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Rozprawy doktorskie na temat „Biochemical Investigations”

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Data publikacji: 6 września 2023

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1

Patel, Vinood Bhagwandas. "Biochemical investigations into experimental alcoholic cardiomyopathy and hypertension". Thesis, King's College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266477.

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2

Taylor, Claire Louise. "Biochemical investigations of defects of the mitochondrial respiratory chain". Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.281706.

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3

Shnoudeh, Abeer Jabra. "Biochemical and biophysical investigations of novel phosphodiesterase-5 inhibitors". Thesis, King's College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429578.

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4

Harrison, Peter J. "Biochemical investigations of the carotenoid cleavage dioxygenase enzyme family". Thesis, University of Warwick, 2014. http://wrap.warwick.ac.uk/66554/.

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The biosynthesis of the plant hormones strigolactone and abscisic acid is, in part, controlled by a family of enzymes known as the carotenoid cleavage dioxygenases (CCDs), which perform an oxidative cleavage reaction on a carotenoid substrate (9’-cis-neoxanthin for abscisic acid and 9-cis-β-carotene and 9-cis-β-apo-10’-carotenal for strigolactone) to form apocarotenoids, which are metabolised to the functional phytohormone. Phenotypic effects on seed dormancy and shoot branching have been observed in Arabidopsis thaliana and Zea mays on the application of a selection of hydroxamic acids based inhibitors, designed to inhibit CCDs, whereby application of the inhibitor to the plant result in a decrease in the time take for germination (abscisic acid mediated) or an increase in the number of lateral shoot branches (strigolactone mediated). However, the biochemical basis of these phenotypes is not understood. In the present thesis, carotenoid cleavage dioxygenases from the abscisic acid biosynthesis pathway (9’-cis-epoxycarotenoid cleavage dioxygenases (NCED)) and strigolactone biosynthesis pathway (CCD7 and CCD8) were produced in vitro and assayed for inhibition against the hydroxamic acid inhibitors. The results show that Z. mays NCED is indeed inhibited by the hydroxamic acid inhibitors (D2: greater than 95% inhibition at 100 μM) in a time dependent fashion, indicating that inhibition of NCED is the basis of the seed germination phenotype. On the strigolactone biosynthesis pathway, recombinant A. thaliana CCD7 is not inhibited by the hydroxamic acids. However, recombinant A. thaliana CCD8 is inhibited by hydroxamic acids that show shoot branching phenotypes (D6 53% inhibition at 10 μM), suggesting that inhibition of CCD8 is the basis of the shoot branching phenotype. Structure activity relationships have also been performed to identify the key features of the hydroxamic acids required to inhibit each enzyme. The biochemistry of several CCDs has also been investigated, along with that of the enzyme Dwarf27, an isomerase enzyme required for the isomerisation of all-trans-β-carotene to 9-cis-β-carotene on the strigolactone biosynthesis pathway. Investigations indicate that Dwarf27 from Oryza sativa could be a novel iron-sulfur protein which isomerises β-carotene via a one electron transfer to or from the β-carotene substrate. D27 is also inhibited to some extent by certain hydroxamic acids (e.g. 41% by D30 at 100 μM). Biochemical characterisation of A. thaliana CCD8 provides evidence for a two-step mechanism involving acid-base catalysis, and evidence is obtained for an active site cysteine residue. A possible mechanism for the double oxidative cleavage reaction catalysed by CCD8 is proposed. These results provide an insight into the biochemistry of the biosynthesis of the phytohormones abscisic acid and strigolactone and demonstrate that enzymes on the biosynthesis pathways of these hormones can be selectively inhibited using a chemical genetics approach. This has potential to aid the development of novel agrochemical compounds which could influence these processes to improve plant architecture and crop yield.

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Haggarty-Weir, Christopher Neil. "Biochemical and biophysical investigations into key malaria parasite proteins". Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/29617.

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Plasmodium falciparum, the most pestilential of the malaria parasite species, is responsible for ~450,000 direct deaths annually. Clinical disease is a consequence of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite interactions. Key to these interactions are the P. falciparum reticulocyte binding-like homologue (PfRh) proteins responsible for binding erythrocyte receptors and gaining entry to host cells. For example, PfRh4 binds to human complement receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another protein important for invasion is the PfRh5-interacting protein (PfRipr), an essential member of the PfRh5-associated invasion complex (PAIN-complex) along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the erythrocyte; essential events during invasion. This study aimed to biochemically and structurally investigate truncated recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr suggested the presence of ten epidermal growth factor-like (EGF) modules, two towards the N-terminus and eight in the C-terminal domain. In this project, monoclonal antibodies made against recombinant PfRh4 were shown, via indirect immunofluorescent assays, to localize to the apical tip of merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase pre-treated erythrocytes. Attempts to produce a stable truncated recombinant PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay using ten alanine-scan mutants suggested the CR1-binding site lies outside of amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based on sequence homology, were produced recombinantly in Escherichia coli and Pichia pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing proteins with disordered regions. EGF-containing PfRipr truncations did not bind recombinant PfRh5 according to ELISA and size-exclusion chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via size-exclusion chromatography or NMR. Crystallisation trials performed on EGF modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three different temperatures, allowed for >86% of backbone assignments. T1/T2 relaxation analysis and heteronuclear NOE data were suggestive of an elongated, rigid protein undergoing intermolecular self-association. Further evidence for EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of denaturation over the temperature range 20 °C - 95 °C indicating a thermally-stable protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical shift perturbations consistent with high-affinity binding. The discovery of inhibitory monoclonal antibodies recognising a conformational epitope on EGF7 provided evidence of the functional importance of this region within PfRipr. The work described in this thesis provides methods for the industrially-scalable production and biophysical investigations of P. pastoris or E. coli-produced disulfide-rich P. falciparum antigens of interest to vaccinologists.

