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Studying murine behaviour and extending the hippocampal place cell model to 3 dimensions
<p>In previous decades, studies focussing on hippocampal place cell activity have resorted to using 2-dimensional simulation models. I argue that such a paradigm proves to be insufficient when extending it to real-world, heavily 3D-biased, applications. As such, in this project, I propose an alternative approach to the study of place cells in which a rat’s neuronal activity is wirelessly monitored while it is allowed to freely explore a lattice maze in all directions of Cartesian space. Most importantly, I aim to show that receptive fields are of similar sizes in the horizontal and vertical directions; I also hypothesise that concatenating receptive fields (RFs) from several place cells will yield a layered organisation with inter-RF distances being larger in the x-z/y-z planes than the x-y plane. Incidentally, this study will also provide data which I hypothesise will confirm the horizontal bias model in murine behaviour proposed by Jovalekic et al. (2011).</p>
Quantifying the activity of cis-regulatory sequences in the hypothalamus using single molecule fluorescence microscopy.
<p>Every cell in the nervous system contains a programme of gene expression that not only determines its morphology and function, but also allows neurons and circuits to respond and adapt to sensory experiences. It is well document that changes in gene expression in response to these stimuli require cis-regulatory DNA sequences (enhancers), which mediate the activation and repression of specific genes. However, how a neuronal network as a whole tunes their transcriptional responses to achieve behavioural changes remain elusive. This project aims to address this by examining how the regulation of the genes arginine vasopressin (AVP) and oxytocin (OT) (neuropeptides that play a role in mammalian social behaviours) can differ between neurons and even gender, by identifying how different enhancer elements contribute to cell-type specific expression. To do this we will combine an enhancer assay with RNA single molecule fluorescence in situ microscopy (smFISH) in the hypothalamus, where these genes are primarily expressed. This will allow us to quantify the activity of specific enhancers in a given context, such as cell type and gender. The results will therefore give an indication of how information is encoded within these enhancer sequences that allows specific expression of the AVP and OT genes.</p>
Investigation of protein CoAlation in the process of apoptosis
<p>CoAlation is a novel post-translational modification to proteins whereby Coenzyme A is covalently attached to proteins. It occurs as part of the oxidative stress response as an alternative mechanism to protein glutathionylation. It is specifically a modification of enzymes involved in cellular metabolism and protects catalytic thiol groups on active site cysteine residues from irreversible damage by reactive oxygen species and reactive nitrogen species. </p> <p> </p> <p>Applying oxidizing agents to cells results in induction of apoptosis. Such agents also induce protein CoAlation. The aims of this project are to monitor induction of apoptosis in HEK293 cells in response to treatment with oxidizing agents using anti-PARP3 and anti-Caspase 3 Western blot and Fluorescence-activated Cell Sorting and to analyse the pattern of CoAlation at different stages of apoptosis using anti-Coenzyme A Western blot.</p>
Developing gene therapy for the inherited childhood epilepsy, Dravet Syndrome
<p>Dravet syndrome is a rare, incurable epilepsy which affects young children. Before two years of age they have seizures, incoordination and cognitive impairment. They carry mutations in the SCN1A gene, which codes for voltage-gated sodium channel Na<sub>V</sub>1.1. This protein is expressed in hippocampal inhibitory interneurons.</p> <p> </p> <p>Gene therapy offers several advantages over conventional drugs. It is a treatment which targets the cause of the disease by delivering the corrected copies of the SCN1A directly to brain cells.</p> <p> </p> <p>Our hypothesis is that we can incorporate a promoter to achieve persistent expression specifically in inhibitory interneurons of the hippocampus. We aim to compare two novel promoters against a pan-neuronal promoter, synapsin. The first is a truncated endogenous promoter, Gad67. The second is a synthetic promoter identified by <em>in silico</em> analysis.</p> <p> </p> <p>Before the project commences, we will clone these two promoters into lentiviral vectors. These, and a synapsin vector, will be injected intracranially into neonatal mice. At the start of the project the brains from four mice will be co-stained with antibodies directed against GFP and inhibitory interneurons to assess colocalisation. To measure persistence of expression from these promoters, the four remaining mice will be subject to whole-body bioluminescence imaging twice-weekly.</p>
Optimisation of carrier materials for the delivery of olfactory ensheathing cells in spinal cord injury
<p>Transplant-mediated repair is a promising method in spinal cord injury (SCI) treatment. This involves transplanting therapeutic cells that promote nerve regeneration at the site of injury. For SCI, one promising therapeutic cell type is olfactory ensheathing cells (OECs). These have been shown to remyelinate demyelinated axons and promote new synapses following injury. They are also easily accessible clinically via trans-nasal endoscopic biopsy, and compelling pre-clinical evidence means that they are now close to being formally tested as part of a first-in-man clinical trial. However, currently these cells are delivered as a simple cell suspension, and this is unlikely to be optimal for creating a permissive and optimised repair environment. </p> <p>Thus, the objective of this project will be to develop and engineer optimised biomaterial scaffolds for OEC delivery. In doing so, it is hoped that a permissive 3D extracellular environment can be created, and the phenotype and behaviour of OECs optimised for spinal cord repair. Promising prospective biomaterials include fibrin, collagen and collagen-fibrin blends. To this end, we will investigate the effect of these promising carrier materials on OEC survival and phenotype, particularly with a focus on changes they may cause on 3D cell morphology.</p>
Developing a behavioural task for measuring the ability of listeners to perform auditory scene analysis.
