Supervisors: Prof Jeremy Henley (University of Bristol), Dr Abderrahmane Kaidi (University of Bristol)

Others in supervisory team/collaborators: Dr Dan Rocca (University of Bristol), Dr Paul Bishop (University of Bristol)

Posttranslational protein modification by ubiquitin regulates the total cellular protein content (proteome) through the timely degradation of specific proteins and is crucial for healthy cellular function. Furthermore, the ubiquitin system also plays roles in regulating the activity of nearly every cellular pathway. Ubiquitination is highly reversible by deubiquitinating enzymes (DUBs). In neurons on specific kind of DUB, UCH-L1 accounts for 1-5% of total cellular protein in the brain and, despite its relatively small size, has one of the most complicated three-dimensional structures yet discovered. The specific functions of UCH-L1 remain enigmatic but the knotted backbone of UCH family proteins been suggested to protect them from proteasomal unfolding and degradation. Thus, UCH-L1 may protect free ubiquitin from degradation and process the retrieved ubiquitin to maintain a global ubiquitin pool.

Interestingly, the major phenotype of dysfunctional UCH-L1 is defective axonal transport and an axonal degeneration, particularly in spinal neurons. Thus, understanding the cell biology and functions of UCH-L1 in neurons represents a significant challenge that will have far-reaching impact.

The aims of this PhD project are to understand the molecular basis of UCH-L1 function by:

1. Using transgenic mice and knockdown and molecular replacement strategies (with UCH-L1 mutants we already have) to identify how manipulating UCH-L1 impacts on axonal formation, maintenance and function. These experiments will use advance molecular biology, neuronal cell culture and confocal imaging to isolate and directly image axons.

2. Identifying the proteins that UCH-L1 interacts with and/or indirectly regulates using state-of-the-art quantitative proteomics approaches. The candidate proteins identified will be ranked according to their roles in axonal function and validated using biochemical and functional assays in the context of aging and axonal degeneration.

Because we already have all of the necessary UCH-L1 tools and have a wealth of expertise in these approaches the student will be ideally placed to make significant progress towards unravelling the mechanisms of action, and physiological and pathophysiological roles of UCH-L1 regulation of ubiquitin homeostasis.

Supervisors: Prof Matthew Crump (University of Bristol), Dr Paul Race (University of Bristol)

Others in supervisory team/collaborators: Prof Bass Hassan (University of Oxford)

Nature’s ability to use an existing blue-print or design and evolve it to serve a new, perhaps unexpected purpose, is a continuing source of fascination and has led to the rich and diverse biology we observe around us. This process scales from the molecular level where changes to proteins might lead to new biochemistry that drives the huge diversity we see at the macroscopic scale. One such example of this process in action can be found in proteins that have a similar three-dimensional shape but are equipped with special hotspots that can adapt to bind or capture different molecules, which may themselves be proteins or simpler chemicals. This is exemplified in a receptor molecule that we study called insulin growth factor 2 receptor (IGF2R) (Crump & Hassan and co-workers 2012, Science 238, 1209-1213.). This 300 kDa protein contains fifteen structurally similar domains but with very different sets of surface loops that have evolved to bind a variety of ligands ranging from simple sugars (mono- and disaccharides) up to larger proteins such as Insulin Growth Factor-2 (see figure). This diversity is unprecedented and reveals the sophistication of this underlying scaffold and the potential for use in biotechnology and biological applications.

Our initial aim is to engineer a super-affinity sugar binding domain (called a lectin) based on domain9 of this receptor that binds a sugar known as mannose-6-phosphate. This domain, for reasons we now understand based on its structure, cannot be purified as a single protein without its neighbouring domains. We do not wish to express all of these together as one of our aims is to maintain the small size of our synthetic receptor. We therefore aim to explore the potential of easily produced domain11 as a synthetic lectin by initially creating a chimeric scaffold that swaps in the binding loops of domain 9 then using molecular selection techniques we have in our lab (yeast surface display, NMR, X-ray and site directed mutagenesis) to explore whether this binding site can be evolved to bind mannose-6-P. If successful this may unlock the potential of these domains for binding other carbohydrates, phosphodiesters and many other ligands of choice. There has been relatively little focus so far on evolving scaffolds to target small molecules but this is changing however and there have been recent reports of using engineered anticalines for binding the well-characterized immunological hapten fluorescein with many biological applications (eg imaging).

Supervisors: Prof Stafford Lightman (University of Bristol), Prof John Terry (University of Exeter)

Others in supervisory team/collaborators: Dr Georgina Russell (University of Bristol), Prof Marcus Munafò (University of Bristol), Dr Nicholas Timpson (University of Bristol), Dr Becky Conway-Campbell (University of Bristol), Prof Catherine Harmer (University of Oxford)

Stress has powerful effects on both emotional and cognitive function as well as on metabolic function. A major factor for these changes is the activation of the hypothalamic-pituitary-adrenal (HPA) axis by stress and the effects of the cortisol released by the adrenal gland on both brain circuits and metabolic pathways. We have found that the pattern in which cortisol is released is critical for these effects and we now want to explore how we can modify the pattern of hormone release and to regulate both brain responses and epigenetic changes important for metabolic and immunological function. This is a multidisciplinary project and the successful applicant will work with a team of scientists in psychology, fMRI scanning, molecular endocrinology and mathematics. The student will organise studies with healthy volunteers, perform psychological testing and investigate functional connectivity from the fMRI data. They will also collaborate in the studies on the epigenetic and regulatory effects on the different patterns of cortisol secretion.

