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Basic Bioscience Underpinning Health Rotation Projects 2014-2015

An experimental approach to investigate the effects of commensal bacteria on host's health

Supervisor: Dr. Anaid Diaz, Veterinary Medicine

Second supervisor: Dr. Olivier Restif, Veterinary Medicine

Project abstract: Microbial communities living inside the digestive tract of animals have a strong influence on health and disease. One of the benefits of commensal associations is that microbes prevent the colonisation of pathogenic bacteria inside the host’s gut, thus reducing disease risks. Despite its importance, we understand little about the dynamics of the interaction of bacteria inside its host or in a population. Using the nematode Caenorhabditis elegans and various bacteria (e.g. Escherichia coli, Salmonella enterica Typhimurium and Pseudomonas aeruginosa) as a model system, we are investigating different aspects of the nematode-bacteria interactions.     We have set up an experimental system in which specific aspects of colonisation by bacteria can be monitored. The existence of fluorescent-tagged bacteria provides markers to track the spread of infection inside a worm and in a population. In parallel, using mathematical modelling techniques, we have constructed demographic models to predict population changes in response to the different bacteria.     This project integrates the next key element: bacteria competition inside the host’s gut and transmission between worms. Using microbiological and microscopy techniques, the student will measure bacteria competition inside hosts and transmission within a population. This will provide important information for the mathematical modeling and understanding of disease dynamics.

Learning outcomes and skills acquired: This project will be beneficial for acquiring skills of experimental design and hypothesis testing. The student will have the opportunity to work on both components of the project (experiments and modeling), with relevant training provided depending on his/her interest and background. The project is part of a BBSRC grant awarded to Olivier Restif. A post-doctoral research associate, Dr Anaid Diaz, is working full-time in the lab on this project, while O.R. focuses on the modeling part. Thus the student will be supervised by experienced researchers on both sides.

Project availability: Michaelmas and Lent Term

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Molecular basis of brown fat differentiation and activation

Supervisor: Dr. Antonio Vidal-Puig, Institute of Metabolic Science

Second supervisor: Julio Saez RoDr.iguez, European Bioinformatics Institute

Project abstract: Brown adipose tissue (BAT) is unique in its ability to generate heat by burning fat rather than storing it.  BAT can expand its mass, ramp up thermogenic gene expression and increase its oxidative capacity very quickly, but the mechanisms regulating these processes are not well understood.     We recently discovered a molecule (bone morphogenetic protein 8b – BMP8B) that is secreted into and around active BAT (Whittle et al. Cell 2012). BMP8B selectively increases BAT sensitivity to adrenergic stimulation, increasing its thermogenic activity. We believe that better understanding of the molecular networks that BMP8b activates will be of great benefit to understanding adrenergic signalling and BAT biology. In this project, the student will generate data in the Vidal-Puig lab to characterizes the pathways triggered by Bmp8b in primary and transformed adipocytes. Phospho-proteomic measurements will be obtained using mass spectrometry, and the effect of Bmp8b on cellular phenotype will be monitored by gene expression arrays. These data will be analyzed using methods developed in the Saez-Rodriguez group at EBI to reconstruct signaling networks by integration of high-throughput omics data and prior knowledge on the pathways, leading to an mechanistic understanding of how Bmp8 controls thermogenesis.    Reduced thermogenesis in humans is increasingly shown to correlate with obesity and ageing, making mechanisms that increase BAT activity extremely relevant to metabolic research. Additionally, knowledge of this previously unknown pathway will have implications for physiological systems controlled by the sympathetic nervous system and be of  relevance to a range of metabolic changes associated with ageing

Learning outcomes and skills acquired: Obtaining knowledge in areas of energy homeostasis, metabolism, stem cells, systems biology. This is part of a multidisciplinary program with an emphasis in integrating biological and physiological responses with moleuclar data using  a systems biology strategy

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Identification of new vaccine targets for the prevention of Strangles

Supervisor: Dr. Andrew Waller, Animal Health Trust

Second supervisor: Duncan Maskell, School of the Biological Sciences

Project abstract: This project exploits TraDIS (transposon-directed insertion site sequencing) to identify genes required by Streptococcus equi (S. equi) to cause Strangles in a susceptible natural host. Strangles remains the most frequently diagnosed infectious disease of horses and is responsible for significant welfare and economic cost. However, vaccine research has been hindered by the time taken to make mutations in individual genes to determine their role in the disease process.     Six collections of S. equi, each comprising 5,000 different mutants (input population) will be used in this project. Each individual mutant contains a copy of the ISS1 transposon inserted randomly in the S. equi genome, disrupting the DNA sequence at the point of insertion. We will infect ponies with the mutant collections and use TraDIS to identify mutants that are recovered from lymph node abscesses (output population). These analyses will provide a numerical measure of the extent to which mutants were negatively- or positively-selected during the infection enabling the identification of genes important for S. equi to cause Strangles. The results of this project will provide new insights into how S. equi causes disease on an unprecedented scale, highlighting novel targets with which to improve the design of vaccines for the benefit of equine health.

Learning outcomes and skills acquired: The student will develop a wide variety of skills in microbiology, bacterial genetics, next generation sequencing and in vivo infection studies.

Project availability: Lent Term Only (January - March 2015)

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Mechanisms underlying formation of axonal endoplasmic reticulum

Supervisor: Dr. Cahir O'Kane, Genetics

Project abstract: Axons contain a continuous longitudinal network of tubular smooth endoplasmic reticulum (ER). Its length and continuity make it like a "neuron-within-a-neuron", a membrane system that could conduct local or long-range signals. Little is understood of how it arises, and the relationship between its form and function. However, many cases of Hereditary Spastic Paraplegia (HSP), with preferential degeneration of longer motor axons, are caused by mutations in proteins that curve ER membrane. The growing number of HSP genes presents opportunities to study this poorly understood compartment, important in axonal homeostasis and maintenance.     We have developed the following Drosophila toolsER axonal markers; GAL4 lines to visualise single axons; and mutant stocks that affect ER integrity in the absence of ER modeling proteins. Phenotypes observed include loss of ER staining from longer but not shorter motor axons, or fragmentation of axonal ER.    

A rotation project could involve any of: 

- Testing additional ER membrane protein mutants for effects on axonal ER 

- Exploring rapid visualisation of axonal ER using novel fluorescent markers in live animals, and feasibility of genetic screens 

- Bioinformatic identification of new ER membrane proteins using algorithms for distinguishing features like intramembrane loops   

Background References:

O’Sullivan, Jahn, Reid, O’Kane (2012) Reticulon-like-1, the Drosophila ortholog of the Hereditary Spastic Paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons. Hum Mol Gen, 21, 3356. 

Blackstone, O’Kane, Reid (2011) Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat Rev Neurosci 12, 31

Learning outcomes and skills acquired: Depending on the project chosen, there is the possibility to learn (1) molecular approaches to neuronal cell biology, including use of Drosophila genetics, genetic databases and resources, immunomicroscopy using laser confocal microscopy, or (2) bioinformatic and programming approaches that make use of protein sequence evolution to identify conserved structural features.    No special skills required beyond basic laboratory techniques and/or computer literacy. Some programming experience is required for the bioinformatics project, but this need not be advanced. The techniques used will be immunocytochemistry, brain dissections, confocal microscopy, image analysis, fly genetics, use of online genetic databases.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Trans-synaptic trafficking of Tau

Supervisor: Professor Clemens Kaminski, Chemical Engineering and Biotechnology

Second supervisor: Dr. Claire Michel, Chemical Engineering and Biotechnology

Project abstract: Amyloid proteins are hallmarks of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, however their behaviour in health remains unclear.   Our lab has developed various optical microscopy techniques to study amyloid protein localisation. In particular, our single molecule localization techniques provide images with a resolution reaching the molecular scale (20nm) and are hence ideal to reveal molecular mechanisms in cells such as the trafficking of proteins in different compartments.  This project aims to study trafficking of Tau between neurons. In particular, we want to define the specific pathways involved in the propagation of Tau through synaptic connections, with the use of custom designed microfluidic devices for the isolation of axons.    For information on our group please see: http://laser.ceb.cam.ac.uk/   

Relevant publications:   

Kaminski Schierle GS, van de Linde S, Erdelyi M, Esbjörner EK, Klein T, Rees E, Bertoncini CW, Dobson CM, Sauer M, Kaminski CF (2011) In situ measurements of the formation and morphology of intracellular β-amyloid fibrils by super-resolution fluorescence imaging. J Am Chem Soc 133:12902–12905.   

Michel CH, Kumar S, Pinotsi D, Tunnacliffe A, St George-Hyslop P, Mandelkow E, Mandelkow E-M, Kaminski CF, Kaminski Schierle GS (2014) Extracellular monomeric tau protein is sufficient to initiate the spread of tau protein pathology. J Biol Chem 289:956–967.

Learning outcomes and skills acquired: Cell culture, immunocytochemistry, fluorescence lifetime imaging microscopy and 2-color super-resolution microscopy (dSTORM) will be extensively used for this project, which will give the student the opportunity to train on advanced fluorescence microscopy techniques.

Project availability: Michaelmas and Lent Term

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Study of Foerster Resonance Energy Transfer (FRET) interactions between fluorescent amyloid fibrils and external fluorophores

Supervisor: Professor Clemens Kaminski, Chemical Engineering and Biotechnology

Second supervisor: Dr. Gabriele Kaminski Chemical Engineering and Biotechnology

Project abstract: Amyloid proteins such as related to many neurodegenerative diseases develop an intrinsic fluorescence upon aggregation (Chan et al., 2014). This phenomenon can be used to construct a FRET based amyloid aggregation sensor for live cell imaging by adding a fluorophore to the amyloid protein of interest. Upon aggregation the reporter fluorophore will act as donor in a FRET interaction between the aggregated amyloid protein and the fluorophore which leads to a decrease in the fluorescence lifetime of the donor fluorophore. In order to optimize the FRET sensor, i.e. the interaction between the donor fluorophore and the intrinsic fluorescence of the amyloid protein, detailed information of the spectral properties of the intrinsic fluorescence of the amyloid protein is required.   The following questions need to be addressed: How do the absorption and the excitation spectra of the amyloid monomer or fibril change with the presence of an attached fluorophore? How does this affect the dynamic range of the fluorescence lifetime sensor?  Chan FTS, Pinotsi D, Kaminski-Schierle GS, Kaminski CF,  "Structure-Specific Intrinsic Fluorescence of Protein Amyloids Used to Study their Kinetics of Aggregation,"  (2014) in Uversky VN, Lyubchenko YL, (eds)  Bio-nanoimaging: Protein Misfolding & Aggregation, Academic Press, pp. 147-155   Kaminski CF, Rees EJ, Kaminski Schierle GS, "A quantitative protocol for intensity-based live cell FRET imaging". Methods Mol Biology (2014), 1076:445-454.

Learning outcomes and skills acquired: Obtain absorption and excitation spectra of amyloid proteins (monomer/vs fibrils).   Perform fluorescence lifetime measurements of labeled amyloid proteins (with different fluorophores attached).  Calculate the FRET efficiency for different amyloid fibrils with different fluorophores attached.  This project will involve aggregation assays in vitro, protein labeling, AFM imaging, training in acquiring emission and excitation spectra, use of (and preparation of samples) Cavity Enhanced Absorption Spectroscopy. Also, the student will acquire in depth knowledge of the FRET theory, will analyse data, plot results, apply Matlab codes and develop simple scripts. Furthermore, the project involves the use of statistical software such as Prism as well as image analysis software (such as ImageJ).

Project availability: Michaelmas and Lent Term

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Dynamic regulation of calcium signalling analyzed by advanced optical microscopy

Supervisor: Professor Colin W Taylor, Pharmacology

Project abstract: Dynamic regulation of calcium signalling analyzed by advanced optical microscopy  ER is the major intracellular Ca2+ store from which receptors that stimulate formation of IP3 evoke calcium release via IP3 receptors. The resulting increases in cytosolic Ca2+ concentration are precisely organized in time and space, and they regulate most cellular activities. ER is a dynamic organelle that associates with the plasma membrane and every other intracellular organelle. We are addressing the contributions of dynamic interactions between intracellular organelles to Ca2+ signalling, using genetically encoded bio-sensors, gene-editing to label endogenous proteins, and super-resolution microscopy. Our work includes extensive interdisciplinary collaborations.

Specific projects for 2014-15: 

1. Development of optical probes for analyses of lysosomal Ca2+ signalling. 

2. Contributions of mobile organelles expressing IP3 receptors to cell migration. 

3. Organization of Ca2+ signals in human astrocytes.   

References:

Seo et al. (2012) Structural and functional conservation of key domains in InsP3 and ryanodine receptors. Nature 483, 108-112.   

Lopez et al. (2013) Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum. J. Cell Sci. 126, 289-300.   

Li et al. (2013) CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions. Proc. Natl. Acad. Sci. USA. 110, 8507-8512.   

Thurley et al. (2104) Reliable encoding of stimulus intensities within random sequences of intracellular Ca2+ spikes. Sci. Signal. 7, ra59.

Learning outcomes and skills acquired: The lab addresses the behaviour of intracellular Ca2+ channels from single molecules (patch-clamp, optical methods, structural biology) to the complex responses of intact cells (imaging, high-throughput analyses) using a combination of approaches (TIRF, PALM and STORM microscopy, electrophysiology, structural and molecular biology) and extensive collaborations (with chemists, mathematicians and experts in NMR and crystallography). You will have the opportunity to apply a variety of these methods and to gain exposure to others through interactions within the lab.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Developing companion animal stem cells for use in veterinary medicine

Supervisor: Dr. Debbie Guest, Animal Health Trust

Project abstract: We have two main areas of research. Firstly, the development of new treatments to aid tendon regeneration in horses. Horses suffer from a high number of tendon injuries. They heal through the formation of scar tissue which leads to a high rate of re-injury. Clinically autologous mesenchymal stem cells (MSCs) are being used to aid tendon regeneration but little is known about their mechanism of action. Our previous work demonstrated a poor long-term tissue integration of MSCs suggesting that they do not make a direct, physical contribution to tissue repair through differentiation. Instead, evidence suggests that MSCs may function through modulation of injury-induced inflammation.  In contrast, pluripotent equine embryo-derived stem cells (ESCs) have a high survival in the injured tendon and appear to undergo differentiation to tenocytes. We are now working to understand the pathways underpinning the different mechanism of stem cell function in tendon repair.     Secondly, we have derived induced pluripotent stem cells (iPSCs) from horses and dogs to allow the in vitro modelling of inherited diseases. For example, fractures occur frequently in racing Thoroughbred horses. Although we have demonstrated that fracture risk has a genetic component, it is a complex disorder with multiple genes involved. We hypothesise that a high risk genotype confers susceptibility to fracture through sub-optimal bone remodelling in response to exercise. We are currently using equine iPSCs to test this hypothesis in vitro by performing bone differentiation of iPSCs derived from horses at high and low risk of fracture.

Learning outcomes and skills acquired: The student will gain experience in a number of molecular biology techniques including cell culture, RNA extraction, quantitative PCR and immunocytochemistry. The research will be carried out in a small, motivated group allowing close supervision by the project leader. The student will have the opportunity to attend departmental laboratory meetings to allow them an insight into the other research which takes place at the Animal Health Trust and the opportunity to develop their presentation skills.

Project availability: Michaelmas and Lent Term

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Structural studies of the nucleosome remodelling and deacetylase complex NuRD

Supervisor: Professor Ernest Laue, Biochemistry

Second supervisor: Dr. Wei Zhang, Biochemistry

Project abstract: We are carrying out structural studies of the NuRD complex, which contains both of the Rb-associated histone chaperone proteins RbAp46 and RbAp48. RbAp46 is also an essential subunit of the histone acetyl-transferase HAT1 complex, whilst RbAp48 is a component of the heterotrimeric p48/p60/p150 chromatin assembly factor CAF-1 that is responsible for deposition of histones H3/H4 in nucleosome assembly.  Several different projects are available to study various aspects of the structure and assembly of the NuRD complex, and its interactions with nucleosomes. Projects could involve tagging different components of the NuRD complex, purifying the complex and carrying out a combination of EM and chemical cross-linking/mass spectrometry (MS) to study the structure of different in vivo NuRD complexes. Secondly, we are systematically co-expressing and purifying different sub- and intact NuRD complexes for structural studies. We would like to study their structures and interactions with nucleosomes using either X-ray crystallography or EM.

