Synthesis and application of novel probes for the detection of cysteine sulphenic acids

Cysteine is a thiol containing amino acid that readily undergoes oxidation by reactive oxygen species (ROS) to form sulphenic (R-SOH) sulphinic (RSO2H) and sulphonic (RSO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signalling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesise and use a chemical probe that selectively recognises the reversible oxidation state of cysteine sulphenic acid to understand more about oxidative signalling. The aim of this thesis was to investigate a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulphenic acids by fluorescence spectroscopy and confocal microscopy. N-[2-(Anthracen-2-ylamino)-2-oxoethyl]-3,5-dioxocyclohexanecarboxamide was synthesised in a multistep synthesis and characterised by nuclear magnetic resonance spectroscopy. The optimisation of conditions needed for sulphenic acid formation in a purified protein using human serum albumin (HSA) and the commercially available biotin tagged probe 3-(2,4-dioxocyclohexyl)propyl-5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate (DCP-Bio1) were identified. This approach was extended to detect sulphenic acids in Jurkat T cells and CD4+ T cells pre- and post-stimulus. Buthionine sulfoximine (BSO) was used to manipulate the endogenous antioxidant glutathione (GSH) in human CD4+ T cells. Then the surface protein thiol levels and sulphenic acid formation was examined. T cells were also activated by the lectin phytohaemagglutinin-L (PHA-L) and formation of sulphenic acid was investigated using SDS-PAGE, western blotting and confocal microscopy. Resting Jurkat cells have two prominent protein bands that have sulphenic acid modifications whereas resting CD4+ T cells have an additional band present. When cells were treated with BSO the number of bands increased whereas activation reduced the number of proteins that were modified. The identities of the protein bands containing sulphenic acids were explored by mass spectrometry. Cysteine oxidation was observed in redox, metabolic and cytoskeletal proteins. In summary, a novel fluorescent probe for detection of cysteine sulphenic acids has been synthesised alongside a model system that introduces cysteine sulphenic acid in primary T cells. This probe has potential application in the subcellular localisation of cysteine oxidation during T cell signalling.

Negative feedback regulation of the ERK1/2 MAPK pathway

The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of de novo gene synthesis of specific pathway inhibitors. We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

The relative contributions of colour and luminance signals towards the visuomotor localisation of targets in human peripheral vision

We sought to determine the extent to which colour (and luminance) signals contribute towards the visuomotor localization of targets. To do so we exploited the movement-related illusory displacement a small stationary window undergoes when it has a continuously moving carrier grating behind it. We used drifting (1.0-4.2 Hz) red/green-modulated isoluminant gratings or yellow/black luminance-modulated gratings as carriers, each curtailed in space by a stationary, two-dimensional window. After each trial, the perceived location of the window was recorded with reference to an on-screen ruler (perceptual task) or the on-screen touch of a ballistic pointing movement made without visual feedback (visuomotor task). Our results showed that the perceptual displacement measures were similar for each stimulus type and weakly dependent on stimulus drift rate. However, while the visuomotor displacement measures were similar for each stimulus type at low drift rates (<4 Hz), they were significantly larger for luminance than colour stimuli at high drift rates (>4 Hz). We show that the latter cannot be attributed to differences in perceived speed between stimulus types. We assume, therefore, that our visuomotor localization judgements were more susceptible to the (carrier) motion of luminance patterns than colour patterns. We suggest that, far from being detrimental, this susceptibility may indicate the operation of mechanisms designed to counter the temporal asynchrony between perceptual experiences and the physical changes in the environment that give rise to them. We propose that perceptual localisation is equally supported by both colour and luminance signals but that visuomotor localisation is predominantly supported by luminance signals. We discuss the neural pathways that may be involved with visuomotor localization. © 2007 Springer-Verlag.

GLM-beamformer method demonstrates stationary field, alpha ERD and gamma ERS co-localisation with fMRI BOLD response in visual cortex

Recently, we introduced a new 'GLM-beamformer' technique for MEG analysis that enables accurate localisation of both phase-locked and non-phase-locked neuromagnetic effects, and their representation as statistical parametric maps (SPMs). This provides a useful framework for comparison of the full range of MEG responses with fMRI BOLD results. This paper reports a 'proof of principle' study using a simple visual paradigm (static checkerboard). The five subjects each underwent both MEG and fMRI paradigms. We demonstrate, for the first time, the presence of a sustained (DC) field in the visual cortex, and its co-localisation with the visual BOLD response. The GLM-beamformer analysis method is also used to investigate the main non-phase-locked oscillatory effects: an event-related desynchronisation (ERD) in the alpha band (8-13 Hz) and an event-related synchronisation (ERS) in the gamma band (55-70 Hz). We show, using SPMs and virtual electrode traces, the spatio-temporal covariance of these effects with the visual BOLD response. Comparisons between MEG and fMRI data sets generally focus on the relationship between the BOLD response and the transient evoked response. Here, we show that the stationary field and changes in oscillatory power are also important contributors to the BOLD response, and should be included in future studies on the relationship between neuronal activation and the haemodynamic response. © 2005 Elsevier Inc. All rights reserved.