Supervisors: Prof Imre Berger (University of Bristol), David Fisher (Astrazeneca)

Others in supervisory team/collaborators: Dr Mark Dillingham (University of Bristol), Robert Roth, Lorenz Mayr (AstraZeneca)

Baculovirus is a highly efficient delivery system for recombinant genes into eukaryotic cells, with great impact on the production of eukaryotic proteins, including high-value drug targets. Vaccines against cervicular cancer and others are produced by this method. More recently, baculovirus has emerged as a versatile tool for gene therapy. We contributed to the field the award-winning MultiBac technology for multiprotein complex research.

These applications, at the forefront of modern biology, rely on a large baculovirus genome (130 kb) derived from AcMNPV. This genome has been intensively researched, mainly by entymologists. Genes essential for propagation in nature and in cell culture were delineated and DNA elements which impede applications in the laboratory. Genetic alterations of the wild-type viral genome have been performed, by classical knock-out technologies, to improve gene insertion, delivery and protein production properties. Such alterations require excessive effort by specialists. Therefore, it is currently not possible to fully exploit the vast potential of the baculovirus system.

In the present project, we boldly propose to fully reverse the current approach. We will design in silico and construct in vitro new, fully synthetic customized baculovirus genomes which will be, for the first time, in a streamlined, highly versatile format for multigene transfer and the production of high-value, next generation recombinant protein targets for drug discovery. We will apply state-of-the-art genome editing tools, notably CRISPR-Cas9, to inform our approach by systematically disrupting genes and non-coding regions including gene regulatory elements. We further aim to address the “scale-up problem” which currently impedes pharma-scale biologics production by baculoviral systems. As proof-of-concept, we already created a partly synthetic hybrid genome by replacing a large part (20 kb) of wild-type with designer DNA. Rigorous testing of this prototype compellingly validated our approach.

The student will:

(1) utilize computational biology, comparative bioinformatics and data mining to create blueprints for optimized minimal baculovirus genomes.

(2) Synthesize designer genomes in large fragments, and use advanced recombination technologies to assemble these into functional genomes (AstraZeneca platform).

(3) Exploit cutting-edge CRISPR-Cas9 tool to edit genes and regulatory DNA elements in the baculoviral genome in a parallelized fashion (AstraZeneca platform) and implement this information in the synthetic design.

(4) Address the “scale-up problem” by reconfiguring the very late phase of baculoviral life cycle (“hyperburst management”).

(5) Rigorously validate novel designer genomes experimentally.

These will be the first fully synthetic baculoviral genomes, with the potential to transform academic and industrial R&D applications.

Supervisors: Dr Ross Anderson (University of Bristol), Prof Adrian Mulholland (University of Bristol)

Others in supervisory team/collaborators: Dr Richard Sessions (University of Bristol), Dr Birte Hoecker (MPI for Developmental Biology, Germany)

At the core of the emerging field of Synthetic Biology 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 undoubtedly provide an incredibly powerful framework for the design and exploitation of augmented or even new biochemical pathways in or ex vivo. To this end, we have previously demonstrated how manmade proteins can be processed through natural biochemical pathways in vivo to produce fully functional manmade c-type cytochromes without the need for further in vitro assembly – a hitherto unrealized feat in de novo protein design. These evolutionarily naive proteins, maquettes, have proven capable of incorporating many engineering elements common to natural oxidoreductase proteins (e.g. reversible O2 binding, electron transport) within a robust but simple protein scaffold lacking the complexity of naturally evolved proteins. The prototype c-type cytochrome maquette (CTM) is capable not only of binding oxygen, but also of assembling nascent electron transfer chains, photoactive light harvesting dyads and engaging in interprotein electron transfer with natural cytochromes. Further engineering of this prototype has resulted in the assembly of a CTM enzyme capable of catalyzing chemistries common to natural heme-containing enzymes with catalytic efficiencies equaling or surpassing those of the current state-of-the-art de novo enzymes and certain naturally evolved enzymes.

The aims of this proposal are to use computational and experimental methods to develop and enhance the catalytic activity of this CTM enzyme scaffold, enabling both the creation of new, powerful enzymes and to enable the understanding of how catalytic activity was incorporated into natural oxidoreductases through their evolutionary history. We will use computer simulations of our CTMs to inform the design of diverse CTM libraries for high-throughput screening and directed evolution. Individual CTM proteins will be subjected to comprehensive biophysical characterization (e.g. circular dichroism, EPR and NMR spectroscopies, redox potentiometry, X-ray crystallography) and the catalytic cycle will be examined using steady-state and pre-steady-state methods (e.g. stopped flow spectrophotometry). QM/MM simulations will also be employed to examine the catalytic cycle in detail and will inform the design of future CTM iterations.

Supervisors: Prof Matthew Crump (University of Bristol), Dr Richard Taylor (UCB Celltech)

Others in supervisory team/collaborators: Dr Richard Sessions (University of Bristol), Dr Christine Prosser (UCB Celltech), Dr Alistair Henry (UCB Celltech), Prof Adrian Mulholland (University of Bristol), Dr Marc van der Kamp (University of Bristol)

Alongside X-ray crystallography, NMR is one of the only techniques capable of delivering protein structural information with atomic resolution. NMR offers the advantage over X-ray that, as a free solution based technique, protein dynamics can be observed, but it is currently more limited in terms of the size of proteins that can be studied and requires that proteins can be labelled with stable isotopes (15N, 13C, 2H) which can place restrictions on the available expression systems. Nevertheless, NMR is established as an invaluable tool for identifying novel chemical starting points for new targets as it can not only confirm binding, even for very weak milli-molar Kd interactions, but also can identify the site of this interaction and report on any conformational changes either induced by, or as a pre-requisite for ligand binding.