Learning outcomes and skills acquired: The student would learn about and become familiar with:    •      Mass spectrometry and proteomics to identify components of in-vivo protein complexes  •          Protein expression and purification  •   Structural studies using Electron Microscopy, X-ray crystallography and NMR spectroscopy  •            Biophysical methods, including Fluoresence spectroscopy

Project availability: Michaelmas and Lent Term

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Neuronal activation by acid: pain and breathing.

Supervisor: Dr. Ewan St. John Smith, Pharmacology

Project abstract: Acid activates a variety of neurones including those that detect noxious stimuli and mediate pain (nociceptors), as well as those that modulate breathing rhythmicity (central chemoreceptors). Painful inflammatory conditions, such as rheumatoid arthritis, are characterised by tissue acidosis, which generates pain. We use a combination of retrograde labelling, pH-imaging, immunohistochemistry and whole-cell electrophysiology to understand how acid activates nociceptors. By contrast, carbon dioxide mediated alterations in pH produce critical changes in neuronal activity to maintain breathing. Unlike mice, naked mole-rats display robust behavioural differences in carbon dioxide sensitivity and we are using immunohistochemistry and electrophysiology to determine the molecular differences that underlie this behavioural difference. Potential projects include assessing the acid-sensitivity of defined sensory neurone populations and exploring mechanisms underlying the differential neuronal carbon dioxide sensitivity between mice and naked mole-rats.    Key words: neurobiology, pain, acid, ion channels and breathing   

Smith, E.S.J., et al. (2011). The Molecular Basis of Acid Insensitivity in the African Naked Mole-Rat. Science 334, 1557 –1560.

Learning outcomes and skills acquired: Students will gain an understanding of molecular neuroscience; in particular how neuronal activity is controlled by changes in tissue pH, which can be both physiological (e.g. the control of breathing) and pathological (e.g. the tissue acidosis associated with inflammatory pain). Students will have the opportunity to gain numerous technical skills: tissue dissection, immunohistochemistry, electrophysiology and pH imaging. In addition, students will participate in group lab meetings and journal clubs, which offer the possibility to develop presentation and analysis skills.

Project availability: Michaelmas and Lent Term

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The role of bacterial symbionts in protecting Drosophila against viruses

Supervisor: Dr. Frank Jiggins, Genetics

Project abstract: The bacterial symbiont Wolbachia protects many insects against viral infections. It can also readily spread through insect populations, and releasing Wolbachia-infected mosquitoes can block the transmission of pathogens like dengue virus. The aim of this project is to understand this poorly characterised form of antiviral protection, using Drosophila as a model system. We have recently established a panel of Wolbachia strains that we have transferred into the same species of Drosophila. This provides a powerful tool to understand the evolution of the interaction and its underlying mechanism. For example, how does the density of Wolbachia in different tissues affect the strength of protection against viruses? Is there a trade-off, such that strains that confer high levels of protection harm insects that have not been exposed to the virus? Depending on the interests of the student, we can also offer projects involving bioinformatics and evolutionary genetic analyses, and projects looking at the genetics of resistance to viruses. Take a look at the lab website for details of what we work on.

Learning outcomes and skills acquired: This project will provide training in experimental design, Drosophila genetics, insect viruses, and data analysis. It will provide insights into the rapidly moving field of how an animal's microbiota can affect disease susceptibility.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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How nutrition affects microglial interactions with neurons in development, ageing and neurodegeneration

Supervisor: Professor Guy Brown, Biochemistry

Project abstract: Microglia are brain macrophages involved in shaping, protecting and killing brain neurons in development, aging and neurodegeneration.  We have been investigating the mechanisms by which microglia become inflamed and damage neurons.  Recently we have found that inflamed microglia can phagocytose (i.e. eat) live neurons, and thereby cause neuronal death and loss. This project will investigating the signals and receptors involved in this process, and whether phagocytosis is beneficial or detrimental in particular pathologies.  The project will use cell culture, fluorescence microscopy and molecular cell biology.   

Brown GC & Neher JJ (2014) Microglial phagocytosis of live neurons. Nat. Rev. Neurosci. 15, 209-216. 

Neher JJ, Emmrich JV, Fricker M, Mander PK, Thery C, Brown GC (2013) Phagocytosis executes delayed neuronal death after focal brain ischemia. Proc Natl Acad Sci 110:E4098-107. 

See:   www.guybrown.net

Learning outcomes and skills acquired: Ability to practise: Molecular Cell Biology,  Cell culture, Imaging and Biochemistry.

Project availability: Michaelmas and Lent Term

Other relevant themes: Food security

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The role of the GIMAP family GTPases in the control of cell growth and survival of lymphocytes in vitro and in vivo

Supervisor: Dr. Geoffrey Butcher, The Babraham Institute

Project abstract: The host laboratory has recently identified a specific interaction between human GIMAP6 and a protein of the autophagy pathway, GABARAPL2. This has opened the possibility of a role for autophagy in the survival of lymphocytes which has previously been shown to depend on the presence of GIMAP family genes.   We have now derived knockouts of the mouse GIMAP6 gene with both global and conditional deletions. During the period of the rotation projects we will be in a position to characterise the phenotypes of these mice with particular attention to the lymphoid compartments. The student will participate in this work which will involve analysis of lymphocyte populations by flow cytometry, in vitro survival assays and in vitro T cell differentiation assays. Further we will compare the autophagic capacity of GIMAP6 knockout lymphocytes with wild type controls to directly address the role of the GIMAPs in this process.

Learning outcomes and skills acquired: Design of experiments to analyse genetically-manipulated animals; exposure to current issues in the complex field of lymphocyte survival and maintenance; preparation of tissues from laboratory mice; establishment of cell cultures; flow cytometry and associated statistical analyses of lymphocyte subpopulations using multiple parameters; autophagic assays.

Project availability: Michaelmas and Lent Term

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Regulation of Receptor Trafficking by P-Rex (Signalling, GPCR, small GTPase, GEF)

Supervisor: Dr. Heidi Welch, The Babraham Institute

Second supervisor: Martin Baker, The Babraham Institute

Project abstract: P-Rex is a protein family that integrate signals such as hormones, growth factors or pathogen-derived molecules inside cells and transmit them by switching-on the small G protein Rac [1]. Active Rac then causes cells to change shape and migrate, produce oxygen radicals or switch-on genes to produce new signals. These are useful processes that can alert immune cells to invading pathogens, get them to migrate towards the pathogens and destroy them [2,3], assure that skin cells move to where they should during development [4], or preserve nerve cell function during ageing [5]. The same processes can, however, derail during illness. For example, cancer cells often have excess P-Rex or mutated forms that are permanently switched-on, leading to the cancer cells moving around the body unhindered, causing metastasis [4, 6-8]. Recently, we found a new role of P-Rex in receptor trafficking. In this project, the student will express mutant P-Rex in cells that harbour a fluorescently labelled G protein-coupled receptor and analyse by fluorescence imaging which domains of P-Rex regulate trafficking, thus revealing mechanistic insight.  

[1] Welch HC et al, 2002, Cell 108, 809-821.

[2] Welch HC et al, 2005, Curr Biol 15, 1867-1873.

[3] Herter J et al, 2013, Blood 121, 2301-2310

[4] Lindsay CR et al, 2011, Nature Commun 2, 555.

[5] Donald S et al, 2008, PNAS 105, 4483-4488.

[6] Fine B et al, 2009, Science 325, 1261-1265.

[7] Sosa MS et al, 2010, Mol Cell 40, 877-892.

[8] Berger MF et al, 2012, Nature 485, 502-506.

Learning outcomes and skills acquired: The rotation student will learn about cell signalling, in particular GPCR and small G protein signalling, as well as receptor trafficking processes. They will acquire a range of widely useful laboratory skills: culture of mammalian cell lines, transient expression of exogenous P-Rex proteins, cell stimulation to induce receptor trafficking, immuno-staining of the various overexpressed proteins, and fluorescence microscopy to monitor the effects of the various P-Rex protein domains on GPCR trafficking, both in fixed cells and by live imaging. He/she will also participate in our Signalling Programme meetings and present their findings at these meeting to hone their presentation skills.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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How the fat flies: understanding how obesity interacts with longeviety in fruit flies

Supervisor: Dr. Julian Griffin, Biochemistry

Second supervisor: Steve Russell, Genetics

Project abstract: As humans age there is a gradual increase in body mass index which contributes to the rise in obesity in adults and the elderly. This increase in adiposity may have detrimental effects to cellular function, and has been suggested as contributing to a variety of chronic diseases including type 2 diabetes, fatty liver disease and atherosclerosis. The Griffin group (Department of Biochemistry, the Cambridge Systems Biology Centre and MRC Human Nutrition Research) has been using mouse models and metabolomics to look at the interaction between obesity and age [1], but these studies are expensive and can take up to two years to complete. The Russell group (Department of Genetics and the Cambridge Systems Biology Centre) have been using flies as a model organism for a variety of studies to investigate gene regulation at the global scale. As part of this rotation project we will investigate the use of fruit flies as a model organism to investigate the interaction between body fat and age. We will investigate fly mutants where lipid metabolism is deregulated using a combination of high resolution mass spectrometry and nuclear magnetic resonances spectroscopy. This project may also provide an alternative to the use of vertebrates in ageing research, addressing the concept of the 3Rs to reduce the use of laboratory animals.   

[1]  Metabolomics of the interaction between PPAR-alpha and age in the PPAR-alpha-null mouse. Atherton HJ, Gulston MK, Bailey NJ, Cheng KK, Zhang W, Clarke K, Griffin JL. Mol Syst Biol. 2009;5:259.

Learning outcomes and skills acquired: The student will be taught a variety of skills related to the use of big data and omic approaches. This includes the use of multivariate statistics for processing large datasets and NMR spectroscopy and mass spectrometry for metabolomics. In addition the student will receive instruction in fly genetics and the use of fruit flies as a model organism.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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New approaches to understanding cellular regulation by palmitoylation

Supervisor: Dr. Julian Rayner, The Wellcome Trust Sanger Institute

Project abstract: Cellular regulation through signaling cascades that culminate in reversible post-translational modification (PTM) of effector proteins are among the most studied areas of basic cell biology. The best understood PTM is phosphorylation, and the enzymes that catalyse it, kinases and phosphatases, are frequently identified as potential therapeutic targets. Palmitoylation, the reversible addition of a long chain fatty acid to cysteine residues, is a much less studied PTM which can control protein trafficking, stability, signaling, and complex formation. The development of mass spectrometry techniques to study protein palmitoylation has radically changed our understanding of the diversity of proteins that are affected by this PTM. These experiments have made clear that, like phosphorylation, palmitoylation is a regulatory tool that has an impact on a wide range of essential eukaryotic processes, with more than 10% of proteins in a given cell type being palmitoylated. However we currently know little about the enzymes that catalyse the removal and addition of palmitoyl groups. This project will use an array of cutting edge biochemical, mass spectroscopy and genetic approaches to study palmitoyl transferases in Apicomplexan parasites, which are ideal models for study because they have a reduced repertoire of palmitoyl transferases relative to other eukaryotic cells. The project will use affinity purification and mass spectrometry to identify protein-protein complexes involving palmitoyl transferases, develop new proteomic techniques to systematically identify palmitoylation sites for the first time, and apply state of the art experimental genetic tools to inducibly delete essential palmitoyl transferases. The student will learn a comprehensive range of cell biological and genetic techniques, and be involved in the development of new proteomic mass spectrometry methods.

Learning outcomes and skills acquired: Experience with generating and working with mass spectrometry data  Eukaryotic cell culture skills  Understanding of experimental genetic approaches

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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The role of reorganisation and plasticity in age-related cognitive changes

Supervisor: Professor Lorraine K Tyler, Psychology

Project abstract: Although many cognitive functions decline as we age (e.g. working memory, fluid intelligence), others are preserved (e.g. language comprehension). This pattern of declines and preservations is seen in the context of widespread changes in the brain with age. Why do some cognitive functions decline while others are preserved? Studies of the relationship between age-related changes in cognition and neural structure and function are underway in the CamCAN (Cambridge Centre for Ageing and Neuroscience) project. This is a population-representative cohort of 700 healthy people aged 18-88 who undergo a large number of hypothesis-driven cognitive tests of attention and cognitive control, language, memory, and emotion, and for whom we also have a variety of neural (both structural and functional [fMRI and MEG]) and life-style data. This extraordinarily rich data-set provides the basis for investigations into how cognitive functions are preserved as we age, and the roles played by neural network reorganisation and functional plasticity. The Cam-CAN data-sets enable us to carry out a variety of multi-dimensional analyses combining a variety of neural and cognitive measures using novel analytical methods to understand the relationship between brain, neural function and cognition across the adult life-span. The Cam-CAN data-set will allow students to work on a wide variety of key issues in aging, which will be guided by the supervisor.

Learning outcomes and skills acquired: Opportunities in the Centre for Speech, Language and the Brain (CSLB) and in the Cam-CAN project more generally include training in several neuroimaging methods including structural MR, DTI including fractional anisotrophy and tractography, and fMRI. CSLB and Cam-CAN personnel include researchers with expertise in cognitive psychology, cognitive neuroscience, neuroimaging, and epidemiology, and come from both within the University of Cambridge and related institutes (MRC-CBSU). In sum, the studentship will combine existing resources, training facilities, and interdisciplinary collaborations to provide training in a key area of research on ageing.

Project availability: Michaelmas and Lent Term

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A genome-wide, single-cell analysis of vascular smooth muscle cell plasticity. (A bioinformatics rotation project with an optional wet-lab component)

Supervisor: Dr. Mikhail Spivakov, The Babraham Institute

Project abstract: Vascular smooth muscle cells (VSMCs) are major components of blood vessel walls, where they regulate blood flow and blood pressure. However, in response to inflammation and injury in the blood vessels, VSMCs can change into a so-called ‘synthetic’ state, in which they become migratory, proliferate and take part in tissue repair. This unusual property of VSMCs, referred to as phenotypic switching, is misregulated in atherosclerosis, during which the ‘synthetic’ cells proliferate excessively and accumulate in the vascular wall, leading to their blockage and subsequent heart attacks and strokes.     Intriguingly, it has been observed that not all arteries are equally susceptible to atherosclerosis, and VSMCs from the atherosclerosis-prone and atherosclerosis-free regions originate from different embryonic tissues. Furthermore, even in the same region, VSMCs appear to have different capacity to switch to a synthetic phenotype. One possibility is that the cells are differentially “primed” for phenotypic switching, which can be reflected by their global gene expression profiles.     In collaboration with Dr Helle Jørgensen at the Department of Medicine, we are studying mouse VMSC heterogeneity by profiling gene expression genome-wide in individual cells using single-cell RNA sequencing (ssRNAseq) followed by extensive bioinformatics analyses. Preliminary analyses of the ssRNAseq data have already led to the identification of interesting cell sub-populations, and more detailed studies are underway. Hypotheses resulting from ssRNAseq analyses will be tested using more targeted techniques such as real-time PCR, immunoflourescence and flow cytometry. The proposed project predominantly focuses on the bioinformatics comparison of single-cell RNA-seq data for mouse VSMCs isolated from the atherosclerosis-prone and atherosclerosis-free regions, but also offers the opportunity to take part in experimental follow-up analyses.

