Simulation and Analysis of Stimulus Evoked and Seizure-like Activity in an Acute Rat Neocortical Brain Slice Preparation

Ph. D. ThesisThis thesis aims to provide tools for the simulation and analysis of acute brain slice experiments that have recorded spontaneous seizure-like activity and activity evoked using electric eld stimulation. The Virtual Electrode Recording Tool for Extracellular Potentials (VERTEX) (Tomsett et al., 2015) is a simulation framework that can act as a sca old for anatomical and physiological knowledge and can be used to test how interventions a ect the dynamics observed in the extracellular potentials. We extend VERTEX so that one can model a greater range of experimental setups, in particular those that involve electric eld stimulation. We also devise a software pipeline for the identi cation and analysis of epileptiform neuronal activity, which we apply to in vitro recordings from acute neocortical slices from a chronic model of epilepsy in rat. In Chapter 1 we look at the intersection of electric eld stimulation, synaptic plasticity and epilepsy. In Chapter 2 we look at the implementation of electric eld stimulation that we have added to the VERTEX simulator. We show that our simulation compares well with simulations using detailed neuron models, and with previously published in vitro data. We also describe our implementations of short term plasticity and spike-timing dependent plasticity. In Chapter 3 we describe some example simulations of focal electric eld stimulation in neocortex, investigating a single pulse of stimulation, a paired pulse of stimulation and the role of short term plasticity in the response. We also use theta burst stimulation to provoke a potentiation of the response when we apply spike-timing dependent plasticity to the synapses of the network. We then look to the experimental context of our framework. Chapter 4 describes an analysis tool for identifying and evaluating epileptiform activity recorded in vitro and outlines a method devised to measure the abruptness of seizure build up. Chapter 5 uses this analysis pipeline to analyse seizure-like events recorded in vitro from slices of motor cortex prepared from rats with chronic seizures induced by injection of tetanus toxin. In this chapter we also describe two VERTEX simulations; one that uses short term plasticity as the vehicle for the breakdown in inhibition and the build up of activity during a seizure-like event, and another of stimulus evoked activity in a seizure-prone neocortical slice. We compare the latter with stimulus evoked potential in an example in vitro multi-electrode array recording from the chronic epilepsy model. In Chapter 6 we discuss the future uses of VERTEX in modelling stimulus evoked activity and epileptiform activity

Production of more stable induced pluripotent stem cells using the Doggybone (dbDNA) vector.

The application of induced pluripotent stem cell (iPSC)-derived cells in clinical trials is in its infancy but the potential is vast. A key asset of iPSCs is the ability to derive autologous cell therapies, but to date most current or approved clinical trials are using fully characterized allogeneic or non-allogeneic cell banks alongside immunosuppressive drugs. Until now, all current or approved clinical trials utilize iPSC generated using EBNA1 expressing plasmids containing the OriP sequence to maintain a self-replicating episome. These vectors are amplified in bacterial hosts and contain bacterial DNA motifs recognized by the transfected cells innate and intrinsic interferon host defense responses. Moreover, the continued forced expression of the Epstein-Barr virus EBNA1 protein is known to cause widespread alterations in gene expression as well as elevated oxidative stress and DNA damage occurrence. Additionally, this method of iPSC derivation incorporates a short-hairpin RNA (shRNA) for the p53 protein; often referred to as the guardian of the genome. The shRNA functions to transiently silence the expression of p53 protein and has been demonstrated to result in an increased persistence of DNA damage in iPSC produced this way. All of these factors have significant implications for the safe clinical use of iPSC generated using oriP/EBNA1 plasmid episomes. The aim of my project was to investigate the function of a novel system in reprogramming and iPSC development. Doggybone DNA (dbDNA) vectors are free of oriP/EBNA1 sequences, bacterial motifs and are produced in a chemically defined, low endotoxin, cGMP compliant manufacture. My results describe efficient iPSC reprogramming by applying equivalent gene sequences transiently expressed from dbDNA vectors in protocols employing both animal-derived and animal-free constituents when using weight equivalents of both systems and not molecular equivalents. In direct comparator experiments with the current state-of-the-art gold standard oriP/EBNA1 episomes, dbDNA vectors produced iPSC colonies with the same efficiency but dbDNA-iPSC displayed evidence of greater stability in terms of maintenance of pluripotency. Differential transcriptomic evaluations by microarray showed that the persistence of oriP/EBNA1 episomes resulted in an elevated interaction with immune system processes and IFN signalling in iPSC when compared to dbDNA generated iPSC. Moreover, an increased susceptibility for DNA damage incitement alongside unwanted spontaneous differentiation in iPSCs incorporating the 4 oriP-EBNA1 were all demonstrated and showed to be intrinsically linked to one-and-other. We propose a potential that utilizing dbDNA vectors presents a safer and more stable approach to iPSC production and development and that this could, with further work, help to bridge the gap between iPSCs and their greater clinical translation

Are family medicine residents trained to counsel patients on physical activity? The Canadian experience and a call to action.

Physical inactivity is a leading risk factor for non-communicable diseases (NCDs) and early mortality. Family physicians have an important role in providing physical activity counselling to patients to help prevent and treat NCDs. Lack of training on physical activity counselling is a barrier in undergraduate medical education, yet little is known regarding physical activity teaching in postgraduate family medicine residency. We assessed the provision, content and future direction of physical activity teaching in Canadian postgraduate family medicine residency programs to address this data gap. Fewer than half of Canadian Family Medicine Residency Programme directors reported providing structured physical activity counselling education to residents. Most directors reported no imminent plans to change the content or amount of teaching. These results reflect significant gaps between the recommendations of WHO, which calls on doctors to prescribe physical activity, and the current curricular content and needs of family medicine residents. Almost all directors agreed that online educational resources developed to assist residents in physical activity prescription would be beneficial. By describing the provision, content and future direction of physical activity training in family medicine, physicians and medical educators can develop competencies and resources to meet this need. When we equip our future physicians with the necessary tools, we can improve patient outcomes and do our part to reduce the global epidemic of physical inactivity and chronic disease