Development of novel therapeutics and in vitro models for spinal cord injury

Spinal cord injury (SCI) is a devastating condition, which results from trauma to the cord, resulting in a primary injury response which leads to a secondary injury cascade, causing damage to both glial and neuronal cells. Following trauma, the central nervous system (CNS) fails to regenerate due to a plethora of both intrinsic and extrinsic factors. Unfortunately, these events lead to loss of both motor and sensory function and lifelong disability and care for sufferers of SCI. There have been tremendous advancements made in our understanding of the mechanisms behind axonal regeneration and remyelination of the damaged cord. These have provided many promising therapeutic targets. However, very few have made it to clinical application, which could potentially be due to inadequate understanding of compound mechanism of action and reliance on poor SCI models. This thesis describes the use of an established neural cell co-culture model of SCI as a medium throughput screen for compounds with potential therapeutic properties. A number of compounds were screened which resulted in a family of compounds, modified heparins, being taken forward for more intense investigation. Modified heparins (mHeps) are made up of the core heparin disaccharide unit with variable sulphation groups on the iduronic acid and glucosamine residues; 2-O-sulphate (C2), 6-O-sulphate (C6) and N-sulphate (N). 2-O-sulphated (mHep6) and N-sulphated (mHep7) heparin isomers were shown to promote both neurite outgrowth and myelination in the SCI model. It was found that both mHeps decreased oligodendrocyte precursor cell (OPC) proliferation and increased oligodendrocyte (OL) number adjacent to the lesion. However, there is a difference in the direct effects on the OL from each of the mHeps; mHep6 increased myelin internode length and mHep7 increased the overall cell size. It was further elucidated that these isoforms interact with and mediate both Wnt and FGF signalling. In OPC monoculture experiments FGF2 treated OPCs displayed increased proliferation but this effect was removed when co-treated with the mHeps. Therefore, suggesting that the mHeps interact with the ligand and inhibit FGF2 signalling. Additionally, it was shown that both mHeps could be partially mediating their effects through the Wnt pathway. mHep effects on both myelination and neurite outgrowth were removed when co-treated with a Wnt signalling inhibitor, suggesting cell signalling mediation by ligand immobilisation and signalling activation as a mechanistic action for the mHeps. However, the initial methods employed in this thesis were not sufficient to provide a more detailed study into the effects the mHeps have on neurite outgrowth. This led to the design and development of a novel microfluidic device (MFD), which provides a platform to study of axonal injury. This novel device is a three chamber device with two chambers converging onto a central open access chamber. This design allows axons from two points of origin to enter a chamber which can be subjected to injury, thus providing a platform in which targeted axonal injury and the regenerative capacity of a compound study can be performed. In conclusion, this thesis contributes to and advances the study of SCI in two ways; 1) identification and investigation of a novel set of compounds with potential therapeutic potential i.e. desulphated modified heparins. These compounds have multiple therapeutic properties and could revolutionise both the understanding of the basic pathological mechanisms underlying SCI but also be a powered therapeutic option. 2) Development of a novel microfluidic device to study in greater detail axonal biology, specifically, targeted axonal injury and treatment, providing a more representative model of SCI than standard in vitro models. Therefore, the MFD could lead to advancements and the identification of factors and compounds relating to axonal regeneration.

An investigation into the folding and assembly of engineered antibodies and novel antibody formats