Learning outcomes and skills acquired: This is an exciting opportunity to learn the principles of biology-driven data analysis in our friendly and motivated team (http://www.regulatorygenomicsgroup.org), while also potentially participate in experimental analyses. Although the project necessarily involves a bioinformatics component, we can adjust the balance between the “wet-lab” and computational work depending on your preferences. Specific bioinformatics training will be provided, although some experience in computer programming will be desirable. A basic knowledge of R (for example, from the Introduction to R course) is advantageous, but not essential.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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A biophysical study on how the actin and microtubule cytoskeletons dynamically collaborate to regulate cellular organization

Supervisor: Dr. Isabel M Palacios, Zoology

Second supervisor: Prof. R. Goldstein, collaboration Applied Mathematics and Theoretical Physics

Project abstract: We are investigating the relation between the fluid mechanical properties of the cytoplasm and the asymmetries in the oocyte. In collaboration with physicist Prof. Goldstein, we have a unique approach that combines interdisciplinary experimental and theoretical parameters in order to answer these questions (Ganguly et al., PNAS 2012). A physicist has also recently joined the Palacios lab. In the oocyte, as the developmental determinants are being asymmetrically localised, motor proteins also induce the vigorous movement of the cytoplasm, known as cytoplasmic streaming. Streaming was discovered in 1774, but many fundamental questions have remained unanswered: How does the fluid motion arise? What is the relationship between the oocyte asymmetries and the underlying forces of the flows? We have engaged in an experimental and theoretical study of fluid dynamical and transport issues, using techniques from microfluidics to functional genetics.   We are currently extending our previous work with a comprehensive study on how the actin and microtubule(MT) cytoskeletons collaborate to regulate cellular reorganization in the oocyte, including flows and asymmetries. An interplay between the actin and MTs is essential for fundamental processes such as cell migration and cell division. These studies on migrating and dividing cells have concentrated in understanding these systems from a molecular, but not from a biophysical, point of view. Also, not much is known about the interplay between actin and MTs in immobile and interphase cells. We are studying the physical properties of this interplay in the oocyte, and its impact on flows and polarity.

Project availability: Michaelmas and Lent Term

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Metabolic requirements for longevity caused by low metabolic rates

Supervisor: Dr. Markus Ralser, Biochemistry

Project abstract: Aging is characterised by cellular damage, predominantly caused by reactive chemical species originating in cellular metabolism. Cells growth and metabolic activity at low temperatures is lowered; in chronological terms, cells life longer.   We screened for yeast mutants with exceptionally increased lifespan at low temperatures, and could identify 90 yeast gene deletion strains which were viable for five years at 4C, a phenotype termed hibernating lifespan. Surprisingly, a large fraction of these strains seem to be deficient in their anti-oxidative machinery, indicating that maintaining a high active ROS defence was not required - had even negative consequences - for survival at low temperatures and with limiting nutrient supply. Our lab is specialised on the combination of quantitative mass spectrometry with functional yeast genomics. The project would be to apply these techniques to elucidate the metabolic requirements which lead to this massive extension of low temperature lifespan.

Learning outcomes and skills acquired: How to use yeast functional genomics to study metabolism of aging, how to use liquid chromatography mass spectrometry to study quantitative metabolic processes, MS data processing in R

Project availability: Michaelmas and Lent Term

Other relevant themes: Bioenergy and industrial biotechnology

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Epigenetic regulation of genome stability during ageing and stem cell reprogramming

Supervisor: Dr. Peter Rugg-Gunn, The Babraham Institute

Project abstract: In mammals, DNA regions surrounding the centromere are abundant in satellite repeats. The repeat regions were thought to be transcriptionally silent, but evidence now reveals that satellite DNA is transcribed into noncoding RNA. Several epigenetic modifications and higher-order chromatin states have been uncovered that are associated with regulation of satellite DNA. Although it is still unclear how the noncoding RNA function, they have been linked to key process including centromere formation and maintenance of genome stability.     Initial observations in mouse and human tissue suggest that satellite transcription increases upon ageing and aberrant accumulation of satellite transcripts is associated with several major diseases. These data raise the possibility that genome instability may be caused by deregulation of satellite transcription during ageing, thereby impacting cellular function in aged tissues.    Induced pluripotent stem cells are a potential source of therapy for the treatment of age-related disease. Abnormal satellite transcription could provide a barrier to these treatments due to the risk of genome instability. It is therefore important to determine whether regulation of satellite DNA is restored upon stem cell reprogramming of aged cells.     In this project, levels and localisation of satellite transcription in specific tissues will be compared between young and old mice. Regulation of transcriptional changes will be investigated by examining the epigenetic status of these regions. Fibroblasts from old mice will be reprogrammed to stem cells and assayed for restoration of satellite control. Results will define the role for satellite noncoding RNA in genome stability during ageing and stem cell reprogramming.

Learning outcomes and skills acquired: The student will receive training in a range of modern laboratory skills that will be applicable to most fields of biological research. He/she will acquire expertise in molecular biology (including gene expression profiling; chromatin immunoprecipitation; Western blotting) and cellular biology (stem cell reprogramming and culture; immunocytochemistry). More broadly, the student will participate and present in lab meetings, attend regular meetings with the supervisor and participate in departmental seminars. Training will focus on experimental design, data interpretation and presentation skills.

Project availability: Michaelmas and Lent Term

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Regulation of Citron kinase by mitotic kinases during cell division

Supervisor: Dr. Pier Paolo D'Avino, Pathology

Project abstract: Cell division controls growth, development and reproduction in all metazoans. Errors during this process can cause human genetic diseases such as chromosomal syndromes, sterility and cancer. Our lab is interested in the last phase of cell division, when the two daughter cells pinch off, a process known as cytokinesis. Cytokinesis is accomplished through the formation and ingression of a cleavage furrow that bisects the dividing cell. Cleavage furrow formation and ingression requires the co-ordinated action of several proteins whose activity is mainly regulated by phosphorylation and/or degradation.  Citron kinase (CIT-K) is a key regulator of the last phase of cytokinesis and our lab has collected preliminary evidence that CIT-K is itself regulated by three other mitotic kinases, Cdk1 (cyclin-dependent kinase 1), Plk1 (Polo-like kinase 1) and Aurora B. To understand the regulation of CIT-K by these kinases, the student will initially compare the phosphorylation pattern of CIT-K in metaphase vs. anaphase cells. She/he will synchronise cells expressing Flag-CIT-K in metaphase and anaphase, purify Flag-CIT-K by immunoprecipitation and identify phosphorylated residues by mass spectrometry. Three replicates of this experiment will also be carried out in the presence of an inhibitor of Cdk1, Plk1, or Aurora B, respectively. The outcome of these experiments will determine whether the phosphorylation status of CIT-K changes during the metaphase-anaphase transition and if any of these phosphorylation events depends on Cdk1, Plk1, or Aurora B.

Learning outcomes and skills acquired: The student will acquire various skills in cutting -edge techniques in cell biology, biochemistry and proteomics. She/he will also learn about the process of cell division, its regulation by mitotic kinases and its involvement in cancer and other genetic diseases.

Project availability: Michaelmas and Lent Term

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Cell biological mechanisms of tissue bending

Supervisor: Dr. Richard Adams, Physiology Development and Neuroscience

Project abstract: Few epithelia in the body are flat. How cell shapes and arrangements relate to tissue curvature is still poorly understood. Furthermore, how these relationships change during development is not understood. We have developed tools to explore these relationships. The project will explore a variety of tissue types to ask how they are organised and how they change as they elaborate their form or express defects.

Learning outcomes and skills acquired: The project will apply microscopy, image analysis, data analysis, computational biology and statistics. An interest in developing or furthering quantitative skills is recommended.

Project availability: Michaelmas and Lent Term

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Investigating the expression and function of a contact-repulsive protein in astrocytes that regulates nerve growth

Supervisor: Professor Roger Keynes, Physiology Development and Neuroscience

Project abstract: Our laboratory has identified a protein in somites that powerfully repels growing axons, by contact at the cell surface, so generating the segmented repeat pattern of outgrowing spinal nerves in birds and mammals. The same molecular system is present in mammalian (rat) grey matter and on the surface of human astrocytes, where we speculate it may regulate neural plasticity. The project will examine this contact-repellent protein in a cell line of human astrocytes derived from induced pluripotent (iPS) stem cells, to assess whether its surface expression is modulated by reagents (cytokines and growth factors) that induce astrocyte activation, and how this correlates with axon-repulsive activity. Techniques will include embryo microdissection, nerve cell culture and assessment of axon repulsion using a bioassay, immunohistochemistry and molecular methods including SDS-PAGE and Western blotting. The project will suit anyone interested in nervous system development and repair. You will also have the opportunity to make original observations as the system under study is cutting new territory.

Learning outcomes and skills acquired: The primary outcome will be experience in the intellectual challenge of designing and executing experiments in the field of developmental neuroscience. Expertise will be gained in the techniques of molecular, developmental and cell biology. These include embryo microdissection (chick embryos), live cell imaging, culture of cell lines and embryonic tissues (explanted nerve cells). Nerve growth will be assessed using a growth cone collapse assay. Molecular techniques will include two-dimensional gel electrophoresis and Western blotting.

Project availability: Michaelmas and Lent Term

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Deregulation of the immune response: Impact of Streptococci superantigens on Toll-like receptor and cytokine expression in the natural host.

Supervisor: Dr. Romain PAILLOT, Animal Health Trust

Second supervisor: Andrew Waller, Animal Health Trust

Project abstract: Superantigens (sAgs) are powerful bacterial pathogenic factors that interfere with the host protective immunity by disruption of antigen-specific T-cell functions and the generation of an overzealous pro-inflammatory response. Superantigens bypass the mechanism of MHC-restricted antigen presentation, which results in the stimulation of a large fraction (5-20%) of the T-cell population and the release of pro-inflammatory cytokines. A recent study has demonstrated that sAg-dependent cytokine expression was partially associated with a CD28 binding motif1. Furthermore, superantigens also activate innate immunity by up-regulating Toll-like receptor (TLR) expression2. This activity significantly increases the induction of pro-inflammatory cytokines in response to TLR ligation by conserved pathogen associated molecular patterns (PAMPs)3.     Our group has recently identified 4 novel sAgs4 in Streptococcus zooepidemicus and characterised the activity of sAgs5 expressed by Streptococcus equi. Most of these sAgs share a common ability to affect the immune response.    This research project aims to determine the impact of these novel sAgs on the equine pattern recognition receptor (PRR) and cytokine expression. Our objectives are to measure the TLR and cytokine expression by equine immune cells after stimulation with sAgs and bacterial endotoxin/LPS or TLR agonist, or after sAgs mutation by site directed mutagenesis. Results will allow better understanding of the immune response deregulation induced by streptococcal pathogens in their natural host.   

Key references 

1.  Arad et al, PlosBiology 2011, vol9(9), e1001149. 

2.  Hopkins et al, Blood 2005, 105:3655-3662. 

3. Kearney et al, J. Immunol 2011, 187:5363-5369.  

4. Paillot et al, Infection & Immunity 2010a, 78(11):4817-4827; 2010b, 78(4):1728-1739.

Learning outcomes and skills acquired: This project will be based in the Immunology Unit at the Animal Health Trust (AHT). The student will acquire and use a broad range of methods (leukocyte isolation, recombinant protein expression, mutagenesis and gene mRNA expression by qRT-PCR), which have all been developed or established at the AHT. The student will have the opportunity to present/discuss results internally and to prepare a poster communication.    It will be possible to develop this research work into a full PhD project that will further investigate the impact of Streptococcal superantigens on equine innate immunity and their overall deregulation of the immune response.

Project availability: Michaelmas and Lent Term

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The role of hypoxic response in macrophage metabolism and polarization

Supervisor: Professor. Randall Johnson, Physiology Development and Neuroscience

Project abstract: Macrophages are key cells of the innate immune system. They accumulate in hypoxic (i.e. a reduction in oxygen below tissue demand) tissues and are essential cells for proper maintenance of tissue structure.    Cellular adaption to hypoxia is mediated in part through a group of transcription factors known as hypoxia-inducible factor α (HIF-α), of which there are two isoforms, HIF-1α and HIF-2α. When oxygen is limited HIF-α is stabilized and binds to its constitutively expressed HIF-1β partner. This active HIF complex induces the transcription of genes involved in cell survival.    The microenvironment dictates the differentiation of immature monocytes towards a mature pro-inflammatory  M1 phenotype or an anti-inflammatory M2 phenotype. During this switch from a quiescent to a mature activation state, intracellular metabolic reprogramming occurs in macrophages. We hypothesize that: HIF-1 and HIF-2 differentially affect the metabolic reprogramming of M1 versus M2 macrophages, in the hypoxic microenvironment, leading to alterations in macrophage function.    This project will build upon ongoing work aimed at understanding the role of the HIF isoforms in the metabolic reprogramming of polarizing macrophages, specifically there will be a special focus on how this impacts upon macrophage function. The student will isolate macrophages from HIF-1 and HIF-2 conditional knockout animals and subsequently perform a series of macrophage functional assays, including phagocytosis, migration and apoptosis.    Understanding the unique metabolic demands of distinct activation states within hypoxic regions has important implications in both normal biology of mammals and in the treatment of a wide range of pathologies.

Learning outcomes and skills acquired: Students will acquire general knowledge of the current understanding and emerging issues within the field of hypoxic inflammation.  They will develop their ability to design experiments, think independently and critically analyze their findings.    This project will utilize a number of different techniques including animal models, molecular biology and cell culture. Over the course of the project students will gain expertise in the isolation of primary cells from animal models and primary cell culture. In addition, they will learn a variety of molecular biology techniques including; microscopy, protein isolation, characterization using functional assays. They will also learn basic statistics and data analysis.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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The role of chromatin modifiers in disease development

Supervisor: Dr. Suzanne Turner, Pathology

Project abstract: The ATRX (alpha thalassemia X-linked intellectual disability syndrome) gene encodes an ATP-dependent helicase involved in chromatin remodelling. The gene was first discovered as being mutated in patients presenting with symptoms of ATRX which include distinctive craniofacial features, genital anomalies, severe developmental delays, hypotonia, intellectual disability, and mild-to-moderate anemia secondary to alpha-thalassemia. Until this time, the ATRX gene was unknown but has since also been implicated in the pathogenesis of neuroblastoma, a paediatric cancer of the sympathetic nervous system that generally arises during the first 2 years of life. In both diseases, the ATRX gene is present in a mutant form displaying mutations within exons 7-9 or 17-20 in at least 85% of ATRX patients. More recently multi-exon deletions and focal mutations have been detected in ATRX in 9.6% of neuroblastoma patient tumours. The functional significance of this mutation remains to be discovered as well as whether it constitutes a ‘driver’ or ‘passenger’ mutation in tumorigenesis. Therefore, this project will seek to understand the disease-related roles of ATRX with a particular emphasis on its role in the modification of chromatin.

Learning outcomes and skills acquired: This project will examine an epigenetic mechanism in the tumorigenic process that could present a novel therapeutic target: ATRX mutants will be assessed for their functional relevance to the process of cellular transformation, tumour cell propagation and survival. In particular, cell proliferation assays, lentiviral transduction, tissue culture, Western blot, chromatin immunoprecipitation, quantitative reverse transcription PCR and DNA sequencing techniques may be employed in this project to evaluate the role of ATRX mutations towards neuroblastoma development.

Project availability: Michaelmas and Lent Term

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Composition and regulation of RNA-protein complexes

Supervisor: Dr. Timothy Weil, Zoology

Project abstract: Localised mRNA translation is a conserved mechanism for the spatial control of gene expression, enabling cells to target protein function in space and time. Functions requiring mRNA regulation include Drosophila and Xenopus axis formation, cell division in budding yeast, motility in chicken fibroblasts and synaptic plasticity in neurons.    The general aim of this project is to understand mRNA translational control. The main technique will be immunoprecipitation using GFP trap beads to pull down GFP labeled trans-acting proteins and GFP tagged mRNA. In addition, advanced imaging will supplement the biochemical results.    Importantly: I am looking for enthusiastic and creative individuals; the project can be adopted to fit the specific interest of the student.

Learning outcomes and skills acquired: Cross-linking immunoprecipitation (CLIP)  Biochemistry  Advanced light microscopy  Fly keeping techniques  Genetics  Dissection and sample preparation  Immuno-fluorescence labeling

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Serotonin and behaviour studied by chemogenetic approaches in rats

Supervisor: Professor TW Robbins, Psychology

Second supervisor: Dr. J Alsio, Psychology

Project abstract: Understanding the roles of the central serotonin (5-HT) systems in behaviour is an important goal requiring valid animal tests with translational value for human studies of well-being under stress. Whilst there are many pharmacological tools for manipulating the 5-HT system in rodents, the availability to us of transgenic rats expressing Cre recombinase selectively in serotonin neurons provides a new way of investigating this system in the intact animal, using virally-mediated gene transfer of Cre-dependent transgenes to the serotoninergic raphé nuclei. We focus on functions of raphé projections to rat orbitofrontal (OFC) and medial prefrontal cortex (mPFC) in two paradigms: (i) reversal learning, which is sensitive to OFC lesions and effects of locally applied 5-HT receptor agents (ii) impulsive behaviour (in the 5-Choice Serial Reaction Time Task) is sensitive to 5-HT manipulations in the mPFC. We plan to express DREADD (Designer Receptors Exclusively Activated by Designer Drugs) transgenes selectively in the dorsal raphé to allow the activation of 5-HT-containing cells, with predictable effects on both reversal learning and impulsivity. To test for regional specificity of effects following 5-HT raphé activation, infusions of ketanserin (a mixed 5-HT receptor antagonist) will be made into either the OFC or the mPFC.    