The development of constrained source localisation algorithms for human brain imaging

This work sets out to evaluate the potential benefits and pit-falls in using a priori information to help solve the Magnetoencephalographic (MEG) inverse problem. In chapter one the forward problem in MEG is introduced, together with a scheme that demonstrates how a priori information can be incorporated into the inverse problem. Chapter two contains a literature review of techniques currently used to solve the inverse problem. Emphasis is put on the kind of a priori information that is used by each of these techniques and the ease with which additional constraints can be applied. The formalism of the FOCUSS algorithm is shown to allow for the incorporation of a priori information in an insightful and straightforward manner. In chapter three it is described how anatomical constraints, in the form of a realistically shaped source space, can be extracted from a subject’s Magnetic Resonance Image (MRI). The use of such constraints relies on accurate co-registration of the MEG and MRI co-ordinate systems. Variations of the two main co-registration approaches, based on fiducial markers or on surface matching, are described and the accuracy and robustness of a surface matching algorithm is evaluated. Figures of merit introduced in chapter four are shown to given insight into the limitations of a typical measurement set-up and potential value of a priori information. It is shown in chapter five that constrained dipole fitting and FOCUSS outperform unconstrained dipole fitting when data with low SNR is used. However, the effect of errors in the constraints can reduce this advantage. Finally, it is demonstrated in chapter six that the results of different localisation techniques give corroborative evidence about the location and activation sequence of the human visual cortical areas underlying the first 125ms of the visual magnetic evoked response recorded with a whole head neuromagnetometer.

Functional localisation of human sensory-motor cortex using magnetoencephalography

The 19 channel Neuromagnetometer system in the Clinical Neurophysiology Unit at Aston University is a multi-channel system, unique in the United Kingdom. A bite bar head localisation and MRI co-registration strategy which enabled accurate and reproducible localisation of MEG data into cortical space was developed. This afforded the opportunity to study magnetic fields of the human cortex generated by stimulation of peripheral nerve, by stimulation of visceral sensory receptors and by those evoked through voluntary finger movement. Initially, a study of sensory-motor evoked data was performed in a healthy control population. The techniques developed were then applied to patients who were to undergo neurosurgical intervention for the treatment of epilepsy and I or space occupying lesions. This enabled both validation of the effective accuracy of source localisation using MEG as well as to determine the clinical value of MEG in presurgical assessment of functional localisation in human cortex. The studies in this thesis have demonstrated that MEG can repeatedly and reliably locate sources contained within a single gyrus and thus potentially differentiate between disparate gyral activation. This ability is critical in the clinical application of any functional imaging technique; which is yet to be fully validated by any other 'non-invasive' functional imaging methodology. The technique was also applied to the study of visceral sensory representation in the cortex which yielded important data about the multiple cortical representation of visceral sensory function.

Multi-class subcellular location prediction for bacterial proteins

Two algorithms, based onBayesian Networks (BNs), for bacterial subcellular location prediction, are explored in this paper: one predicts all locations for Gram+ bacteria and the other all locations for Gram- bacteria. Methods were evaluated using different numbers of residues (from the N-terminal 10 residues to the whole sequence) and residue representation (amino acid-composition, percentage amino acid-composition or normalised amino acid-composition). The accuracy of the best resulting BN was compared to PSORTB. The accuracy of this multi-location BN was roughly comparable to PSORTB; the difference in predictions is low, often less than 2%. The BN method thus represents both an important new avenue of methodological development for subcellular location prediction and a potentially value new tool of true utilitarian value for candidate subunit vaccine selection.

Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries

Background: Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. The existence of single or multiple VEGF isoforms and receptors suggests that these proteins may have overlapping but distinct functions, which may be reflected in their cell expression and distribution. Methods: The localisation of VEGFs A–C and their receptors (VEGFRs 1–3, respectively) in 30 fresh human atherosclerotic arteries, 15 normal uterine arteries, and 15 saphenous veins using immunohistochemistry and western blotting. Results: Saphenous veins showed no staining for VEGF-B or VEGFR-2. Smooth muscle cells (SMCs) showed the strongest staining for VEGF-A, VEGF-B, VEGFR-1, and VEGFR-2 in all specimens. Conversely, VEGFR-3 and VEGF-C were predominately localised to the endothelial vasa vasorum in normal arteries, whereas medial SMCs showed the strongest staining in atherosclerotic arteries. Western blotting showed variations in VEGF protein localisation, with lower amounts of VEGF-B and VEGF-C in saphenous veins, compared with arterial tissue. Amounts of VEGF-C were lower than those of VEGF-A and VEGF-B in all specimens. Conclusion: This study provides direct evidence of the presence of VEGF proteins and receptors in human physiology and pathology, with variations in both the amounts of VEGF proteins expressed and their cellular distribution in normal arteries compared with atherosclerotic arteries. The presence of VEGFs A–C and their receptors in normal arterial tissue implies that VEGF functions may extend beyond endothelial cell proliferation. Reduced VEGFR-2 staining in atherosclerotic arteries may have implications for the atherosclerosis process and the development of vascular disease and its complications.

