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PhD Projects for Basic Biosciences Underpinning Health 2014-2015

Isabel M Palacios, Department of Zoology

PhD Project Description: A biophysical study on how the actin and microtubule cytoskeletons dynamically collaborate to regulate cellular organization. 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.

Link: http://www.zoo.cam.ac.uk/zoostaff/palacios/

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Pier Paolo D'Avino, Department of Pathology

PhD Project Description: Dissecting the roles of Polo and Aurora kinases during cytokinesis. Animal growth, development and reproduction depend on the accurate process of cell division, which faithfully partitions the genomic information between the two daughter cells. The overarching aim of this project is to dissect the functions of two mitotic kinases – Polo-like kinase 1 (Plk1) and Aurora-B – during the last phase of cell division, cytokinesis. Plk1 and Aurora B play multiple roles during mitosis, including spindle assembly, centrosome maturation, chromosome condensation, alignment, and segregation (1, 2). Because of these early mitotic functions, it has so far been difficult to dissect the roles of these kinases during cytokinesis. To overcome this limitation, the student will employ a combination of chemical genetics, mass spectrometry, time-lapse imaging and drug treatments to investigate the possible cross talk between these two kinases and identify their targets during cytokinesis. The specific aims of this project are: • Characterise the localisation patterns of Plk1 and Aurora B during cytokinesis, using both immuno-fluorescence and time-lapse imaging. Plk1 and Aurora B specific inhibitors will also be used to investigate the localisation dependency of these two kinases, including their active forms, and their effects on the distribution of other cytokinesis proteins. • Initial studies have indicated that Plk1 and Aurora B localisations are slightly different, leading to the hypothesis that their distribution might be functionally relevant. To address this, we will engineer a Plk1 mutant able to mimic Aurora B localisation and vice versa and then study the effects of these Plk1 and Aurora B variants on cytokinesis. • To identify substrates phosphorylated by Plk1 and Aurora B specifically during cytokinesis in vivo, we propose to employ a chemical genetic screen that has already been successfully used to identify AMPK targets (3). We will engineer the Plk1 and Aurora B kinase domains in order to enable these mutants to accept ‘bulky’ ATP analogues. We will then express these mutants in HeLa cells and after synchronisation in telophase we will incubate them with ‘bulky’ ATPS. Thio-phosphorylated substrates will be immuno-precipitated using a thiophosphate ester-specific antibody and identified by mass spectrometry.

Referees:

1. V. Archambault, D. M. Glover. Nat Rev Mol Cell Biol 10, 265-275 (2009)

2. S. Ruchaud, et al. Nat Rev Mol Cell Biol 8, 798-812 (2007)

3. M. R. Banko, et al. Mol Cell 44, 878-892 (2011)

Link: http://www.path.cam.ac.uk/research/investigators/davino/research.html

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Rafael E. Carazo Salas, Dept. of Genetics

Second Supervisor/Collaborator: Ludovic Vallier, Cambridge Stem Cell Institute and Sanger Institute

High-Content Microscopy and Human Stem Cells: Investigating how stem cells’ differentiation propensity differs through the cell cycle and from cell to cell

PhD Project Description: Cellular pluripotency and differentiation are traditionally thought of as regulated by transcription factors and chromatin epigenetic states. By contrast, surprisingly little is known of the role that actual cell biological processes (cell cycle, cytoskeletal/polarity control, cell-cell interactions, growth/size control), and cell-to-cell heterogeneity in those processes, play in imposing or relieving ‘bottlenecks’ on differentiation. Identifying and understanding such bottlenecks is key to unleash the full potential of stem cell-based therapeutics. In collaboration with Ludovic Vallier (Cambridge Stem Cell Institute/Sanger Institute), we want to examine in detail one such bottleneck: that imposed by the cell cycle. His group showed that cells’ propensity to undergo differentiation along specific lineages differs throughout the cell cycle and that, conversely, cell cycle manipulation can increase the efficiency of differentiation into specific lineages. This interdisciplinary PhD project will investigate in detail how stem cells’ differentiation propensity differs through the cell cycle and between cells by: a) Helping complete the establishment of a time-lapse, high-content microscopy pipeline to phenotype human pluripotent (ES/IPS) cells at the single cell level and at large scale b) Using that pipeline to investigate how cell-cycle controlled differentiation varies from cell to cell, by single cell lineage tracing and quantitative phenotypic analysis. This will be done using ‘live’ fluorescent reporters of the cell cycle, pluripotency/differentiation, and other relevant cell biological features (cell size, polarity, shape…) c) Exploiting cellular heterogeneity to clarify the molecular/causative linkage(s) between differentiation, fate specification and cell cycle control d) Characterising in detail the linkage(s) identified by chemical or genetic strategies

Referees:

A genomic Multiprocess survey of machineries that control and link cell shape, microtubule organization, and cell-cycle progression. Graml V, Studera X, Lawson JL, Chessel A, Geymonat M, Bortfeld-Miller M, Walter T, Wagstaff L, Piddini E, Carazo-Salas RE. Dev Cell. 2014 Oct 27;31(2):227-39.

