Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment

Excluded-Volume Effects in Tethered-Particle Experiments: Bead Size Matters

The tethered-particle method is a single-molecule technique that has been used to explore the dynamics of a variety of macromolecules of biological interest. We give a theoretical analysis of the particle motions in such experiments. Our analysis reveals that the proximity of the tethered bead to a nearby surface (the microscope slide) gives rise to a volume-exclusion effect, resulting in an entropic force on the molecule. This force stretches the molecule, changing its statistical properties. In particular, the proximity of bead and surface brings about intriguing scaling relations between key observables (statistical moments of the bead) and parameters such as the bead size and contour length of the molecule. We present both approximate analytic solutions and numerical results for these effects in both flexible and semiflexible tethers. Finally, our results give a precise, experimentally-testable prediction for the probability distribution of the distance between the polymer attachment point and the center of the mobile bead.Comment: 4 pages, 3 figure

An improved method for surface immobilisation of RNA: application to small Non-Coding RNA - mRNA pairing

Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time. Whilst a number of approaches exist to label RNAs for surface-immobilisation, the method documented here is simple, quick, efficient, and utilises the high-affinity streptavidin-biotin interaction. Specifically, we ligate a biotinylated nucleotide to the 3' end of RNA using T4 RNA ligase. Although this is a previously recognised approach, we have optimised the method by our discovery that the incorporation of four or more adenine nucleotides at the 3' end of the RNA (a poly-A-tail) is required in order to achieve high ligation efficiencies. We use this method within the context of investigating small non-coding RNA (sRNA)-mRNA interactions through the application of surface technologies, including quantitative SPR assays. We first focus on validating the method using the recently characterised Escherichia coli sRNA-mRNA pair, MicA-ompA, specifically demonstrating that the addition of the poly-A-tail to either RNA does not affect its subsequent binding interactions with partner molecules. We then apply this method to investigate the novel interactions of a Vibrio cholerae Qrr sRNA with partner mRNAs, hapR and vca0939; RNA-RNA pairings that are important in mediating pathogenic virulence. The calculated binding parameters allow insights to be drawn regarding sRNA-mRNA interaction mechanisms

Volume-exclusion effects in tethered-particle experiments: bead size matters

We give a theoretical analysis of bead motion in tethered-particle experiments, a single-molecule technique that has been used to explore the dynamics of a variety of macromolecules of biological interest. Our analysis reveals that the proximity of the tethered bead to a nearby surface gives rise to a volume-exclusion effect, resulting in an entropic stretching-force on the molecule that changes its statistical properties. In addition, volume exclusion brings about intriguing scaling relations between key observables (statistical moments of the bead) and parameters such as bead size and contour length of the molecule. We present analytic and numerical results for these effects in both flexible and semiflexible tethers. Finally, our results give a precise, experimentally testable prediction for the probability distribution of the bead center measured from the polymer attachment point

The potential for CO \u3c inf\u3e 2 -induced acidification in freshwater: A great lakes case study

Ocean acidification will likely result in a drop of 0.3–0.4 pH units in the surface ocean by 2100, assuming anthropogenic CO2 emissions continue at the current rate. Impacts of increasing atmospheric pCO2 on pH in freshwater systems have scarcely been addressed. In this study, the Laurentian Great Lakes are used as a case study for the potential for CO2-induced acidification in freshwater systems as well as for assessment of the ability of current water quality monitoring to detect pH trends. If increasing atmospheric pCO2 is the only forcing, pH will decline in the Laurentian Great Lakes at the same rate and magnitude as the surface ocean through 2100. High-resolution numerical models and one high-resolution time series of data illustrate that the pH of the Great Lakes has significant spatio-temporal variability. Because of this variability, data from existing monitoring systems are insufficient to accurately resolve annual mean trends. Significant measurement uncertainty also impedes the ability to assess trends. To elucidate the effects of increasing atmospheric CO2 in the Great Lakes requires pH monitoring by collecting more accurate measurements with greater spatial and temporal coverage