References   

Bari A, Theobald DE, Caprioli D, Mar AC, Aidoo-Micah A, Dalley JW, Robbins TW (2010) Serotonin modulates sensitivity to reward and negative feedback in a  probabilistic reversal learning task in rats. Neuropsychopharmacology. 2010  May;35(6):1290-301.   

Harrison AA, Everitt BJ, Robbins TW (1997) Central 5-HT depletion enhances impulsive responding without affecting the accuracy of attentional performance: interactions with dopaminergic mechanisms. Psychopharmacology 133:329–342   

Weber T, Schonig K, Tews B, Bartsch D (2011) Inducible gene manipulations in brain serotonergic neurons of transgenic rats. PLOS ONE 6(11): e28283.

Learning outcomes and skills acquired: This project would require a student to learn how to run computer-controlled behavioural paradigms and analyse behavioural data, employ stereotactic surgery for cannulation implants, perform behavioural pharmacology experiments, and learn basic rodent histology and fluorescent immunohistochemistry.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Remodelling of the mammalian host cell cytoskeleton by the intracellular bacterial pathogen Salmonella

Supervisor: Professor Vassilis Koronakis, Pathology

Project abstract: The WAVE regulatory complex (WRC) drives formation of newly polymerised actin filaments at the mammalian cell plasma membrane to generate lamellipodia and membrane ruffles that are pivotal to cell processes (e.g. movement) and pathology (e.g. cancer). The WRC is also crucial to host cell invasion by the bacterial pathogen Salmonella Typhimurium that delivers subversive virulence effectors into cells to trigger membrane ruffling and uptake. How WRC is regulated fundamentally in the cell and how it is manipulated by Salmonella remains unclear.     Our laboratory has identified a novel cellular activator of the WRC known as Arf GTPase that cooperates with Rac1 GTPase to control WRC(1). We uncovered that Salmonella targets this pathway through pathogen and host guanine nucleotide exchange factors (GEFs) that activate Arf and Rac1 to remodel the actin cytoskeleton via WRC and drive invasion into host cells(2, 3). Multiple parallel projects in the laboratory are investigating the biochemistry and cell biology of WRC activation and actin filament assembly via small GTPases (i.e. Arf, Rac1), and how these cellular signalling networks are manipulated by Salmonella to establish infection. You will work alongside PhDs and postdocs within the group to gain new insight into WRC control and Salmonella infection.   

1. Koronakis V, Hume PJ,Humphreys D, et al. (2011). Proc Natl Acad Sci U S A. 

2. Humphreys D, Davidson A, Hume PJ, & Koronakis V (2012). Cell Host Microbe 11(2):129-139. 

3. Humphreys D, Davidson AC, Hume PJ, Makin LE, & Koronakis V (2013). Proc Natl Acad Sci U S A 110(42):16880-16885.

Learning outcomes and skills acquired: The student will learn about:  (a) cellular small GTPase signalling pathways and their reconstitution in vitro  (b) Pathogen virulence subversion of cellular pathways  (c) fluorescence microscopy, cell culture and infections, a range of biochemical techniques including recombinant protein purification, and protein-protein interactions at model membranes in vitro.

Project availability: Michaelmas and Lent Term

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Reprogramming the epigenome

Supervisor: Professor Wolf Reik, Babraham Institute

Project abstract: Epigenetic information in the genome is important for normal development and ageing and its deregulation can be associated with various diseases. Epigenetic information is generally stable in differentiated cells in the adult organism, though it degrades during ageing. However in germ cells, early embryos, embryonic stem cells and iPS cells genome-wide reprogramming of epigenetic information takes place. Epigenetic reprogramming is associated with the return of the genome to pluripotency or totipotency, the erasure of epimutations, resetting parental imprints, and possibly the repression of retransposons in the germline. Epigenetic reprogramming is also critical for experimental reprogramming such as cloning by nuclear transfer, cell fusion, and iPS cell generation.    You will be working in an internationally leading lab that studies the mechanisms and functional consequences of epigenetic reprogramming. You will study the molecular pathways of reprogramming especially of demethylation of DNA which includes passive and active demethylation. Passive demethylation can occur by regulation of Dnmt1 and Uhrf1 including by cell signaling principles. Active demethylation occurs by oxidation of methylcytosine (5mC) to 5hmC, 5fC, and 5caC by the TET enzymes or by deamination by AID/APOBEC enzymes. You will be using mouse knockout and cell models, or biochemical approaches, to study these pathways. Our approaches include epigenomics, bioinformatics and cell signalling which you will have the opportunity to engage with, including through the affiliated Single Cell Genomics Centre (http://www.sanger.ac.uk/research/projects/singlecellcentre, at the nearby Sanger Institute). The functional consequences of manipulating reprogramming pathways for normal development, ageing, and iPS cell generation will also be investigated. You will be joining an enthusiastic and collaborative team of students and postdocs embedded in one of the largest programmes of Epigenetics and Nuclear Dynamics science within Europe.   

References: 

Popp et al 2010 Nature, Ficz et al 2011 Nature

Booth et al 2012 Science

Seisenberger et al 2012 Molecular Cell

Ficz et al 2013 Cell Stem Cell

Learning outcomes and skills acquired: You will be learning epigenomics technologies including genome-wide bisulfite sequencing and RNA sequencing in germ cells, early embryos, stem cells and iPS cells. You will culture and manipulate various pluripotent cell types and potentially assess their functions. You will also be learning bioinformatics skills that allow interpretation of complex epigenomics datasets, and potentially be exposed to single cell epigenomics approaches.

Project availability: Michaelmas and Lent Term

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Cellular and molecular characterization of breast epithelial heterogeneity

Supervisor: Dr. Walid Khaled, Pharmacology

Project abstract: Breast cancer is the most common type of cancer in the UK with an estimated 4000 new cases every month. However, the heterogeneous nature of the disease means that an effective “one size fits all” treatment remains elusive. Thus understanding the cellular and molecular basis of breast cancer heterogeneity is essential for the development of more effective treatments.  Microarray studies have shown that there are multiple subtypes of breast cancer with varying degrees of prognosis. It is known that the luminal subtype has the best prognostic outcome while the basal like breast cancer (BLBC) subtype has the worst outcome. There is evidence that such tumour heterogeneity could be attributed to the tumour’s cell of origin. For example, luminal breast cancer is thought to arise from differentiated luminal cells while BLBC is thought to arise from undifferentiated progenitor and stem like cells. Such model would necessitate the expression and function of cell type specific factors, which determine the cells’ fate.  Research in our lab aims to understand the cellular and molecular mechanisms behind breast cancer heterogeneity (funded by CRUK). We have recently identified the transcription regulator BCL11A as a BLBC oncogene and a mammary stem cell regulator (in press). Part of the research effort in the lab will be to understand the role of BCL11A at the cellular and molecular levels. Then exploit this understanding to develop anti-BCL11A therapeutic approaches for the treatment of BLBC.  The proposed PhD project will be part of this effort and will focus on the use of novel proteomics, genomics and genetic technologies to investigate the role of BCL11A in BLBC and mammary gland development. The student will join an enthusiastic and committed team of researchers and will be expected to contribute and fully engage with our collaborators at the Sanger Institute and CRUK, Cambridge Institute to drive this project forward.

Learning outcomes and skills acquired: Proteomics,  Cell culture,  Cloning,  shRNA/Crispr technology to KD/KO genes

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Live long and prosper – lessons from the Naked Mole Rat

Supervisor: Dr. Walid Khaled, Pharmacology

Second supervisor: Ewan St. John Smith, Pharmacology

Project abstract: Naked mole-rats (NMR) are highly unusual mammals that look like cocktail sausages with rather large, protruding teeth. They are roughly the same size as a mouse, but are cold-blooded, eusocial (the queen is the only breeding female), cancer resistant and exceptionally long-lived – approximately 30 years, 10 times longer than a mouse. Understanding the molecular, cellular and physiological basis for all of these highly unusual phenotypes is of great interest, especially considering that globally we are faced with an aging population – understanding more about how NMRs successfully age will enable us to better understand how aging occurs in humans. In this project we NMR cell lines and novel gene editing technologies (CRISPR/CAS9) to identify genes, which are key to the NMR’s resistance to transformation. Dr. Ewan Smith and Dr. Walid Khaled will jointly supervise this project.

Learning outcomes and skills acquired: Techniques used: Cell culture, clonogenic assays, high throughput Genetic screens, molecular cloning and CRISPR/Cas9.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Learning-to-learn across the lifespan

Supervisor: Professor Zoe Kourtzi, Psychology

Project abstract: We aim to understand how humans develop insightful behaviour across the lifespan by learning-to-learn; i.e. learning to extract the underlying structure of variable environments (i.e. common features and principles of organisation) and generalise this previously acquired knowledge to solve new problems (i.e. tasks with similar underlying structure as the trained task). Investigating the mechanisms of this structural learning is critical for understanding how we generalise learning to novel situations and developing training applications that transfer to real life situations (e.g. success at school and work). Previous work has contributed to the understanding of the mechanisms that support this structural learning in perceptual and conceptual domains. However, the development of these mechanisms across the life course and their maintenance as we age remains largely unknown. We aim to develop paradigms that will test structural learning in the context of temporal regularities (i.e. learning sequences of probabilistic events) and categorical structures based on inferences about conceptual similarities. Using mathematical modelling, we ask whether young and older learners use the same or different strategies to achieve structural learning and generalise learning to new domains. Using brain imaging, we aim to understand the mechanisms that mediate the ability of individuals to learn across the lifespan; that is, extract principles of organisation in our environments, integrate them into existing knowledge schemes and generalise to novel problem solving situations.

Learning outcomes and skills acquired: -Behavioural and cognitive testing  -Brain Imaging: data acquisition and analysis  -Computational modelling

Project availability: Michaelmas and Lent Term

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Visualising stress responses at the single cell level

Supervisor: Dr Brian Ferguson, Pathology

Project abstract: The cellular response to exogenous stress is critical for the maintenance of tissue homeostasis and hence underpins the maintenance of health during the lifetime of any multicellular organism. Two exogenous stresses that profoundly affect life-long health are chronic inflammation and DNA damage. Information about how cells respond to such stresses has traditionally come from studies of cell populations, ignoring differences between individual cells despite evidence for variable and stochastic responses at the single-cell level. The ability to understand how single cells respond to stresses relies on visualisation of those systems. Taking advantage of novel gene editing technology combined with high resolution imaging allows us to study how specific proteins respond to stresses in individual cells. The aim of this project is to modify the 5’ end of specific genes in human cells such that the endogenous proteins are expressed with tags allowing high resolution fluorescence-imaging. The transcription factors (TFs) interferon-regulatory factor 3 (IRF-3), nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) act as or signalling hubs for numerous stresses including inflammatory and DNA damage stimuli. Endogenous tagging of IRF-3, AP-1 and NF-κB in human fibroblasts will be followed by treatment with inflammatory and DNA damage stimuli. The dynamics of how the TFs in each cell respond to these stimuli will be monitored by fluorescence microscopy and analysed using image analysis software. These data will provide a platform for future studies to produce models of stress responses allowing the effect of age, cell type and other variables to be incorporated.

Learning outcomes and skills acquired: The student will learn a variety of cutting edge molecular and cellular biology techniques including the CRISPR/cas9 genome editing systems, molecular cloning and cell culture in order to generate modified cell lines. Subsequently, live-cell confocal microscopy and software-based image analysis will be used to monitor the responses of the generated cell lines to various exogenous stresses. In addition to laboratory-based skill advancement, the student will learn transferable skills. Presentation, communication and interpersonal skills will be improved by active involvement in lab-meetings, journal clubs and in the course of working as part of a growing and vibrant team.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Does TopBP1 play a role in mitotic chromosome segregation in vertebrate cells?

Supervisor: Dr Christine Farr, Genetics

Project abstract: Faithful chromosome segregation during cell division is critical to the maintenance of a stable genome. The type II topoisomerases play key roles in ensuring that after DNA replication the entwined sister chromatids are disentangled (1). This function is ongoing even during anaphase, when persistent ultrafine DNA bridges (UFBs) between the separating chromosome masses have to be resolved (2). A small number of proteins has been shown to localise to these anaphase ultrafine DNA threads: BLM helicase, PICH and most recently TopBP1 (2, 3). TopBP1 was identified originally as a Topo 2 interacting protein (4) and has since been shown to have multiple roles in DNA replication and in replication checkpoint control (5). However, a role during M-phase itself has only recently been uncovered (3). This study will investigate how TopBP1 and Topo 2 influence each other’s function during M-phase. A human cell line, retrofitted with an auxin-responsive degron, from which Topo 2 can be rapidly depleted (6,7) will be used, in conjunction with RNAi, to elucidate the role of Topo 2 and TopBP1 at the mitotic centromere, and in the resolution of anaphase UFBs, using immunoblotting, immunoprecipitation and fluorescence microscopy.   

1. Wang, JC (1996) DNA topoisomerases. Annu Rev Biochem, 65, 635-692. 

2. Liu Y, Nielsen CF, Yao Q, Hickson ID (2014) The origins and processing of ultra fine anaphase DNA bridges. Curr Opin Gen Dev 26: 1-5. 

3.Germann SM, Schramke V, Pedersen RT, Gallian I, Eckert-Boulet N, Oestergaard VH, Lisby M (2013) TopBP1/DPB11 binds DNA anaphase bridges to prevent genome instability. JCB 204: 45-59. 

4. Yamane K, Kawabata M, Tsuruo T (1997) A DNA topoisomerase II binding protein with eight repeating regions similar to DNA repair enzymes and to a cell cycle regulator. Eur J Biochem 250: 794-799. 

5. Wang J, Chen J, Gong Z (2013) TopBP1 controls BLM protein level to maintain genome stability. Mol Cell 52: 667-678. 

6. Spence, JM, Phua, HH, Mills, W, Carpenter, AJ, Porter, ACG and Farr, CJ  (2007) Depletion of topoisomerase IIalpha leads to shortening of the metaphase interkinetochore distance and abnormal persistence of PICH-coated anaphase threads. J. Cell Sci. 120: 3952-3964. 

7. Farr CJ, Antoniou-Kourounioti M, Mimmack ML, Volkov A, Porter ACG (2014) The alpha isoform of topoiosmerase II is required for hypercompaction of mitotic chromosomes in human cells. NAR 42: 4414-26.

Learning outcomes and skills acquired: This work will allow experience to be acquired in a range of wet-lab techniques, including molecular and protein biology (site-directed mutagenisis, cloning and plasmid construction, western blotting), mammalian tissue culture (including stable and transient transfection protocols) and fluorescence microscopy (including indirect immunofluorescence).

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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The role of Roma/Zfp157 in DNA replication

Supervisor: Professor Christine J Watson, Pathology

Project abstract: We have recently identified a novel KRAB-domain zinc finger protein, which we called Roma/Zfp157, that is a regulated by Stat6. Deletion of the gene for Roma results in hyper-proliferation of mammary epithelial cells. Interestingly, this is associated with unscheduled DNA replication which results in stalling of replication forks and subsequent DNA damage. Thus, Roma is a novel regulator of genomic stability. We have derived mouse embryonic fibroblasts from Roma knockout mice and have shown that proteins involved in DNA replication licensing have dysregulated expression in these cells and also in mammary epithelial cells in vivo. In this project, the student will investigate how Roma regulates replication licensing by carrying out cell cycle analyses, investigating fork stalling using DNA fiber analysis, and validate Roma interacting proteins by co-immunoprecipitation and, if time permits, gene knockdown.

