-afbc-2ee6b8078cec).html.
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Endogenous cannabinoids, such as anandamide and 2-arachidonoyl-glycerol, are compounds naturally produced in the body that act as important signalling modulators, particularly, in the brain. Endocannabinoids bind to cannabinoid receptors (the CB1 and CB2 receptors), the same receptors to which the psychoactive tetrahydrocannabinol ingredient of Cannabis saliva derivatives bind. Endocannabinoids, together with their receptors, and the protein network involved in endocannabinoid synthesis, uptake, release and degradation constitute the endogenous cannabinoid system. The endogenous cannabinoid system is an emerging target in pharmacotherapy for the treatment of several diseases. In the context of a project aimed at studying important proteins of the ECS attention was focused on a 19kDa protein called Cannabinoid Receptor Interacting Protein la (CRIP1a). CRIP1a has been recently discovered, using a yeast-two-hybrid approach, as a binding partner of the CB1 receptor, but not the CB2 receptor. CRIP1a has been suggested to bind to the C-terminal cytoplasmatic tail of the CB1 receptor and to reduce the CB1 receptor-mediated tonic inhibition of voltage-gated calcium currents. The CRIP1a rat ortholog, RnCRIP1a, was over-expressed in E. coli and purified to homogeneity. Single crystals of wild-type and selenomethionine-labelled RnCRIP1a were grown using the microseeding technique and diffracted to high resolution using synchrotron radiation. The structure of RnCRlP1a was solved using the multiple anomalous diffraction technique at a resolution of 1.8 Å. RnCRIP1a is a domain-swapped dimer in the crystal. Its C-terminal region is in an extended β-strand conformation, 'crossing over' to engage in a β-sheet symmetry-related molecule, thus resulting in the formation of a homodimer. Analytical ultracentrifugation and NMR experiments show, however, that RnCRIP1a is a monomer in solution. On the basis of homonuclear NOE signals, in solution RnCRIP1a appears to have its C-terminus folded back into the β-sandwich. In consideration that RnCRIP1a C-terminal region harbours a PDZ-binding consensus sequence, the possibility of a functional role for the opening of its C-terminal is intriguing. The RnCRIP1a monomer displays a modified immunoglobulin fold equipped with a 'lid' domain above its β-sandwich scaffold. From a topological point of view, RnCRIP1a resembles members of the Ig-fold family of DNA binding proteins. RnCRIP1a has been proposed to bind to the last 55 amino acids of the CB1 receptor. This was tested by in-vitro pull-down and NMR titration experiments and failed any interaction between RnCRIP1a and the C-terminal tail of the CB1 receptor. Subcellular localisation assessed by confocal microscopy using COS7 cells reveals that RnCRIP1a is predominantly a cytoplasmatic protein.
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Johansson, Conny M. "Biophysical characterisation of the hepatocyte growth factor-glycosaminoglycan interaction." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/9904.
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Glycosaminoglycans (GAGs) such as heparin, heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS) are sulfated polysaccharides that exist on animal cell surfaces and in the extracellular matrix. GAGs are important in providing structural and hydrating support and interaction points for proteins of varied functions, for example growth factors and homeostasis regulatory proteins. Hepatocyte Growth Factor (HGF) is a protein growth factor that regulates cell growth, survival, proliferation, chemotaxis, cell morphology, tissue regeneration and angiogenesis. It is involved in embryogenesis, wound healing and many cancers. In this project, the interactions between the GAG binding N and NK -domains of HGF (HGF-N and HGF-NK) and different types of GAGs are characterised with biophysical techniques. GAG oligosaccharides were produced by enzymatic digestion and purified by preparative gel filtration and ion exchange chromatography. Different constructs of HGF were cloned from human cDNA, expressed with the Pichia pastoris expression system, purified to homogeneity and characterised by mass spectrometry and nuclear magnetic resonance (NMR). The dissociation constants between the different HGF protein constructs, different heparin oligosaccharide lengths and the drug Fondaparinux were shown by isothermal calorimetry (ITC) to vary between 0.35 and 9.26 μM. It was found that the entropy contribution was favourable for short oligosaccharides and disfavourable for long oligosaccharides and that the enthalpy contribution was less important for shorter oligosaccharides than for longer oligosaccharides. NMR titrations of CS, DS, heparin, Fondaparinux and sulfated maltose into 15N labelled protein samples showed that all ligands bind to the same HGF-N binding site, but different binding modes exists. The binding site consists of three regions, with the α2-helix and L2 loops playing key roles (residues 70-84). All GAGs also utilise the N-terminal residues 32-42, whereas long heparin oligosaccharides can also utilise a binding region formed mainly by the β2-strand (residues 59-64, 66, 95, 96). The GAG binding mode changes if HGF-N has an N-terminal truncation and the β2- strand residues become more important, emphasising the role of the N-terminal residues in the HGF-GAG interaction. Spin-labelled fully sulfated heparin-derived hexasaccharide was used to determine its binding direction on the HGF-N surface. Affinity chromatography confirmed the importance of the N-terminal residues and that HGF binds to all investigated GAGs. The oligomeric states of HGF-N and HGF-NK were investigated by AUC, gel filtration and ITC. The results suggest that the proteins oligomerise like beads on a string for long oligosaccharides. An HGF-N self-associating dimer with a slow on/off rate was characterised by affinity chromatography, gel filtration and NMR.
