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You searched for +publisher:"University of Manchester" +contributor:("FORD, ROBERT RC"). Showing records 1 – 3 of 3 total matches.

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University of Manchester

1. Ramsay, Ewan. Structural and Mutational Characterisation of Human Retinoschisin.

Degree: 2017, University of Manchester

X-Linked Retinoschisis (XLRS) is a currently incurable, progressive retinal degeneration that affects approximately 1:20,000 males. Sufferers have a loss of retinal structure and visual acuity, leading to blindness. The condition is caused by mutation of the RS1 gene encoding the retinal-specific protein retinoschisin. Retinoschisin is critical in maintaining the normal, ordered retinal architecture, with deletion in mice models leading to loss of both structure and visual processing, analogous to XLRS sufferers. However, re-introduction of retinoschisin using adeno-associated viral vectors leads to complete rescue in these models. Despite the importance of retinoschisin in maintaining retinal architecture, the mechanism by which it maintains this structure remains unknown. As a result, this study aimed to structurally characterise retinoschisin and XLRS-associated point mutants R141H and H207Q to gain insight into the mechanism of retinoschisin action. To this end, retinoschisin was expressed and purified from HEK 293-EBNA cells and the structure of both monomeric and octameric retinoschisin was investigated using Small-Angle X-Ray Scattering (SAXS) and Cryo-electron microscopy (Cryo-EM). Monomeric retinoschisin was found to adopt an elongated structure that allowed for the tight association of the subunits into a planer propeller structure. However, in solution conditions the octamer also stably self-assembled into a dimer of octamers, for which the structure was solved using cryo-EM. This allowed for construction of a quasi-atomic model, enabling mapping of XLRS-associated point mutations on the complex. Two major classes of mutation were identified, in the intra-octamer and inter-octamer interfaces, suggesting a mechanism of pathology for these mutants. Observation of clustered conservative mutations at the inter-octamer interface suggested the dimer of octamers may be physiologically relevant. Furthermore, comparison of the R141H mutant to the wild-type revealed an additional mutated site in the propeller tips. Here, R141H was suggested to induce a small conformational change and alter an interaction site. Another mutant, H207Q, however, induced a destabilization of the assembled retinoschisin molecule.In conclusion, we purified and structurally characterised human retinoschisin, identifying a new hexadecameric oligomer. The structure of this allowed for identification of distinct classes of mutations on the assembled molecule and a hypothesis of the mechanism of retinoschisin action in the retina. Advisors/Committee Members: FORD, ROBERT RC, Ford, Robert, Baldock, Clair.

Subjects/Keywords: Retinoschisin; X-Linked Retinoschsis; Cryo-EM; Small Angle X-Ray Scattering

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Ramsay, E. (2017). Structural and Mutational Characterisation of Human Retinoschisin. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:306992

Chicago Manual of Style (16th Edition):

Ramsay, Ewan. “Structural and Mutational Characterisation of Human Retinoschisin.” 2017. Doctoral Dissertation, University of Manchester. Accessed September 21, 2020. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:306992.

MLA Handbook (7th Edition):

Ramsay, Ewan. “Structural and Mutational Characterisation of Human Retinoschisin.” 2017. Web. 21 Sep 2020.

Vancouver:

Ramsay E. Structural and Mutational Characterisation of Human Retinoschisin. [Internet] [Doctoral dissertation]. University of Manchester; 2017. [cited 2020 Sep 21]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:306992.

Council of Science Editors:

Ramsay E. Structural and Mutational Characterisation of Human Retinoschisin. [Doctoral Dissertation]. University of Manchester; 2017. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:306992