Learning outcomes and skills acquired: Understanding the biology of DNA replication and DNA damage, acquiring skills in basic molecular biology techniques including FACS, RNA and protein analyses, cell culture, and genetic alteration approaches.

Project availability: Michaelmas and Lent Term

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The role of cell density and  mechanotransduction in cell competition

Supervisor: Dr Eugenia Piddini, Zoology

Project abstract: Cells have the ability to monitor the fitness levels of their surrounding cells and eliminate subfit members in a process termed cell competition. Cell competition probably acts as a quality control mechanism to ensure that the fittest cells contribute to a tissue, likely contributing to tissue health. The mechanisms of cell competition are still poorly understood and it is unclear how cells compare fitness and how detection translates into the elimination of subfit cells, named losers. While some molecular pathways have been implicated in cell competition, recent advances from our lab indicate the involvement of mechanical insults, such as cell compression or density, in this process. We find that cell competition is preceded by mechanical squeezing of loser cells to a higher local density. This is then followed by extrusion and cell death. Remarkably, live imaging shows that compression results from activation of a migratory chase-and-run behaviour where, upon cell contact, wild-type cells ‘chase’ loser cells, which ‘run’ away. These observations open several interesting questions. For example what pathways are triggered by mechanical compression that lead to cell competition? What cell surface molecules are involved in the recognition among winners and losers that lead to wild-type cells chasing losers?  Exploring the involvement of candidate signalling pathways (e.g. Hippo, p53) and adhesion molecules (e.g. Cadherin, integrins) in the above processes could form the basis of a rotation project and could potentially be expanded into a PhD project.      

Vivarelli et al., Essays in Biochemistry, (2012)

Wagstaff et al., Trends Cell Biology, (2013)

Learning outcomes and skills acquired: During this rotation the student will be trained in:  mammalian cell culture, live cell imaging, transfections, RNAi mediated silencing, immunofluorescence techniques, confocal microscopy,   western blotting etc.

Project availability: Michaelmas and Lent Term

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Regulation of synapse growth and neuronal homeostatic adjustment by Reactive Oxygen Species, metabolic by-products of mitochondrial respiration.

Supervisor: Dr Matthias Landgraf, Zoology

Project abstract: Neurons exhibit extensive developmental and homeostatic plasticity. Adjustments in connectivity, excitability and morphology within individual neurons conspire to maintain a pre-determined activity set-point and to remain within a physiologically appropriate function range of function.    We previously discovered that neurons respond to changes in activity not only by changing their electrical properties, but also by adjusting the sizes of their synaptic terminals, called ‘structural homeostasis’ (Tripodi et al., 2008). We since developed a genetic expression system that allows us to study nerve cells in their entirety, revealing how structural changes of postsynaptic dendrites in the CNS precede those at neuromuscular junctions in the periphery.     Asking how neurons sense their activity levels, we recently discovered a novel and fundamental mechanism: neurons use Reactive Oxygen Species (ROS) as a proxy for neuronal activity. ROS are by-products of ATP production, generated by electron leakage from mitochondrial complexes I and III. ROS act as second messengers that are necessary and sufficient for driving homeostatic synaptic terminal adjustments. We found that Parkinson’s disease-linked, DJ-1b acts as a redox sensor, which binds to the lipid phosphatase PTEN and thus regulates PI3K-signalling, a known regulator of neuronal morphology.     Using state of the art genetic methods, high resolution & calcium imaging and computer aided 3D reconstructions to quantify complex neuronal shapes, we are now investigating a range of questions. How does ROS signalling intersect with other synaptic growth pathways? And is abnormal ROS signalling at the synapse causal to many phenotypes seen in neurodegenerative conditions, including prion-induced neurotoxicity?

Learning outcomes and skills acquired: You will become part of an interactive group and receive expert training in:    - immunofluorescence methods for visualisation of proteins and transcripts;   - imaging, using widefield, point and field scanning confocal microscopes;  - laser based cell manipulations (cutting axons; cell ablation);  - computer aided 3D digital reconstructions of neurons for accurate quantitative measurements of complex morphologies;  - molecular biology techniques; PCR and cloning; generate transgenes and transgenics;  - Drosophila husbandry and advanced genetics: working with complex genotypes that allow targeted genetic manipulation and visualisation of specific cells in the nervous system;  - dissections methods for embryos and larval nerve cords;

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Getting to the right place: how leukocytes find their way to sites of infection

Supervisor: Dr. Milka Sarris, Physiology Development and Neuroscience

Project abstract: In order to fight infections, white blood cells (leukocytes) must navigate through complex tissue environments and locate sites of microbial presence. A crucial element in this process is their ability to interpret external input signals, in the form of attractive or repulsive cues. We are interested in the intracellular information processing that allows accurate leukocyte navigation. Although leukocyte behaviour has been extensively interrogated in vitro, remarkably little is known about how these cells search tissues and read guidance cues in live tissues, in situ. We exploit the zebrafish as a model, whose transparency allows us to monitor leukocyte behaviour and signalling in vivo with high resolution, through advanced light microscopy techniques. We use quantitative and statistical methods to analyse the dynamics of leukocyte guidance processes and genetic, chemical or optogenetic perturbations to dissect the underlying mechanisms.    The goal of the project is to monitor the dynamics of chosen components of the cytoskeleton during leukocyte migration to sites of bacterial infection and establish how local gradients of attractant molecules (chemokines) influence these dynamics. The student will use infection assays in zebrafish, genetic perturbation of chemokine signaling and transgenic zebrafish expressing fluorescent probes for cytoskeletal markers. Using fast, high resolution confocal imaging and quantitative analyses, the aim is to define cytoskeletal changes in leukocytes that result from chemokine gradient sensing. There will also be an opportunity to test novel fluorescent probes for chemokine signalling currently under development in the lab.

Learning outcomes and skills acquired: The project provides background in cell biology with emphasis on how the cytoskeleton and cell signaling produce directed cell movements. In addition the project will introduce the student to mechanisms of innate immunity.     The student will gain practical experience in:  - Live microscopy (laser-scanning and spinning disk confocal microscopy)  - Quantitative analyses of cell behaviours and signalling   - Molecular biology (DNA and RNA manipulation)  - Microinjection techniques (injection of small molecules or bacteria into zebrafish larvae)

Project availability: Lent Term Only (January - March 2015)

Other relevant themes: Food security

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Why has evolution conserved the fatty acyl composition of inositol phospholipids?

Supervisor: Dr Phillip Hawkins, The Babraham Institute

Second supervisor:  

Project abstract: Inositol phospholipids are critical regulators of membrane biology. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol headgroups to specific protein domains eg PH, FYVE and PX domains. This interaction serves to both recruit and regulate the activity of proteins which act on membrane surfaces, including key components of intracellular signalling networks (1).    Little attention has been paid to the function of the fatty acids in these lipids. Most classes of phospholipids comprise a large collection of molecular species that differ in their fatty acyl composition.  Remarkably, recent studies in mammals (2)  and dictyostelids (3) have indicated that inositol phospholipids are much more molecularly homogenous than other phospholipid classes, indicating that their fatty acyl composition must play some evolutionary conserved function. Given that the pathways regulating the production of these lipids are central drivers of cancer and other diseases it is important to discover what this function is.    In collaboration with Jonathan Clark’s group at BI we are currently synthesising inositol lipids with different fatty acyl compositions and heavy-isotopes. This project will establish a liposome-based delivery method to incorporate these lipids into cellular pools. This approach has great potential to interrogate the function of the acyl chains in these lipids and make a major contribution to understanding phospholipid biology and its relationship to diet.    This is an interdisciplinary project involving cell culture, liposomes, confocal microscopy and mass spectrometry.  

(1) Balla T (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93: 1019-1137   

(2) Clark J, Anderson KE, Juvin V, Smith TS, Karpe F, Wakelam MJ, Stephens LR, Hawkins PT (2011) Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry. Nat Meth 8: 267-272   

(3) Clark J, Kay RR, Kielkowska A, Niewczas I, Fets  L, Oxley D, Stephens L,  Hawkins PT (2014) Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling. EMBO J In Press

Learning outcomes and skills acquired: Understanding how chemists and biochemists can combine knowledge and skills to tackle an important question.    Understanding intracellular signalling pathways.    Understanding and using cutting-edge mass spectrometry methods to measure cellular metabolites.    Understanding the use of heavy isotopes, combined with mass spectrometry, to trace the fate of molecules in cells.    Developing skills in cell culture, confocal microscopy, lipid extraction and preparing liposomes.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Cell fate signalling by the DYRK protein kinases, relatives of the MAP Kinases

Supervisor: Dr Simon Cook, The Babraham Institute

Project abstract: The dual-specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are a family of five protein kinases related to the MAP kinases (e.g. ERK).  The DYRKs appear to be important in normal development/homeostasis and may be de-regulated in certain pathological states but their precise functions remain enigmatic. Based on our success in MAPK biology we are increasingly interested in the DYRKs and their role in control of cell cycle, survival, differentiation and autophagy(1-3). However, few DYRK substrates or DYRK target genes have been defined to explain how these enzymes regulate such cell fate decisions.  We have now established a panel of cell lines that exhibit Tet-regulated expression of the different DYRK protein kinases. In addition we have a panel of cells in which the individual DYRKs have been knocked out by CRISPR/Cas9 technology and have access to unique, proprietary DYRK inhibitors.  We are using this ‘DYRK toolkit’ to investigate the cellular consequences of DYRK activation/inactivation and to apply transcriptomics/phosphoproteomics approaches to understand the biological functions of the DYRKs. For example, we have used this resource to show that DYRK1B (1) and DYRK1A (2) phosphorylate cyclin D1 to control the G1 phase of the cell cycle and have used mass spectrometry to identify and validate completely new DYRK1B substrates (3).  The student will use the DYRK toolkit to investigate how individual DYRKs influence cell cycle, survival and autophagy. The long-term goals of the project will be to integrate this knowledge with genomic and proteomics data to define the biological functions of the DYRK protein kinases.  

1.  Ashford et al. (2014) Biochem J. 

2.  Najas et al. (2014) Manuscript in review 

3.  Ashford & Cook, unpublished observations

Learning outcomes and skills acquired: The student will join a motivated research group of nine (2 Snr Scientists, 4 post-docs and 3 PhD students) who are taking a multidisciplinary approach to interrogate protein kinase signalling. They will culture their own cells and treat them to activate/inhibit specific DYRKs. They will use SDS-PAGE and immunoblots to analyse signalling downstream of DYRKs. They will use flow cytometry and confocal microscopy to examine cell cycle, cell death and autophagy. They will learn how to interpret their results and formulate and test new hypotheses. They will attend and present their own work at weekly Cook lab meetings.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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How does the cancer-associated ubiquitin-conjugating enzyme, UbcH10/Vihar, trigger chromosome instability?

Supervisor: Dr Yuu Kimata, Genetics

Project abstract: The ubiquitin conjugating enzyme, UbcH10/Ube2c, cooperates with the ubiquitin ligase, Anaphase promoting complex (APC/C), to regulate the progression of mitosis. Its cellular level is highly regulated during the cell cycle. In the majority of human carcinomas UbcH10 is highly expressed and its over-expression indeed induces chromosome instability (CIN) and tumour formation.   We are trying to understand the cause and consequence of UbcH10 over-expression utilising Drosophila melanogaster as the experimental model system. We have found that over-expression of Vihar, Drosophila orthologue of UbcH10, induces chromosome missegregation in embryos and disrupts tissue structures in various organs. We recently obtained the evidence that suggests that the primal cause of Vihar-induced CIN might be DNA replication stress. In this rotation project we will investigate the potential role of Vihar in DNA replication and how its over-expression might deregulate this process. We will exploit various molecular and cell biological techniques, imaging as well as mass spectrometry.

Learning outcomes and skills acquired: In the best outcome the produced data will establish that the primal cause of the CIN induced by UbcH10/Vihar is DNA replication defects. The student will learn basic cell biology, Drosophila genetics and imaging techniques and will gain experience in mass spectrometric analysis.

Project availability: Michaelmas and Lent Term

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Mechanisms of integrin adhesion in morphogenesis and mechanotransduction

Supervisor: Dr. Nick Brown, Physiology Development and Neuroscience

Project abstract: Integrins are transmembrane receptors that enable cell migration and maintain cohesion between cell layers, making them crucial in development and disease. When activated and clustered, integrins nucleate a cytoplasmic complex of proteins that links the cytoskeleton to the extracellular matrix. Integrin adhesions play a crucial role in mechanotransduction, as they allow the cell to sense and respond to the stiffness of its environment, and integrin adhesions strengthen in response to increases in the force acting on the adhesion sites. We use the powerful molecular genetics of Drosophila in combination with imaging and computer modelling to discover what factors control the assembly of this complex, and the distinct developmental roles of each component. Diverse projects are available to explore the assembly and function of this mechanosensitive machine from different angles.

Project availability: Michaelmas and Lent Term

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Early origins of chronic human lung diseases of ageing

Supervisor: Dr. Emma Rawlins, Pathology

Project abstract: It is now well established that chronic lung diseases which place a huge burden on our ageing population have their origins in early life events: either genetic/developmental factors, or early exposures to specific environmental factors. These can be studied at a population level, or modelled in mice, but can we develop human cellular systems for studying these complex processes in the lab? Our lab is interested in the cellular and molecular mechanisms controlling the development and maintenance of the respiratory system. We have 2 particular questions: 1. How do adult lung epithelial stem cells function to maintain epithelial organisation throughout the lifetime of the individual? 2. How are specific cell fate decisions made in normal lung development? Our long-term goal is to manipulate stem cell behaviour at will in order to ameliorate the effects of ageing or disease processes. Lung development and ageing are almost always studied in animal models, or in human cells which are grown as monolayers in culture. Can we develop better human cell-based systems for studying these questions? This would both provide better biology (healthy ageing) and a reduction in animal use (3Rs). We have developed culture conditions for human embryonic lung samples. The short project would be to test the factors which we have shown can promote alveolar fate in the mouse lung for their ability to do so in the human system.

Learning outcomes and skills acquired: Generation of hypotheses from interpretation of transcriptome data and available literature; primary cell/organ culture and complementary histological techniques; western blot.

Project availability: Michaelmas and Lent Term

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Signalling between human axons and OPCs

Supervisor: Dr. Ragnhildur Thora Karadottir, Veterinary Medicine

Project abstract: The brain’s white matter provides an information superhighway that links ~100 billion neurons situated in the grey matter. Its function depends on oligodendrocytes wrapping myelin around axons to provide fast neurotransmission, synchronization and maintenance of neuronal function. Despite its importance, the regulation of myelination is poorly understood. White matter plasticity is increasingly invoked as a mechanism for learning, and in disease destruction of myelin disrupts cognitive and motor function.  Important recent findings have revealed the presence of functional synaptic connections between unmyelinated axons and CNS stem cells called oligodendrocyte progenitor cells (OPCs). In recent years it has also become evident that OPCs differentiate throughout life into myelinating oligodendrocytes, perhaps for maintaining myelin or as a response to learning. Furthermore, recent developments in the lab have shown that after myelin injury demyelinated axons generate de novo synapse to recruited OPCs to direct OPCs differentiation.    In this project we will investigate whether human OPCs (derived from iPS cells) sense axonal activity, and if neurotransmitters are released from active human axons. This will involve imaging OPCs filled with calcium dye (or expressing iGluSNfr on the surface of OPCs and or the calcium indicator GCaMP6s) and stimulate axons in human OPC-neuronal co-cultures.

Learning outcomes and skills acquired: Main Techniques acquired:  The project will involve cell culture work, imaging (both time lapse and calcium), immunohistochemistry and electrophysiology.

Project availability: Lent Term

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Mechanism of induced pluripotency

Supervisor: Dr. Jose Silva, Biochemistry

Project abstract: TInvestigation of the molecular mechanisms by which the gene expression network and epigenetic landscape are reprogrammed during the establishment of naïve pluripotency.