The focus of this collaboration is to address two of the perceived shortcomings of applying NMR in early drug discovery: First that NMR experiments consume too much protein, and second that the experiments are slow to perform, being too time consuming and therefore insufficiently high throughput either to impact the Medicinal Chemistry design iteration cycle or to support fragment screening activities.

The aim of this Ph.D. studentship at Bristol University is, therefore, to enable rapid delivery of information rich multidimensional NMR data to confirm the location of fragment ligand binding, using minimal amounts of protein but without compromising the data quality. This will be achieved by a combination of computational modelling to prioritise the most promising chemical leads and advanced mathematical non-uniform data sampling and reconstruction methods applied to the acquisition and processing of NMR data recorded using state-of the- art low volume NMR technology for samples of fragment ligand mixed with proteins of therapeutic interest in drug discovery. NMR data will be collected on a Bruker 700 MHz spectrometer located in the School of Chemistry at UoB and equipped with a 1.7 mm cryoprobe using only 35 L of sample. Purchase of this instrument was part funded by UCB. The computational aspects of the project will then be extended to using this NMR data to further refine the positioning of this fragment in the protein binding site and will be undertaken in collaboration with the School of Biochemistry at UoB (Sessions). The studenstship will also benefit from access to UCB’s extensive Structural Biology infrastructure, including NMR facilities, via the industrial placement component of the project.

Supervisors: Dr Paul Curnow (University of Bristol), Dr Paul Race (University of Bristol)

Others in supervisory team/collaborators: Dr Richard Sessions (University of Bristol)

Volatile esters are produced in yeast and fungi during fermentation, in plants during fruit ripening, and in higher organisms such as humans in response to ethanol stress. These compounds play an important role in the flavour of fruits and the taste of fermented beverages such as beer and wine. For example, isoamyl acetate tastes of banana; ethyl hexanoate tastes of apples. These esters are produced by enzymes that act as acyl—CoA:ethanol O— acyltransferases AATases), and understanding the molecular biochemistry of yeast and plant AATases may lead to designer yeast strains with tailored fermentation products and improvements to fruit crops, fruit flavour and shelf life. There is also increasing interest in using AATases for metabolic engineering to produce renewable biofuels and fine chemicals.

However, these enzymes have only been partially characterized to date because of a lack of methods for isolating recombinant protein and for studying enzyme function. We have recently established methods that can be used to purify and study these proteins (Knight et al, (2014) Yeast 31:464; Nancolas et al, (2015) in preparation). The student will use and extend these methods to pursue two linked research strands: (i) Use molecular biochemistry and biophysics to characterize the structure and function of two yeast AATases from different protein families. This will use X–‐ray crystallography and classical enzyme kinetics, combined with computational ligand docking and site—directed mutagenesis. The outcome of this strand will be to define the location and features of the catalytic active site of these proteins. This will provide the first detailed insights into enzyme mechanism, including how these enzymes discriminate between different substrates. (ii) Use our established ‘toolkit’ of methods to begin studying other AATases associated with the ripening of commercial fruits such as strawberry, banana, apple, papaya and kiwifruit. This will include cloning these genes prior to protein expression in a recombinant host. The recombinant protein will be purified and analysed by a suite of biochemical and biophysical methods. The outcome of this project strand will be the first detailed study of this enzyme family. Skills training for the student will include: Protein Biochemistry (immobilized metal affinity chromatography, gel filtration, SDS—PAGE and native—PAGE, circular dichroism, site–‐directed mutagenesis, enzyme kinetics); Structural Biology (including high—throughput crystallization screening with state-of-the art liquid handling robotics and structure determination using synchrotron radiation); and Computational Biochemistry (ligand docking). The student will be introduced to each of these methods during laboratory rotation projects.Volatile esters are produced in yeast and fungi during fermentation, in plants during fruit ripening, and in higher organisms such as humans in response to ethanol stress. These compounds play an important role in the flavour of fruits and the taste of fermented beverages such as beer and wine. For example, isoamyl acetate tastes of banana; ethyl hexanoate tastes of apples. These esters are produced by enzymes that act as acyl—CoA:ethanol O— acyltransferases AATases), and understanding the molecular biochemistry of yeast and plant AATases may lead to designer yeast strains with tailored fermentation products and improvements to fruit crops, fruit flavour and shelf life. There is also increasing interest in using AATases for metabolic engineering to produce renewable biofuels and fine chemicals. However, these enzymes have only been partially characterized to date because of a lack of methods for isolating recombinant protein and for studying enzyme function. We have recently established methods that can be used to purify and study these proteins (Knight et al, (2014) Yeast 31:464; Nancolas et al, (2015) in preparation).

The student will use and extend these methods to pursue two linked research strands:

(i) Use molecular biochemistry and biophysics to characterize the structure and function of two yeast AATases from different protein families. This will use X—ray crystallography and classical enzyme kinetics, combined with computational ligand docking and site—directed mutagenesis. The outcome of this strand will be to define the location and features of the catalytic active site of these proteins. This will provide the first detailed insights into enzyme mechanism, including how these enzymes discriminate between different substrates.

