The retinal explant as a model to investigate neuronal pathology and neuroprotective strategies

Despite the association of neuronal cell loss with a wide range of neurodegenerative disorders, the mechanisms leading to this cell death remain poorly understood. In this thesis I have investigated these mechanisms and tested whether they represent viable targets for therapeutic intervention. The adult mouse retinal explant is a popular model of axotomy-induced neuronal degeneration but has been limited by the lack of morphometric data. Since dendritic pruning is well-evidenced to precede cell loss in neurodegenerative diseases, including glaucoma and Alzheimer’s disease, I investigated whether the quantification of dendritic morphology of retinal ganglion cells in the retinal explant could be used as a more sensitive measure of neuronal health after axotomy. I report here that retinal ganglion cell dendrite loss precedes cell loss by at least 7 days and that this retraction is substantially retarded following treatment with brain-derived neurotrophic factor applied at the time of explantation. Perhaps most importantly, I demonstrate for the first time in this model that delayed application of brain-derived neurotrophic factor significantly protects against dendritic retraction of retinal ganglion cells. The work outlined in this thesis thus supports the targeting of dendritic outgrowth and/or synaptic connectivity for the treatment of neurodegenerative disorders

Sholl analysis: A quantitative comparison of semi-automated methods

Background: Sholl analysis remains one of the most commonly used methods to quantify neuronal dendritic complexity and is therefore a key analysis tool in neurobiology. While initially proposed when the quantification of neuronal structure was undertaken manually, the advent of software packages allowing automated analysis has resulted in the introduction of several semi and fully automated methods to quantify dendritic complexity. Unfortunately results from these methods have not in all cases been consistent. We therefore compared the results of five commonly used methods (Simple Neurite Tracer, manual, Fast Sholl, Bitmap, and Ghosh lab) using manual analysis as a ground truth. New method: Comparison of four semi-automated methods to the manual method using diolistically labelled mouse retinal ganglion cells. Results: We report consistency across a range of published techniques. While the majority perform well (Simple Neurite Tracer and Fast Sholl profiles have areas under the curve within 4.5% of the profile derived using the manual method), we highlight two areas in two of the methods (Bitmap and Ghosh lab methods) where errors may occur, namely undercounting (>20% relative to the manual profile) and a second peak. Comparison with existing methods: Our results support published validation of the Fast Sholl method. Conclusions: Our study highlights the importance of manual calibration of automated analysis software

Flow heterogeneity in the fractured Chalk aquifer of southern England

The aim of the current work is to investigate the heterogeneity of flow in the Chalk aquifer of southern England. The rock mass properties and hydraulic characteristics of the aquifer have been characterised using a suite of geological and geophysical surveys and hydraulic tests. Fracture logs have been produced based on core logging and using optical images of boreholes. Flow has been characterised using borehole flow logs and dilution tests and hydraulic conductivity measured using packer tests. Fractures have been recorded with apertures in the range <1mm to about 30cm (sub-karstic enlargement) in diameter, however, hydrogeologically significant flow is not restricted to the enlarged fractures and is affected by the local groundwater head distribution. The work is being undertaken as part of the LOCAR Programme. LOCAR is a multi-project programme with the aim of measuring and modelling processes controlling water and material fluxes within lowland permeable catchments in the UK

Fully human agonist antibodies to TrkB using autocrine cell-based selection from a combinatorial antibody library

The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation

Genomic reconstruction of the SARS-CoV-2 epidemic in England

AbstractThe evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021.</jats:p