Similarities on Graphs: Kernels versus Proximity Measures

We analytically study proximity and distance properties of various kernels and similarity measures on graphs. This helps to understand the mathematical nature of such measures and can potentially be useful for recommending the adoption of specific similarity measures in data analysis.Comment: 16 page

Kernels on Graphs as Proximity Measures

International audienceKernels and, broadly speaking, similarity measures on graphs are extensively used in graph-based unsupervised and semi-supervised learning algorithms as well as in the link prediction problem. We analytically study proximity and distance properties of various kernels and similarity measures on graphs. This can potentially be useful for recommending the adoption of one or another similarity measure in a machine learning method. Also, we numerically compare various similarity measures in the context of spectral clustering and observe that normalized heat-type similarity measures with log modification generally perform the best

Structural and functional analysis of CD81-Claudin-1, a hepatitis C virus receptor complex

Many viruses initiate infection through a multistep process involving host cell membrane proteins. Hepatitis C virus (HCV) is an important human pathogen that infects more than 185 million people worldwide and results in progressive liver disease. Recent advances have identified an essential role for tetraspanin CD81 and tight junction protein Claudin-1 in HCV entry into hepatocytes in the liver. CD81 associates with Claudin-1 and this complex is necessary for virus internalisation; defining the full length interface of this membrane protein interaction is therefore important for the design of future anti-viral therapies. Structural information is lacking for CD81: indeed, there is no high resolution structure for any full-length tetraspanin. This thesis describes an analysis of the protein-protein interaction interface between CD81 and Claudin-1 full-length proteins using a split-ubiquitin yeast assay. Also, using recombinant protein production of CD81, this thesis describes work towards successful crystallisation trials of a full length tetraspanin. CD81 homotypic and heterotypic interactions with Claudin-1 were analysed in a high-throughput format in yeast, showing that this interaction is specific and does not require other mammalian cell factors. This work demonstrates that the CD81 large extracellular loop and its first transmembrane domain are involved in the CD81-Claudin-1 interaction: a novel full length molecular model predicted interacting amino acid residues that were confirmed in vivo using yeast assays. Thermal stability assays used to investigate recombinant membrane protein found that both detergent and buffer components are vital for the stability of recombinant CD81, which shows increased thermostability in the presence of cholesteryl hemisuccinate. Using the improved protein solution environment found here, and the increased understanding of the tetraspanin interaction interface; this work paves the way for CD81 structural characterisation alone or in combination with Claudin-1

Design with the Desert

Typical development in the American Southwest often resulted in scraping the desert lands of the ancient living landscape, to be replaced with one that is human-made and dependent on a large consumption of energy and natural resources. This transdisciplinary book explores the natural and built environment of this desert region and introduces development tools for shaping its future in a more sustainable way. It offers valuable insights to help promote ecological balance between nature and the built environment in the American Southwest-and in other ecologically fragile regions around the world

Doctor of Philosophy

dissertationNeural recording devices are a therapeutic and diagnostic option for central nervous system (CNS) diseases and a vital component of neuroscience research. However, poor functional longevity is a major hurdle facing this broad class of devices. Decreases in functionality are associated, in part, with the foreign body response (FBR) surrounding chronically implanted recording devices; which includes chronic inflammation, astrogliosis, blood-brain barrier (BBB) leakiness, and neuronal cell death. Two potential areas for intervention were explored including the initial hemorrhage that results from device insertion and the neuroinflammatory sequela. Researchers have shown that cellular interactions with extracellular matrix (ECM) are able to affect both of these aspects of the FBR. The central hypothesis driving this work is that ECM coatings which target the initial hemorrhage, should decrease the FBR. This was investigated by coating silicon microelectrode arrays (MEAs) with ECM and implanting them into motor cortex of rats. Two ECM coatings were investigated, including the xenogeneic clinically-used Avitene Microfibrillar Collagen Hemostat and allogeneic astrocyte-derived ECM. Results show that the allogeneic astrocyte-derived ECM decreased astrogliosis within the recording zone at the 8-week time point. This decrease in astrogliosis may improve device functionality, as indicated by previous studies that correlated recording metrics to histology. Interestingly, the xenogeneic Avitene coating increased IgG within the recording zone at the 8-week time point. Collectively, these results show that ECM coatings with different genetic backgrounds and compositions are able to differentially affect specific aspects of the FBR. To broaden the knowledge on the FBR to neural recording devices, the FBR of headstage components used to anchor CNS devices to the skull was analyzed. Results showed that the FBR to fixation screws and fixation anchoring adhesive illicit a chronic FBR that has all of the hallmarks described for MEAs implanted in brain tissue. Moreover, results show evidence of persistent neuroinflammation below a variety of fixation screws including chronic macrophage activation, demyelination, and neural tissue loss. Understanding the FBR of fixation techniques, which is common to a wide variety of CNS devices, may improve the biocompatibility of existing devices and provide a reference for future biologically-informed device designs

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs

Proteomic analysis of biomarkers associated with immunotherapy in murine tumour models