Monoclonal antibodies and novel antibody formats are currently one of the principal therapeutic in the biopharmaceutical industry worldwide and are widely used in the treatment of autoimmune diseases and cancer. It is for this reason that the productivity and quality of antibody production requires improvement; specifically investigations into the engineering of antibodies and any issues that may arise from the production of these therapeutics. The work presented in this thesis describes an investigation into the folding and assembly of seven antibodies plus the novel antibody format FabFv. IgG is comprised of two identical HCs and two identical LCs. The folding process of immunoglobulin is controlled by the CH1 domain within the HC. The CH1 domain remains in a disordered state and is sequestered by BiP in the endoplasmic reticulum. Upon the addition of a folded CL domain, BiP is displaced, the CH1 domain is able to fold and the complete IgG protein can then be secreted from the cell. The results presented in this thesis however, have outlined an additional mechanism for the folding of the CH1 domain. We have shown that the CH1 domain is able to fold in the absence of LC resulting in the secretion of HC dimers in a VH dependent manner. The proposed mechanism for the secretion of HC dimers suggests that some VH domains can interact with each other in order to bring the CH1 domains in close proximity to enable folding to occur. As HC dimer secretion is a hindrance in antibody production, this result has highlighted an engineering target to improve antibody yield. Examination of the folding of IgG4 with the variable region A33 has revealed the inability to secrete LC dimers, cleavage of the HC during expression and secretion of HC dimers in the Fab, FabFv and full length forms. The attributes described have also been shown to be variable region dependent. This has introduced a new concept that the variable domain is important in determining the expression and secretion of antibodies and their individual chains. Pulse chase and 2D gel electrophoresis analysis of the novel antibody format FabFv has revealed that the folding and expression of the LC and HC causes multimeric species of FabFv to be secreted, as opposed to the monomeric form which is the desired therapeutic. Our hypothesis is that this process occurs via a LC dependent mechanism. The proposed hypothesis suggests that further engineering to the LC could diminish the formation and secretion of FabFv multimers. The results from these investigations can be applied to increase the productivity of therapeutics and increase the biological understanding of the domain interactions of IgG during folding, assembly and secretion.

Radiogenic isotopes in the Mediterranean Sea: water mass exchange and precessional variability during the Messinian

During the late Miocene, exchange between the Mediterranean Sea and Atlantic Ocean changed dramatically, culminating in the Messinian Salinity Crisis (MSC). Understanding Mediterranean-Atlantic exchange at that time could answer the enigmatic question of how so much salt built up within the Mediterranean, while furthering the development of a framework for future studies attempting to understand how changes may have impacted global thermohaline circulation. Due to their association with specific water masses at different scales, radiogenic Sr, Pb, and Nd isotope records were generated from various archives contained within marine deposits to endeavour to understand better late Miocene Mediterranean-Atlantic exchange. The archives used include foraminiferal calcite (Sr), fish teeth and bone (Nd), dispersed authigenic ferromanganese oxyhydroxides (Nd, Pb), and a ferromanganese crust (Pb). The primary focus is on sediments preserved at one end of the Betic corridor, a gateway that once connected the Mediterranean to the Atlantic through southern Spain, although other locations are investigated. The Betic gateway terminated within several marginal sub-basins before entering the Western Mediterranean; one of these is the Sorbas Basin, a well-studied location whose sediments have been astronomically tuned at high temporal resolution, providing the necessary age control for sub-precessional resolution records. Since the climatic history of the Mediterranean is strongly controlled by precessional changes in regional climate, the aim was to produce records at high (sub-precessional) temporal resolution, to be able to observe clearly any precessional cyclicity driven by regional climate which could be superimposed over longer trends. This goal was achieved for all records except the ferromanganese crust record. The 87Sr/86Sr isotope record (Ch. 3) shows precessional frequency excursions away from the global seawater curve. As precessional frequency oscillations are unexpected for this setting, a numerical box model was used to determine the mechanisms causing the excursions. To enable parameterisation of model variables, regional Sr characteristics, data from general circulation model HadCM3L, and new benthic foraminiferal assemblage data are employed. The model results imply that the Sorbas Basin likely had a positive hydrologic budget in the late Miocene, very different to that of today. Moreover, the model indicates that the mechanism controlling the Sr isotope ratio of Sorbas Basin seawater was not restriction, but a lack of density-driven exchange with the Mediterranean. Beyond improving our understanding of how marginal Mediterranean sub-basins may evolve different isotope signatures, these results have implications for astronomical tuning and stratigraphy in the region, findings which are crucial considering the geological and climatic history of the late Miocene Mediterranean is based entirely on marginal deposits. An improved estimate for the Nd isotope signature of late Miocene Mediterranean Outflow (MO) was determined by comparing Nd isotope signatures preserved in the deeper Alborán Sea at ODP Site 978 with literature data as well as the signature preserved in the Sorbas Basin (Ch. 4; -9.34 to -9.92 ± 0.37 εNd(t)). It was also inferred that it is unlikely that Nd isotopes can be used reliably to track changes in circulation within the shallow settings characteristic of the Mediterranean-Atlantic connections; this is significant in light of a recent publication documenting corridor closure using Nd isotopes. Both conclusions will prove useful for future studies attempting to understand changes in Mediterranean-Atlantic exchange. Excursions to high values, with precessional frequency, are also observed in the radiogenic Pb isotope record for the Sorbas Basin (Ch. 5). Widening the scope to include locations further away from the gateways, records were produced for late Miocene sections on Sicily and Northern Italy, and similar precessional frequency cyclicity was observed in the Pb isotope records for these sites as well. Comparing these records to proxies for Saharan dust and available whole rock data indicates that, while further analysis is necessary to draw strong conclusions, enhanced dust production during insolation minima may be driving the observed signal. These records also have implications for astronomical tuning; peaks in Pb isotope records driven by Saharan dust may be easier to connect directly to the insolation cycle, providing improved astronomical tuning points. Finally, a Pb isotope record derived using in-situ laser ablation performed on ferromanganese crust 3514-6 from the Lion Seamount, located west of Gibraltar within the MO plume, has provided evidence that plume depth shifted during the Pliocene. The record also suggests that Pb isotopes may not be a suitable proxy for changes in late Miocene Mediterranean-Atlantic exchange, since the Pb isotope signatures of regional water masses are too similar. To develop this record, the first published instance of laser ablation derived 230Thexcess measurements are combined with 10Be dating.