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6

Berrisford, John Mark. "Biochemical and structural investigations on phosphoglucose isomerase from Pyrococcus furiosus". Thesis, University of Sheffield, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434961.

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7

Konrad, Josef. "In vitro culture of Babesia divergens : biochemical and immunological investigations". Thesis, Imperial College London, 1986. http://hdl.handle.net/10044/1/38073.

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8

Lee, Jenny Swee Chin. "Biochemical and physiological investigations of Brassica oleracea var. botrytis L". Thesis, University of Bath, 1985. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355378.

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The curds of cauliflower (Brassica oleracea var.botrytis L.) which are representative of the European biennials, European annuals and Australian types were used to extract aspartate aminotransferase (EC 2.6.1.1) and acid phosphatase (EC 3.1.3.2). These enzymes were separated into their isoenzymes using polyacrylamide gel electrophoresis and were shown to have different numbers of isoenzymes depending upon which of the three main groups of cauliflower cultivars were used. The enzymes examined showed evolutionary divergence of the cauliflower types. Growth and development, storage and frost damage in cauliflowers were examined in relation to peroxidase activity. Physiological age in different parts of the curds and between different size curds was shown to have very little effect on peroxidase activity. Total peroxidase activity in the stem was about 2 times the value in the curds. A gradual increase of peroxidase was found to occur with duration of storage period at 7°C. Peroxidase activity remained low when cauliflowers were kept at low temperatures, namely both -1 + 1°C and -5 + 1°C, but increased rapidly on warming up at 7°C for 2 days. Isolation and partial purification of the peroxidase were carried out using conventional methods, including ammonium sulphate precipitation, Sephadex G-100 molecular sieve and DEAE Cellulose ion exchange chromatography. Further purification was achieved using Phenyl Sepharose hydrophobic interaction chromatography. Peroxidase activity was localized in the 50-95% ammonium sulphate precipitation. Molecular sieve chromatography showed a broad peak of peroxidase activity and molecular weight was estimated to be about 42,000. Polyacrylamide gel electrophoresis and isoelectric focusing of this fraction showed the existence of two peroxidase isoenzymes with P 6.7 and 7.3 using guaiacol as substrate; one of the isoenzymes also possessed a simultaneous IAA oxidase activity. Optimal pH and temperature with guaiacol as a substrate was found to be pH 5.6 and 50°C. Kinetic studies indicated an apparent Km of 8.3 mM for guaiacol at an optimal hydrogen peroxide concentration and a Km of 6.25 mM for hydrogen peroxide at an optimal guaiacol concentration. By including hydrophobic interaction chromatography in the purification scheme, a homogeneous cauliflower peroxidase isoenzyme was obtained. It showed as a single band in polyacrylamide gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Molecular weight estimation of this peroxidase isoenzyme was 41,000+1,000. The elution profiles of white and green cauliflowers and white sprouting broccoli peroxidases by hydrophobic interaction chromatography were found to be different. Green cauliflowers with high frost resistance were found to have high percentage of peroxidase activity bound very tightly to the hydrophobic resin. Similar peroxidase activity patterns using guaiacol, o-dianisidine and pyrogallol were obtained. Antiserum specific for the homogeneous peroxidase from cauliflower (cv. Milreef) was used to examine homology among cauliflower peroxidases and among peroxidases from green and white cauliflowers, white sprouting broccoli and horseradish. The antiserum cross reacted with all cauliflower peroxidases as well as peroxidases from green cauliflower, white sprouting broccoli and horseradish, indicating the antigenic sites were similar.

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9

Coates, Christopher J. "Hemocyanin-derived phenoloxidase : biochemical and cellular investigations of innate immunity". Thesis, University of Stirling, 2012. http://hdl.handle.net/1893/12228.

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Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.

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10

Tormey, William Patrick. "The provision of biochemical investigations in forensic toxicology for coroners". Thesis, Ulster University, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.646399.