(ii) Use our established ‘toolkit’ of methods to begin studying other AATases associated with the ripening of commercial fruits such as strawberry, banana, apple, papaya and kiwifruit. This will include cloning these genes prior to protein expression in a recombinant host. The recombinant protein will be purified and analysed by a suite of biochemical and biophysical methods. The outcome of this project strand will be the first detailed study of this enzyme family.

Skills training for the student will include: Protein Biochemistry (immobilized metal affinity chromatography, gel filtration, SDS—PAGE and native—PAGE, circular dichroism, site—directed mutagenesis, enzyme kinetics); Structural Biology (including high—throughput crystallization screening with state—of—the art liquid handling robotics and structure determination using synchrotron radiation); and Computational Biochemistry (ligand docking). The student will be introduced to each of these methods during laboratory rotation projects.

Supervisors: Dr Jonathan Hanley (University of Bristol), Prof Krasimira Tsaneva-Atanasova (University of Exeter/University of Bristol)

Others in supervisory team/collaborators: Dr Mark Jepson (University of Bristol), Dr Dominic Alibhai (University of Bristol)

Long-term synaptic plasticity underlies learning and memory and the tuning of neural circuitry. Two major postsynaptic processes are involved in plasticity of excitatory synapses: modification of AMPA receptors (AMPARs), which mediate fast synaptic excitation in the brain, and alterations in the size and shape of dendritic spines. These protrusions from the dendritic shaft compartmentalise the postsynaptic protein machinery, and concentrate biochemical signals such as Ca2+ ions. Dendritic spines shrink following the induction of long-term depression (LTD), and grow during long-term potentiation (LTP). In addition, aberrant spine morphology is emerging as a critical factor in brain disorders such as autism spectrum disorders, schizophrenia and stroke. Dendritic spine structural plasticity involves an elaborate network of signalling pathways converging on protein complexes that regulate the actin cytoskeleton.

Protein interactions can be analysed by live cell imaging using FLIM-FRET (Fluorescence Lifetime Imaging – Förster Resonance Energy Transfer), which is an advanced cell imaging technique that provides a dynamic measure of the proximity of two fluorophores, and hence of two proteins with appropriate fluorescent tags.

The aim of this project is to analyse relevant protein-protein interactions in dendritic spines using FLIM-FRET in response to stimuli that induce plasticity. The study will focus on PICK1, which inhibits actin polymerisation and is essential for LTD. PICK1 interacts with a number of proteins that are crucial for plasticity, such as small GTPases, PKC, calcineurin, actin and the Arp2/3 complex. FLIM-FRET will be used to analyse interactions between PICK1 and each of these proteins in turn, with simultaneous recording of spine size. This will be done in real time, following neuronal stimulation to induce plasticity.

Our hypothesis is that the extent of these specific protein interactions governs the degree of spine shrinkage. Computational methods will be employed to test this hypothesis and build a model of dendritic spine dynamics based on specific protein-protein interactions.

This project will be carried out under the expert supervision of a multi-disciplinary team covering neuronal cell biology (Dr. Jonathan Hanley), specific expertise in FLIM-FRET (Dr. Dominic Alibhai/ Dr. Mark Jepson), and computational biology (Dr. Krasimira Tsaneva-Atanasova). The cell imaging and image analysis will be carried out in the state-of-the-art Wolfson Bioimaging Facility at the University of Bristol.

Supervisors: Prof Imre Berger (University of Bristol), Prof Ian Collinson (University of Bristol)

Others in supervisory team/collaborators: Prof Christiane Schaffitzel (University of Bristol)

A third of the cellular proteome is transported into or across a membrane, through the ubiquitous Sec-machinery. Associated factors facilitate protein translocation, insertion and folding. The bacterial core SecYEG channel assembles with four further membrane proteins, SecD, SecF, YajC and YidC to form the holo-translocon secretase/insertase supercomplex. Knowledge of holo-translocon structure and mechanism is vital, but elusive in spite of intense efforts. We developed ACEMBL, a synthetic biology-based combinatorial multiprotein expression tool to produce functional SecYEG-SecDFYajC-YidC holo-translocon. Biochemical characterisation revealed new mechanisms of membrane protein insertion and proton-motive-force-dependent secretion. Recently, we obtained first medium resolution density for holo-translocon by cryo-EM compellingly underscoring the high quality of our recombinant complex. We propose to determine the structure and mechanism of holo-translocon protein secretase/insertase at atomic resolution. A range of biochemical, biophysical and engineering approaches are combined to enable this. Atomic information can be obtained by X-ray crystallography and, through implementation of revolutionary methods, by cryo-electron microscopy, and we will pursue both synergistically to discover holo-translocon architecture, substrate polypeptide translocation and membrane insertion, and reveal the underlying mechanisms. We will subject holo-translocon to high-throughput crystallization using our state-of-the-art biosuite (BBSRC funded). Specific antibodies and nanobodies we already prepared will be used as crystallization aids and conformational stabilizers. Crystal data will be collected at high-brilliance X-ray beamlines at the national facility Diamond and structure solution, refinement and model building performed at Bristol. In parallel, also to reveal distinct conformational states, high-resolution cryo-EM will be pursued. Chemical cross-linking and nanobodies will be utilized to map subunit locations and to stabilize specific conformations, to decisively improve the current medium resolution EM map for near atomic interpretation.