Rapid tonicity induced re-localisation of endogenous aquaporin 4 in primary rat astrocytes - a therapeutic target for cytotoxic brain oedema?

It is estimated that 69-75 million people worldwide will suffer a traumatic brain injury (TBI) or stroke each year. Brain oedema caused by TBI or following a stroke, together with other disorders of the brain cost Europe €770 billion in 2014. Aquaporins (AQP) are transmembrane water channels involved in many physiologies and are responsible for the maintenance of water homeostasis. They react rapidly to changes in osmolarity by transporting water through their highly selective central pore to maintain tonicity and aid in cell volume regulation. We have previously shown that recombinant AQP1-GFP trafficking occurs in a proteinkinase C-microtubule dependant manner in HEK-293 cells in response to hypotonicity. This trafficking mechanism is also reliant on the presence of calcium and its messenger-binding protein calmodulin and results in increased cell surface expression of AQP1 in a time-scale of ~30 seconds. There is currently very little research into the trafficking mechanisms of endogenous AQPs in primary cells. AQP4 is the most abundantly expressed AQP within the brain, it is localised to the astrocytic end-feet, in contact with the blood vessels at the blood-brain-barrier. In situations where the exquisitely-tuned osmotic balance is disturbed, high water permeability can become detrimental. AQP4-mediated water influx causes rapid brain swelling, resulting in death or long term brain damage. Previous research has shown that AQP4 knock-out mice were protected from the formation of cytotoxic brain oedema in a stroke model, highlighting AQP4 as a key drug target for this pathology. As there are currently no treatments available to restrict the flow of water through AQP4 as all known inhibitors are either cytotoxic or non-specific, controlling the mechanisms involved in the regulation of AQP4 in the brain could provide a therapeutic solution to such diseases. Using cell surface biontinylation of endogenous AQP4 in primary rat astrocytes followed by neutraavidin based ELISA we have shown that AQP4 cell surface localisation increases by 2.7 fold after 5 minutes hypotonic treatment at around 85 mOsm/kg H2O. We have also shown that this rapid relocalisation of AQP4 is regulated by PKA, calmodulin, extra-cellular calcium and actin. In summary we have shown that rapid translocation of endogenous AQP4 occurs in primary rat astrocytes in response to hypotonic stimuli; this mechanism is PKA, calcium, actin and calmodulin dependant. AQP4 has the potential to provide a treatment for the development of brain oedema.

Localising the auditory N1m with event-related beamformers:localisation accuracy following bilateral and unilateral stimulation

The auditory evoked N1m-P2m response complex presents a challenging case for MEG source-modelling, because symmetrical, phase-locked activity occurs in the hemispheres both contralateral and ipsilateral to stimulation. Beamformer methods, in particular, can be susceptible to localisation bias and spurious sources under these conditions. This study explored the accuracy and efficiency of event-related beamformer source models for auditory MEG data under typical experimental conditions: monaural and diotic stimulation; and whole-head beamformer analysis compared to a half-head analysis using only sensors from the hemisphere contralateral to stimulation. Event-related beamformer localisations were also compared with more traditional single-dipole models. At the group level, the event-related beamformer performed equally well as the single-dipole models in terms of accuracy for both the N1m and the P2m, and in terms of efficiency (number of successful source models) for the N1m. The results yielded by the half-head analysis did not differ significantly from those produced by the traditional whole-head analysis. Any localisation bias caused by the presence of correlated sources is minimal in the context of the inter-individual variability in source localisations. In conclusion, event-related beamformers provide a useful alternative to equivalent-current dipole models in localisation of auditory evoked responses.

Disorder-induced light localisation:from random to artificial

Light localisation in one-dimensional (1D) randomly disordered medium is usually characterized by randomly distributed resonances with fluctuating transmission values, instead of selectively distributed resonances with close-to-unity transmission values that are needed in real application fields. By a resonance tuning scheme developed recently, opening of favorable resonances or closing of unfavorable resonances are achieved by disorder micro-modification, both on the layered medium and the fibre Bragg grating (FBG) array. And furthermore, it is shown that those disorder-induced resonances are independently tunable. Therefore, selected resonances and arranged light localisation can be achieved via artificial disorder, and thus meet the demand of various application fields.