‘Mo Rùn am Fearann’ – ‘My Love is the Land’: Gaelic landscapes of the 18th and 19th centuries

The period of the 18th and 19th centuries was one of great change in the Highlands and Islands of Scotland. Improvement, the Jacobite Rebellions, and the Clearances transformed its communities and landscapes. These events have rightly been a focus of research. However, archaeological approaches have often sought simply to illustrate these processes, rather than create new narratives about life in the past. The resulting picture of the period can over-emphasise economic change whilst failing to reflect the richness and variety of everyday life. This thesis aims to suggest a new approach to the place and period, one which addresses matters often ignored in previous work. Whilst it has an archaeological sensibility, it draws on ideas from outside archaeology, such as landscape theory and on Gaelic oral tradition, an underused resource, to create a novel and broad-based approach to the period. An important part of the method is a synchronic approach that seeks to reconstruct the experience of the landscape at very particular times, engaging fully with the everyday experience of landscape rather than grand historical narratives. Two Hebridean case studies are utilised: Hiort (St Kilda) and Loch Aoineart, South Uist. Thematic discussions drawn from these landscapes are intended as critical assessments of the efficacy of the approach, as well as new narratives about life in the past in themselves. The thesis concludes by comparing the two case studies, reflecting on the merits of the approach, discussing recurrent themes in the work, and considering its wider context and implications. It is concluded that taking a novel approach to the case study landscapes can create narratives that often contrast or expand upon those produced by previous scholars, allow for a more detailed consideration of everyday life in the period, and open up new areas for archaeological enquiry. The extensive and critical use of evidence from Gaelic oral tradition is highlighted as crucial in understanding life and society in the period. The thesis questions the utility of grand historical narratives as a framework for archaeological study of post-medieval Gaeldom and suggests that our understanding of the past is best served by approaching the evidence in ways which allows for many different voices and stories from the past to emerge.