The project will offer unique opportunities for training in state-of-the-art experimental strategies. Complementary execution of these approaches will offer very high training potential and acquisition of a unique breadth of laboratory skills for the prospective student in the leading methods for molecular structure determination. Success prospects are maximized by body of data available and opportunity to train in crystallography and cryo-EM of a transmembrane complex in a synergistic approach.

Supervisors: Dr Jack Mellor (University of Bristol), Dr Emma Robinson (University of Bristol)

Others in supervisory team/collaborators: Prof Krasimira Tsaneva-Atanasova (University of Exeter/University of Bristol)

The early life experiences of newborn babies and infants are a key determinant in their future mental health. Early life adversity within the mother-infant relationship is highly significant in determining a child’s future susceptibility to a range of psychiatric disorders including anxiety and depression. Early life adversity causes stress in infants which raises cortisol levels and activity in the hypothalamic-pituitary-adrenal (HPA) axis. However, we know very little about the changes in brain circuit development caused by early life adversity and stress.

The circuits controlling positive and negative affect (or emotions) and those that regulate the stress response to emotional situations are thought to reside principally in the amygdala and hippocampus. In particular, positive and negative affective behaviour is thought to be encoded by the strength of synaptic inputs to genetically and anatomically defined subsets of neurons in the hippocampus and amygdala. Thus we propose that adverse early life events will lead to altered synaptic strengths in these hippocampal and amygdala circuits compared to normal early life experiences. Furthermore, reversing these changes in synaptic strength could ameliorate the behavioural effects of early life adversity in adults.

This project will test this hypothesis using rodent models of maternal separation and behavioural tests of positive versus negative affective behaviour developed by the Robinson group. The primary objective will be to determine how these early life effects on developing circuits impact on adult behaviour, particularly affective behaviour and decision-making. By making electrophysiological measurements of synaptic transmission coupled with genetic and anatomical identification of neuronal subtypes we will investigate how these circuits are altered by the model of early life adversity. The aim is to subsequently reverse these circuit changes using optogenetics or pharmacology guided by a mathematical model of the circuit dynamics. The ultimate goal will be to find out if manipulating synapses within the circuits underlying behaviour using pharmacological or optogenetic tools is capable of changing the balance of positive and negative affect in adult animals.

The student will be trained in animal behavioural paradigms, in vitro and in vivo electrophysiology and genetic manipulation of neuronal subtypes. In addition, through collaboration with Krasimira Tsaneva-Atanasova the project also aims to use computational models to predict the likely outcome of synaptic modifications on behaviour.

Supervisors: Prof Eshwar Mahenthiralingam (Cardiff University), Dr Denise Donoghue (Unilever R&D Port Sunlight)

Others in supervisory team/collaborators: Dr Thomas Connor (Cardiff University), Prof Ed Feil (University of Bath)

Preventing microbial contamination of industrial products and processes is a major area of industrial biotechnology. Preservatives and detergents are added to products to ensure sterility, stable shelf-life, and reduce potential risk of infection to consumers. The manufacturing industry is one of the largest users of antimicrobial agents, however, with regulatory changes and a global need to reduce environmental impact, the sector is moving towards greener, minimal formulations. Certain bacteria show reduced susceptibility to antimicrobial agents and may also evolve greater resistance upon exposure to preservatives. Bacteria such as Pseudomonas, Burkholderia, Klebsiella, Enterobacter and Acinetobacter bacteria may persist during manufacture of consumer products causing disruptive plant closure or products recalls. Understanding the basis for resistance and anticipating adaptation are important for industry to develop novel product preservation strategies.

This PhD will work with Unilever Research and Development, Port Sunlight, to examine how bacteria adapt to overcome preservative strategies used by industry. State-of-the-art genomic approaches of bacterial whole genome sequencing (WGS) and global gene expression (transcriptomics via RNA-sequencing) will be used to map antimicrobial resistance genes, metabolic pathways and mutations that underpin preservative resistance in bacteria such as Pseudomonas, Burkholderia, Klebsiella, Enterobacter and Acinetobacter. The project will: (i) build custom genomic databases of each bacterial industrial contaminant species to enable their accurate identification and analysis; (ii) systematically map the resistance genes, metabolic pathways, and mutational signatures relevant to preservative resistance in each species; (iii) examine the genetic basis for adaptation to preservative resistance by analysis of bacteria strains which have been exposed to existing or novel preservative formulations. The project will provide an interdisciplinary training in molecular microbiology, industrial microbiology, bioinformatics and statistical analysis of big datasets. Placements with the industrial partner will facilitate training in the applied aspects of industrial biotechnology, and the business strategies behind the home and personal care industry.

Supervisors: Dr Ian Fallis (Cardiff University), Prof David Williams (Cardiff University)

Others in supervisory team/collaborators: Dr Athanasia Dervisi (Cardiff University)

In recent years the increase in antimicrobial resistance has become a growing concern, and hence understanding the multi-facetted process of acquired resistance is an important issue in UK and global healthcare. In this project we propose to prepare Transition Metal Masked Antibiotics (TMMAs), in which our own class of silver N-heterocyclic carbene (Ag-NHC) complexes will be combined with antibiotics of proven efficacy, affording pre-existing antibacterials masked within a metal complex. These constructs may themselves possess anti-proliferative properties, and the presence of the metal may protect the antibiotic from acquired bacterial resistance mechanisms. We will therefore use our TMMAs as mechanistic probes for both 1) acquired soft metal resistance; and 2) the circumvention of antibiotic resistance mechanisms by metallo-adducts of pre-existing agents.

