Design and implementation of transgenic tools to visualise cell cycle progression in mammalian development

Cell cycle progression is the series of steps a cell has to take in order to duplicate its DNA and produce two daughter cells. Correct spatial and temporal coordination of the cell cycle is key for the normal development of any organ or tissue and is stringently controlled during embryogenesis and homeostasis. Misregulation of cell cycle progression is causal in many developmental disorders and diseases such as microcephaly and cancer. Fucci (Fluorescent Ubiquitination based Cell Cycle Indicator) is a system that allows for the visualisation of cell cycle progression by the use of two differently coloured fluorescent probes whose abundance is regulated reciprocally during the cell cycle. The probes contain the E3 ligase recognition domains of Cdt1 and Geminin fused to the fluorophores mCherry (red fluorescence) and mVenus (yellow fluorescence) respectively. Cells are therefore labelled red during G1, yellow in the G1/S transition and green during late S/G2 and M phases of the cell cycle. In order to study development and tissue homoeostasis a Fucci expressing mouse line was developed however this has several key limitations: First, the two Fucci probes are expressed from separate loci complicating mouse colony maintenance. Second, the constructs were not inducible, making it impossible to follow cell cycle progression in specific cell lineages and third the mice were generated by random transgenesis which is prone to silencing and can exhibit variation in expression between different tissues. Here I have characterised an improved version of the original Fucci system known as Fucci2a designed by Dr Richard Mort (University of Edinburgh) to overcome these limitations. The Fucci2a genetic construct contains both Fucci probes fused with the Thosea asigna virus self-cleaving peptide sequence T2A. This allows expression of both probes as a single bicistronic mRNA with subsequent cleavage by ribosomal ‘skipping’ during translation to yield separate proteins. A Fucci2a mouse (R26Fucc2aR) was generated by homologous recombination into the ROSA26 locus using the strong, ubiquitous CAG promoter to drive expression and incorporating a floxed-Neo stop cassette. This allows tissue specific activation by Cre recombinase when combined with a second Cre expressing mouse line. Building on the bicistronic Fucci2a technology I have gone on to develop and characterise four new tricistronic reporter constructs which allow for the dual visualisation of cell cycle progression with apoptosis, cytokinesis and ciliogenesis. In each case an additional fluorescent probe was added to the original Fucci2a construct separated by the self-cleaving peptide P2A and the construct characterised in 3T3 stable cell lines. The combination of a dual cilia and cell cycle reporter construct proved fruitful and I have gone on to investigate the relationship between cell cycle progression and ciliogenesis in 3T3 cells and have generated and characterised the R26Arl13b-Fucci2aR mouse line. I have also illustrated the utility of the R26Fucci2aR mouse for generating quantitative data in development research in two development situations; melanocyte development and lung branching morphogenesis. Melanocytes are specialised melanin producing cells responsible for the pigmentation of the hair, skin and eyes. Their precursors, melanoblasts, are derived from the neural crest where they migrate and proliferate before becoming localised to hair follicles and their study provides a good model for understanding the development of other neural crest derived lineages such as the peripheral nervous system. Using time-lapse imaging of ex vivo skin cultures in which melanoblasts are labelled with the Fucci probes I have characterised melanoblast migration and proliferation. In addition, I have shown that Kit signalling, which is necessary for melanoblast migration and survival, controls melanoblast proliferation in a density dependent manner and that melanoblast migration is more persistent in S/G2/M phases of the cell cycle. Lung branching morphogenesis requires constant proliferation at the apical tip of a growing epithelial branch. Loss of epithelial symmetry through an unidentified mechanism (requiring BMP, FgF10, Shh and Wnt signalling) within a branch is required to initiate branching either latterly from the side of a elongating branch by domain branching or by bifurcation of the tip. In the final section of this thesis I performed a comparative analysis of the behaviour of the developing lung epithelium using proliferative status (Fucci2a expression) to categorise each cell. Using a combination of live imaging and immunohistochemistry I have identified a transition zone 100-150μm from the tip of the branching lung epithelium where epithelial cells become stationary and drop out of the cell cycle corresponding with the onset of proximal bronchial progenitor marker Sox2. A comparative gene expression analysis of the proliferating and non-proliferating regions using Fucci2a to distinguish them has eluded to several interesting genes which could influence branching morphogenesis during lung development

Initial results from an IPv6 Darknet

A darknet is an advertised and routed portion of Internet address space that contains no advertised services. Any traffic observed on a darknet is therefore illegitimate and darknets are useful tools for observing the level of background ‘noise’ on a larger network. Darknets have been used in existing IPv4 networks to help to identify malicious traffic, malware trends, or the consequences of misconfiguration. We have created what may be the world’s first IPv6 darknet to help us observe the ‘noise’ present on the IPv6 Internet and to see how this differs from the IPv4 Internet. Initial results suggest that the level of undirected malicious software active on the IPv6 Internet is currently minimal and there is no apparent undirected port-scanning activity. We suspect this is partially a (predicted) consequence of the larger IPv6 address space and also an indication of the immaturity of the IPv6 Internet at the present time