2. Hughes, Gareth William. INVESTIGATING THE SUPRAMOLECULAR ORGANISATION OF RESPIRATORY MUCINS IN HEALTH AND DISEASE.

Degree: 2018, University of Manchester

The polymeric mucins, MUC5B and MUC5AC provide the structural and functional backbone of the respiratory mucus barrier, conferring both viscoelastic and antimicrobial properties onto these complex secretions. Within the mucus barrier, polymeric mucins are believed to be highly organised, arranged in a network-like formation. Structural details regarding how mucins are packaged, secreted and expand to form a functional barrier remain elusive. In addition, despite recent investigations into mucin secondary structure, it is unclear as to how the heavy glycosylation found within mucin domains can influence polymer conformation. Through the use of electron microscopy techniques in combination with biophysical analyses we performed an in depth structural analysis on purified MUC5B and MUC5AC, we then utilised these same techniques to investigate mucin packaging and expansion in response to changes in calcium concentration and pH levels. Finally through structural investigations into the native organisation of the salivary mucus barrier we aimed to investigate the hierarchical mucin topology present within mucosal systems, thus enabling us to draw conclusions about how this organisation may be compromised in disease states such as Cystic Fibrosis (CF). Through transmission electron microscopy (TEM) combined with single particle analysis, we provide evidence to suggest that MUC5B contains bead-like structures, which repeat along the polymer axis and suggest that MUC5B may consist of repeating motifs derived from distinct glycosylation patterns, rather than the disorganised random coil conformation previously assumed. Moreover, through TEM and rate zonal centrifugation, we demonstrate that respiratory mucins form highly entangled linear polymers, the structures of which are sensitive to calcium concentration and fluctuations in pH. In the presence of Ca2+ at pH 5.0, MUC5B adopted a compact conformation which was lost upon removal of calcium, via EGTA, and through increasing the pH to 7.4. We further suggest that the expansion of mucins following secretion occurs via two-step process, with an initial rapid expansion followed by a slower protease-independent maturation process, which is driven by distinct changes to the mucin interactome. Through isolation of mucins derived from CF sputum we have assessed the structural and biophysical changes that occur to the mucin network during CF progression, we suggest that the CF mucin network is severely degraded, with mucin glycopeptides predominating. Furthermore, we suggest that poly (acetyl, arginyl) glucosamine (PAAG) may represent an attractive treatment for CF lung disease, here we highlight its ability to directly bind MUC5B and induce its linearisation, resulting in a less viscous mucin network. Here we provide novel insights into the structural organisation of MUC5B and MUC5AC on both intra- and intermolecular levels, and shed light onto the processes required for mucin packaging and expansion, which have allowed us to understand the mechanisms behind aberrant mucus transport… Advisors/Committee Members: FORD, ROBERT RC, Thornton, David, Ford, Robert.

Subjects/Keywords: Mucus; Mucin; MUC5B; MUC5AC; Cystic Fibrosis; Electron Microscopy; Saliva

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Hughes, G. W. (2018). INVESTIGATING THE SUPRAMOLECULAR ORGANISATION OF RESPIRATORY MUCINS IN HEALTH AND DISEASE. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312990

Chicago Manual of Style (16th Edition):

Hughes, Gareth William. “INVESTIGATING THE SUPRAMOLECULAR ORGANISATION OF RESPIRATORY MUCINS IN HEALTH AND DISEASE.” 2018. Doctoral Dissertation, University of Manchester. Accessed September 21, 2020. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312990.

MLA Handbook (7th Edition):

Hughes, Gareth William. “INVESTIGATING THE SUPRAMOLECULAR ORGANISATION OF RESPIRATORY MUCINS IN HEALTH AND DISEASE.” 2018. Web. 21 Sep 2020.

Vancouver:

Hughes GW. INVESTIGATING THE SUPRAMOLECULAR ORGANISATION OF RESPIRATORY MUCINS IN HEALTH AND DISEASE. [Internet] [Doctoral dissertation]. University of Manchester; 2018. [cited 2020 Sep 21]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312990.

Council of Science Editors:

Hughes GW. INVESTIGATING THE SUPRAMOLECULAR ORGANISATION OF RESPIRATORY MUCINS IN HEALTH AND DISEASE. [Doctoral Dissertation]. University of Manchester; 2018. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312990

3. Rodzli, Nazahiyah Binti Ahmad. STRUCTURAL AND INTERACTION STUDIES OF PSD95 PDZ DOMAIN-MEDIATED KIR2.1 CLUSTERING MECHANISMS.