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Jutton, Mark Robert. "Biophysical and biological characterisation of a soluble human CD23 pigment." Thesis, King's College London (University of London), 2007. https://kclpure.kcl.ac.uk/portal/en/theses/biophysical-and-biological-characterisation-of-a-soluble-human-cd23-pigment(4a13a8ad-ea7a-4ba1-877d-ddd3811786a6).html.
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C3-antigen complexes induce B cell proliferation and the synthesis of specific antibodies in the immune response. The mechanism is understood to involve the co-ligation of antigen receptor (lgM) and CD21 on the membrane of antigen-specific B cells. CD23 is the low-affinity IgE receptor and also binds to CD21. It has been shown by NMR spectroscopy that the binding sites for IgE and CD21 on CD23 are distinct and nonoverlapping. CD23 is expressed as a homotrimer on the membrane of B cells and is cleaved by an endogenous metalloprotease to release soluble fragments that contain the f binding sites for both IgE and CD21. It is suggested that these fragments, like the C3antigen complexes, can co-ligate membrane IgE (mlgE) and CD21 on the surface of B cells to up-regulate IgE synthesis. This would enhance allergic responses in vivo. To test this hypothesis, a construct was generated to express a trimeric fragment of soluble human CD23 (LZ-CD23) and provide evidence for the protein's oligomeric state in solution using analytical ultracentrifugation. Surface plasmon resonance studies also showed that LZ-CD23 binds IgE with a high affinity. Confocal microscopy was used to demonstrate that LZ-CD23 caused the time-dependent re-distribution of fluorescentlylabelled mlgE and CD21 on human tonsillar B cells, stimulated with anti-CD40 and IL-4 to induce heavy-chain switching to 19B. As early as 15 minutes following incubation with LZ-CD23, rnIgE and CD21 were seen to co-cap in large aggregated clusters on the B cell surface. Additionally, the specificity of LZ-CD23 towards these markers was demonstrated as LZ-CD23 had no effect on the surface distribution of CD38. LZ-CD23 was shown to be biologically active by enhancing the secretion of IgE from B cells. Moreover, evidence is presented that CD23 exerts its IgE-potentiating action by specifically targeting cells that are pre-committed to 19B. The mechanism thus appears to resemble that of the C3-antigen complex in the immune response, except that it is isotype rather than antigen-specific. These results shed light on the role of CD23 in IgE homeostasis and suggest a novel means of targeting CD23 for intervention in the allergic response.
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林志明 and Chi-ming Lam. "Characterisation of the biophysical and pathophysiological effects of hepatic cryosurgery." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1996. http://hub.hku.hk/bib/B31979580.
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Streeter, Simon David. "Biochemical and biophysical characterisation of the gene regulatory protein C.AhdI." Thesis, University of Portsmouth, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402283.
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Schlegel, K. "Next generation biosensors for biophysical characterisation and detection of viruses." Thesis, University College London (University of London), 2016. http://discovery.ucl.ac.uk/1492686/.
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In 2015, an estimated 425 million people were chronically infected with Hepatitis B, Hepatitis C or Human Immunodeficiency Virus. Many of these infections are preventable but remain undiagnosed and entirely off the record. This is driving new technologies in the space of vaccines and diagnostics. A current bottleneck in making vaccines accessible is the ease and cost of manufacturing, which depends significantly on adequate process control technology. Major limitations of current diagnostic tools concern their speed, ease-of-use and cost. In short, there is a lack of biosensing tools which are fit for use in point-of-care settings as well as for quality control in viral vaccine manufacturing. The aim of this thesis is to explore the potential for micro- and nanotechnologies to fight infectious diseases, with a specific focus on new vaccines and mobile phone connected tests. The first chapter explores the potential of dielectrophoresis in microfluidic channels for rapid biophysical characterisation of whole virus in vaccine development. The first bioapplication of dielectrophoretic sensing based PAT and surface conduction of bovine Herpes virus is reported. The second chapter is concerned with innovative paper microfluidic diagnostics, which are inherently low cost and can be read out by a smart phone app. Capture ligands were sourced and characterised to detect acute Hepatitis B and C biomarkers as well as first key demonstrations of a new sensitivity enhancement strategy using dendrimers to detect down to low ng/ml level within 2 minutes. The third chapter evolved out of a collaboration with our industrial partner OJ-Bio and contains a proof of concept for detection of Hepatitis C biomarkers on a surface acoustic wave immunosensor. Each chapter highlights a landmark step in the development of different biosensor approaches, while also demonstrating what some of the real challenges are before they can be adopted into either clinical or industrial use. Our findings afford important insights into how these technologies can be further developed to become the next generation of biosensors, to help diagnose more people, monitor outbreaks of infectious diseases better and widen access to affordable vaccines.
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Chamberlain, Jonathan Michael Garrard. "Biophysical characterisation of the serum amyloid P component-DNA interaction." Thesis, University of Portsmouth, 2016. https://researchportal.port.ac.uk/portal/en/theses/biophysical-characterisation-of-the-serum-amyloid-p-component--dna-interaction(992877ff-9c22-4fab-9e29-b88dda3dff1b).html.