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Death certification plays a central role in health service planning therefore identification of specific causes of death is critical. The requirements of biochemical toxicology as set out in the Coroners Acts in Ireland, Northern Ireland and England and Wales will be parsed to facilitate the construction of a modern best practice template for coroners' toxicology. The Royal College of Pathologists (RCPath) provides published standard guidelines for pathologists reporting to coroners. Their adequacy will be critically evaluated to facilitate reform with the intention of maximising the accuracy of death certification. The roles of psychological factors, tobacco smoking, non steroidal anti-inflammatory drugs, and cannabis in cardiac death will be detailed. The potential for adverse drug reactions to prescription medication to cause death by misadventure will be explored. The role for the expert witness in the inquisitorial coroners system to improve the accuracy of the causes of death and thus the verdict will be explored. My experience has shown that misinterpretation of presence of cannabis in autopsy blood and urine samples is common and this underlines the need for true expert guidance for the coroner. The current practice in biochemical toxicology of screening blood, urine and vitreous humor will be critically evaluated and the necessity for a wide ranging screen of potential toxins as a contributor to the cause of death examined. The appropriate analytes on the screening menu will be determined by local cultural factors. Gas and liquid chromatography with mass spectrometry are the methods of choice. The interpretation of isopropanol, ethanol and ketones in post-mortem blood will be considered as will t~e role of alcohol in death. The apparent population exposure to poisons as reported by Poisons Information Services will be used to explore the dichotomy between the usually benign outcome of common poisons and the often lethal consequences of poisoning by prescription and illicit drugs. The aim of this research is to use the template of the current legal requirements and routine laboratory procedures to suggest reforms which will improve the analytical protocol and reporting of biochemical toxicology in the coronial system resulting in greater accuracy in delineating the causes of death The narrative of this thesis travels through the areas of the coroners acts especially in the Republic of Ireland and the United Kingdom where forensic biochemistry plays a role in the specification of the causes of deaths. There is a deconstruction of the place of doctors in the coronial system and in the new arrangements following the passage of the Coroners and Justice Act 2009 in England and Wales and the potential for change in the Coroners Bill in the Republic of Ireland which fell with the dissolution of the Dail in20ll. There is an examination of the autopsy guidelines issued by the RCPath and suggestions for change which have been published. There is an analysis of the place of cannabinoids in coroners ' cases and publications setting out the position are included. A series of recommendations are made regarding improvement in practice for the reporting of biochemical toxicology in the coronial system. Two cases where there appears to be potential misinterpretation of the toxicological evidence, which may result in the review of the causes of death, are detailed as relevant clinical examples. Some laboratory pitfalls in relation to alcohol analysis have been demonstrated and the consequences of a protocol free service have been detailed with a prescription for improvement and practical solutions to improve outcomes. The under-estimation of the impact of tobacco toxicity is also addressed as is the potential for error due to lack of appreciation of drug-drug interactions. The role of biochemistry in the post-mortem diagnosis of alcoholic and diabetic ketoacidosis is discussed. Multidisciplinary reviews of biochemical toxicology for the coroners' court are suggested as the best safeguard of accurate interpretation to assist coronial enquiry. Conclusions suggesting standard operating procedures for post-mortem scenarios are detailed where possible.

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Huggins, Anthony. "Investigations into the role of endogenous Annexin-A1 in dendritic cell biology". Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8302.

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A school of literature has shown that Annexin-A1 (Anx-A1) is an endogenous anti-inflammatory protein that exerts a regulatory control over the innate immune system in order to restore homeostasis after an inflammatory reaction. Surprisingly, recent published works have highlighted that Anx-A1 has an alternate role in the adaptive immune system by positively modulating the strength of TCR signalling and biasing helper-subset differentiation. Dendritic cells are a class of innate leukocytes, poised at the environmental interface, that are the essential immune cells responsible in the initiation of T-cell driven responses. These findings provided the foundation for this PhD project, the principal aim of which is to provide a link between the disparate effects of Annexin-A1 in innate and adaptive immunity by investigating the role of endogenous Annexin-A1 in dendritic cell biology and its effector function as an antigen-presenting cell towards T cell activation and differentiation. To address this hypothesis, I cultured bone marrowderived dendritic cells from AnxA1-deficient mice or control littermates and stimulated with LPS (100ng/ml) then compared phenotypic and functional characteristics. My results demonstrate that Anx-A1-/- bone marrow derived dendritic cells show an increased number of CD11c+ cells expressing high levels of some maturation markers such as CD40, CD54 and CD80 and a decreased capacity to take up antigen compared to control Anx-A1+/+ cells. However, analysis of LPS-treated dendritic cells from Anx-A1-/- mice demonstrated a diminished up-regulation of maturation markers, a decreased migratory activity in vivo and an attenuated production of the inflammatory cytokines Interleukin (IL)- 1β, Tumour Necrosis Factor (TNF)-α and IL-12. This defect was resultant of an impaired Nuclear Factor (NF)-κB/DNAbinding activity due to lack of Anx-A1 signalling as demonstrated by the reduced activation of Extracellular-signal Regulated Kinase (ERK) 1/2 and protein kinase B (PKB)/Akt compared to cells from control littermates. As a consequence of these defects, I assessed the antigenpresenting/ T-cell activating capabilities of these DC. Anx-A1-/- DC showed an impaired capacity to stimulate T cell proliferation and differentiation in allogeneic mixed leukocyte reaction. To dissect this biologically relevant phenomenon further, I employed an antigenspecific, T-cell restricted model; a co-culture system of chicken ovalbumin peptide-pulsed, LPS-matured bone marrow-derived DC incubated with transgenic TCR T cells from OT-I/RAG-1-/- (OT-I, OTI/ CD8+) or OT-II/ RAG-1-/- (OT-II, OT-II/CD4+) mice. Peptide-pulsed, LPS-matured AnxA1-/- DC failed to initiate an appropriate T cell activation in both OT-I and OT-II T cells indicated by reduced cell proliferation when compared to T cells co-cultured with peptide6 pulsed, LPS-matured AnxA1+/+ DC. Additionally, comparison of peptide-pulsed, LPS-matured AnxA1-/- DC with AnxA1+/+ DC counterparts detected severely diminished levels of IL-2 from cocultures with OT-I T cells and ablated IFN-γ production from cocultures with both OT-I and OT-II T cells. In conclusion, AnxA1 seems to act as a positive modulator of immunogenic activation of DC, whereby the AnxA1 signal pathway has a probable synergism with the TLR4 signalling cascade. DCderived AxnA1 appears to contribute in promoting T cell activation with a larger influence on OT-I/CD8+ T cells than OT-II/CD4+ T cells. Altogether these findings suggest that inhibition of Anx-A1 expression or function in dendritic cells might represent a useful way to modulate the adaptive immune response and pathogen-induced T cell-driven immune diseases.