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes

On the Measurement of the Magnitude and Orientation of the Magnetic Field in Molecular Clouds

We demonstrate that the combination of Zeeman, polarimetry and ion-to-neutral molecular line width ratio measurements permits the determination of the magnitude and orientation of the magnetic field in the weakly ionized parts of molecular clouds. Zeeman measurements provide the strength of the magnetic field along the line of sight, polarimetry measurements give the field orientation in the plane of the sky and the ion-to-neutral molecular line width ratio determines the angle between the magnetic field and the line of sight. We apply the technique to the M17 star-forming region using a HERTZ 350 um polarimetry map and HCO+-to-HCN molecular line width ratios to provide the first three-dimensional view of the magnetic field in M17.Comment: 37 pages, 12 figures, 3 table

The DEEP2 Galaxy Redshift Survey: Spectral classification of galaxies at z~1

We present a Principal Component Analysis (PCA)-based spectral classification, eta, for the first 5600 galaxies observed in the DEEP2 Redshift Survey. This parameter provides a very pronounced separation between absorption and emission dominated galaxy spectra - corresponding to passively evolving and actively star-forming galaxies in the survey respectively. In addition it is shown that despite the high resolution of the observed spectra, this parameter alone can be used to quite accurately reconstruct any given galaxy spectrum, suggesting there are not many `degrees of freedom' in the observed spectra of this galaxy population. It is argued that this form of classification, eta, will be particularly valuable in making future comparisons between high and low-redshift galaxy surveys for which very large spectroscopic samples are now readily available, particularly when used in conjunction with high-resolution spectral synthesis models which will be made public in the near future. We also discuss the relative advantages of this approach to distant galaxy classification compared to other methods such as colors and morphologies. Finally, we compare the classification derived here with that adopted for the 2dF Galaxy Redshift Survey and in so doing show that the two systems are very similar. This will be particularly useful in subsequent analyses when making comparisons between results from each of these surveys to study evolution in the galaxy populations and large-scale structure.Comment: 10 pages, 9 figures, Accepted for publication in Ap

Petrogenesis of Siletzia: the world’s youngest oceanic plateau

Siletzia is an accreted Palaeocene-Eocene Large Igneous Province, preserved in the northwest United States and southern Vancouver Island. Although previous workers have suggested that components of Siletzia were formed in tectonic settings including back arc basins, island arcs and ocean islands, more recent work has presented evidence for parts of Siletzia to have formed in response to partial melting of a mantle plume. In this paper, we integrate geochemical and geochronological data to investigate the petrogenetic evolution of the province. The major element geochemistry of the Siletzia lava flows is used to determine the compositions of the primary magmas of the province, as well as the conditions of mantle melting. These primary magmas are compositionally similar to modern Ocean Island and Mid-Ocean Ridge lavas. Geochemical modelling of these magmas indicates they predominantly evolved through fractional crystallisation of olivine, pyroxenes, plagioclase, spinel and apatite in shallow magma chambers, and experienced limited interaction with crustal components. Further modelling indicates that Siletzia magmatism was derived from anomalously hot mantle, consistent with an origin in a mantle plume. This plume has been suggested to have been the same as that responsible for magmatism within the Yellowstone Plateau. Trace element compositions of the most primitive Siletzia lavas are similar to suites associated with the Yellowstone Mantle Plume, suggesting that the two provinces were derived from compositionally similar sources. Radiogenic isotope systematics for Siletzia consistently overlap with some of the oldest suites of the Yellowstone Magmatic Province. Therefore, we suggest Siletzia and the Yellowstone Mantle Plume are part of the same, evolving mantle plume system. Our new geochronological data show the province was emplaced during the time when Eocene sea surface temperatures were their highest. The size of Siletzia makes the province a potential contributing factor to the biospheric perturbation observed in the early Eocene