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Serum amyloid P component (SAP) is a conserved, constitutively-expressed, plasma protein and member of the pentraxin family of secreted pattern recognition receptors. It is present in all individuals at concentrations of 30 to 50 mg/L, and was first discovered in 1965 as a ubiquitous constituent of amyloid plaques. Its physiological function remains unclear despite considerable work to elucidate its pathological role. SAP has been identified as the only serum protein that exhibits calcium-dependent DNA-binding and has been shown to bind and solubilise native long chromatin. Due to its abundance in the serum it has been suggested that SAP’s normal role may be as a scavenger of extracellular DNA. However, there is very little information regarding the precise molecular nature of this interaction. Several biophysical methods were employed to investigate the affinity, stoichiometry, specificity and structure of the SAP-DNA interaction. SAP was found to bind with high affinity to double stranded DNA of 25 bp or longer, regardless of sequence, although binding is not entirely sequence independent. Mutational analysis using HEK 293 cell expressed recombinant protein has identified a number of positively charged amino acids that play a key role in the formation of the complex. The stoichiometry of SAP-DNA binding has been shown to be complicated, with little evidence for the formation of a discrete complex, but rather an indication that multiple SAP molecules decorate longer DNA strands. This may explain the difficulties in obtaining a crystal structure of the complex. SAP has also been shown to bind to nucleosome core particles, albeit with significantly weaker affinity than to naked DNA. The apparent promiscuity of SAP-DNA binding appears to support claims that SAP acts as a serum DNA scavenger, providing further evidence that SAP’s main physiological role is in the prevention of autoimmune reactions against nuclear material.
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Daga, Sunil Kumar Omprakash. "Biophysical characterisation and profile of HLA-specific antibodies in transplantation." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/70964/.
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Following five decades of kidney transplantation, increasingly high risk immunological kidney transplantation (which previously was considered as sub-optimal) are carried out. The risk stratification with the current available assays have allowed safe transplantation in low risk non-sensitised patients and direct transplantation in high risk highly sensitised patients by removal of circulating donor specific antibodies (DSA) with reasonable outcomes. However, a large number of patients with chronic kidney disease and with low or intermediate antibody levels measured by current assay, the best way forward is uncertain resulting in denial of transplantation in some cases. Whilst in other cases, the solid phase Luminex assay may under or overestimate the risks of rejection and graft failure following direct kidney transplantation. Currently only IgG-class of DSA is considered immunologically important and routinely measured in clinical laboratories. Other bio-physiological characteristics such as class, sub-class and binding kinetics of DSA may be more specific for risk stratification of immunological risks. In this thesis, we studied effect of de novo IgM class of HLA-specific antibodies on outcome of kidney transplantation and characterised binding kinetics and strength of HLA-specific antibodies. De novo IgM or IgG HLA-specific responses alone were not associated with adverse outcomes following kidney transplantation. Presence of both IgM and IgG responses, however, was associated with poor graft function at 36 months. There was no temporal relationship of antibody response and episodes of rejections. De novo Donor specific responses were less frequent compared to non-specific responses. A shorter follow-up and use of modern triple immunosuppressant therapy (Tacrolimus, Mycophenolate and Steroid) may explain this. Binding kinetics measured by biosensor assay- surface plasmon resonance (SPR) on purified monoclonal HLA-specific antibodies showed binding kinetics and strength differed between HLA alleles despite same epitope and paratope interactions. There was a tendency towards higher affinity and faster association rate for HLA protein that was the initial immunizing antigen for the corresponding monoclonal HLA-specific antibodies. The dissociation constant (KD) of human monoclonal HLA-specific antibodies range between 10-8 to 10-10 M. Thermodynamic analysis showed higher Gibbs free energy released for interactions with higher binding strength. The binding strength of mixed monoclonal HLA-specific antibodies is generally average of the strength of individual monoclonal HLA-specific antibodies. Enriched polyclonal HLA-specific antibodies from clinical sample gave distinct binding response on bio-sensor based on SPR assay. Quantification of polyclonal HLA-specific antibodies using sandwich ELISA and SPR allowed quantitative measurement of binding kinetics and strengths. A range of binding strength was observed between patients and within same patient antibodies of different affinities was observed. Thus the antibodies could be grouped in four groups based on the strength of binding and this can serve as additional biomarker for risk stratifications.
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Lam, Chi-ming. "Characterisation of the biophysical and pathophysiological effects of hepatic cryosurgery." Hong Kong : University of Hong Kong, 1996. http://sunzi.lib.hku.hk/hkuto/record.jsp?B19712091.
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Henning, Karen. "Structural, biochemical and biophysical characterisation of human transcription factor RBP-Jκ." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-59860.
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Gretton, Sarah N. "Topology and biophysical characterisation of the hepatitis C virus NS4B protein." Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433078.
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Fung, Kar Ho Herman. "Biophysical and structural characterisation of the bacteriophage HK97 DNA packaging system." Thesis, University of York, 2017. http://etheses.whiterose.ac.uk/19153/.