References:

Stuart HT, van Oosten AL, Radzisheuskaya A, Martello G, Miller A, Dietmann S, Nichols J, Silva JCR. (2014). NANOG amplifies STAT3 activation and they synergistically induce the naïve pluripotent program. Current Biology 24, 1–7

Buecker C, Srinivasan R, Wu Z, Calo E, Acampora D, Faial T, Simeone A, Tan M, Swigutemail T, Wysocka J. (2014). Reorganization of Enhancer Patterns in Transition from Naive to Primed Pluripotency. Cell Stem Cell 14, 838-853

Factor DC, Corradin O, Zentner GE, Saiakhova A, Song L, Chenoweth JG, McKay RD, Crawford GE, Scacheri PC, Tesar PJ. Epigenomic Comparison Reveals Activation of “Seed” Enhancers during Transition from Naive to Primed Pluripotency. Cell Stem Cell 14, 854-863

Whyte, WA, Orlando, DA, Hnisz, D, Abraham, BJ, Lin, CY, Kagey, MH, Rahl, PB, Lee, TI, and Young, RA. (2013). Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319

Guo, G., Yang, J., Nichols, J., Hall, J.S., Eyres, I., Mansfield, W., and Smith, A. (2009). Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063–1069

Learning outcomes and skills acquired: 

Cell culture (ESCs, EpiSCs, iPSCs), including maintenance, expansion, genetic manipulation, reprogramming, timecourses, flow cytometry.

RNA extraction for RNAseq and RT-qPCR.

Genotyping.

ChIP

Project availability: Michaelmas and Lent Term

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Holding on and letting go: the role of the Synaptonemal Complex in the dynamic pairing of meiotic chromosomes

Supervisor: Dr. Luca Pellegrini, Biochemistry

Project abstract: Human fertility and genetic diversity depend on the correct execution of the genetic programme of meiosis. At the physical and functional centre of meiosis is the Synaptonemal Complex (SC), a large proteinaceous structure that holds together homologous chromosomes, providing the structural framework for meiotic recombination and crossover formation. SC function is important for our reproductive health, as failure in SC formation is associated with miscarriage and infertility. Despite its importance in meiosis, the physical features of the SC are known only in rudimentary detail. Recent biological research has identified the principal protein components of the SC and has paved the way for the biochemical analysis of the SC structure and mechanism of assembly.

The project focuses on the structural role of the SYCP3 protein in the establishment of the SC lateral element, an essential component of the meiotic chromosome axis. Based on our recent determination of the crystal structure of human SYCP31, the project aims to investigate important biochemical and biophysical properties of recombinant SYCP3, such as its ability to bind DNA and to assemble in regular filamentous ultra-structures. In addition, the project will include a search for interacting SYCP3 partners by yeast two-hybrid screen of cDNA libraries.

 References:

1. Syrjanen J. L. et al., A molecular model for the role of SYCP3 in meiotic chromosome organization Elife. 2014 Jun 20:e02963

Learning outcomes and skills acquired: The successful completion of the proposed experiments will provide important insights into the mechanism of SYCP3-dependent SC assembly on the axis of meiotic chromosomes. The student will become familiar with essential preparative biochemistry skills such as protein expression and purification, as well as biophysical and genetic tools designed for the study of protein-protein and protein-nucleic acid interactions. The student will have a chance to test and validate hypotheses of SYCP3 function by site-directed mutagenesis and protein engineering. Finally, the student will acquire basic crystallographic skills by getting involved with the on-going determination of SYCP3 crystal structures, in unliganded form and bound to DNA.

Project availability: Michaelmas and Lent Term

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Macromolecular mechanisms of genome duplication and stability

Supervisor: Dr. Luca Pellegrini, Biochemistry

Project abstract: Correct genetic inheritance relies on the biochemical process of DNA replication, responsible for the accurate duplication of our genome before mitosis. In the cell, a dedicated collection of protein molecules, collectively known as the replisome, cooperate in space and time to execute the complex programme of DNA replication. The overall aim of this project is to use the tools of structural biology to understand at atomic level how these proteins carry out their vital task. Such knowledge is important, because faulty DNA replication is a major predisposing cause for cancer and neurodegenerative disease.

Possible projects focus on three related questions, each addressing a critical step of genomic duplication, which is currently poorly understood:

- Initiation of nucleic synthesis by the DNA polymerase a/primase complex1,2.

- Replisome architecture in normal conditions and under replicative stress3.

- Assembly and activation of the replicative helicase complex.

References:

1: Perera RL et al., Mechanism for priming DNA synthesis by yeast DNA Polymerase á. Elife. 2013 Apr 2;2:e00482.

2. Kilkenny ML et al., Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering. Proc Natl Acad Sci U S A. 2013 110:15961-6.

3. Simon AC et al., A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome. Nature. 2014 510:293-7.

Learning outcomes and skills acquired: A successful project outcome will contribute to our understanding of the molecular mechanisms of genomic duplication and maintenance of genome integrity. The student will learn a range of biochemical and biophysical techniques, including recombinant DNA work, protein expression and purification, protein engineering, quantitative analysis of macromolecular interactions and assemblies, enzymatic assays, X-ray crystallography of protein complexes.

Project availability: Michaelmas and Lent Term

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Control of the dominant time constant of response recovery in zebrafish cone photoreceptors.

Supervisor: Dr. Hugh R Matthews

Project abstract: Recovery of the photoreceptor light response requires shutoff of both intermediates in the phototransduction cascade: the photopigment and the transducin-phosphodiesterase complex. Whichever quenches more slowly will govern overall recovery with a characteristic dominant time constant. While rods and cones share similar transduction cascades, significant differences exist in the control of their responses by calcium, a crucial messenger in light adaptation. In amphibian rods, the calcium-dependent quenching of rhodopsin does not dominate recovery of the saturating flash response, which is determined instead by the GTP-ase activity of transducin. In contrast, the dominant time constant in all spectral classes of salamander cone is greatly prolonged when the calcium concentration is prevented from falling during the light response, providing an additional mechanism by which light can modulate the photoresponse. Removal of external chloride, which contributes to the stabilising complex counterion in the red cone photopigment, accelerates the dominant time constant even without changes in calcium concentration. A similar acceleration in the dominant time constant is induced by a reduction in external pH indicating an external action of protons on the red cone opsin. This role for calcium has recently been extended to red-sensitive zebrafish cones. This project will investigate control of the dominant time constant by calcium in zebrafish cones using the suction pipette technique to record their electrical responses during rapid superfusion with a solution designed to prevent calcium concentration changes. Manipulation of the external chloride and proton concentrations will reveal whether these experimental modulators both act via the complex counterion.

Learning outcomes and skills acquired: Electrophysiological methods for recording responses of isolated sensory receptor cells.  Methods for rapid external superfusion of single cells.  General methods for electrophysiological data recording, reduction and analysis.

Project availability: Lent Term (January - March 2015)

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Structural analysis of Gpr54 mutant placentae

Supervisor: Professor William Colledge

Project abstract: INTRODUCTION. Kisspeptins, encoded by the Kiss1 gene, are a set of related neuropeptides that are required for activation of the mammalian reproductive axis at puberty and to maintain fertility. In addition, kisspeptin signalling via the G-protein coupled receptor GPR54 (KISS1R) has been suggested to regulate human placental formation and correlations have been found between altered kisspeptin levels in the maternal blood and the development of pre-eclampsia. METHODS. We have used Kiss1 and Gpr54 mutant mice to investigate the role of kisspeptin signalling in the structure and function of the mouse placenta. RESULTS. Expression of Kiss1 and Gpr54 was confirmed in the mouse placenta but no differences in birth weight were found in mice that had been supported by a mutant placenta during fetal development. Stereological measurements found no differences between Kiss 1 mutant and wild-type placentas. Measurement of amino-acid and glucose transport across the Kiss1 mutant placentas at E15.5 days did not reveal any functional defects. DISCUSSION. This data indicates that mouse placentas can develop a normal structure and function without kisspeptin signalling and can support normal fetal development and growth.

Project availability: Lent Term (January - March 2015)

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Understanding the nature and role of placental endocrine function in materno-fetal resource allocation during pregnancy

Supervisor: Dr Amanda Sferruzzi-Perri

Project abstract: During pregnancy, adequate nutrients must be supplied to the fetus for growth with sufficient resources also partitioned to the mother to maintain her health. The placenta is central to this as it secretes a plethora of factors which are thought to adapt maternal metabolism to favour nutrient delivery to the fetus. Failures in placental function and maternal adaptation may result in pregnancy complications including abnormal birth weight, premature delivery and maternal diabetes. Moreover, babies that are born of abnormal weight are more likely to die as neonates and/or develop metabolic disease postnatally. The overall aim of this work is to understand the nature and role of placental endocrine function in materno-fetal resource allocation during pregnancy and determine its importance for fetal growth, maternal health and offspring outcome. The proposed study will combine cell-specific genetic manipulation to selectively increase or decrease the formation of endocrine cells in the mouse placenta, with in vivo functional assays and in vitro stereological, molecular and proteomic methodologies. The overall goal of this project is to discover novel factors secreted by the placenta that regulate maternal-fetal resource allocation with a view to extend into translational studies assessing whether such factors are altered in pregnancies complicated by placental mal-function. For details on what a specific rotation project would entail, please contact me directly.

Project availability: Lent Term (January - March 2015)

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Developmental programming of heart disease

Supervisor: Professor Dino A. Giussani

Second supervisor: Dr. Emily Camm

Project abstract: In addition to genetics and lifestyle risk factors, it is now accepted that the quality of the intrauterine environment also plays a role in shaping the risk of heart disease in adult life (1).  However, the mechanisms underlying this developmental programming of cardiovascular disease in complicated pregnancy remain unknown.    Recently, we have shown in rats that chronic fetal hypoxia, one of the most common consequences of complicated pregnancy, induces oxidative stress in the fetal heart and circulation and programmes cardiovascular dysfunction at adulthood.  Furthermore, maternal treatment with vitamin C in hypoxic pregnancy prevents these effects (1-3).  While this discovery implicates developmental oxidative stress as a causative mechanism and targets for intervention, only high doses of vitamin C incompatible with human treatment, were effective. Mitochondria are a major source of free radical production; therefore they are a key target of antioxidant therapies. This new project will test the hypothesis that mitochondrial targeted antioxidant therapy is an effective intervention against programmed cardiovascular dysfunction by developmental hypoxia.      During the rottion project, questions will be addressed at the molecular level or the isolated organ level (Langendorff heart preparation and in vitro wire myography) using tissue obtained from rodent pregnancy or chick embryo incubation under hypoxic conditions.     http://news.sciencemag.org/sciencenow/2012/02/embryos-starved-of-oxygen-may-be.html    1.              Giussani et al. PLoS One 2012; 7(2):e31017.  2.    Richter et al. J Physiol. 2012; 590(Pt 6):1377-87.  3.                Herrera et al. J Vasc Res. 2012; 49(1):50-8.

Learning outcomes and skills acquired: Isolated Langendorff preparation to assess cardiac function;  In vitro wire myography to determine changes in vascular reactivity;  Western and Southern blots

Project availability: Lent Term (January - March 2015)

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Germline:  Specification and epigenetic programming for totipotency and mammalian development

Supervisor: Dr. Azim Surani

Second supervisor:

Project abstract: The mechanism of the specification of primordial germ cells (PGC) in mice, and the identity of the key determinants of the mouse germ cell lineage have been identified from studies in vivo.  PGC specification is linked with the initiation of extensive epigenetic reprogramming involving histone modifications and DNA demethylation towards generating the totipotent state.  The recently established in vitro system for the derivation of PGCs directly from ES cells allows for generating large numbers of PGCs, which provides opportunities to carry out detailed mechanistic investigations on events leading to PGC specification and reprogramming.  The aim of the project will be to determine the function of a key candidate involved in the establishment of subsequent events of reprogramming.  Following fertilisation, the parental pronuclei also undergo distinct and specific epigenetic reprogramming events despite being present in the same cytoplasm in the zygote.  The maternally inherited factors play an important role in these events and are crucial for the establishment of totipotency and preimplantation development.

Project availability: Lent Term (January - March 2015)

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To what extent are males and females epigenetically different? Characterisation of autosomal and sex chromosome specific transcriptomes and epigenomes from purified populations of mammalian cell types'

Supervisor: Professor Anne Ferguson-Smith

Project availability: Lent Term (January - March 2015)

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Cardiovascular adaptation of the mother during pregnancy; a role for phosphoinositol 3 kinase (PI3K) p110a?

Supervisor: Dr Amanda Sferruzzi-Perri

Second supervisor:

Project abstract: During pregnancy, the mother has to undergo major cardiovascular changes during pregnancy which favour maternal blood flow, particularly to the gravid uterus to support fetal growth. If such changes don’t occur, this can place strain on the maternal heart, which may adopt mechanisms to cope with the increased load during pregnancy (such as expansion of cardiac mass). PI3K p110a is a central regulator of growth and function. We have recently identified that a genetic deficiency in p110a is associated with increased weight of the maternal heart during pregnancy, which is not apparent in female mice prior to pregnancy. Stereology reveals that alterations in maternal heart weight are related to an enlarged left ventrical. This suggests that in response to a lack of p110a, the mother fails of to adapt her cardiovascular system during pregnancy. Although the nature of these cardiovascular changes and mechanism by which this occurs in pregnant p110a mutant mice remains unknown. This project will determine whether an increase in heart weight of pregnant p110a mutant mice may be caused by an increase in the number or size of left ventricular cardiomyocytes. It will also assess the expression of genes and proteins involved in hypertrophy, hyperplasia and remodelling, in the heart of p110a pregnant mutant hearts. Thus, a rotation student would perform in vitro analyses of heart tissues which have been recovered from mice which are deficient in p110a (such as immunohistochemistry, western blotting and real time PCR). This will ultimately lead onto further analyses of the mutant heart employing functional tests. For details on what a specific rotation project would entail, please contact me directly.

Project availability: Lent Term (January - March 2015)

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Dietary modification, nitrogen oxides and tissue fat oxidation

Supervisor: Dr. Andrew Murray

Second supervisor:

Project abstract: Nitrate is a ubiquitous dietary component, present in large quantities in leafy green vegetables, and the stable end-product of nitric oxide (NO) biosynthesis. Nitrate supplementation has been shown to improve mitochondrial efficiency in man, increasing ATP production for a given amount of oxygen consumed. Work in our laboratory has shown that nitrate supplementation preserves normal mitochondrial function in heart and muscle in the face of tissue hypoxia, preventing oxidative damage, and is able to enhance fatty acid oxidation in both hypoxia and normoxia. Dietary nitrate supplementation might therefore be beneficial in all humans supporting healthy ageing via improvements in tissue energy metabolism and redox homeostasis. Pilot studies have suggested that a role for the renin-angiotensin system, with ACE inhibition potentially eliciting similar effects, perhaps through a common pathway which increases NO bioavailability. eNOS knockout mice have an impaired capacity to synthesise NO and therefore develop the metabolic syndrome of obesity, hypertension, dyslipidemia and insulin resistance, yet dietary nitrate supplementation has been shown to be highly effective in preventing this. The aim of this project is therefore to investigate whether dietary nitrogen oxides or ACE inhibition enhance cardiac and skeletal muscle mitochondrial function and fatty acid oxidation, and the mechanisms underlying this. Practical work will involve the use of enzyme assays, western analysis of proteins and mitochondrial respiratory measurements.    Carlström M, et al. Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice. Proc Natl Acad Sci U S A. 2010, 107(41):17716-20.

Learning outcomes and skills acquired: General background and advanced understanding of metabolic regulation, cardiac physiology, mitochondrial biology, nitric oxide biology.    Skills acquired: molecular biology (PCR, western blotting), enzyme assays, high-resolution respirometry for the measurement of mitochondrial function.