Four classes of compounds, of increasing complexity, will be investigated: 1. Simple silver(I) and copper(I) complexes of non-antibacterial co-ligands (e.g. methionine, thioxane {Walton, Inorg. Chem., 1966, 5(4), 643–649}). These samples provide controls to determine the antibacterial action of soft metal ions in the presence of a carrier ligand. 2. Bimodular TMMAs: Ag-NHCs based on our previous work. These may have antibacterial activity in themselves, but crucially they decompose to release two antibacterial components: silver(I) ions and imidazolium cations. 3. Trimodular TMMAs: Ag-NHCs which incorporate an existing clinical antibiotic into the structure. It is expected that adding the Ag-NHC component will reactivate the clinical antibiotic against acquired resistance mechanisms, as well as incorporating the additional effect of the Ag-NHC itself. 4. Multimodular TMMAs: antibiotic-Ag-NHCs in which the NHC decomposes following release of silver(I) ions, to release a non-toxic imidazole and at least one additional antibacterial component.

We anticipate that this approach will provide a method to understand the role of metal chelation in reactivating antibiotics against multi-drug-resistant bacteria. A student working on this project will enjoy high quality training in synthetic organic and inorganic chemistry, microbiology (including handling of pathogenic bacteria), and in the use of cutting edge spectroscopic techniques.

Supervisors: Prof Andrew Quantock (Cardiff University), Prof Bruce Caterson (Cardiff University)

Others in supervisory team/collaborators: Dr Robert Young (Cardiff University), Dr Elena Koudouna, Prof Keith Meek (Cardiff University), Prof Shigeru Kinoshita (Kyoto, Japan), Prof Virender Sangwan (Hyderabad, India)

The corneal epithelium is vital for healthy vision and its malfunction owing to injury or disease leads to irreversible blindness. The epithelium is maintained by a population of slow-cycling adult stem/progenitor cells located at the edge of the cornea. These exist basally and within radial infoldings of the epithelial basement membrane.

This studentship will involve an in-depth investigation of the stem/progenitor cell niche at the edge of the human cornea. Immunofluorescence microscopy of post-mortem tissue will be conducted using a battery of monoclonal antibodies developed in the Caterson laboratory against variously sulphated chondroitin sulphate epitopes. This will allow identification of matrix markers of the human corneal stem/progenitor cell niche. Co-localisation with putative identifiers of corneal stem/progenitor cells (e.g. CD-90, CD-105, p63 and β-catenin) will clarify specific co-associations between cells and their immediate environment. Higher resolution imaging by immunoelectron microscopy with nanogold particles will be conducted to distinguish cell surface-associated, or matrix-associated, localisation of the chondroitin sulphate biomarkers.

The stem cell niche will also be investigated at high magnification and in three-dimensions using the emerging technique of serial block face scanning electron microscopy to ascertain the nature and extent of these proposed intercellular connections in, and away from, the niche. Our group have pioneered the use of serial block face scanning electron microscopy for the study of cornea (Young RD, Knupp C, Pinali C, Png KMY, Ralphs JR, Bushby AJ, Starborg T, Kadler KE, Quantock AJ. Three-dimensional aspects of matrix assembly by cells in the developing cornea. Proc Natl Acad Sci USA 2014;111:687-692; Bushby AJ, P’ng KM, Young RD, Pinali C, Knupp C, Quantock AJ. Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy. Nature Protocols 2011;6:845-858).

Finally, to identify the role of the stem/progenitor cell niche in corneal epithelial dysfunction, changes in the presence, distribution and sulphation specificity of chondroitin sulphate — and of the pattern of cell-cell connections across the epithelial basement membrane — will be investigated in tissue obtained postoperatively from eyes with corneal epithelial stem cell deficiencies, caused either by disease or chemical injury.

Supervisors: Dr Thomas Richard Connor (Cardiff University), Dr Stefano Pagliara (University of Exeter)

Others in supervisory team/collaborators: Prof Andy Sewell (Cardiff University), Dr Konrad Paszkiewicz (University of Exeter), Dr Bryan Charleston (The Pirbright Institute)

Sequencing technologies have enabled us to explore the variation within human and animal populations at unprecedented scale. However, while it is now possible to routinely sequence hundreds of mammalian genomes, a huge amount of genetic variation within mammalian populations remains unexplored by focusing only upon DNA that is encoded in the germline.

The T-cell is the Swiss army knife of the immune system; orchestrating immune responses to a variety of threats – from infections to cancer. Each T-cell expresses a receptor (TCR) that can see hundreds of targets, and mammals are each born with a fixed TCR repertoire that is quasi-randomly generated in utero. It is becoming increasingly obvious that understanding how the TCR repertoire – and the “extended genome” it represents – is formed and how it varies between individuals is critical for fully understanding human and animal health.

The TCR itself is encoded by a pair of loci formed by a rearrangement event that occurs during development. Both loci are located on separate chromosomes. As a result it is currently not possible to unambiguously pair these two chains, meaning it is currently impossible to accurately dissect the TCR repertoire at scale. This is a serious limitation as it means that we have little idea of how a TCR repertoire is structured in most mammals; limiting our ability to develop vaccines to key diseases that can have an enormous effect on the health, welfare and productivity of livestock.