<p>The auditory brain separates simultaneous sounds arriving at the ear into identifiable and localisable sources by a process known as Auditory Scene Analysis (ASA). The two steps that are involved in ASA are i) segregation of the simultaneous auditory information and ii) the integration of the sounds from the same source into one stream. To understand how these two steps are connected and how different auditory cues interact to shape the scene, this project will develop a behavioural task and analyse the performance of human listeners. A target vowel will be presented alongside with a distractor vowel, and human listeners will identify what the target is. Listeners will only be able to identify the target if they can separate the two sounds: changing the location and pitch of target and distractor will help this. In order find out whether the separation of competing sounds is facilitated by the formation of perceptual streams, the vowels will also be presented as part of a sound sequence. Our hypothesis is that the ability to identify a target vowel will be improved by the formation of two perceptual streams. The long-term goal is to develop a behavioural paradigm suitable for humans and animals.</p>
Up and down regulation of MafB gene in the developing chick hindbrain.
<p>The project's aim is to up and down regulate MafB gene, that is expressed in the Nucleus Laminaris (NL) and Nucleus Angularis (NA), in the developing chick hindbrain and ask questions about:</p> <p>1) formation of nucleus Laminaris and nucleus Angularis in the dorsal hindbrain;</p> <p>2) other effects on hindbrain development e.g interfering with fgf8 molecule expression, which in turn would affect the development of the cranial motor nerves VI, VII and nVIa.</p> <p>Such experimental techniques as <em>in </em><em>ovo</em> electroporation, immunofluorescence and <em>in situ</em> hybridization will be used to look at the genes expressed in the auditory brainstem. The<em> in </em><em>ovo</em><em> </em>electroporation constructs used will overexpress MafB and also express a dominant negative version of MafB and immunofluoresce analysis will be carried out to test whether the electroporation was successful. The <em>in situ </em>hybridization analysis will be performed to establish the effect of MafB on the expression of such genes like FGF8, Pou6F2, N-cadherin, gamma catenin, cadherin-13 and cadherin-22 in the hindbrain. These techniques would also allow the analysis of the formation of the nucleus Laminaris in the developing hindbrain.</p>
Identifying Integrative and Conjugative Elements using DLIGHT
<p>Integrative and conjugative elements (ICEs) are mobile genetic elements present in both gram-positive and gram-negative bacteria. They mostly reside in the host chromosome and under certain conditions, will excise and transfer to a new host via the conjugation machinery. ICEs have been found to provide the host with a wide range of phenotypes, including antibiotic and heavy metal resistance and the ability to colonise a eukaryotic host, promote virulence and biofilm formation. The ability of ICE to spread to different species of bacteria through horizontal gene transfer is a major factor in bacterial evolution. Bioinformatics approaches have been increasingly used to identify possible ICEs through sequence similarity. In this project, we aim to find out the effectiveness of using an algorithm, DLIGHT (Distance Likelihood based Inference of Genes Horizontally Transferred) that was originally used to detect lateral gene transfer, to identify integrative and conjugative elements. We will achieve this by assessing DLIGHT's ability to recover already documented ICEs. We will also use DLIGHT to test certain sequences which we suspect to contain ICEs. The predictions of new ICEs will then be vetted through manual analysis and collaboration with experimentalists.</p>
Investigating the sleep modulating effect of oxytocin in zebrafish models of autism.