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Aldosary, Mazhor Salem K. "Biochemical and molecular genetic investigations of patients with mitochondrial DNA disease". Thesis, University of Newcastle Upon Tyne, 2011. http://hdl.handle.net/10443/1121.

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Mitochondria are organelles present in all nucleated cells and are the only location of extra-chromosomal DNA within mammalian cells. They are responsible for the generation of ATP by oxidative phosphorylation (OXPHOS). An extensive range of molecular defects have been identified in the human mitochondrial genome, many associated with wellcharacterised, progressive neurological syndromes. Diseases due to mutations in the mitochondrial genome (mtDNA) are clinically, genetically and biochemically diverse. I have investigated the molecular genetic basis of mitochondrial disease in a cohort of patients with biochemical deficiencies, using a range of histochemical, biochemical, molecular genetic and cell biological techniques. Whole genome sequencing results revealed the identification of novel and recurrent pathogenic mutations in different subunits of complex I (m.11453G>A, m.11777C>A, m 13051G>A, m.13513G>A and m.14453G>A). To assign pathogenicity of these identified mutations, several approaches were carried out to distinguish between pathogenic and neutral mtDNA changes. Investigation of different tissues from patients and their relatives explored mtDNA heteroplasmy, and determined whether mutations had a risen sporadically or been maternally-inherited. Transmitochondrial cybrids were established as an in vitro model to assess the functional consequences of the m.11777C>A mutation, revealing significant defects in these cells supporting the role of this mutation in causing disease in patient. The m.11777C>A and m.13051G>A mutations were associated with increased reactive oxygen species (ROS) production as a consequence of the effect of these mutations on complex I function. Moreover, the expression of several mitochondrial complex I subunits was differentially affected in fibroblasts from patients based on western blotting analysis. In addition, 3 novel, pathogenic mt-tRNA mutations were identified; m.618T>G MTTF gene mutation, a m.12261T>C MTTS2 gene mutation and a m.12283G>A MTTL2 gene mutation. These mutations were shown to be pathogenic as they segregated with the biochemical defect within individual cytochrome c oxidase (COX)-deficient fibres. Interestingly, the m.12283G>A mutation was present at very low levels in mature muscle as compared to other mt-tRNA mutations, and exhibited an unusual pattern of segregation in single muscle fibres. The MitoChip (V2.0) is an oligonucleotide tiling array for the resequencing of the human mitochondrial genome, which has been proposed as an alternative diagnostic screening tool given it is rapid and relatively cheap. A comparative study between conventional dideoxy Sanger sequencing and MitoChip (V2.0) analysis revealed an inability of this technology to detect single nucleotide insertion and deletion mtDNA mutations.

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13

Zainuddin, Siti Lailatul Akmar. "Immunohistochemical and biochemical investigations into the spatial distribution of amelogenin isoforms". Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522918.

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Hansen, R. K. "Biophysical and biochemical investigations of the M2 peptide of influenza A". Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603662.

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Membrane proteins, especially passive ion channels, are very difficult to characterise using normal biophysical methods. This thesis used the transmembrane domain of the M2 proton channel of Influenza A (M2-TM) as a model system for biophysical investigations. Patch clamping is the most accurate method for assessing ion channel function, but requires rigid liposomes that can withstand mechanical stress, but are not usually optimal for structural investigations. Here a new hydrogen deuterium exchange (HDX) method has been optimised for the analysis of a membrane spanning peptides in lipid vesicles. A simple electrospray ionisation mass spectrometry method is introduced for HDX studies of carefully prepared aqueous proteoliposome samples. 11 backbone amide sites of M2-TM in lipid vesicles were shown to be resistant to HDX for several weeks. By contrast, HDX of M2-TM in methanol or in detergent micelles was complete within an hour, indicating that the peptide adopts a non-nativee conformation. A solution state nuclear magnetic resonance (NMR) strategy relying on quenching the exchange reaction at low pH, lyophilisation and resuspension in acidified methanol was applied to studies of HDX in a transmembrane peptide for the first and revealed site specific information. His16 and Leu17 exchanged much faster than neighbouring residues consistent with a "flip-open" mechanism for proton conduction. Binding of the drug amantadine weakly perturbs the HDX of Ala8 and Ala9 in agreement with neutron scattering data showing an interaction with the N-terminus of the channel. NMR studies investigated details of the binding and release of fluoroamantadine to liposomes in the presence and absence of M2-TM consistent with the drug binding only to the closed state of the channel in DMPC vesicles. HDX studies have great potential to reveal details of the structure, dynamics and intermolecular interactions of membrane proteins.

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Koudouna, Elena. "Structural and biochemical investigations of the cornea and the trabecular meshwork". Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/56775/.