Degree: 2017, University of Manchester

PSD95 is the canonical member of the Membrane Associated Guanylate Kinase class of scaffold proteins. PSD95 is a five-domain major scaffolding protein abundant in the postsynaptic density (PSD) of the neuronal excitatory synapse. Within PSD95 three PDZ domains modulate protein-protein interactions by selectively binding to short peptide motifs of target proteins. Under the direction of the multivalent PDZ domain interactions, the interacting proteins tend to cluster at the PSD, a phenomenon that is critical for synaptic signalling regulation. Earlier studies have shown that the N-terminal PDZ domains of PSD95 are obligatory for the clustering to occur. This thesis focuses on the strong inwardly rectifying potassium channel, Kir2.1 as the PSD95 binding partner. Kir2.1 is known to maintain membrane resting potential and control cell excitability. Previous studies have reported that Kir2.1 clustered into ordered tetrad complexes upon association with PSD95.This study investigates the detailed clustering mechanisms of Kir2.1 by PDZ domains. To achieve this, components that are involved in the formation of a complex namely PSD95 sub-domains comprising single PDZ and the tandem N terminal PDZ double domain (PDZ1–2), and Kir2.1 cytoplasmic domains(Kir2.1NC) are studied in detail via different structural and biophysical approaches; 1) PDZ1–2 is examined in apo- and bound ligand form with a Kir2.1 Cterminal peptide in crystal and solution via X-ray crystallography and small angle X-ray scattering; 2) the tandem and the single PDZ domain interaction with ligand are measured thermodynamically via isothermal calorimetry (ITC); 3) the complex of full length PSD95 with Kir2.1NC is analyzed with electron microscopy (EM). The protein components are produced in high quality by protein expression and multiple-step protein purification techniques. PDZ1–2 crystallographic structures were solved at 2.02Å and 2.19Å in theapo- and the liganded forms respectively. The solution state analysis showed domain separation and structural extension of the tandem domain when incorporated with the ligand. The ITC experiment revealed PDZ1–2 to have greater affinity towards the peptide ligand relative to the single PDZ domains. These combinatorial outcomes lead to the conclusion that PSD95 clusters Kir2.1 by adopting an enhanced binding interaction which is associated with increased PDZ1–2 inter-domain separation. The preliminary analysis of PSD95-Kir2.1NC complex with cryo-EM showed the establishment of a tetrad and led to a reconstruction at 40Å resolution. The work in obtaining a higher resolution complex structure is promising with further data collection required to allow the employment of more sophisticated model reconstruction processes. Advisors/Committee Members: FORD, ROBERT RC, Ford, Robert, Prince, Steve.

Subjects/Keywords: PDZ domain, PSD95; Inwardly rectifying potassium channel, Kir; X-ray crystallography, SAXS, ITC, cryoEM

…screwrotation TM Transmembrane domain 19 THE UNIVERSITY OF MANCHESTER Candidate : Nazahiyah… …given The University of Manchester certain rights to use such Copyright, including for… 

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Rodzli, N. B. A. (2017). STRUCTURAL AND INTERACTION STUDIES OF PSD95 PDZ DOMAIN-MEDIATED KIR2.1 CLUSTERING MECHANISMS. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:308198

Chicago Manual of Style (16th Edition):

Rodzli, Nazahiyah Binti Ahmad. “STRUCTURAL AND INTERACTION STUDIES OF PSD95 PDZ DOMAIN-MEDIATED KIR2.1 CLUSTERING MECHANISMS.” 2017. Doctoral Dissertation, University of Manchester. Accessed September 21, 2020. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:308198.

MLA Handbook (7th Edition):

Rodzli, Nazahiyah Binti Ahmad. “STRUCTURAL AND INTERACTION STUDIES OF PSD95 PDZ DOMAIN-MEDIATED KIR2.1 CLUSTERING MECHANISMS.” 2017. Web. 21 Sep 2020.

Vancouver:

Rodzli NBA. STRUCTURAL AND INTERACTION STUDIES OF PSD95 PDZ DOMAIN-MEDIATED KIR2.1 CLUSTERING MECHANISMS. [Internet] [Doctoral dissertation]. University of Manchester; 2017. [cited 2020 Sep 21]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:308198.

Council of Science Editors:

Rodzli NBA. STRUCTURAL AND INTERACTION STUDIES OF PSD95 PDZ DOMAIN-MEDIATED KIR2.1 CLUSTERING MECHANISMS. [Doctoral Dissertation]. University of Manchester; 2017. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:308198

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