Student: Kate Petty

Supervisors: Dr Paula Kover (Department of Biology & Biochemistry, University of Bath),  Dr Simon Hiscock (School of Biological Sciences, University of Bristol)

Changes in the environment are expected to challenge the persistence of populations and the yield of current crop varieties. Plants will need to adapt genetically or phenotypically to new environmental conditions to persist. However, the relative importance of genetic adaptation and phenotypic plasticity (the ability of a genotype to change its phenotype according with environmental conditions) to coping with environmental change is controversial. Current understanding of the molecular mechanisms behind plastic responses and how much genetic variation is available to respond to changes in temperature is currently very limited. Understanding the genetic basis of potential evolutionary and plastic responses to predicted increases in temperature, would improve our ability to improve crops to insure food security, and to determine the role of evolutionary processes in conservation. The goal of this project is to use a genetically well characterized population of Arabidopsis to perform field experiments with gradients in temperature, and perform QTL and selection analysis to address the following questions:

• Is there genetic variation for plastic responses in plant development and fitness (yield)? And are the observed plastic responses adaptive?
• Can small populations repond to changes in the environment fast enough to maintain its fitness?
• What is the genetic basis of fitness (fruit production, seed size) under different temperatures and of plasticity for these traits?

Student: Josephine Palandri

Supervisors: Dr Chris Bailey (Dept of Pharmacy & Pharmacology, University of Bath), Professor Sue Wonnacott (Department of Biology & Biochemistry, University of Bath), Dr Alexis Bailey (Department of Biochemistry and Physiology, University of Surrey)

Numerous learning and memory processes are key to life. One such process is motivational learning. Motivation or ‘drive’ is a critical determinant of success in life and is governed, in part, by the prefrontal cortex (PFC). However, this process can also be hijacked by adverse external reinforcers such as drugs of abuse, gambling etc. Motivational learning can be modelled in laboratory animals using the Conditioned Place Preference technique. This exploits a form of Pavlovian conditioning whereby an animal learns to associate a particular environment with a rewarding or aversive stimulus.

Mammalian learning and memory processes are complex systems involving interplay between different neuronal populations, neurotransmitters, receptors and downstream signalling cascades. The mesocorticolimbic pathway (consisting of dopaminergic neurones projecting to the nucleus accumbens and PFC) is heavily implicated in motivational behaviour. This pathway interacts with glutamatergic inputs at multiple levels. Plasticity at glutamate synapses provides a paradigm for molecular and cellular changes underlying memory formation. Modulation of this plasticity by other transmitter systems (acetylcholine, dopamine) is less well defined and forms the basis of this proposal.

Nicotinic acetylcholine receptors (nAChRs) modulate the crosstalk between glutamate and dopamine pathways in the PFC (Livingstone & Wonnacott (2009) Biochem Pharmacol 78:744-55). Moreover, nAChRs have been implicated in learning and memory mechanisms in many brain regions including the PFC (dos Santos Coura & Granon (2012) Psychopharmacology 221:1-18).

This project will take a trans-disciplinary approach, integrating in vivo behavioural learning paradigms, brain-slice electrophysiology and ex vivo neurochemical techniques to elucidate the specific roles, and mechanisms, of nAChRs in mediating or modulating motivational learning processes. The project will build upon recent findings from Chris Bailey’s lab that showed inhibition of the α7 subtype of nAChR to play a distinct role in motivational learning in vivo. In separate experiments using brain-slice electrophysiology, we have discovered that the α7 nAChR plays a key modulatory role in synaptic plasticity processes in the prefrontal cortex. The proposed project aims to integrate these approaches.

Student: Catherine Beedie

Supervisors: Professor Zafar Bashir (School of Physiology and Pharmacology, University of Bristol), Dr Denize Atan (School of Clinical Sciences, University of Bristol), Professor John Aggleton (School of Psychology, Cardiff University)

The PR/SET domain-containing protein, Prdm8, is a transcriptional regulator of neural circuit assembly. In recent work, one of the supervisors (DA) has made the novel discovery that Prdm8-/- mice lack specific populations of interneurons in distinct regions of the CNS.