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The experiments which comprise this thesis focused on structure-function relationships in two distinct collagen-rich connective tissues of the eye, the cornea and the trabecular meshwork. The cornea is the transparent tissue that covers the front of the eye and has the ability to transmit and refract light. Corneal transparency is the result of the unique organisation of collagen fibrils in the corneal stroma matrix, of which sulphated proteoglycans are key regulators, owing to the presumed importance of the sulfation pattern of corneal proteoglycans. The trabecular meshwork is the sponge-like tissue located around the cornea through which the bulk of the aqueous humor flows towards the juxtacanalicular tissue and the inner wall of Schlemm’s canal to exit the eye and control intraocular pressure. First part of the current research examined the chemical composition and sulphur speciation during corneal embryogenesis in order to elucidate important changes in the biochemical signature of the corneal matrix associated with the acquisition of transparency. It also investigated the content and distribution of distinct sulphur species through the depth of the mature corneal stroma and assessed biochemical-functional relationships that ultimate render tissue transparency. The research also studied the three-dimensional ultrastructure of the human trabecular meshwork, particularly the ultrastructure of the juxtacanalicular tissue that lies adjacent to the inner wall of Schlemm’s canal and the three-dimensional assembly of collagen type VI in the trabecular meshwork itself. X-ray fluorescence microscopy revealed key differences in the chemical composition of the cornea of the developing chick. In particular, the chemical signature of phosphorus, chlorine, sulphur, potassium and calcium were observably different during the developmental period from day 12 to day 16. S k-edge x-ray near edge structure spectroscopy showed that the main sulphur species present in the embryonic cornea were thiols, organic monosulfides, ester sulphate and inorganic sulphate. The chemical signature of these sulphur species was also noticeably different during embryonic corneal development. The changes in the chemical signature of phosphorus with development are believed to underline changes in the presumptive keratocyte population within the embryonic corneal stroma. Chlorine, potassium and calcium are important elements involved in the regulation and balance of the net negative or positive charge of the embryonic cornea and may influence the interactions of corneal matrix molecules. The changes in the sulphur speciation character amongst different developmental corneas are associated with changes in the sulphation status of corneal proteoglycans which play a fundamental role in governing tissue structure and function, and thus transparency. With regards to the sulphur speciation across the depth of the mature corneal stroma, it was found that there is an inconsistency in the sulphur content and distribution throughout the depth of the tissue, from the stromal region closest to the epithelium against the deeper stromal regions near the endothelium. The heterogeneity of the sulphur species in the most anterior part of the mature corneal stroma, at the interface with the Bowman’s layer supports the view that the differentiation and the transition between these two corneal layers is not very abrupt. The rest of the mature corneal stroma depth does not show any differences regarding its content in the sulphur-containing compounds indicating that the distribution and sulfation status of the corneal glycosaminoglycans have very little impact on the overall sulfur speciation. The three-dimensional ultrastructure of the human trabecular meshwork in large volumes and at high resolution identified giant vacuoles in the endothelial cell layer of the inner wall of Schlemm’s canal and these were grouped into four categories based on whether they formed pores, basal and apical, or not. Interestingly, the distribution of these vacuoles was found to be non-uniform. It was discovered that the juxtacanalicular tissue was not homogenous with respect to the proportion of the electron lucent, matrix free spaces throughout the tissue’s depth away from the inner wall of Schlemm’s canal. Three-dimensional reconstructions of collagen type VI in the trabecular meshwork showed that there is no structural regularity in the organisation of type VI collagen assemblies, or their association with sulphated proteoglycans, suggesting a role in aqueous humor outflow. This data allow us to propose a model of aqueous humor outflow and how this is funneled through the juxtacanalicular tissue towards the lumen of Sclemm’s canal.

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16

Imtiaz, Faiqa. "Biochemical and genetic investigations on patients with congenital disorders of glycosylation". Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269733.

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Chang, Michelle M. (Michelle Miran). "Biochemical and biophysical investigations of N-linked glycosylation pathways in archaea". Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/97981.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, February 2015.
Cataloged from PDF version of thesis. "December 2014."
Includes bibliographical references.
Asparagine-linked glycosylation is an abundant and complex protein modification conserved among all three domains of life. Much is known about N-glycan assembly in eukaryotes and selected bacteria, in which the oligosaccharyltransferase (OTase) carries out the en bloc transfer of glycans from polyprenyl-PP-linked donors onto asparagine side chains of acceptor proteins. The first aim of this thesis is to elucidate the biochemical details of archaeal N-linked glycosylation, specifically through in vitro analysis of the polyprenyl-P-dependent pathway of the methanogenic archaeon Methanococcus voltae. The archaeal OTase, known as AglB, utilizes a-linked dolichyl-P-trisaccharide substrate as the glycosyl donor for transfer to the acceptor protein. This dolichyl-P-glycan is generated by an initial retaining glycosyltransferase (AglK) and elaborated by additional glycosyltransferases (AglC and AgIA) to afford Dol-P-GlcNAc- Glc-2,3-diNAcA-ManNAc(6Thr)A. Despite the homology to other bacterial or eukaryotic OTases that exploit polyprenyl-PP-linked substrates, the M. voltae AglB efficiently transfers disaccharide to model peptides from the Dol-P-GlcNAc-Glc-2,3-diNAcA monophosphate. While this archaeal pathway affords the same asparagine-linked P-glycosyl amide products generated in bacteria and eukaryotes, these studies provide the first biochemical evidence revealing that despite the apparent similarities of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life. A second focus of this thesis involves biophysical studies to probe structural features and conformational dynamics of AglB. An intramolecular LRET experimental system was developed to report on substrate binding and the resulting structural transformations in AgIB. There is a strong need for detailed studies on the mechanistic and functional significance of archaeal adaptations of N-linked glycosylation, especially exploring differences between AglB and other OTases that allow AglB to utilize these unique polyprenyl-P-linked substrates. Lastly, a cell-free expression system was established for the efficient synthesis of Alg5, a yeast dolichyl-phosphate glucosyltransferase that shares high sequence similarity to AglK, the first glycosyltransferase in the M. voltae pathway. Dol-P-Glc was generated and examined to unambiguously characterize the stereochemistry of the product of Alg5.
by Michelle M. Chang.
Ph. D.