This project will put you at the forefront of exploration to help us understand how the TCR repertoire is constructed in pigs and how it can be utilised to enable protection against key pathogens. This project will combine cutting edge single cell sequencing and bioinformatics method development to dissect the TCR repertoire of pigs.

Working with world-class researchers at the Universities of Cardiff and Exeter, the student will design microfluidic devices to capture individual cells prior to sequencing. The student will then improve existing methodologies to extract nucleic acids, generate high throughput sequencing libraries, comparing these approaches using cutting-edge bioinformatics, developing a bioinformatics framework to characterise TCR repertoires. Using this framework, the student will then characterise TCR repertoires from multiple pigs to examine how the repertoire differs between individuals that respond differently to viral disease, revolutionising our understanding of the immune system of this organism and demonstrating a ground-breaking new large mammalian model for the study of the immune system and human/animal disease.

Supervisors: Prof Rosalind John (Cardiff University), Dr Anthony Isles (Cardiff University)

Others in supervisory team/collaborators: Prof Ian Jones (Cardiff University), Prof Andrew Ward (University of Bath), Dr Araxia Urrutia (University of Bath)

Maternal behaviour is one of the most important features of the female mammal, ensuring the appropriate postnatal development and survival of her offspring. Conversely, aberrant maternal care can result in aggression and the killing of young while young exposed to poor maternal care early in life can exhibit life long changes in their behaviour, a feature also reported in humans. This project seeks to employ a systems biology approach to explore the mechanisms that induce maternal behaviour and then use this information to make further predictions that will then be tested in vivo. The work will be done using a novel model in which maternal behaviour is genetically mis-programmed. Data on behaviour, biochemistry and the transcriptome of key brain regions has been gathered. The student will interrogate the transcriptomic data to identify the neural systems underpinning maternal responses in the context of the phenotypes we have identified. They will use this information to predict behaviours and ask whether these behaviours are consistent with the data available to support their predictions. Once this phase of the work is complete, the student will apply their bioinformatics approach to a second model, as yet uncharacterised, initially generating RNAseq data and integrating this to direct the behavioural characterisation of the new model based on just the transcriptomic data. They will then validate their predictions in vivo.

This project will combine experimental work (wet lab training) with the integration, interrogation and analysis of large transcriptomic datasets (advanced computational training) to provide the student with an advantageous skill set appropriate to 21st century science. The student will be rigorously trained in good experimental design and statistical analysis, training in molecular biology, immunohistochemistry, classic and state-of-the-art behavioural assays, and bioinformatics.

Supervisors: Dr Edze Westra (University of Exeter), Prof Mark Szczelkun (University of Bristol)

Others in supervisory team/collaborators: Prof Angus Buckling (University of Exeter)

Bacteria in the rhizosphere can benefit agriculture by making nutrients available to plants. However, their beneficial effect is thought to be limited by viruses (phage) in soil. CRISPR-Cas adaptive immune systems of bacteria provide phage resistance but it is frequently very difficult to have bacteria evolve this type of phage resistance, and when it evolves, phage can readily escape CRISPR-Cas by point mutation. Recent studies suggest phage resistance may be improved when the adaptive immune system is integrated with an innate immune system, known as restriction-modification (RM). However, synergism between CRISPR and RM has not been clearly demonstrated yet, and because RM systems are highly diverse, it is also unclear whether such synergism would apply to all or only some RM systems. The key aim of this project is therefore to investigate the existence of synergy between different types of RM and CRISPR-Cas, and examine how this synergistic interaction affects bacteria-phage co-evolution. We already have a bacterial host that evolves high levels of CRISPR immunity in response to some, but not all phages. We will use this experimental system to first examine if levels of evolved CRISPR immunity increase when bacteria are equipped with different types of RM systems, to reveal putative synergistic RM-CRISPR interactions. We will then compare the genetic changes that accompany the evolution of CRISPR immunity in the presence and absence of RM, in order to reconstruct the mechanistic basis underlying the synergistic RM-CRISPR interactions. This will be complemented with in vitro biochemical studies that examine how purified RM components digest DNA molecules. Finally, we will investigate how an integrated RM-CRISPR immune response impacts bacteria-phage co-evolution, specifically the ability of phage to overcome immunity, compared to bacteria carrying a single immune mechanism and examine trade-offs associated with encoding multiple immune mechanisms.

The student will receive training in experimental evolution, biochemistry, molecular microbiology and genetics. This interdisciplinary project integrates expertise on CRISPR-Cas (Westra, Exeter), RM (Sczcelkun, Bristol) and bacteria-phage co-evolution (Buckling, Exeter).

Supervisors: Dr Alan Brown (University of Exeter), Dr Steve Porter (University of Exeter)

The Burkholderia cepacia complex (BCC) is a versatile group of closely-related bacterial species which occupy diverse environmental and clinical niches. They are frequently isolated from sources in which heavy metals are elevated, including heavy metal-contaminated soils and the sputum of cystic fibrosis patients. Consequently, BCC have robust heavy metal resistance mechanisms. This project will focus on defining the regulators and effectors of this resistance, and how they contribute to BCC’s success in diverse niches. We have characterized the CzcRS two-component system (TCS) of BCC, which confers zinc and cadmium resistance by regulating the CzcCBA efflux pump and a separate gene cluster encoding a novel putative heavy metal resistance determinant. Strikingly, CzcRS also plays a role in virulence and intracellular survival, and has been implicated in antibiotic resistance, highlighting the importance of this single metal-responsive TCS in both natural and clinical environments. Clinical relevance is heightened by the increasing incorporation of zinc (and other metals) into medical devices (e.g. catheters), which may result in the activation of CzcRS-related TCSs during infection. We have also identified two other putative heavy metal-responsive TCSs that we predict form a novel multi-kinase network with CzcRS and may enable cells to modulate their responses to certain metals depending on the other stressors present.