Hypothesis

Our hypothesis is that Prdm8 is required for normal formation of hippocampal neuronal circuits. This project, will combine expertise from three labs to test the role of Prdm8 in: development of interneurones in hippocampus, hippocampal circuitry, synaptic transmission and learning and memory by using Prdm8-/- mice.

Research Plan

The student will be exposed to a wide variety of skills and training in electrophysiology, molecular biology, behavioural and computational neuroscience.

Aim 1: What is the cellular impact of Prdm8 knockout on hippocampal circuitry?

These experiments will define morphological changes in hippocampal circuitry in Prdm8-/- mice vs litter-matched controls, using neuronal stains (Nissl, Golgi, NeuN) and cell-type specific markers to identify changes in populations of hippocampal neurons. Selected targets of Prdm8 in retina (identified by DA) will be investigated for expression in hippocampus using molecular techniques, such as rt-PCR, Western blotting, immunohistochemistry and chromatin immunoprecipitation of direct Prdm8 targets. These results will further define the genetic network in which Prdm8 functions to regulate hippocampal circuit formation.

Aim 2: What is the functional impact of Prdm8 knockout on hippocampal synaptic transmission?

Experiments will be carried out using electrophysiological recording from hippocampal slices in vitro from Prdm8-/- mice vs litter-matched controls. Basal synaptic transmission from field potential recordings of CA3, CA2 and CA1 regions of the hippocampus in response to stimulation of the afferent input (mossy fibres to CA3; Schaffer collaterals to CA2 and CA1) will be recorded. These experiments will provide data on any functional changes in synaptic transmission resulting from Prdm8 knockout.

During the remainder of the PhD the student will use structural equation modelling to investigate alterations in activity through hippocampal circuits during learning and memory tasks. In addition the student will model changes in network activity in hippocampus resulting from altered interneuron function in Prdm8-/- mice.

Student: Matt Brewer

Supervisors: Professor Leo Brady (School of Biochemistry, University of Bristol), Dr Darryl Hill (School of Cellular and Molecular Medicine, University of Bristol), Dr David Dymock (School of Oral and Dental Science, University of Bristol), Professor Ed Feil (Department of Biology and Biochemistry, University of Bath)

Fusobacteria (Fn) are anaerobic bacteria associated with a range of farm animal and human conditions, including digital necrobacillosis in domestic ruminants, liver abscesses and necrotizing stomatitis in cattle, and foot abscesses and abortion in sheep. The population structure of Fn is complex: F. necrophorum, which affects both animals and humans, has 2 major subspecies (necrophorum and funduliforme), and F. nucleatum, which principally affects humans, has 5 subspecies (nucleatum, fusiforme, vincentii, polymorphum and animalis). With multi-locus sequence type (MLST) genome-based schemes still under development for Fn, there is little known about the virulence factors that are responsible for Fn colonisation and disease.

Immune responses to pathogens are largely initiated and regulated by membrane surface proteins. We have recently identified an immunoglobulin superfamily (IgSF)-binding group of variable adhesins termed CProoF from the Fn surface, homologues of which are present in diverse strains and species of Fn. Diversity amongst such proteins is driven by selective pressures imposed, for example, by the immune response during infection. Mapping molecular diversity and conservation of these highly immune-system visible proteins is likely to prove extremely valuable for novel vaccine design.

This study will evaluate adhesive and structural diversity of CProoF-related proteins from fusobacterial disease isolates and compare protein diversity to population diversity using an established MLST system. Studies on the structural basis of bacteria-host interactions have recently been established between our laboratories and will be extended to use matrix proteins and specific IgSF constructs from canine, bovine and human Fn species. These studies are crucial in order to provide a molecular basis for the interpretation of inter-species variations and to evaluate the basis of species-specific adhesive events. The identified functional adhesive domains will be used to exploit their potential as preventive agents, including novel vaccine components, effective against diverse diseases caused by Fn.