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DNA packaging is a key step in the assembly of dsDNA viruses such as tailed bacteriophages and herpes viruses, whereby empty capsids are filled with a copy of the viral genome. The task is mediated by a DNA packaging motor, made of terminase proteins interacting with the portal vertex of a capsid. Cos phages use a defined signal recognised by the terminase machinery to mark the beginning and end of their genome in newly replicated, concatemeric DNA. For cos phages, the structures of assemblies that initiate, perform and terminate packaging are unknown. The structures of individual terminase proteins are also unknown. To further elucidate the mechanisms of DNA packaging in cos phages, a new packaging motor was assembled in vitro based on Escherichia coli bacteriophage HK97. Structural, biochemical and biophysical evidence suggests that cos, pac and φ29-like motors share a common mechanism for DNA translocation, despite their different initiation and termination behaviours.
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Yang, Su jeong. "Biochemical and biophysical characterisation of allelic variants of ovine prion protein." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611255.
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Michalke, Kerstin. "Expression, purification and biophysical characterisation of mammalian GPCRs for structural studies." Aix-Marseille 1, 2008. http://www.theses.fr/2008AIX11018.
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Les récepteurs couplés aux protéines G (RCPG) représentent la plus large famille de récepteurs transmembranaires. En raison de leur rôle central dans la transduction des messages, les RCPG sont impliqués dans de nombreuses pathologies et représentent donc des cibles de la plus haute importance pour l’industrie pharmaceutique. La résolution de leur structure tridimensionnelle devrait faciliter le développement de nouvelles familles de médicaments. Cependant, en raison de leur nature hydrophobe et de leur faible abondance dans les tissus, seules 3 structures de ces RCPG ont pu être résolues. Dans le cadre du projet MePNet, nous avons donc entrepris de produire et de caractériser un certain nombre de ces récepteurs pour les soumettre à des essais de cristallogenèse. Nous avons exprimé sous forme de corps d’inclusion dans E. Coli 45 des 100 RCPG que contient notre collection. Presque la moitié d’entre eux ont été produits en grande quantité. Leur expression n’a pas été plus dépendante de la température d’induction que de leur composition en cystéine ou leur taille. Les vecteurs Gateway se sont révélés être bien plus efficaces que les vecteurs de la famille pET et la souche C43 a été la plus prometteuse. Les récepteurs ont ensuite été produits à l’échelle préparative en fioles et dans des bioréacteurs. Le récepteur de l’hormone parathyroïdienne PTH1R (huPTH1R) et le récepteur cannabinoïde (muCB1R) exprimés en fermenteur ont ensuite été solubilisés avec du SDS et renaturés à l’aide de cyclodextrine (« artificial chaperon assisted refolding »). 280 mg de PTH1R et 370 mg de CB1R ont ainsi été obtenus en partant d’un litre de culture. L’analyse par BIAcore et par spectroscopie de fluorescence des interactions entre le PTH1R et son ligand, le PTH(1-34), ont permis de trouver un Kd de 6,5 nM et 56 nM, respectivement. Des tests de fixation à l’équilibre entre le CB1R et deux de ses ligands marqués au tritium, l’agoniste [3H]CP55,940 et l’inverse agoniste [3H]SR141716A, ont révélé des affinités de 38,2 nM et 19,4 nM, respectivement. Au moins 30 % des récepteurs CB1R renaturés sont donc apparus comme étant pleinement fonctionnels. Des analyses par chromatographie d’exclusion couplée à une détection UV-visible, la spectroscopie infrarouge et la mesure dynamique de la lumière ont permis de montrer que ces récepteurs étaient contaminés par des agrégats. On pouvait réduire la quantité d’agrégats par ultracentrifugation et chromatographie d’affinité ou bien en modifiant les conditions de renaturation et de gel filtration. Des fragments d'anticorps à chaîne lourde (VHH) de camélidés spécifiques de ces deux récepteurs ont été synthétisés et caractérisés par BIAcore. Tous ces fragments ont présenté des affinités de l’ordre du nanomolaire (de 12 à 70 nM). Les épitopes des différents VHH générés pour le PTH1R, tous distincts les uns des autres, n’ont pas semblé se lier au site actif, ce qui nous a permis d’utiliser différents VHH pour des essais de co-cristallisation en présence également du peptide PTH(1-34). Ces essais, ~ 1400 conditions de cristallisation au total, sont restés jusqu’à présent infructueuxG protein–coupled receptors represent the largest family of eukaryotic transmembrane signal transduction proteins. Because of their central role in the body, GPCRs are linked to many human diseases and are therefore important targets for therapeutics. The knowledge of high resolution x-ray structures for GPCRs would facilitate the development of those therapeutics. However, because of their hydrophobic nature and their low abundance in the body, only a few GPCR x-ray structures are known. Within the project MePNet, we therefore produced and characterised GPCRs to subject them to cristallisation assays. We have expressed 45 out of 100 GPCRs in inclusion bodies, almost the half of them with high yields. Expression is neither dependent on the expression temperature nor the cystein content nor the size of individual receptors. Gateway vectors appeared to be superiour to pET vectors and C43 proved to be the most successful expression strain. Expression was successfully scaled up in flasks and fermentors. Fermentor expressed huPTH1R and muCB1R were solubilised in SDS and refolded by artificial chaperone assisted refolding, yielding 280mg and 370mg of refolded PTH1R and CB1R per litre fermentor culture, respectively. Native receptor folding was verified by binding tests. For PTH1R/PTH(1-34) interaction a Kd of 6,5 nM was measured by BIAcore and 56 nM by tryptophan fluorescence quenching assay. CB1R showed an affinity of 38,2 nM for inverse agonist [3H]SR141716A and 19,4 nM for agonist [3H]CP 55,940 in the radioligand binding assay. The CB1R preparation contained 30% of active receptor. Analytic SEC coupled to LS/UV/RI detectors showed that the receptor preparations were contaminated with aggregates, which could be reduced by ultracentrifugation and ligand affinity chromatography, and by changing refolding and gelfiltration conditions. PTH1R and CB1R specific camel heavy chain antibodies fragments (vHHs) were generated and characterised by BIAcore. All tested fragments showed receptor binding activities in the nanomolar range (12- 70nM). Epitopes of PTH1R specific vHHs seem neither to overlap one another nor the binding site of ligand PTH (1-34), which might allow the usage of PTH1R/ PTH (1-34)/ vHH complexes with more than one vHH in crystallisation. PTH1R and CB1R were tested in up to 1400 crystallisation condition, none of these leading to the formation of crystals
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Widdowson, Philip. "Biochemical and biophysical characterisation of Anopheles gambiae NADPH-cytochrome P450 reductase." Thesis, University of Liverpool, 2010. http://livrepository.liverpool.ac.uk/1496/.