Through a multidisciplinary programme of research combining biochemistry, molecular bacteriology and genomics, the student will:

• define the role of individual CzcRS-regulated genes;
• define the hierarchy and interconnectedness of the heavy metal-responsive TCSs;
• identify & characterize the resistance determinants controlled by each TCS.

The first year will comprise two rotation projects. The first (with Dr Steve Porter) will use bioinformatics to predict metal-responsive multi-kinase networks in BCC, and perform experimental validation on selected systems using phosphorylation assays. The second (with Dr Alan Brown) will investigate the putative novel heavy metal resistance determinant referred to above through targeted gene deletion and phenotyping of resulting mutants.

Thereafter (years 2-4), the student will (a) apply genomics (ChIP-seq) to define the regulon of each TCS; (b) perform targeted mutagenesis and phenotypic characterization of relevant regulators and effectors of resistance; (c) use fluorescent-reporter strains to assess the responsiveness of each TCS and their interdependence on each other; (d) biochemically investigate direct interactions within the putative multi-kinase network.

The project, which offers outstanding training opportunities in diverse methodologies, will reveal the complex regulatory networks and downstream effectors of heavy metal resistance in clinically- and environmentally-important BCC.

Supervisors: Dr Alastair Wilson (University of Exeter), Dr Alex Thornton (University of Exeter)

Cognitive processes are vital for carrying out the day-to-day behaviours needed for survival and reproduction. Comparative psychologists have made great progress in determining the cognitive mechanisms underpinning behaviour, showing that many species are capable of performing more sophisticated cognitive tasks than previously thought. However, it is also becoming clear that cognitive performance varies can vary a lot among individuals within populations of the same species. This variation is hugely significant as it is a prerequisite for both natural selection and genetic variation – the two ingredients for ongoing adaptive evolution.

Currently our understanding of how cognition evolves in animals is limited. How does cognitive ability impact fitness and how do genes mediate these effects? One widely suggested idea is that differences in cognitive processes are linked to repeatable differences in individual “personality” traits (e.g. boldness, aggressiveness) that are known to be under natural selection in many animals. However though intuitive, the existence of such relationships remains largely speculative and directions of causality are unclear. Fitness could potentially trade-offs between distinct “domains” of cognitive performance. For instance, an individual’s ability to learn novel useful information might conceivably be negatively correlated with its ability to remember it. In humans, correlations tend to be positive across domains, consistent with a single underlying “general intelligence” factor (g) though whether this is true for other species is unclear. If so, then selection for higher intelligence could lead to rapid evolution, but only if genes contributes importantly to variation in g. In humans, a large body of (sometimes controversial) research suggests this is likely, but comparable studies of other taxa are scarce.

This project will address these gaps in our knowledge, taking an experimental approach to determine the genetic causes and consequences of among-individual variation in cognition in guppies, Poecilia reticulata. It will do this by combining lab-based behavioural studies with quantitative genetic modelling to frame and test evolutionary hypotheses about the causes and consequences of (genetic) variation in cognition. Specific objectives will depend on the direction and interests of the successful applicant but might include

1- Demonstrating among-individual variation in cognitive performance and determining whether this reflects general intelligence (g).

2- Determining whether variation in g can explains animal personality.

3- Characterising the genetic architecture of g.

4- Determining how g shapes the genetic relationships among fitness-related traits.

Supervisors: Dr Timothy Etheridge (University of Exeter), Prof Christian Soeller (University of Exeter)

Others in supervisory team/collaborators: Dr Ryan Ames (University of Exeter)

As people grow older skeletal muscle gradually becomes smaller and weaker. This progressive muscle weakness results in reduced mobility, independence and quality of life, and increased incidence of frailty-related falls and injury in ageing populations. A commonly observed feature that might contribute to age-related muscle decline is blunted growth responses to exercise training, and a likely impairment in muscular regeneration from individual bouts of activity. Whilst the mechanisms underpinning this phenomenon are poorly understood, failure of exercise-responsive molecular signals are logical candidates for investigation. However, many cross-talking signalling pathways are involved in the post-exercise remodelling process, and systematic evaluation of all associated regulatory molecules and their dysregulation in ageing muscle is not feasible in humans. This project will therefore employ an interdisciplinary approach to predict novel signalling networks regulating exercise-induced metabolic/functional adaptation, and examine the physiological role of these networks directly in ageing people during acute post-exercise muscle remodelling. Bioinformatic predictive modelling will be used to filter candidate network components involved in muscle adaptation to exercise. The relevance of these networks to ageing muscle decline will then be examined using state-of-the-art immunofluorescent imaging techniques, applied to human muscle biopsy samples collected during the acute post-exercise remodelling period. The dynamic temporo-spatial responses of putative regulators of muscle regeneration, overlaid onto the direct functional responses to exercise in humans, will provide new insight into the mechanisms underpinning exercise-mediated muscle adaptation and its deregulation during the ageing process.