Initial results from an IPv6 Darknet

A darknet is an advertised and routed portion of Internet address space that contains no advertised services. Any traffic observed on a darknet is therefore illegitimate and darknets are useful tools for observing the level of background ‘noise’ on a larger network. Darknets have been used in existing IPv4 networks to help to identify malicious traffic, malware trends, or the consequences of misconfiguration. We have created what may be the world’s first IPv6 darknet to help us observe the ‘noise’ present on the IPv6 Internet and to see how this differs from the IPv4 Internet. Initial results suggest that the level of undirected malicious software active on the IPv6 Internet is currently minimal and there is no apparent undirected port-scanning activity. We suspect this is partially a (predicted) consequence of the larger IPv6 address space and also an indication of the immaturity of the IPv6 Internet at the present time

The Global academic research organization network: Data sharing to cure diseases and enable learning health systems.

Introduction:Global data sharing is essential. This is the premise of the Academic Research Organization (ARO) Council, which was initiated in Japan in 2013 and has since been expanding throughout Asia and into Europe and the United States. The volume of data is growing exponentially, providing not only challenges but also the clear opportunity to understand and treat diseases in ways not previously considered. Harnessing the knowledge within the data in a successful way can provide researchers and clinicians with new ideas for therapies while avoiding repeats of failed experiments. This knowledge transfer from research into clinical care is at the heart of a learning health system. Methods:The ARO Council wishes to form a worldwide complementary system for the benefit of all patients and investigators, catalyzing more efficient and innovative medical research processes. Thus, they have organized Global ARO Network Workshops to bring interested parties together, focusing on the aspects necessary to make such a global effort successful. One such workshop was held in Austin, Texas, in November 2017. Representatives from Japan, Taiwan, Singapore, Europe, and the United States reported on their efforts to encourage data sharing and to use research to inform care through learning health systems. Results:This experience report summarizes presentations and discussions at the Global ARO Network Workshop held in November 2017 in Austin, TX, with representatives from Japan, Korea, Singapore, Taiwan, Europe, and the United States. Themes and recommendations to progress their efforts are explored. Standardization and harmonization are at the heart of these discussions to enable data sharing. In addition, the transformation of clinical research processes through disruptive innovation, while ensuring integrity and ethics, will be key to achieving the ARO Council goal to overcome diseases such that people not only live longer but also are healthier and happier as they age. Conclusions:The achievement of global learning health systems will require further exploration, consensus-building, funding aligned with incentives for data sharing, standardization, harmonization, and actions that support global interests for the benefit of patients

Working Paper 1 : Electricity storage and electric vehicles

The purpose of this review is to analyse the evidence and gaps in the policy and regulatory landscape of (smart) local energy systems in the UK

Working Paper 3 : Decarbonisation of heat : how smart local energy systems can contribute

This review on how Smart Local Energy Systems can contribute to the decarbonisation of heat. The purpose of the review is to analyse current policy, regulation and market structures in the UK, and use the evidence to identify gaps and barriers to the emergence and success of SLES

NLTT 41135: a field M-dwarf + brown dwarf eclipsing binary in a triple system, discovered by the MEarth observatory

We report the discovery of an eclipsing companion to NLTT 41135, a nearby M5 dwarf that was already known to have a wider, slightly more massive common proper motion companion, NLTT 41136, at 2.4 arcsec separation. Analysis of combined-light and radial velocity curves of the system indicates that NLTT 41135B is a 31-34 +/- 3 MJup brown dwarf (where the range depends on the unknown metallicity of the host star) on a circular orbit. The visual M-dwarf pair appears to be physically bound, so the system forms a hierarchical triple, with masses approximately in the ratio 8:6:1. The eclipses are grazing, preventing an unambiguous measurement of the secondary radius, but follow-up observations of the secondary eclipse (e.g. with the James Webb Space Telescope) could permit measurements of the surface brightness ratio between the two objects, and thus place constraints on models of brown dwarfs.Comment: 15 pages, 6 figures, 10 tables, emulateapj format. Accepted for publication in Ap

Five Kepler target stars that show multiple transiting exoplanet candidates

We present and discuss five candidate exoplanetary systems identified with the Kepler spacecraft. These five systems show transits from multiple exoplanet candidates. Should these objects prove to be planetary in nature, then these five systems open new opportunities for the field of exoplanets and provide new insights into the formation and dynamical evolution of planetary systems. We discuss the methods used to identify multiple transiting objects from the Kepler photometry as well as the false-positive rejection methods that have been applied to these data. One system shows transits from three distinct objects while the remaining four systems show transits from two objects. Three systems have planet candidates that are near mean motion commensurabilities---two near 2:1 and one just outside 5:2. We discuss the implications that multitransiting systems have on the distribution of orbital inclinations in planetary systems, and hence their dynamical histories; as well as their likely masses and chemical compositions. A Monte Carlo study indicates that, with additional data, most of these systems should exhibit detectable transit timing variations (TTV) due to gravitational interactions---though none are apparent in these data. We also discuss new challenges that arise in TTV analyses due to the presence of more than two planets in a system.Comment: Accepted to Ap