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As the principal vector for the transmission of the Plasmodium falciparum parasite, and hence the spread of malaria in Sub-Saharan Africa, Anopheles gambiae is a globally significant species of mosquito. Over recent years, the efficacy of established insecticides has waned and there is a constant need for novel effective compounds. Cytochrome P450 reductase (CPR) is a diflavoprotein known to have a central role in phase I metabolism of xenobiotic compounds and, in mosquitoes, this involves the detoxification of insecticides. Due to an inherent lack of understanding regarding the mechanisms of action of A. gambiae CPR, the selective inhibition of this enzyme is a previously untried approach. This project aims to biochemically characterise A. gambiae CPR in direct comparison to the human enzyme. It was found that A. gambiae CPR was deficient in bound FMN, and to a lesser extent FAD, relative to human CPR with 20 % less FMN bound to the purified mosquito protein. Following the dissection of A. gambiae CPR into its constituent FMN- and FAD-binding domains, and using Isothermal Titration Calorimetry (ITC), a 4-fold decreased was observed for the binding affinity for FMN in the A. gambiae FMN-binding domain as compared to the equivalent human protein. The redox potential of the oxidised/semiquinone transition of the A. gambiae FMN-binding domain was -92 mV, much more negative than the published value for human FMN-binding domain. These data suggest a clear difference between these enzymes in the binding strength of FMN and its propensity to accept electrons. The binding characteristics of NADPH nucleotides were probed in some detail. Comparison of the binding of NAD+ and NADP+ revealed a strong bias for the phosphate containing NADP+. In addition, the position of the phosphate was important as 3’-AMP bound very poorly whilst 2’-AMP bound more strongly. 2’, 5’-ADP binding highlighted the importance of additional stabilising interactions involving the 5’-phosphate. Comparison of 2’, 5’-ADP and NADP+ binding confirmed that the 2’-phosphate interaction was the principal site for NADPH recognition and provided the majority of the binding energy for this interaction. A. gambiae CPR was shown to bind NADPH nucleotide analogues 2’-AMP, 2’, 5’-ADP and NADP+ much less strongly than the human enzyme highlighting a potentially significant difference in coenzyme binding. Binding affinities for the nucleotide ligand to intact CPR and the isolated FAD domain showed that the FAD-binding site is fully contained within the FAD-binding domain However, differences in the thermodynamic parameters between the intact enzyme and the isolated FAD-binding domain suggest that, although not directly involved in NADPH binding, the presence of the FMN binding domain had an effect on the overall binding energetics. Despite an apparent difference between A. gambiae and human CPR in flavin incorporation and NADPH binding affinity, it was interesting that the activity of cytochrome c reduction of both enzymes was similar. The measured Km with respect to NADPH corroborated the ITC data by suggesting a stronger interaction of the coenzyme with human CPR compared to A. gambiae CPR. There was an approximate 2-fold increase in potassium ferricyanide reduction with the isolated A. gambiae FAD-binding domain compared to the intact enzyme with the presence of the FMN-binding domain again seemingly imparting an effect of events involving the FAD-binding domain. In order to fully understand and rationalise all of the data, a comprehensive structural determination of A. gambiae CPR is required. With this in mind, isotopic labelling and subsequent biophysical analysis was carried out on the intact CPR and its FMN- and FAD-binding domains. Successful labelling was achieved for all samples, including the deuteration of the intact CPR and FAD-binding domain However, the greatest success involved the FMN-binding domain with sufficient triple resonance spectra collected for backbone assignments. Although this success could not be matched for the intact CPR and FAD-binding domain, the work has provided a solid base for more a comprehensive study in the future.
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Lappin, Sarah Crawford. "Biophysical and pharmacological characterisation of recombinant and native rat P2X7 receptors." Thesis, Imperial College London, 2009. http://hdl.handle.net/10044/1/4360.