Student: James Clark

Supervisors: Professor Philip Donoghue (School of Earth Sciences, University of Bristol), Professor Simon Hiscock (School of Biological Sciences, University of Bristol), Professor Harald Schneider (Department of Life Sciences, The Natural History Museum)

Whole-genome duplication (WGD, polyploidy) has been a recurrent phenomenon in the evolutionary history of animals, fungi, and especially plants where it has been implicated as a causal factor in effecting macroevolutionary change, such as in the origin of seed plants and flowering plants (Jiao et al. 2011), and extinction resistance (Fawcett et al. 2009). Under this hypothesis, whole genome duplication produces a redundant set of protein coding genes that can be co-opted to new functions, in physiology, development and, ultimately, effecting new phenotype. However, the prevalence and relative timing of WGD events within plant phylogeny are poorly known – and so their role in effecting phenotypic evolution has not been tested even at this most simplistic of levels. Furthermore, our understanding of the timing and tempo of phenotypic evolution is equally poorly constrained since fossil evidence has largely been ignored.

The project’s aim is to resolve the timing and evolutionary impact of WGD events that have occurred within the fundamental (deep) evolutionary lineages of plants. Integrating evidence from living and fossil plants, you will derive an holistic understanding of plant phenotypic evolution and, drawing on molecular sequence data from living plants, and phenotypic data from both living and fossil plants, establish a timescale for the plant phenotypic evolution. In parallel, you will elucidate ploidy history within the fundamental plant lineages through flow cytometry (which determines ploidy level directly) and microRNA paralogy (which reveals ploidy history through the number and relations within microRNA gene families). The timing of WGD events will be determined through divergence time analysis of gene families, for which databases of well-annotated gene paralogy are already in place. Ultimately, you will establish, within a probabilistic framework, whether WGD has been formative in plant phenotypic evolution, exacting both temporal tests of causality between WGD events and phenotypic evolution, and through parametric characterization of increases in phenotypic evolution across WGD events.

• Fawcett, J.A., Maere, S., and Van de Peer, Y. (2009). Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event: Proceedings of the National Academy of Sciences, 106:5737-5742.
• Jiao, Y. et al. (2011). Ancestral polyploidy in seed plants and angiosperms. Nature 473: 97-100.

Student: Henry Darch

Supervisors: Professor Richard Apps (School of Physiology and Pharmacology, University of Bristol), Professor Iain D. Gilchrist (Bristol Clinical Research and Imaging Centre (CRICBristol) and School of Experimental Psychology, University of Bristol)

The ability to modify movements and actions in response to changes in an environment is critical to an animal’s success. There are still several key gaps in our understanding of how our brains are able to flexibly and rapidly modify our behaviours (motor plasticity). It is clear that there is a network of brain regions supporting these abilities, and some of the key areas include the cerebellum, motor, and prefrontal cortices. This project will aim to study whether these areas interact, and how the dynamics of neural activity supports motor plasticity with the use of advanced neural analysis techniques in both human and animal models performing a simple adaptation task, in which a reaching arm must overcome a targeting error, introduced by the experimenter.

Student: Alen Eapen

Supervisors: Professor David Jane (School of Physiology and Pharmacology, University of Bristol), Professor Stafford Lightman (School of Clinical Sciences, University of Bristol)