Project availability: Lent Term (January - March 2015)

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Molecular mechanism and regulation of neuro-inflammation mediated by the Toll-like receptors and protein kinase LRRK2

Supervisor: Professor Nick Gay

Project abstract: In this project we will investigate the role played by microglia in the initiation and progression of neurodegeneration in PD. Microglia are specialised immune system cells that are restricted to the nervous system. We will study the respective roles of inflammation initiated by the Toll-like receptors and of LRRK2, a protein kinase, variants of which cause a predisposition to PD in the human (1,2). We will determine how LRRK2 and TLRs are activated in microglia and how activation is linked to neurotoxicity. Specifically we will analyse what cellular factors activate TLRs in the absence of infection and what proteins associate with LRRK2 in the presence and absence of signalling. We also use transcriptomic and proteomic approaches to identify which genes are expressed when microglia are activated and what proteins are substrates for the LRRK2 kinase. An understanding of the events that initiate and maintain neurodegeneration will lead to the development of novel therapies aimed at inhibiting these processes.  1.\tMoehle, M.S., Webber, P.J., Tse, T., Sukar, N., Standaert, D.G., DeSilva, T.M., Cowell, R.M. & West, A.B. LRRK2 inhibition attenuates microglial inflammatory responses.  J. Neurosci. 32, 1602-11 (2012). 2.\tGillardon, F., Schmid, R. & Draheim, H. Parkinsons disease-linked leucine-rich repeat kinase 2(R1441G) mutation increases proinflammatory cytokine release from activated primary microglial cells and resultant neurotoxicity. Neuroscience 208, 41-8 (2012). .

Learning outcomes and skills acquired: (1) Gene cloning for protein expressio (2) Protein purification, biophysical/structural analysis (3) Biased and unbiased methods to characterize protein-protein interactions (4) Lipidomics

Project availability: Lent Term (January - March 2015)

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Gene expression genomics in T cells

Supervisor: Dr Sarah Teichmann

Project abstract: While the genomic DNA template is static, dynamic changes in gene expression lead to specification of cells and tissues. The immune system is a particular dramatic example of this, where cells differentiate very rapidly in response to a challenge. We exploit the power of next-generation sequencing to discover global principles of gene expression regulation, and focus on immune responses.

Learning outcomes and skills acquired: Computational approaches to analysing NGS data, modeling, single cell transcriptome analysis, TCR-seq analysis

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Mechanism of multi drug efflux

Supervisor: Ben Luisi

Project abstract: The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters, In Gram-negative bacteria, may of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. We have obtained a pseudo atomic model of a complete multi-drug efflux pump that identifies the quaternary organisation of the pump, identifies key domain interactions and suggests a cooperative process for channel assembly and opening. The project will apply a similar approach to structurally and functionally characterise related protein transporter machines.

Learning outcomes and skills acquired: Experience in protein purification, functional assays and X-ray crystallography.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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A "Three Pipe Problem": Quorum Sensing in an Opportunistic Human Pathogen

Supervisor: Dr Martin Welch

Second supervisor:

Project abstract: The opportunistic human pathogen, Pseudomonas aeruginosa (PA), is a common cause of respiratory infections, especially among the elderly (who frequently suffer predisposing co-morbidities such as diabetes or structural lung disease) and the very young. PA uses "quorum sensing" (QS) to regulate the expression of many virulence factors during infection. The QS system in PA exploys a two-tiered N-acyl homoserine lactone-dependent mechanism. Located between the upper ("las") and lower ("rhl") tiers is the Pseudomonas Quinolone Signal (PQS). Although much is now known about how the PQS signal is generated and perceived, far less (in fact, next-to-nothing) is known about how this signalling system impinges on virulence. However, we *do* know that the protein denoted PqsE is required for virulence to be manifest, and is the most likely "effector" of PQS signalling. In this project, we will adopt a biochemical approach towards identifying PqsE targets. The pqsE gene will be PCR-cloned and the protein His-tagged for over-expression and purification. The purified protein will be immobilized (either via the tag, or directly onto CNBr-activated beads) and used for pull-down analyses. Any binders will be identified and themselves cloned, over-expressed for further in vitro analysis of the binding interaction (via calorimetry, cross-linking, gel filtration chromatography, biacore etc) . This is a simple, well-defined project that employs techniques commonly used in the host lab, yet the results (if promising) should form the basis for significant further study/publication.

Learning outcomes and skills acquired: PCR-based cloning  Protein purification  Protein-protein interaction analysis  Biophysical analyses

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Neurocognitive characterisation of ageing and human memory

Supervisor: Dr Jon Simons

Project abstract: Many older adults are concerned about declines in their memory abilities, which may be associated with degeneration in a number of brain regions, most notably the frontal lobes.  However, the ageing brain may be more resilient than previously thought, with evidence of activity increases in other cortical regions that may reflect compensation, with preserved areas taking over the function of declining regions, or might indicate shifts in the way older people perform some cognitive tasks.  By capitalising on the cognitive abilities that are comparatively resistant to the ageing process, it may be possible for older adults to develop effective strategies that will allow them to make the most of their memories as they age.  Our work aims to investigate potential strategies, including the use of deep, elaborative encoding tasks that can promote the formation of rich, vivid memory traces.  For example, participants can be trained to process studied material in a meaning-based, associative manner, or to focus on self-referential or emotional value-based details.  Neuroimaging data can be used to explore the brain networks that might support age-resistant elaborative memory processes, investigating the hypothesis that frontal lobe regions which have been linked to elaborative processing may be, in relative terms at least, less disrupted by the ageing process than other areas.  If successful, the results of this work could be applied to develop memory-related cognitive training techniques that could produce potentially long-lasting enhancement of remembering in older adults.

Learning outcomes and skills acquired: Research in the laboratory uses a number of methods, including behavioural studies, functional neuroimaging (fMRI), electrophysiology (EEG/MEG), and brain stimulation (TMS/tDCS).

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Cardiovascular adaptation of the mother during pregnancy; a role for phosphoinositol 3 kinase (PI3K) p110alpha?

Supervisor: Dr Amanda Sferruzzi-Perri

Project abstract: During pregnancy, the mother has to undergo major changes in her cardiovascular system, which favour maternal blood flow, particularly to the gravid uterus to support fetal growth. If such changes don’t occur, this can place strain on the maternal heart, which may adopt mechanisms to cope with the increased load during pregnancy (such as expansion of cardiac mass). PI3K p110alpha is a central regulator of growth and function. We have recently identified that a genetic deficiency in p110alpha is associated with increased weight of the maternal heart during pregnancy, which is not apparent in female mice prior to pregnancy. Stereology reveals that alterations in maternal heart weight are related to an enlarged left ventricle. Although the molecular mechanisms governing these cardiovascular changes in pregnant p110alpha mutant mice remains unknown. This project will determine whether an increase in heart weight of pregnant p110alpha mutant mice may be caused by an increase in the number or size of left ventricular cardiomyocytes. It will also assess the expression of genes and proteins involved in hypertrophy, hyperplasia and remodelling, in the heart of p110alpha pregnant mutant hearts.

Learning outcomes and skills acquired: In vitro analyses of the heart which have been recovered from mice which are deficient in p110alpha (immunohistochemistry, western blotting and quantitative real time PCR). This work will ultimately lead onto further analyses of the mutant heart, largely employing functional tests.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Viral discovery in complex diseases of humans and animals

Supervisor: Professor Jonathan Heeney

Second supervisor: Dr Barbara Blacklaws

Project abstract: The identification of novel infectious agents, or unrecognised infectious triggers of inflammatory diseases has recently been possible by the combination of Next Generation Sequencing and high throughput bioinformatics pipelines capable of discriminating host from pathogen sequences. Large datasets from diseases of animals and humans have been generated in our lab and we are looking for highly motivated individuals to help us identify novel infectious agents within these lesions. The skill-sets and experience you will gain will cover a spectrum of cutting edge technologies including: Next generation sequencing (NGS), Bioinformatics, Innate Immunity, Pathology, Evolution, In Situ Hybridisation, Immunohistochemistry, Immunosupressive diseases, new diagnostics technologies, Single cell flow cytometry.

Learning outcomes and skills acquired: You will learn about next generation sequencing and the analysis of human and animal RNAseq datasets from healthy control as well as disease specimens. You will search for long contiguous sequences of known or potentially unknown new viruses and will design probes and primers to genome-walk to identify full length genomes from humans or animals that have complex inflammatory diseases for which a virus is suspected, but no known agent has been identified.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Food Security, World class underpinning bioscience

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Equine cartilage stem cell responses to joint damage

Supervisor: Dr Frances Henson

Second supervisor:

Project abstract: Joint diseases, such as osteoarthritis, in horses cause lameness and discomfort.  This lameness is a significant welfare and economic problem.  Many of the joint diseases that affect horses arise as defects in the articular cartilage.  Healing of these articular cartilage defects is a difficult problem due to the poor intrinsic healing capacity of the tissue. It has been demonstrated that cartilage does contain small numbers of progenitor (stem) cells that have the potential to heal their parent tissue – so why does this not happen?  We hypothesise that there may be inhibitory factors in the environment of the damaged tissue that prevents effective functioning of the progenitor cells. Identification of inhibitory inflammatory mediators could lead to potential therapies for joint disease.    This project will focus on how cartilage progenitor cells respond to inflammatory mediators released from damaged tissues.  High efficiency colony forming cells will be isolated from equine cartilage and their responses to inflammatory mediators characterised.

Learning outcomes and skills acquired: The student will learn cell culture, flow cytometry, histology, western blotting and qPCR techniques in this project.  They will learn how to analyse and contextualise results and, by participation in regular laboratory meetings, learn communication and presentation skills.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Epigenetic stability in intestinal stem cells during ageing and in inflammation

Supervisor: Dr Patrick VARGA-WEISZ

Project abstract: Epigenetic stability in intestinal stem cells during ageing and in inflammation    Patrick Varga-Weisz   Adult stem cells, such as the intestinal stem cells, need to be highly proliferative to maintain tissue integrity but also need to maintain their epigenome through the lifetime of an organism. How this is accomplished is an important question.  We have shown that chromatin remodelling factors play an important role in the maintenance of the epigenome. Recently in collaboration with the group of Marc Veldhoen (Babraham Institute), we found that such a factor plays a role of interest in the maintenance of correct histone modification profile and gene expression in intestinal stem cells and loss of this factor leads to aberrant regulation of genes normally expressed in other tissues. Furthermore, loss of this factor leads to defects in intestinal immunity.  In the proposed programme, we will examine if aging, inflammation and inflammation linked to aging (inflammaging) also lead to short and long-term changes in the epigenomes and gene expression programme of intestinal stem cells. Furthermore, using transcriptome and proteome analysis we will find out what are the mechanisms and pathways that lead to such changes. We will use the mouse as model system and will collaborate with several teams at Babraham Institute and elsewhere.  References:  Mermoud, J.E., Rowbotham, S.P., and Varga-Weisz, P. (2011) Keeping chromatin quiet: How nucleosome remodeling restores heterochromatin after replication. Cell Cycle 10 (23) 4017 – 4025.    Rowbotham, S.P., Barki, L., Neves-Costa, A., Santos, F., Dean, W., Hawkes, N., Choudhary, P., Will, W.R., Webster, J., Oxley, D.,Green, C.M.,Varga-Weisz, P.* and Mermoud, J.E.* (2011). Maintenance of silent chromatin through replication requires SWI/SNF-like chromatin remodeler SMARCAD1. Mol Cell 42, 285-296. (*, corresponding authors, J. Mermoud was senior postdoctoral fellow in my lab)

Learning outcomes and skills acquired: You will learn how to apply epigenomic approaches (ChIP-seq, genome-wide mapping of histone modifications and other chromatin features). You might also apply organoid cultures. Skills that would be beneficial to start in my lab, but not essential: basic molecular biology, some basic bioinformatics

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Biochemical analysis of novel Fbxo7/PARK15 substrates

Supervisor: Dr. Heike Laman

Project abstract: Defects in the ubiquitin-proteasome system have been implicated in both cancer and neurodegenerative disease, two diseases whose incidence is linked to aging. This project focuses on an F-box protein, Fbxo7, which is the substrate-specifying subunit of an SCF-type E3 ubiquitin ligase.  Fbxo7 is a protein of clinical relevance in a number of different cell types.  It is over-expressed in human epithelial cancers, different SNPs are associated with variations in haematological parameters, and point mutations in Fbxo7 have been identified as causative for an atypical, early-onset Parkinson’s disease.  In cycling cells, Fbxo7 can affect the proliferation, differentiation, and viability.  In addition, we have discovered that Fbxo7 acts in a common pathway with two other Parkinson’s disease genes, Parkin and PINK1, to regulate stress-induced mitophagy.  However, no ubiquitinated substrates for Fbxo7 have been shown to be critical mediators of its various functions. We have identified a large number of proteins that are ubiquitinated by Fbxo7 using a protoarray screen.  This project will involve the study of these new substrates, including testing for the types of ubiquitination linkages catalysed by the SCF(Fbxo7) E3 ligase and the functional consequences of ubiquitination for the substrates.     Nelson DE, Randle SJ, Laman H. Beyond ubiquitination: the atypical functions of Fbxo7 and other F-box proteins. Open Biol. 2013 Oct 9;3(10):130131.    Burchell VS*, Nelson DE*, Sanchez-Martinez A*, Delgado-Camprubi M, Ivatt RM, Pogson JH, Randle SJ, Wray S, Lewis PA, Houlden H, Abramov AY, Hardy J, Wood NW, Whitworth AJ&, Laman H&, Plun-Favreau H&. The Parkinson's disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy. Nat Neurosci. 2013 Sep;16(9):1257-65.    Laman H, Funes JM, Ye H, Henderson S, Galinanes-Garcia L, Hara E, Knowles P, McDonald N, Boshoff C. Transforming activity of Fbxo7 is mediated specifically through regulation of cyclin D/cdk6. EMBO J. 2005 Sep 7;24(17):3104-16.

Learning outcomes and skills acquired: The student will use information from our Protoarray screen and reference this with proteomic analysis of primary tissues from our Fbxo7 mouse model.  Selected substrates will be analyzed for their ubiquitination profiles.  The project will utilize mainly biochemical, proteomic and cell biology techniques to understand the capacity of this F-box protein scaffolds proteins to integrate pathways and affect cell outcomes.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Delineating the roles of the interferon induced proteins with tetratricopeptide repeats (IFIT) family of anti-viral proteins

Supervisor: Dr Trevor Sweeney

Second supervisor: Prof Ian Goodfellow

Project abstract: IFITs are expressed at very high levels in response to infection and have been implicated in restricting viral replication in part by interfering with translation of viral mRNAs. Most mammals express IFITs 1, 2, 3 and 5 while different species also possess extra IFIT genes such as IFIT1B and 1C. Cellular ‘self’ mRNAs (cap1) are marked by 2’-O-methylation of the first and second bases. We recently demonstrated that the recognition of ‘non-self’ cap0 (no 2’-O-methylation) and 5’-ppp (no cap) containing RNAs by IFIT1 and IFIT5 respectively, directly inhibited translation initiation (1).  This identified a dual detector/effector role for these IFITs in regulating viral replication. We also showed that IFIT1B can bind and inhibit the translation of cap1 containing RNAs (1). IFIT1 interacts with IFIT2 and 3 in infected cells with the formation of hetero-oligomeric complexes suggested as a requirement for full anti viral activity. In contrast, IFIT5 was found not to interact with other IFIT proteins (2,3). Induced expression of IFIT1 has been shown to restrict replication and translation of a parainfluenza variant that encodes a functional 2’-O-methlytransferase in interferon deficient cells (4). This project will examine the downstream effects of ‘non-self’ RNA recognition by different IFITs. These studies will greatly enhance our understanding of the role of IFITs as important regulators of viral replication.    1) Sweeney and Kumar et al., NAR (2014) doi:10.1093/nar/gkt1321   2) Pichlmair et al., Nat. Immunology (2011) doi:10.1038/ni.2048  3) Habjan et al., PLoS Path. (2013) doi:10.1371/journal.ppat.1003663  4) Andrejeva et al., J. Gen. Vir. (2013) doi:10.1099/vir.0.046797-0

Learning outcomes and skills acquired: In this short rotation project the student will learn valuable transferable molecular biology skills in cloning, mutagenesis and recombinant protein production. These proteins will be used to study protein/RNA interactions using various techniques. They will also learn to use tissue culture models and siRNA techniques to knock down cellular proteins to analyze their function. Problem solving, data analysis and experimental planning skills will be developed. Lab meetings and divisional research reports will also provide the student with excellent opportunities to develop data presentation skills.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience, Food Security

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Purification of recombinant Drosophila UbcH10-Apc11 complex: a potential anti-tumour target

Supervisor: Dr Yuu Kimata

Project abstract: The ubiquitin conjugating enzyme, UbcH10/Ube2c, cooperates with the ubiquitin ligase, the anaphase promoting complex (APC/C), to regulate cell cycle progression. In a great number of human carcinomas, UbcH10 is highly expressed and its over-expression (OE) induces genomic instability and tumour formation in mouse. To gain insight into the tumorigenic mechanism associated with UbcH10 OE, we have been investigating the effect of OE of its Drosophila orthologue, Vihar (Vih). We have found that Vih OE induces genomic instability in embryos and cultured cells, which is in part mediated by APC/C activity. We have also demonstarted that Vih OE can lead to over-proliferation of cells within the developing notum, pointing to the conservation of the oncogenic activity of UbcH10 across species. We are currently investigating a primal cause of genomic instability induced by Vih OE.  In this rotation project, we aim to express and purify the recombinant Vih-Apc11 complex to solve the crystal structure of the complex. Thus far, the structure of the human counterparts remain unsolved. The successful completion of this project would be an initial step towards our long term goal of developing small molecules that specifically target UbcH10 over-expressing cancer cells.