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P2X7 receptors exhibit a mainly non-neuronal localisation on immune and glial cells and primarily function as non-selective cation channels. After prolonged or repeated exposure to agonist, functional and cellular changes occur: the formation of a large diameter pore, cell lysis and the release of mature, biologically active interleukin-1β (IL-1β) a potent inflammatory cytokine. It is this repertoire of functions, along with its localisation that underlies the hypothesis for its involvement in pain processing. The biophysics and pharmacology of rat P2X7 receptors were investigated using stable cell lines. Increases in the current amplitude were shown to be dependent upon the agonist concentration and current deactivation was agonist application number and voltage dependent. These results increased our understanding of the receptor, but have also had implications for the design of protocols to investigate antagonist potency and efficacy. GSK31418A was identified as a potent, reversible and voltage-independent antagonist of rat and human P2X7 receptors. GSK314181A was >10000 fold selective over P2X4 receptors and >1000 fold selective over P2X2/3 receptors. GSK314181A produced a significant reversal of FCA-induced hypersensitivity when profiled in vivo, providing further validation of the role of P2X7 receptors in inflammatory pain. Although the influence of glia cells on neuronal activity in the CNS is now well documented, the role of peripheral glia, Schwann cells and satellite cells of sensory ganglia, is less well established. Non-neuronal cells in DRG cultures were shown to express P2X7 receptors by pharmacological, biophysical and immunofluorescence techniques. Native P2X7 receptors expressed on these cells were shown to have many of the properties of recombinant P2X7 receptors, in regards to the response to agonist activation and pharmacology. Finally, I have shown that Lamotrigine is an effective inhibitor of recombinant rat and human P2X7 receptors and native P2X7 receptors expressed in DRG. The potent inhibition of human P2X7 by Lamotrigine was replicated with the chemical analogue and neuroprotective agent Sipatrigine. However, little effect was recorded for a P2X7 antagonist in two models of epileptiform activity studies.
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O'Brien, Fiona Margaret. "Biophysical characterisation of the mechanisms regulating sarcoplasmic reticulum K+ channel gating." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:17e51bc1-5b1f-4b52-8e46-98c888a91e00.
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Intracellular Ca2+-release from the sarcoplasmic reticulum (SR) occurs through highly specialised Ca2+-release channels called ryanodine receptors (RyR) in a process known as excitation-contraction coupling (EC-coupling) in striated muscle. The RyR is the main ion-channel mediating Ca2+-release from the SR but the SR also contains many other ion-channels and proteins, with many of unknown physiological role. For example, two SR K+ channels, TRIC-A and TRIC-B, have recently been identified and are thought to play a key role in supporting intracellular Ca2+ movements across the SR. The TRIC double knockout (DKO) mouse dies in embryonic heart failure and the TRIC-B knockout (KO) dies in respiratory failure immediately after birth, highlighting their important roles in different tissue types. The TRIC-A KO mouse survives until adulthood, however, there are SR structural abnormalities and compromised Ca2+ release. I have focused on characterising and comparing the single-channel properties of the native SR K+ channels from WT and TRIC-A KO mouse skeletal muscle. I have investigated if the SR K+ channels can gate in a cooperative manner when multiple SR K+ channels are present in the bilayer. Since TRIC and RyR channels are both located in high abundance in the junctional SR regions, I have examined whether the SR K+ and RyR channels also gate in a cooperative manner when both are present in a bilayer. In addition to voltage-regulation of native SR K+ channels, I have characterised the pH sensitivity of the SR K+ channels from WT and TRIC-A KO tissue. Finally, I investigated if the mouse isoform of TRIC-A could be purified using the Wheat germ cell-free system and incorporated into bilayers without loss of function. In summary, this thesis describes novel mechanisms of regulation of ion-channels that are involved in the process of Ca2+-release from the SR. By focusing on the biophysical properties of SR K+ channels, I have highlighted the complexity of the ionic fluxes that are thought to be required to maintain normal EC-coupling in striated muscle.
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Huang, Hai. "Biophysical Characterisation of Two Mutations Causing Long QT Syndrome and Brugada Syndrome." Thesis, Université Laval, 2006. http://www.theses.ulaval.ca/2006/23724/23724.pdf.
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Brazer, Stephen Paul. "Structural characterisation of recombinant and synthetic proteins and peptides by biophysical techniques." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285159.
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Murton, Ben Lewis. "A biophysical characterisation of the complex of proteins associated with Set1 (COMPASS)." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611449.
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Martino, Angela. "A new serogroup B meningococcal vaccine : biophysical characterisation of its protein components." Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/59409/.