A major issue with ageing is the loss of cognitive function. Most humans develop impairments in the ability to learn and remember information as they age. The study of synaptic plasticity, in particular long-term potentiation (LTP) and long-term depression (LTD), is providing major insights into the molecular basis of learning and memory. Bristol scientists discovered the N-methyl-D-aspartate receptor (NMDAR) and its pivotal role as the trigger for many forms of synaptic plasticity. Recently we have identified a novel allosteric regulation site on the NMDAR and have just been awarded BBSRC funding to develop more potent and selective compounds to modulate NMDAR function in a subtype-dependent manner. In unpublished work we have found that one of these compounds, UBP709, restores NMDAR-dependent synaptic plasticity that is lost in old rats. The purpose of this proposal is to (i) use computer-aided design to synthesise novel analogues of UBP709 with improved potency and subtype selectivity and (ii) to study the ability of UBP709 and the novel analogues to restore synaptic plasticity in aged rats, by performing experiments in vivo. Professor David Jane, a chemist in the FMVS will supervise the computer modelling and synthetic chemistry. Professor Stafford Lightman, a clinician in the FMD, will supervise the in vivo experiments. Key to these experiments will be the monitoring of the levels of stress in the animals, since NMDAR-dependent synaptic plasticity is extremely sensitive to stress.

Student: Jonathan Jenkins

Supervisors: Dr Ross Anderson (School of Biochemistry, University of Bristol), Professor Adrian Mulholland (School of Chemistry, University of Bristol), Professor Ron Koder (Physics, City College of New York)

At the core of the emergent Synthetic Biology field lies a fundamental goal to construct new functional and bio-compatible parts and devices for incorporation into explicitly biological organisms or systems. Such a synthesis of artificial and biological components will provide an incredibly powerful framework for the design and exploitation of augmented or even new biochemical pathways in or ex vivo. Taking inspiration from nature and the simple engineering rules gleaned from the analysis of natural c-type cytochromes, we have demonstrated that a manmade protein (maquette) can be designed, expressed and processed through natural biochemical pathways in vivo to produce a fully functional, manmade c-type cytochrome (CTM) without the need for further in vitro assembly – a hitherto unrealized feat in de novo protein design. We have since developed a thermophilic CTM containing a c-type heme primed for oxygenic catalysis and have demonstrated nascent catalytic activity – the oxidation of small organic molecules such as styrene. Since the CTMs are expressed and fully assembled in vivo, this represents a powerful system for the development and laboratory evolution of manmade, cofactor-containing enzymes.

The goal of this project is to construct functional oxygen-activating CTMs using a powerful combination of complementary approaches to de novo protein design, marrying our stripped-down, iterative maquette approach with high-level computational modeling by QM/MM and MD methodologies.

Student: Melissa Smith

Supervisors: Dr Jo Murrell (Langford Veterinary Sciences, University of Bristol), Professor Mike Mendl (Langford Veterinary Sciences, University of Bristol)

Clinical pain in animals is currently considered to be a single uniform entity, regardless of cause. Similarly, until recently, pain in man was also considered to be a homogeneous entity; however it is now recognized that distinct types of chronic pain exist that have different underlying neurobiological mechanisms and therefore have different susceptibilities to analgesics. This has led to the concept of pain phenotypes in man; specific pain patterns as defined by quantitative sensory testing (QST) that are markers of different underlying pain mechanisms. There are no parallel studies in animals and the concept of pain phenotypes in animals has, until now, been largely neglected. However knowledge of the relationship between pain phenotype and the underlying aetiopathogenesis of pain is required to advance our understanding of pain mechanisms in animals with spontaneous disease. Furthermore, the development of diagnostic tools to identify the aetiology of pain in an individual animal combined with pharmacotherapy that acts specifically on these mechanisms is a new strategy to ensure a rational, mechanism based and personalized approach to analgesia.

The student will investigate the relationship between pain phenotype and pain mechanisms using spontaneous osteoarthritis (OA) as a chronic pain model. Within a multi-disciplinary team, training will be provided in animal behaviour, pain assessment, in vivo neurophysiology and models of osteoarthritis.

Student: Philip Smith

Supervisors: Dr James Hodge (School of Physiology and Pharmacology, University of Bristol), Dr Krasi Tsaneva (College of Engineering, Exeter University)

All eukaryotes including humans express endogenous 24hr circadian rhythms in their physiology and behaviour, allowing them to be optimally adapted to daily changes in environmental light and temperature. These rhythms arise from an intrinsic intracellular molecular clock oscillating in their clock neurons. The molecular clock is remarkably conserved across the animal kingdom, and controls 24hr rhythms in gene expression that regulate electrical activity rhythms in the clock circuit. In both flies and mammals, clock neurons show similar changes in electrical activity across the circadian day. Such changes in electrical activity help communicate clock information to the rest of the brain and body; inhibition of such neural activity impairs clock control of behaviour and physiology. Such electrical changes are lost in clock neurons lacking a molecular clock. Conversely electrical activity can impose time of day on the circadian transcriptome of clock neurons.