Learning outcomes and skills acquired: Through this project the student will learn basic molecular biological and biochemical techniques such as plasmid construction, protein expression in bacteria and beculovirus expression, affinity purification of recombinant proteins.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience, Food Security

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Genetic manipulation of neurons to prevent axon degeneration

Supervisor: Dr Michael Coleman

Second supervisor:

Project abstract: We and collaborating groups have identified two proteins with strong, opposing effects on axon degeneration and shown they are positive and negative regulators of the same pathway (Conforti, Gilley et al, Nat Rev Neurosci 2014). The NAD synthesizing enzyme NMNAT blocks the pathway, while the Toll-like receptor protein SARM1 is required for its completion (Mack et al, Nat Neurosi 2001; Osterloh et al, Science 2012).  Long term, we aim to elucidate the entire pathway and identify key steps for intervention in axonal disorders and age-related axon loss.  The more immediate goal of this project is to understand how these two proteins interact with one another.     The student will work alongside an experienced postdoc carrying out some steps in this process. Neurons will be grown in primary cell culture and genetically manipulated by microinjecting variant forms of the SARM1 protein, generated using molecular cloning methodology. Axons will be transected in culture the following day and the rate of degeneration measured using fluorescent microscopy and quantified using custom software. This will lead to an understanding of the structure-function relationship of SARM1, enabling us to answer questions such as ‘how does it signal or respond to other molecules in this pathway?’ and ‘what is the precise subcellular site in which it acts?’.       The student will also have the opportunity to learn about other projects in the group on age-related axon loss, live imaging of axonal transport, and the roles of axon pathology in Alzheimer’s disease, motor neuron disease and peripheral neuropathies.

Learning outcomes and skills acquired: The student will learn the following methods: dissection of various neuronal subtypes, primary neuronal culture, manipulation and cloning of recombinant DNA, DNA microinjection, fluorescent, phase contrast and confocal microscopy, and automated quantification of axon degeneration. There will also be chance to learn about organotypic slice culture, live imaging, immunohistochemistry and mouse and Drosophila genetics.    Scientifically, the student will gain knowledge in neuronal, and especially axon survival mechanisms, axon loss in age-related neurodegenerative disorders, trafficking of proteins and organelles in axonal transport and cell death mechanisms.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Imprinted loci in maternal behaviour

Supervisor: Dr Carole A Sargent

Second supervisor: Prof Nabeel Affara

Project abstract: Abnormal infanticidal behaviour in pigs shows strong mother-daughter inheritance. It is a feasible model for human puerperal psychosis, an extreme condition thought to be on the bipolar disorder spectrum. Recent deep sequencing of brain samples from our lab indicates that several of the most significant genes with altered expression between matched cases and controls are either imprinted, or have monoallelic expression (but it is unclear if the gene is always expressed from the paternal or maternal chromosome, or randomly selected).   Deep sequencing data are being combined with genome wide association, family association and linkage, and trio analysis to filter the most promising candidates for targeted DNA analysis in affecteds. However, the involvement of genes carrying an imprint or epigenetic mark means that deep sequencing of target intervals to study the methylation and hydroxymethylation status will give greater insight into the mechanisms that could be involved in the phenotype.   We have tissue and DNA samples from the brains of affected and control animals, plus DNA from ear/ tail tags of over 2000 animals collected on-farm. The project would involve making libraries from the brain samples to compare the methylation and hydroxymethylation status of the genomes through a deep sequencing approach. Results will be analysed and integrated with the genetic analyses to prioritise which loci are the most promising.

Learning outcomes and skills acquired: The student will acquire skills in library preparation for next generation sequencing, and analysis of sequence output through bioinformatics packages. The student will also liaise with other team members to understand how the sequencing approaches are integrated with the genetic analyses.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Food Security

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Investigation of the molecular mechanisms of metastatic dissemination of canine mast cell tumours

Supervisor: Dr Mike Starkey

Project abstract: The spread of tumour cells to distant sites is responsible for the majority of cancer-associated deaths. Understanding the molecular mechanisms by which tumour cells gain the capability to escape the constraints of interactions with neighbouring cells and the extracellular matrix, migrate to, and colonise remote organs is pivotal to the development of rational therapeutics to target metastatic spread.     Mast cell tumours are the most common skin tumour in dogs, affecting dogs of all breeds. The majority of affected dogs are cured by surgery and local radiotherapy, but around 30% of tumours spread to local lymph nodes, spleen, liver, and bone marrow. In this project we will elucidate the processes altered in benign neoplastic canine mast cells which are associated with the acquisition of metastatic potential.    Bioinformatic analyses will be undertaken to identify over-represented biological functions and cellular pathways shared by genes (identified by transcriptome analysis) that display statistically significant differential expression between metastasising and non-metastasising canine mast cell tumours. Putative molecular markers of metastatic potential will subsequently be evaluated by reverse transcription-quantitative PCR gene expression assay of a large cohort of archival canine mast cell tumours.

Learning outcomes and skills acquired: The student will undertake an intensive period of training and work in a research laboratory, gaining a taste of life as a member of a small team at a research establishment. He/she will obtain experience in the large scale analysis of gene expression data, learning how to interpret the data in order to assess biological implications. The student will also acquire proficiency in measurement of gene expression by reverse transcription-quantitative PCR.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Cellular mechanisms underlying left-right asymmetry in the brain

Supervisor: Professor Ole Paulsen

Project abstract: Using ontogenetic stimulation techniques, we have earlier reported that hippocampal synaptic plasticity in the mouse shows hemispheric asymmetry, specifically, synaptic long-term potentiation (LTP) is present in hippocampal afferents originating in the left, but not the right, hippocampus (Kohl et al., 2011). Recently, using ontogenetic silencing techniques, we demonstrated a functional correlate of such asymmetry by showing that hippocampus-dependent long-term memory requires input from the left, but not the right hippocampus (Shipton et al., 2014). We now want to understand how such left-right asymmetry may develop. Towards this aim, we will try to reproduce left-right asymmetry in a cell culture system, growing neurons from either the left or right hippocampus or both, following ontogenetic labelling of the neurons originating from either the left or right hippocampus. Using this system, you will test whether neurons from left and right  hippocampus are intrinsically different, whether they are different during competitive interactions, or whether they require differential activity to develop left-right asymmetry. The techniques you will use include optogenetics and patch-clamp recordings.     References:   Kohl MM, Shipton OA, Deacon RM, Rawlins JNP, Deisseroth K and Paulsen O (2011) Hemisphere-specific optogenetic stimulation reveals left-right asymmetry of hippocampal plasticity. Nat Neurosci 14: 1413-1415.      Shipton OA, Apergis-Schoute J, Deisseroth K, Bannerman DM, Paulsen O and Kohl MM (2014) Left-right dissociation of hippocampal memory processes in mice. Proc Natl Acad Sci USA 111: 15238-15243.

Learning outcomes and skills acquired: In addition to generic learning outcomes, such as experimental design, data analysis, statistics, and report writing, the specific skills that you would be trained in when doing this project would be neuronal tissue culture techniques, optogenetics and patch-clamp recordings.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Rho GTPase cytoskeleton networks and subversion by pathogenic bacteria

Supervisor: Dr. Daniel Humphreys

Second supervisor: Prof. Vassilis Koronakis

Project abstract: Mammalian cells polymerise actin filaments at the membrane to create a driving force for cells to move, remodel their architecture and divide, and to provide a structural framework that defines cell shape and polarity. The formation of actin structures at the membrane requires a network of actin machineries that are controlled through master regulators of the actin cytoskeleton known as Rho GTPases (20 isoforms).     A central paradigm in bacterial pathogenicity is Rho GTPase subversion by an enormous repertoire of pathogen-encoded virulence proteins that are injected into the target host cell. We recently uncovered how bacterial effectors hijack co-incident lipid and GTPase signals that cooperate to facilitate infection, e.g. Salmonella uptake into host cells (Humphreys et al, 2012, 2013). This has opened my eyes to the fact that while we know many of the Rho targets, we do not know how the components interact with each other nor how virulence proteins manipulate the complex Rho cytoskeleton networks in the membrane environment.     The interdisciplinary PhD project will combine live cell imaging of the actin cytoskeleton, infection biology and biochemical reconstitution of virulence-driven actin polymerisation to understand how virulence effectors function at the membrane and hijack Rho cytoskeleton networks during infection. This is especially important given the health threat posed to humans and farmed food chain animals by bacterial pathogens that continue to develop multidrug resistance. Understanding the basis of disease has the potential to identify new therapeutic targets and augment our anti-infectives arsenal.

Learning outcomes and skills acquired: The student will immerse themselves in the field of bacterial pathogenesis, learning how injected virulence effectors function and hijack cell biology. They will acquire a spectrum of interdisciplinary skills that will enable molecular mechanisms underpinning disease to be resolved. In particular, the student will generate purified fluorescent derivatives of virulence effectors, and reconstitute protein-protein using pioneering liposome-based technology, fluorescent actin polymerisation in cell-free extracts, and will use live confocal microscopy of effector-manipulated cells, fluorescence-recovery after photobleaching (FRAP) to assess cytoskeleton manipulation, and exploit a range of cell infection models. Training will focus on experimental design, data interpretation and presentation skills.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience, Food Security

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Exploring the epigenetic mechanisms behind transgenerational inheritance of developmental phenotypes

Supervisor: Dr Erica Watson

Project abstract: During their lifetime, a person is exposed to environmental stressors that may influence the epigenetic profile of their genome. These changes in epigenetic patterns (e.g., in DNA or histone methylation) might contribute to an increased risk in disease later in life. Furthermore, if the so-called ‘epimutations’ occur within the germ cell population, it is possible to pass them down to subsequent generations in a process called transgenerational inheritance. This non-conventional type of inheritance occurs independent to changes in DNA sequence. However, epimutations have the potential to change patterns of gene expression that can ultimately disrupt normal cellular states and developmental programmes. In the lab, we work on a mouse model of transgenerational inheritance whereby genetic disruption in folate metabolism in the first generation leads to severe developmental phenotypes up to four wildtype generations later (Padmanabhan et al, 2013 Cell). Folate metabolism is necessary to transfer one-carbon methyl groups to cell substrates that undergo methylation (e.g., DNA). When folate metabolism is disrupted, fewer methyl groups are available resulting in hypomethylation of the cell. The aim of this project will be to use next generation sequencing technology to assess the DNA methylation profiles in germ cells derived from this model of abnormal folate metabolism and compare to profiles in wildtype germ cells. Ultimately, we would like to identify epimutations that are passed between generations through the germ line to help us to understand the mechanism behind transgenerational epigenetic inheritance.

Learning outcomes and skills acquired: Using this model, the student will trace how epigenetic instability caused by metabolic disruption or nutritional deficiency leads to dysregulation of gene expression that triggers the formation of developmental phenotypes. Importantly, the student will study the mechanism of how epigenetic instability is inherited between generations by focusing on the germline. Little is known about how transgenerational inheritance occurs and so their findings will contribute to the overall field. They will gain hands-on experience in molecular biology techniques required for epigenetic analysis (e.g., methylated DNA immunoprecipitation, pyrosequencing, qPCR) and be involved in bioinformatics analysis of data collected from next generation sequencing.

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Complex trait association analysis in isolated populations using very low depth whole genome sequencing data

Supervisor: Prof. Eleftheria Zeggini

Second supervisor: Arthur Gilly

Project abstract: There is increasing evidence that rare variants play a role in complex traits and common diseases. The genetic architecture of isolated populations makes them particularly suited for rare variant association analysis (Hatzikotoulas et al. 2014), because rare variants may have drifted up in frequency, and neutral rare variation is lost from the haplotype pool. Very low-depth sequencing (1x or less) has been proposed as an approach to increase sample size in WGS studies and therefore boost power (Pasaniuc et al. 2012). However, this study paradigm has not yet been empirically put to the test.    In this project, the student will work on very low-depth (1x) WGS as part of the HELIC project (www.helic.org), which focuses on 2,500 samples from two Greek population isolates, combined with genome-wide genotyping and deep phenotyping data on a wide range of cardiometabolic-related quantitative traits (e.g. Tachmazidou et al, 2013; Panoutsopoulou, Hatzikotoulas et al, 2014). During the past year, a robust pipeline for calling accurate genotypes from low-depth sequence data has been established and both cohorts have completed variant calling. The student will be involved in running sequence-based genome-wide association analysis of medically-relevant quantitative traits, in signal prioritisation and follow-up, and in dealing with the specific challenges posed with this new type of data. Moreover, the scope of the project offers the opportunity to get involved in all steps of the discovery process, including but not limited to variant quality control, genotype refinement and imputation, and haplotype phasing in the context of WGS data.

Learning outcomes and skills acquired: The student should have working experience using the UNIX command line and should be familiar with at least one scripting language, such as Perl and Python. General knowledge about human genetics and statistics is essential. During the rotation, the student will become familiar with the file formats and computational approaches used in analysing whole genome sequencing data, from variant calling to association analysis, and with interpreting association signals.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health (ageing research: lifelong health)

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The role of Adherens Junctions in pluripotency

Supervisor: Dr Alfonso Martinez Arias

Project abstract: 

Embryonic Stem (ES) cells are clonal populations derived from the blastocyst of mammalian embryos that can be differentiated into all cell types of an organism in culture and renew this ability indefinitely i.e. they are pluripotent. Over the last few years, their potential for regenerative medicine has made them a subject of intense scrutiny but they are also an excellent system for research in cell and developmental biology. Studies of the molecular basis of their properties have focused, for the most part, on their transcriptomes, epigenetics and underlying gene regulatory networks, and have created a foundation for understanding pluripotency and the transition to differentiation. However, there is evidence that non-transcriptional events, such as protein degradation, the organization of the cell cycle and of the cytoskeleton, also have significant influence on the state of these cells. The aim of this project is to to focus on one of these events, the emergence of cell polarity, to understand how it relates to pluripotency.

There are two kinds of pluripotent cells in mouse: ES cells, derived from the preimplantation blastocyst at E3.5-4.0 and Epi Stem cells (EpiSC) which are derived from the postimplantation blastocyst at E5.5. ES cells can be transited in culture into EpiSCs. A major difference between these two cell types is their structure and, associated with this, the organization of E-Cadherin. EpiScs exhibit a clear apico basal organization with E-Cadherin localized to the Adherens Junctions in contrast with ES cells which also contain E-Cadherin but lack apico-basal polarity and adherens junctions. A number of studies, including some from our laboratory, suggest that E-Cadherin is important form the maintenance of the ES cells and it is not know whether it is required for the EpiSCs.

In this project, we shall study the evolution in expression and subcellular localization of the proteins associated with the adherens junctions during the transition from ES to EpiS cells. We shall also correlate this with their function by analysing the phenotypes and structures of E-Cadherin mutant ES and EpiS cells. An important tool of this project will be a cell line in which E-Cadherin has been tagged with GFP which allows us to film the construction and deconstruction of the junctions.

Learning outcomes and skills acquired: The laboratory is well equipped for the experiments as it has experience in the growth and analysis of ES cells. The project will involve some biochemical analysis but, above all, imaging and image analysis of immunostainings and live recordings of cells under different conditions.

Project availability: Lent Term (January - March 2015)

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