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Meningococcal infection due to serogroup B disease is the leading cause of meningitis across Europe, particularly in infants. Prevention through vaccination will soon be possible utilising a new multicomponent vaccine (4CMenB or Bexsero®) principally directed against serogroup B Neisseria meningitidis. In addition to OMVs, the vaccine contains three novel recombinant proteins discovered by reverse vaccinology. Studies were performed on the biophysical characteristics of these antigens to better understand their structural properties in solution and aspects of their immunogenicity. Initially, the antibody responses to the vaccine components were assessed in a low-dose mouse model. This extreme immunogenicity model was chosen to highlight any subtle differences in the immune response to individual antigens which may have been overlooked by previously high-dose immunisation studies. The recombinant vaccine components are synthetic homologues of proteins naturally present on the bacterial cell and changes in their structure can induce loss of functional responses. By deliberately stressing the antigens to lose functional immune responses, it was possible to study the structural changes at the molecular level. Using molecular modeling and protein-specific monoclonal and polyclonal antibody binding in parallel with UV circular dichroism (CD) spectroscopy, it was possible to assess at which point changes in the structure of proteins affected their ability to induce functional antibody responses in vivo. The two fusion proteins present unique biophysical characteristics derived from their component parts. As a result, they were shown to possess distinctive profiles by CD. When combined with fluorescence unfolding studies, in which the tryptophan residues were used as reporters for integrity, CD uncovered significant structural differences among the vaccine antigens. The NHBA-FP comprises two domains having different thermodynamics while in the fHbp-fusion both halves cooperate in the unfolding process. The choice of including a β-barrel protein in the chimeric constructs proved successful as both the NHBA and fHbp exhibited excellent solvent accessibility profiles due to the numerous inter- and intra-strand hydrogen bonds stabilising the structure. This had a direct effect on their immunogenicity which could only be adversely affected by prolonged harsh treatments. Maintenance of the α-helical profile was found to be critical for the immunogenicity of NadAΔ351–405 component as was the maintenance of its trimeric organisation. Overall, results confirmed the requirement and importance of the three additional protein antigens to the bactericidal response, even when directed against the homologous OMV bacterial strain. Structural studies confirmed the quality of the vaccines antigens and supported their critical contribution to the vaccine.
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Ono, Shusuke. "The biophysical characterisation of the Enterobacterial nucleoid proteins H-NS and StpA." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1445757/.
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H-NS is a major protein component of the nucleoid, found in many Gram-negative bacterial species. H-NS is involved in the modulation of expression of a wide range of genes, as well as contributing towards nucleoid structure. There are two structur ally independent domains in H-NS, one involved in protein-protein interactions (the N-terminal oligomerisation domain), and the other involved in DNA-binding (C- terminal). These are joined via a flexible linker sequence. H-NS both self-associates and interacts with other nucleoid-associated proteins to form specific oligomeric complexes that bind DNA, allowing a precise level of control in gene expression. The protein StpA, a paralogue of H-NS with 58% sequence identity, was originally identified by its RNA chaperone activity. Subsequent studies have suggested struc tural similarities between H-NS and StpA, with a degree of overlap in their function in vivo. StpA self-associates in a similar manner to H-NS, and has been shown to in teract with H-NS. Whilst small differences in the properties of H-NS and StpA have been identified, no clear distinctions have been made between the two proteins. This work investigates several key issues regarding the properties of the StpA protein. A number of biophysical techniques have been used to investigate the interaction of H-NS and StpA. The results of these experiments are consistent with a model whereby StpA self-associates via a 'head-to-taif interaction. Furthermore, StpA ex hibits a different affinity and kinetic behaviour of association in comparison to H-NS. The properties of self-association and interaction of H-NS and StpA are fully consis tent with studies that highlight the intimate relationship between the two proteins in vivo. Two independent structures of the N-terminal oligomerisation domain of H-NS have been reported. These structures were derived at different temperatures (i.e. 25 C and 35 C). To investigate the implications of these model structures on the thermoregula tory functions of H-NS, the oligomerisation properties of H-NS were investigated over a temperature range. Both the oligomerisation and DNA-binding properties of H-NS were found to vary within a physiologically relevant range of temperatures. To characterise the interaction of StpA with DNA and to allow comparison with H- NS, the solution structure of the C-terminal domain of StpA was determined, solved by NMR. The interaction of DNA with this domain was characterised using both NMR and calorimetric methods. Statement The work described in this thesis was carried out in the Department of Biochemistry and Molecular Biology, University College London between 2000 and 2004. The NMR spectra were acquired with the assistance of Dr. M. Williams, Dr. R. Harris or J. Taylor. All experiments involving H-NS 1.39 were conducted with the assistance of T. Olsson. All other work was carried out by the author. This project was funded by the Biotechnology and Biological Sciences Research Council (BBSRC). Some of the work detailed in this thesis has been published elsewhere: Esposito, D., Petrovic, A., Harris, R., Ono, S., Eccleston, J. F., Mbabaali, A., Haq, I., Higgins, C. F., Hinton, J. C, Driscoll, P. C, and Ladbury, J. E. (2002) H-NS oli gomerisation domain structure reveals the mechanism for high order self-association of the intact protein. J Mol. Biol. 324, 841-850.
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McDevitt, Christopher A. "The molecular analysis and biophysical characterisation of DMS dehydrogenase from Rhodovulum sulfidophilum /." St. Lucia, Qld, 2002. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16102.pdf.
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Lehner, Ines [Verfasser]. "Biophysical and biochemical characterisation of the SMR proteins Hsmr and EmrE / Ines Lehner." Frankfurt : Universitätsbibliothek Frankfurt am Main, 2011. http://d-nb.info/1011595397/34.
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Bullard, Desmond. "The biochemical and biophysical characterisation of DNA and RNA ligases from bacteriophage T4." Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441566.