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18

Krämer, Franziska. "Molecular and biochemical investigations into VMD2, the gene associated with Best disease". [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968349293.

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[Verfasser], Fahrurrozi, i Bernward [Akademischer Betreuer] Bisping. "Microbiological and biochemical investigations of cocoa bean fermentation / Fahrurrozi. Betreuer: Bernward Bisping". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/1072713268/34.

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20

Sacho, Elizabeth J. Erie Dorothy A. "Biophysical and biochemical investigations of proteins involved in genomic maintenance and stability". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,1431.

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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Apr. 25, 2008). " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

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21

Xu, Hui. "Molecular biological and biochemical investigations of the biosynthesis of the aminocoumarin antibiotics". [S.l. : s.n.], 2004. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11195194.

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22

Fan, Aili [Verfasser], i Shu-Ming [Akademischer Betreuer] Li. "Biochemical investigations on bacterial and fungal prenyltransferases / Aili Fan. Betreuer: Shu-Ming Li". Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/1082379085/34.

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23

Collins, Jane E. "Biochemical and physiological investigations of volatile oil production in Monarda citriodora var. citriodora". Thesis, Liverpool John Moores University, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337898.

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24

Slivka, Eric John. "Structural and biochemical investigations of the thyroid hormone receptor and v-erbA oncoprotein". Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3352469.

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25

Kar, Rekha. "Investigations of the mechanisms of cell death induced by 15-deoxy delta (12,14) prostaglandin J2 a dissertation /". San Antonio : UTHSC, 2008. http://proquest.umi.com.libproxy.uthscsa.edu/pqdweb?did=1663069051&sid=2&Fmt=2&clientId=70986&RQT=309&VName=PQD.

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26

Winkelblech, Julia [Verfasser], i Shu-Ming [Akademischer Betreuer] Li. "Biochemical investigations on bacterial and fungal dimethylallyltryptophan synthases / Julia Winkelblech ; Betreuer: Shu-Ming Li". Marburg : Philipps-Universität Marburg, 2016. http://d-nb.info/1120583756/34.

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Bonitz, Tobias [Verfasser], i Lutz [Akademischer Betreuer] Heide. "Biochemical Investigations of Aromatic Prenyltransferases from the ABBA Superfamily / Tobias Bonitz ; Betreuer: Lutz Heide". Tübingen : Universitätsbibliothek Tübingen, 2013. http://d-nb.info/1162844345/34.

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28

Mohabir, G. "Biochemical investigations of yield-limitations in potatoes (Solanum tuberosum L.) under warm tropical conditions". Thesis, University of Reading, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380838.

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29

Gray, Julie Anne. "Investigations into the use of the biochemical markers of bone metabolism in the horse". Thesis, Open University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336810.

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30

Chan, Susan L. F. "Biochemical and pharmacological investigations into the role of α2-adrenoceptors in rat islets of Langerhans". Thesis, Keele University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292754.

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31

Davis, Aubrey K. "Biochemical, molecular, and genomic investigations into the response of the diatom Thalassiosira pseudonana to copper stress /". Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3191991.

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32

Ponnusamy, Rajesh [Verfasser]. "Crystallographic and biochemical investigations on coronarvirus replication proteins - non-structural proteins 8 and 9 / Rajesh Ponnusamy". Lübeck : Zentrale Hochschulbibliothek Lübeck, 2010. http://d-nb.info/1004773706/34.

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33

Kunze, Kurt, Hendrik Niemann, Susanne Ueberlein, Renate Schulze, Hermann Ehrlich, Eike Brunner, Peter Proksch i Karl-Heinz van Pée. "Brominated Skeletal Components of the Marine Demosponges, Aplysina cavernicola and Ianthella basta: Analytical and Biochemical Investigations". Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-127045.

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Demosponges possess a skeleton made of a composite material with various organic constituents and/or siliceous spicules. Chitin is an integral part of the skeleton of different sponges of the order Verongida. Moreover, sponges of the order Verongida, such as Aplysina cavernicola or Ianthella basta, are well-known for the biosynthesis of brominated tyrosine derivates, characteristic bioactive natural products. It has been unknown so far whether these compounds are exclusively present in the cellular matrix or whether they may also be incorporated into the chitin-based skeletons. In the present study, we therefore examined the skeletons of A. cavernicola and I. basta with respect to the presence of bromotyrosine metabolites. The chitin-based-skeletons isolated from these sponges indeed contain significant amounts of brominated compounds, which are not easily extractable from the skeletons by common solvents, such as MeOH, as shown by HPLC analyses in combination with NMR and IR spectroscopic measurements. Quantitative potentiometric analyses confirm that the skeleton-associated bromine mainly withstands the MeOH-based extraction. This observation suggests that the respective, but yet unidentified, brominated compounds are strongly bound to the sponge skeletons, possibly by covalent bonding. Moreover, gene fragments of halogenases suggested to be responsible for the incorporation of bromine into organic molecules could be amplified from DNA isolated from sponge samples enriched for sponge-associated bacteria.