<p>In this project I will test the hypothesis that oxytocin expression and development of oxytocin-expressing neurons are altered in zebrafish with mutations in the ASD risk genes <em>cntnap2</em> and <em>chd8</em>. I hope to find evidence for the sleep modulating effects of oxytocin, and posit whether deficiencies in oxytocin signalling pathways may contribute to sleep disorders in autism mutants.</p> <p>I will examine oxytocin mRNA levels across the day/night cycle for both wild-type and mutant fish established in the Rihel lab. I will then analyse the pattern of oxytocin expression in the brains of mutant embryos and their wild-type siblings. From the findings in related studies with <em>cntnap2</em> mutant mice and the Rihel lab zebrafish models of autism (see references <sup> </sup>and ), I expect to see an alteration in the amount of oxytocin mRNA for day/night between the wild-type and mutant embryos, and a change in the number of neurons expressing oxytocin. If such changes are found, they could explain the sleep phenotype observed in <em>cntnap2</em> autism mutants, and elucidate a link between neuronal circuit dysfunction and behavioural perturbation in this animal model.</p>
Evaluation of antimicrobial resistance and intrahospital transmission of respiratory pathogens in antibody-deficient patients.
<p>I will be studying the respiratory microbiome of antibody-deficient patients to determine whether the number of bacterial species that are resistant to common antibiotics correlates with antibiotic usage, and whether transmission of these bacteria occurs between patients whilst attending hospital for immunoglobulin infusions. Immunocompromised patients provide a highly permissive environment for pathogen evolution as the lack of immune pressure allows resistance to develop without an associated fitness cost. Many of these patients take long-term prophylactic antibiotics together with frequent treatment courses, which we hypothesise acts as a selection pressure to further increase the number of resistant bacterial species in their microbiome. By analysing sputum samples with conventional microbiology techniques and MALDI-TOFF mass spectrometry, I will identify the bacterial species present in each sample and determine how many are resistant to common antibiotics, comparing this to questionnaires detailing the patients’ antibiotic usage. Additionally, for any resistant species identified in multiple patients, I will compare the antibiograms from each sample and extract DNA for 16S next generation sequencing to determine whether the presence of these species is due to intrahospital transmission. This project could inform clinical management of these patients as well as other situations where immunocompromised patients share hospital facilities. </p>
Pathways of nanopore assembly by bacterial toxins and immune effectors
<p>To attack cells in our body, bacteria make use of toxins that drill holes in the cell membranes. Following a similar strategy, our immune system makes use of such pore forming proteins to target cancerous, virus-infected and bacterial cells. In the course of their action, pore forming proteins are first secreted as monomers, bind to the membrane, and next self-assemble into oligomeric pores. Some of the various open questions are how these pore assembly processes take place on more complicated, composite membranes such as bacterial envelopes. This project will aim to contribute to answering these questions, while providing the student research expertise in nanoscale microscopy methods applied to process that is essential for bacterial attack and immune defence.</p> <p>More precisely, the student will image live bacteria (E. coli) as they are attacked by the membrane attack complex. This is part of on-going atomic force microscopy experiments in the supervisors lab, which offer the possibility to visualise bacterial cell wall degradation in real time. Time permitting, the student will also be exposed to computational approaches to analyse such new data as well as past data on assembly and membrane insertion of immune effector perforin.</p> <p> </p> <p> </p>
Microtubule organisation and role of LL5-beta in lymphoid stromal fibroblasts
<p>The lymph node is a meeting point for lymphocytes with antigen-presenting cells, and rapidly expands during immune responses. Lymph node structure is highly compartmentalised, and the complex internal architecture is maintained during lymph node expansion. Therefore, mechanisms must exist to balance lymph node integrity with the need to remodel very rapidly. Fibroblastic reticular cells (FRCs) are the most abundant lymphoid stromal cell population, and span the full volume of the tissue. They provide structural support and are highly contractile. FRCs ensheathes bundles of extracellular matrix, termed the conduit, which filters draining lymph. The Acton lab works to understand how lymph nodes are remodeled during expansion and has shown that interaction between FRCs and dendritic cells change FRC behaviour. This project asks how the microtubule networks within FRCs are reorganised as the FRC network expands. Phosphoproteomic screening has revealed that LL5-beta, a protein targetting microtubules to adhesion sites is regulated by interactions between FRCs and dendritic cells. This may provide a mechanism by which FRCs uncouple from underlying matrix, and target secretion of proteases or new matrix to the expanding network. This project will investigate whether LL5-beta coordinates organization of microtubules in FRCs and whether dendritic cell contact changes LL5-beta activity.</p>
The putative propriospinal contribution to feedforward control mechanisms during skilled grasp
<p>The corticospinal pathway is the major direct pathway contributing to hand and motor function, after stroke or spinal cord injury this pathway can become irreversibly damaged. However, other parallel pathways may still function and are accessible to the motor system. The propriospinal network is an interneuronal system that is located at the mid-cervical levels (C3-C4), which transmits and alters descending commands for targeted reaching and grasping. Lesion studies have illuminated the role of this system in the recovery of reach and grasp movements.The aim of this project is to investigate the role of this connection in healthy humans. Specifically, our objective is to show the contribution of this system in feedforward grasping mechanisms. We will employ a motor task that involves grasping an object between the index finger and thumb where the task demands change prior to contact. Paired low-intensity peripheral nerve stimulation (PNS) and transcranial magnetic stimulation (TMS) will be used to probe the propriospinal modulation of corticospinal output during the task. Studying the propriospinal system in healthy humans will show how motor commands are updated at a spinal premotoneuronal level and could provide a novel pathway to target for neurorehabilitation after lesions of the central nervous system.</p>
Determining the allosteric network in HDAC8 using NMR and molecular dynamics.