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Bacteriophage T4 encodes three polynucleotide ligases that seal phosphodiester backbones during infection of E. coli, referred to as T4Dnl, T4Rnll and T4Rn12. A detailed biochemical examination of the nucleic acid substrate specificity of each ligase was performed using recombinant proteins and a variety of double stranded substrates comprised of a mixture of DNA and RNA, with a single nick. The RNA ligases sealed a broad range of substrates, with the activity of T4Rnll being 50-1000 fold less than that of T4Rn12. Mutagenesis identified regions of the RNA ligases that are important for nick-joining activity. T4Dni had a greater specificity than both RNA ligases, since it was not active on some substrates containing RNA. All proteins joined a double stranded substrate with the 3'-hydroxyl group at the nick being RNA, but with the 5'phosphate group at the nick and the complementary strand being DNA. The conserved use of this substrate questions whether it may be important during nucleic acid metabolism. In assays under identical conditions, the rates of nick-joining by all three recombinant ligases were best at 37°C. For all substrates, optimal1igation was at pH 8.0 for T4Dni and T4Rnll, and pH 7.0 for T4Rn12, therefore showing that the pH optima of ligation is determined by the protein. Changes to buffer conditions showed that all three proteins are dependent upon ATP and Mi+ in order for ligation to occur, although Mn2+ can provide an alternative to Mg2+. Furthermore, Ml+ and ATP along with DTT influenced intrinsic fluorescence emission by the proteins, suggesting that these constituents influence the structure of the proteins. The genome of Streptomyces avermitilis encodes a possible RNA ligase, which was cloned and purified for biochemical analysis. The recombinant protein exhibited weak nick joining activity, although further study is required to provide a better insight into this novel nucleic acid repair process in bacteria.
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Fox, Daniel George. "Expression and biophysical characterisation of the domains of the Pf1 gene 5 protein." Thesis, University of Portsmouth, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386252.
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Dioletis, E. "Biochemical and biophysical characterisation of the death domain of death associated protein kinase." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1338144/.
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Death associated protein kinase (DAPk) is a multidomain protein kinase that plays a key role in the promotion of programmed cell death. A number of different stimuli such as TΝF-α, TGF-β, IFN-γ, ceramide, c-Myc oncogenic transformation and matrix detachment are all known to activate DAPk, suggesting that DAPk lies at an important convergent point for the different cell death signalling pathways. DAPk-induced cell death is not entirely dependent on the presence of functional p53 or caspases and is highly conserved in C. elegans, rodents and human. The pro-apoptotic functions of DAPk are dependent on its kinase domain as well as the death domain (DAPk-DD). The structure of the kinase domain has been solved, but no experimental structural information is available for the DAPk-DD. The death domain belongs to the death fold superfamily that also includes the death effector domain, the caspase recruitment domain, and the Pyrin domain. These domains all share a common architecture of a six α-helix bundle arranged in an antiparallel manner. The death domain is a protein-protein interaction motif found in different signalling proteins involved in apoptosis, inflammation and NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) signalling. The role of the death domain is to promote the assembly of large multimember protein complexes, such as the DISC and the apoptosome, required for signal transduction purposes. This project aimed to tackle the specific structural parameters of the DAPk-DD; an important step in understanding the biological function of this enzyme. An efficient protocol was developed for the production of milligram quantities of human DAPk-DD in a stable and soluble form. Purification and buffer optimization provided the necessary sample homogeneity and high concentration solubility required for downstream applications, respectively. The multimeric state of DAPk-DD was assessed hydrodynamically, while biophysical techniques such as NMR, CD spectroscopy and fluorimetry were used to explore the 3D shape of DAPk-DD. Further analysis was performed on the in vitro interaction of the DAPk-DD with a newly identified partner, extracellular signal-regulated kinase (ERK2). GST pull-down assays highlighted the necessity of an intact DAPk-DD for interaction with ERK2, while ITC estimated the ratio and affinity of the complex. CD spectroscopy was also performed on the complex to monitor structural changes upon mutual binding. Overall, the DAPk-DD proved to have an unusual structural behaviour not previously seen in other members of the death domain superfamily that puts into question its classification as a classic DD.
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Rabu, Amir. "Biochemical and biophysical characterisation of heat shock protein 90 and its domain interactions." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/11286.
Full textAbstract:
This thesis describes the study of the Hsp90 proteins from Homo sapiens (Hsp90α) and Caenorhabditis elegans. The attempt to crystallise the C-terminal proteins is also described. The overall goal of the project was to biochemically and biophysically characterise various Hsp90 constructs and use this information to elucidate the biological role of this important family of proteins. The C-terminal domain of human Hsp90α. The work involved over-expression, purification and characterisation of the C-terminus of human Hsp90 α protein. The over-expression and purification led to the production of two reproducibly pure C-terminal human Hsp90 α protein constructs. The characterisation of these proteins focused on the interaction of the C-terminus with the immunophilin Cyp40 and also with ligands such as ATP and novobiocin. C. elegans Hsp90. The work involved the cloning, over-expression and purification of the N and C-terminus of C. elegans Hsp90. The cloning of genes for the N and C-termini was successful. Purification of the proteins only led to the production of one C-terminal protein but no purified N-terminal protein could be obtained. Similar characterisation studies were carried out to the C-terminus of C. elegans Hsp90. As for the human C-terminal protein, the C. elegans C-terminal protein was found to bind to Cyp40 with a dissociation constant in the micromolar concentration range. The protein also binds to ATP and novobiocin with an affinity very similar to that of the C-terminal proteins of human Hsp90α. The C-terminus of C. elegans also exhibits ATPase activity but the activity was ten-fold lower than that of the C-terminal of human Hsp90α. The work presented in this thesis provides conclusive evidence of the existence of an ATP binding site in the C-terminal domains of the Hsp90 class of proteins. The biological relevance of this finding is also discussed.