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34

Philpott, Amelia. "Investigations into the biochemical and cellular biology of a cytoplasmic dynein mutation, abnormal rear leg (Arl)". Thesis, University of Sussex, 2011. http://sro.sussex.ac.uk/id/eprint/6977/.

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The aim of this project was to investigate the effects of a novel mouse cytoplasmic dynein mutation; Abnormal rear leg (Arl). Cytoplasmic dynein is a microtubule (MT) based motor protein important for diverse cellular processes including Golgi maintenance and retrograde transport of organelles. Arl is a mouse point mutation in the heavy chain subunit of dynein (Dync1h1). Homozygous Dync1h1Arl/Arl die at embryonic day 10. Dync1h1Arl/+ heterozygotes have a normal life span, but exhibit abnormal gait and hindlimb clasping during tail suspension, typical of neuronal dysfunction. Protein purification from wildtype and heterozygous brain tissue showed increased MT binding in Dync1h1Arl/+ compared to wildtype. Delayed endosomal trafficking was observed in EGF stimulated Dync1h1Arl/+ mouse embryonic fibroblasts (MEFs) compared to wildtype, in both fixed cells and using live cell imaging. Similarly, a delay in the reassembly of the Golgi complex after disruption with a MT depolymerisation agent, nocodazole, was observed in Dync1h1Arl/+ MEFs compared to wildtype. In addition, the Golgi complex was observed as being structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons using transmission electron microscopy (TEM) compared to the wildtype. TEM also revealed that the mitochondria were structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons compared to wildtype, and O2 consumption assays investigating their function showed the Dync1h1Arl/+ mitochondria to have increased respiration rates compared to wildtype. Thus, these data highlight the Arl mouse as an invaluable model for studying the mechanism of dynein function and the subsequent outcomes when they are compromised.

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35

Warburton, Sarah. "Biochemical Investigations of Macular Degeneration: The Significance of Protein Oxidation including Novel Methods for Its Study". Diss., CLICK HERE for online access, 2006. http://contentdm.lib.byu.edu/ETD/image/etd1583.pdf.

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36

Pockrandt, Daniel [Verfasser], i Shu-Ming [Akademischer Betreuer] Li. "Molecular biological and biochemical investigations of prenyltransferases from different Aspergillus species / Daniel Pockrandt. Betreuer: Shu-Ming Li". Marburg : Philipps-Universität Marburg, 2014. http://d-nb.info/1052994768/34.

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37

Duz, Marco. "Biochemical and epidemiological investigations of non-steroidal anti-inflammatory drug usage and related side effects in equids". Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7581/.

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Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in equine veterinary practice. These drugs exert their effect by inhibiting cyclooxygenase (COX) enzymes, which control prostaglandin production, a major regulator of tissue perfusion. Two isoforms of COX enzymes exist: COX-1 is physiologically present in tissues, while COX-2 is up-regulated during inflammation and has been indicated as responsible for the negative effects of an inflammatory response. Evidence suggests that NSAIDs that inhibit only COX-2, preserving the physiological function of COX-1 might have a safer profile. Studies that evaluate the effect of NSAIDs on COX enzymes are all performed under experimental conditions and none uses actual clinical patients. The biochemical investigations in this work focus on describing the effect on COX enzymes activity of flunixin meglumine and phenylbutazone, two non-selective COX inhibitors and firocoxib, a COX-2 selective inhibitor, in clinical patients undergoing elective surgery. A separate epidemiological investigation was aimed at describing the impact that the findings of biochemical data have on a large population of equids. Electronic medical records (EMRs) from 454,153 equids were obtained from practices in the United Kingdom, United States of America and Canada. Information on prevalence and indications for NSAIDs use was extracted from the EMRs via a text mining technique, improved from the literature and described and validated within this Thesis. Further the prevalence of a clinical sign compatible with NSAID toxicity, such as diarrhoea, is reported along with analysis evaluating NSAID administration in light of concurrent administration of other drugs and comorbidities. This work confirms findings from experimental settings that NSAIDs firocoxib is COX-2 selective and that flunixin meglumine and phenylbutazone are non-selective COX inhibitors and therefore their administration carries a greater risk of toxicity. However the impact of this finding needs to be interpreted with caution as epidemiological data suggest that the prevalence of toxicity is in fact small and the use of these drugs at the labelled dose is quite safe.

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38

Maratos, Eleni Chryssa. "Behavioural and biochemical investigations into dyskinesia by levodopa or dopamine agonists in the MPTP-treated common marmoset". Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271323.

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Liebhold, Mike Patrick [Verfasser], i Shu-Ming [Akademischer Betreuer] Li. "Biochemical Investigations on Microbial Prenyltransferases in the Presence of DMAPP Analogues / Mike Patrick Liebhold. Betreuer: Shu-Ming Li". Marburg : Philipps-Universität Marburg, 2015. http://d-nb.info/1071947915/34.