During the first rotation they will be trained by a BBSRC-funded postdoc to whole-cell patch-clamp clock neurons identified using the clock-specific PDF promoter expressing GFP (PDF>GFP). This will allow them to measure how membrane parameters including individual channel currents vary across the circadian day.

During the second rotation they will be taught biophysical, mathematical and computational modeling of membrane potential and ionic currents by Dr Krasi Tsaneva and BBSRC-funded researchers. The membrane parameters will be used to generate a model of the neuron’s circadian electrophysiology.

The student will be taught fly molecular genetics by the host lab. Brains of PDF>GFP flies taken across the circadian day will be GFP-FAC-sorted to isolate clock neuron RNA that will be subjected to microarray analysis. Of the mRNAs oscillating in abundance, gene ontology will be used to extract those involved in membrane function and ion transport. The change in abundance of these circadian ion channels (confirmed by quantitative RT-PCR) will be fed into the model to see how they would change the behavior of the clock neuron. As a team we have set up dynamic clamp that allows the experimenter to computationally add or subtract individual currents to a living neuron to determine the contribution of each to the different electrophysiological parameters of the cell. The student will use dynamic clamp to systematically test all predictions of their model. Key changes will be confirmed using channel specific pharmacology and electrophysiology while circadian rhythms will be assessed behaviourly and using period-luciferase analysis of flies with genetically altered levels of specific channels in their clock

Student: Ashley Winter

Supervisors: Professor Matthew P Crump (School of Chemistry, University of Bristol), Professor Richard W Titball (College of Life and Environmental Sciences, Exeter University)

Toxin-antitoxin systems regulate metabolic activity at a systems level in bacteria. Entry into a metabolically inactive state is determined by toxin excess and relieved by antitoxin. Therefore, the behaviour of a cell population is determined by the relative levels of toxin-antitoxin. In a metabolically inactive state the bacteria can survive a range of otherwise lethal stresses and insults. This may be important for survival in the environment and for the establishment of chronic infectious disease (i.e. Burkholderia pseudomallei).

Our aim is to investigate how these system-level switches function at a molecular level.

Previously we have identified a number of toxin-antitoxin systems in Burkholderia thailandensis, a model bacterium that can persist for long periods in soil and water. In contrast to dogma, our preliminary results indicate that the toxins are able to cross-regulate the activity of a range of antitoxins. This has important implications for the mechanisms that allow bacteria to modulate their global metabolic activity. Investigation into the microbiology of these toxin-antitoxin interactions is funded by a BBSRC project grant ((4) below) that also seeks to develop mathematical models to explain switching. However, to understand how toxins and antitoxins function at a global level we aim to determine the solution structures of toxin-antitoxin complexes by NMR (~15 kDa). We have shown that the toxin (BPSS0390) is amenable to this approach and have now solved the structure of the toxin and used a combination of site directed mutagenesis and growth assays to identify functionally important regions of this structure (The HicA toxin from Burkholderia pseudomallei plays a role in antibiotic mediated persistence. We have recently shown that the anti-toxin protein can be produced in protease deficient strains of E. coli suggesting we are well placed to begin studying the toxin-antitoxin complex by NMR. The ability to perform NMR studies on individual domains, larger complexes and perform structure-function across both sites will provide an excellent multi-disciplinary training.