Emergence of proteomics and high-throughput technologies has allowed the identification of protein expression patterns of disease that potentially hold clinical importance in predictive medicine. The analysis of complex data generated by these technologies incorporates the use of computer algorithms for data mining and identification of important protein biomarkers. Such candidate biomarkers can potentially be used for diagnosis, prognosis and monitoring a variety of diseases as well as the prediction of therapy response. Mass spectrometry has been used widely, for the discovery and quantitation of disease associated biomarkers using a variety of samples such as serum and tissue. In particular, matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) has been used to generate proteomic profiles or “fingerprints” from serum to distinguish patients at different clinical stages of disease. Currently, early stage disease is difficult to diagnose in most cancers as current cancer markers have limited sensitivity and specificity. In advanced stage metastatic disease, treatment options are limited, although it is recognised that some patients may benefit from immunotherapy and in particular vaccine therapy. The use of animal models is critical to evaluate the efficacy of immunotherapies and to investigate tumour immunity in general and the mechanisms involved in tumour progression. These models provide an in vivo environment which cannot be reproduced in vitro, which results in more accurate and reliable information on the host response to immunotherapy and the mechanisms involved

Design with the Desert

The modern southwestern cities of Phoenix, Tucson, Las Vegas, Albuquerque, and El Paso occupy lands that once supported rich desert ecosystems. Typical development activities often resulted in scraping these desert lands of an ancient living landscape, to be replaced with one that is human-made and dependent on a large consumption of energy and natural resources. Design with the Desert: Conservation and Sustainable Development explores the natural and built environment of the American Southwest and introduces development tools for shaping the future of the region in a more sustainable way. Explore the Desert Landscape and Ecology This transdisciplinary collaboration draws on insights from leading authorities in their fields, spanning science, ecology, planning, landscape development, architecture, and urban design. Organized into five parts, the book begins by introducing the physical aspects of the desert realm: the land, geology, water, and climate. The second part deals with the "living" and ecological aspects, from plants and animals to ecosystems. The third part, on planning in the desert, covers the ecological and social issues surrounding water, natural resource planning, and community development. Bring the Desert into the City The fourth part looks at how to bring nature into the built environment through the use of native plants, the creation of habitats for nature in urban settings, and the design of buildings, communities, and projects that create life. The final part of the book focuses on urban sustainability and how to design urban systems that provide a secure future for community development. Topics include water security, sustainable building practices, and bold architecture and community designs. Design Solutions That Work with the Local Environment This book will inspire discussion and contemplation for anyone interested in desert development, from developers and environmentalists to planners, community leaders, and those who live in desert regions. Throughout this volume, the contributors present solutions to help promote ecological balance between nature and the built environment in the American Southwest—and offer valuable insights for other ecologically fragile regions around the world

Characterisation of styrene-maleic acid-solubilised G protein-coupled receptors

Detergents have historically been used to solubilise membrane proteins for structural studies and pharmacological research, however detergents can alter the lipid environment surrounding a membrane protein. The styrene-maleic acid (SMA) copolymer has been designed to solubilise membrane proteins from the cell membrane, with the lipid bilayer intact, thus forming styrene-maleic acid lipid particles (SMALP). This would retain the native conformation of the protein and therefore suitable for applications such as drug discovery. In this project, the adenosine 2A receptor (A2AR) and the calcitonin gene-related peptide receptor (CGRPR) were solubilised into SMALPs. Various techniques were used to characterise the SMA-solubilised receptors.Out of the SMA copolymers tested, SMA2000 was chosen as the copolymer to solubilise the GPCRs. The copolymer was also compared with the new diisobutylene-maleic acid (DIBMA) copolymer, which has better resistance to divalent cations than the SMA copolymer. Molecular techniques confirmed the expression of the GPCRs in membranes and after solubilisation into SMALPs. Radioligand binding assays demonstrated that the A2AR retained its binding capability when solubilised and purified. The binding assay showed that the A2AR was more stable in SMALPs than DIBMA lipid particles (DIBMALP). Various techniques were used to characterise the A2AR-SMALP, providing novel properties of GPCRs in SMALP. The x-ray radiolytic footprinting (XRF) was used to detect regions of the GPCR-SMALP which were exposed to hydroxyl modification. The transmembrane domain, and the intracellular surface of the SMA-solubilised A2AR were exposed to water, demonstrating the SMALPs can successfully be used in XRF. Fluorescence correlation spectroscopy (FCS) was implemented to characterise the pharmacology of a single ligand binding to a single receptor, where the pharmacological profile of the A2AR was successfully characterised when in a SMALP.SMALPs were also tested for their applicability in phage display in order to generate GPCR-specific nanobodies against receptors in their native conformation. The M13 phage used in this project were conjugated with a VHH nanobody. The A2AR-SMALP was immobilised onto ELISA plates for phage binding, where approximately 22% of the total phage were A2AR-SMALP specific, which was lower than the control Fab protein. Avi-tagged A2AR and CGRPR constructs were designed to improve the immobilisation of the SMALPs, to yield a higher enrichment of phage, specific to the GPCR of interest.Finally, the photoaffinity cross-linking assay was implemented in this project, which has potential implications in drug discovery as receptors can be locked into a particular conformation when cross-linked with a ligand. The technique can theoretically be applied to receptors in SMALPs. Residues of the extracellular loops 1 and 3 of the CGRPR were studied. The assay showed residues A199, N200 and N201 of the extracellular loop 1 forming crosslinks with the ligand, when substituted with azidophenylalanine. Overall, the project demonstrated techniques which are applicable to study SMA-solubilised receptors. Using the various techniques revealed novel properties of the GPCRs in SMALPs. SMALPs were also applied to the drug discovery technique, phage display, with limited success. Techniques were incorporated into this study to improve the applicability of SMALPs in phage display