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40

Lange, Christiane [Verfasser], i Dieter [Akademischer Betreuer] Jahn. "Structural and biochemical investigations of the late enzymes of (bacterio)chlorophyll a biosynthesis / Christiane Lange ; Betreuer: Dieter Jahn". Braunschweig : Technische Universität Braunschweig, 2016. http://d-nb.info/1175818798/34.

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41

Chahyadi, Agus [Verfasser], i Maike [Akademischer Betreuer] Petersen. "Biochemical and Molecular Investigations of Arabidopsis thaliana Transformed with Genes of Rosmarinic Acid Biosynthesis / Agus Chahyadi ; Betreuer: Maike Petersen". Marburg : Philipps-Universität Marburg, 2017. http://d-nb.info/1136718508/34.

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42

Guymer, David. "Biochemical and physiological investigations of the activities of TorD family chaperones in Escherichia coli and Salmonella enterica serovar Typhimurium". Thesis, University of Dundee, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505653.

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43

Singh, Poonam Verfasser], Ludger [Akademischer Betreuer] Beerhues i Robert [Akademischer Betreuer] [Hänsch. "Biochemical and molecular investigations of benzoic acid metabolism in Hypericum calycinum cell cultures / Poonam Singh ; Ludger Beerhues, Robert Hänsch". Braunschweig : Technische Universität Braunschweig, 2020. http://d-nb.info/1203798792/34.

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44

Sequeira, Suzanne Simone. "'1 NMR spectroscopic investigations into the metabolism and biochemical effects of model drugs and enzyme inducers in the rat". Thesis, Birkbeck (University of London), 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342211.

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45

Wanaguru, Madushi Kaushla. "Biochemical investigations of Plasmodium falciparum erythrocyte invasion pathways using recombinant merozoite surface proteins produced in a mammalian expression system". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607835.

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46

Giannetti, Anthony Michael Rees Douglas C. "Biochemical, biophysical, and cellular investigations of the interactions of transferrin receptor with transferrin and the hereditary hemochromatosis protein, HFE /". Diss., Pasadena, Calif. : California Institute of Technology, 2004. http://resolver.caltech.edu/CaltechETD:etd-05262004-173612.

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47

Peng, Chen. "Synthesis of very-long-chain bifunctional and isotope-labeled compounds for biochemical investigations into novel compounds in plant cuticular waxes". Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43752.

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Plant cuticles are the interface for plant-environment interactions, and the first barrier protecting plants from environmental stresses such as water loss and pathogens. Structurally, the cuticle consists of a hydrophobic polymer lattice, cutin, and cuticular waxes deposited inside and outside cutin. The major components of the cuticular waxes are aliphatics derived from very-long-chain (VLC) fatty acids, such as alkanes and aldehydes. Besides compounds with primary functionalities, some compounds with two or more functional groups have also been identified in cuticular waxes. However, only limited knowledge about them has been acquired so far. The current work was to identify novel 1,2- and 1,3-bifunctional wax compounds from various plant species, in order to expand our current understanding of their structure, biosynthesis and function in plant cuticles. Synthetic methods were first developed to produce various VLC 1,2- and 1,3-bifunctionalized standard compounds for current and future structure elucidation studies. Unknown compounds found in Cosmos bipinnatus petal wax were identified as alkane-1,2-diol monoacetates by GC-MS, with chain lengths ranging from C20 to C24. The ratio between the primary and the secondary monoacetates was quantified to be 3:5, as opposed to the thermodynamic equilibrium ratio of 7:3. Novel β-hydroxy acid methyl esters were also identified from Aloe arborescens leaf wax, with chain lengths ranging from C26 to C30. In addition, two NMR-based methods were established to study the stereoconfigurations of alkane-1,2-diols from C. bipinnatus petal wax, and the carbons bearing secondary hydroxyl functionality were determined to have predominately the R-configuration. Apart from the research on bifunctional compounds, synthetic methods to produce β-deuterium labeled VLC substrates were also established. The resulting C30 fatty acid methyl ester was double-labeled and can be used directly or indirectly as substrates in future biochemical assays. At last, the hypothetic substrate for the CER1 enzyme implicated in wax alkane biosynthesis, C30 aldehyde, was synthesized and used in in vivo assays with heterologously expressed protein.

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48

Mikołajka, Aleksandra. "Functional and structural studies of the FGFR1 oncogene partner protein and biochemical investigations of the retinoblastoma protein and its binding partners". [S.l.] : [s.n.], 2007. http://mediatum2.ub.tum.de/doc/614708/document.pdf.

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Yu, Xia [Verfasser], i Shu-Ming [Akademischer Betreuer] Li. "Molecular biological and biochemical investigations on the biosynthetic enzymes of prenylated indole alkaloids from fungi / Xia Yu. Betreuer: Shu-Ming Li". Marburg : Philipps-Universität Marburg, 2013. http://d-nb.info/1038786347/34.

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Seifert, Florian Ulrich [Verfasser], i Heinrich [Akademischer Betreuer] Leonhardt. "Structural and biochemical investigations of the eukaryotic DNA double-strand break repair complex Mre11-Rad50-Nbs1 / Florian Ulrich Seifert ; Betreuer: Heinrich Leonhardt". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/111907388X/34.

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