Student: Adam Thomas

Supervisors: Dr Nicholas Harmer (Department of Biosciences, University of Exeter), Dr Mark Van Der Giezen (Department of Biosciences, University of Exeter)

The development of Synthetic Biology requires toolkits for the preparation of novel biomolecules, and for using diverse inputs to synthetic pathways. One attractive approach is the use of ancestral gene reconstruction. This method, which has recently enjoyed a renaissance, has been used principally to establish the original partners for proteins at earlier points in evolution. There is an expectation that a proto-enzyme will be less substrate selective than more highly evolved enzymes. This offers the potential for exploitation by reconstructing groups of ancestral genes, which when combined will support novel biosynthetic pathways.

This project will apply ancestral gene reconstruction to glycoscience synthetic biology. There is a significant need for a synthetic biology toolkit for preparing polysaccharides for medicine and biotechnology. The ancestral gene method offers the opportunity to dramatically accelerate the development of such toolkits, as this would expedite the use of transferases with relaxed specificity to facilitate modular biosynthetic systems.

This project will explore two specific, relevant examples.

Deoxyheptose sugars are critical for the polysaccharides of the food-borne pathogens Campylobacter jejuni and Yersinia pseudotuberculosis and the Tier 1 select agent Burkholderia pseudomallei. There are no characterised glycosyltransferases for adding these sugars to polysaccharides. We have recently engineered the precursor biosynthetic pathway into E. coli. The student will prepare an ancestral transferase gene predicted to show relaxed specificity, to expedite the addition of these sugars to existing E. coli polysaccharides for vaccine development.

Many human and animal pathogens use the poorly characterised Group II capsular polysaccharide biosynthesis mechanism. Two defining members of this pathway, kpsC and kpsS, are critical for biosynthesis as they initiate polymerisation. The ancestral versions of these genes will be reconstructed to identify likely ancestral sugars used for capsule biosynthesis. This will then be exploited to expand the range of initiating sugars available for designing polysaccharides through synthetic biology.

Student: Emily Sheppard

Supervisor: Dr Richard Chahwan (College of Life and Environmental Sciences, University of Exeter), Dr Nicholas Harmer (College of Life and Environmental Sciences, University of Exeter)

Mammals are constantly required to safeguard their genomic integrity from environmental genotoxins and defend themselves from lethal foreign agents. An adequate DNA damage response (DDR) achieves the former; while the latter is ensured by the acquired immune system, especially the antibody response in B cells. Interestingly, the DDR also contributes to the generation of immune diversity needed to protect against foreign antigens. That is why a defective DDR can have various adverse effects including the development of immunodeficiencies.

Chromatin modifications have been recently established to play an important role in both DDR and antibody diversity. We have previously shown that histone mono- and poly- ubiquitination by RNF8 and RNF168, respectively, are important for both the maintenance of genomic integrity (Science 2007) and the prevention of the Riddle Syndrome immunodeficiency (OMIM# 611943) – harbouring a defective RNF168 protein (PNAS 2010). We have now conducted 2 independent genome-wide screens in collaboration with colleagues in NYC and Toronto and have found that a new histone modification (H3K36me3; active transcription mark) might offer the missing mechanistic link between the requirement for both transcription and DDR for antibody hypermutations. Recent published work seem to further support our results (Cell 2013;153(3):590-600). The proposed project aims to elucidate the contribution of H3K36me3 to basic DDR, antibody response, and immunodeficiencies in mammals.

The planned project will rely on various computational, molecular, and cellular techniques that I have optimized. These include: deep-sequencing and meta-analysis of the NCBI, mod/ENCODE, and IMMGEN databases; isolation and culture of various cell lines; genetic manipulation of mammalian cells using the newly developed CRISPR/Cas9 technology; various methods to assess the efficacy of the cell cycle checkpoint, DDR, and apoptosis; and various methods to measure the efficacy of the immune response including somatic hyermutation and class switch recombination. The project will also rely on the biochemical expertise of the Harmer lab where we aim to purify proteins from bacterial or mammalian cells under different conditions and perform in vitro binding assays and catalytic experiments. We also aim to reconstitute nucleosomes in vitro to test the binding efficiency of various proteins modules to the histone modification of interest.