<p>The main goal of this project is to study the allosteric regulation of Histone deacetylase 8 (HDAC8). HDAC8 is an enzyme involved in transcriptional regulation and diseases such as acute myeloid leukaemia. Recent work in the group has shown that there are changes in chemical shifts in the helix 1, loop 1 and helix 2 region of HDAC8 when the inhibitor TSA binds to the active site – chemical shift changes are observed over 28 Å from the inhibitor binding site. This is of particular interest as recent work by J. Schwabe's group has shown that this region binds co-repressors in other class 1 HDACs modulating their activity. This suggests that the information transfer between the active site and the region around helix 1, loop 1 and helix 2 is a general allosteric pathway in class 1 HDACs and is important for their regulation. In order to derive a mechanism for the allosteric regulation we will use side-chains as probes in NMR experiments. In conjunction with this we hope to characterise the transition between the drug-bound structure and the apo HDAC8 using meta-dynamics. In doing so we hope to identify key motions and residues, which mediate this transfer of information.</p>
Toxin-Drug Conjugates: structural and functional studies of Protoxin-II
<p>Chronic pain affects a large number of patients worldwide but the available treatment options are often far from adequate. The voltage-gated sodium channel Na<sub>v</sub>1.7 has been identified as a target for drugs to treat nociceptive chronic pain, but as yet no clinical candidates have been identified. Protoxin-II is a small protein found in the venom of the Peruvian green velvet tarantula, and is a potent and specific inhibitor of Na<sub>v</sub>1.7; it is therefore a promising lead as a drug for chronic pain. </p> <p>However, many crucial aspects of the interaction of Protoxin-II with Na<sub>v</sub>1.7 remain unknown: it is not known where the toxin binds to Na<sub>v</sub>1.7 and how this binding mode effects inhibition of the channel, or how Protoxin-II is able to select for Na<sub>v</sub>1.7 over other voltage-gated sodium channels.</p> <p>This project will attempt to develop an efficient and versatile synthetic route to Protoxin-II and analogues, and to use these analogues to test the structure-function relationship in Protoxin-II. This information will be used to probe its method and site of action against Na<sub>v</sub>1.7, and to design ‘toxin-drug conjugates’, analogous with antibody-drug conjugates used as treatments for cancer, with high therapeutic potential.</p>
Homeostatic gene networks in Drosophila models of epilepsy and dyskinesia
<p>Transcriptional and translation control in neurons is highly plastic, allowing firing frequency and synaptic output to be regulated with high temporal precision. Recent research has demonstrated that the complement of ion channels within a neuron can undergo homeostatic remodelling in response to altered neuronal excitability. However, the extent to which this occurs in neurological diseases is unknown, as are the alterations in ion channel expression that may buffer disease-linked mutations to the greatest degree. We aim to investigate these questions using the fruit fly, <em>Drosophila melanogaster</em>. Using homologous recombination, we will generate a novel knock-in fly model of Generalized Epilepsy and Paroxysmal Dyskinesia (GEPD). This disorder is caused by a gain-of-function mutation in the <em>KCNMA1</em> BK potassium channel – the mammalian homologue of <em>Drosophila</em> <em>slowpoke</em> (<em>slo</em>). We will characterise changes in ion channel expression in GEPD <em>slo</em> knock-in flies through RNAseq, and using this data, perform a modifier screen to determine which alterations are compensatory or pathogenic. Genetic suppressors identified via this strategy will represent promising targets for future therapeutic interventions.</p>