The cell-cycle state of stem cells determines cell fate propensity. Pauklin S, Vallier L. Cell. 2013 Sep 26;155(1):135-47.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.gen.cam.ac.uk/research-groups/carazo-salas

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Dr. Simone Schnall, Dept. of Psychology

Embodied Perception: Exploring the Mechanism behind Action-Relevant Perceptual Judgments

PhD Project Description: The Cambridge Embodied Cognition and Emotion Laboratory explores the relationship between cognitive and emotional processes. We combine methods from social psychology and cognitive science to understand how people represent the world, and how they make judgments and decisions. One line of research involves perceptions of the physical environment. We showed that after having consumed glucose people find a hill less steep than after having consumed noncaloric sweetener, presumably because glucose provides energy (Schnall, Zadra & Proffitt, 2010). In addition to physiological influences, similar effects are obtained with respect to a psychosocial resource, namely social support. In the presence of a friend, or even when simply thinking about a significant other, people perceive a hill to be less steep than when they are alone (Schnall, Harber, Stefanucci & Proffitt, 2008). Resources considerations are also relevant when it comes to social power: Indeed, the definition of having power is having control over one's own and other people's resources. We showed that people who feel powerful literally experience a heavy burden to be less heavy than people who feel powerless (Lee & Schnall, 2014). Thus, whether or not you have resources available changes how you see the physical world. The PhD Project will explore whether there is a common underlying mechanism behind those findings, such that different kinds of resources lead to similar action-relevant perceptual changes. Depending on the student’s background and expertise, methods may include psycho-physiological measurements or other techniques. Interested applicants are encouraged to discuss potential project ideas with Dr. Schnall.

Referees:

Schnall, S., Zadra, J., & Proffitt, D. R. (2010). Direct evidence for the economy of action: Glucose and the perception of geographical slant. Perception, 39, 464-482.

Schnall, S., Harber, K., Stefanucci, J. & Proffitt, D. R. (2008). Social support and the perception of geographical slant. Journal of Experimental Social Psychology, 44, 1246-1255.

Lee, E. H., & Schnall, S. (2014). The influence of social power on weight perception. Journal of Experimental Psychology: General, 143, 1719-1725.

Link: http://www.psychol.cam.ac.uk/cece

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Nabeel Affara, Dept. of Pathology

Epigenetic and transcriptional correlates of early life exposures in a rat model of the metabolic syndrome

PhD Project Description: The DOHaD (Developmental Origins of Health and Disease) hypothesis proposes that symptoms of metabolic syndrome (e.g. coronary heart disease, hypertension, type 2 diabetes) originate through developmental programming of the epigenome in response to undernutrition in early life. Work using a rat model has characterised expression changes in male rat livers associated with in-utero undernutrition via manipulation of maternal diet. Further work in this model, studying female offspring, and their treatment with leptin, demonstrated complex interactions between diet, metabolism and immune system function, likely involving coordination from the central nervous system. In our previous study of this rat model 249 genes were detected with different expression levels in liver between control and maternally undernourished male rats at day 55 after birth. No differences were detected using microarrays in muscle or white adipose tissue. Undernourished animals develop symptoms of metabolic syndrome as early as day 110. Consequently, we know that differences between the groups are set up early in their development, including changes in expression of genes related to lipid metabolism and mitochondrial function, before the disease phenotype manifests. These changes presumably include epigenetic components. The student will re-examine the three tissues from these animals for transcriptional and epigenetic differences using RNA-Seq, MeDIP-Seq and ChIP-Seq approaches. These will inform us about the changes set up during development and the hierarchy of transcriptional effects, and will be correlated these with the measured phenotype data.

Referees:

1. Morris TJ, Vickers M, Gluckman P, Gilmour S, and Affara N. (2009). Transcriptional profiling of rats subjected to gestational undernourishment: implications for the developmental variations in metabolic traits. PLoS One Sep 29;4(9):e7271.

2. Ellis PJI, Morris TJ, Skinner BM, Sargent CA, Vickers MH, Gluckman PD, Gilmour S and Affara NA. (2014) "Thrifty" metabolic programming in rats is induced by both maternal undernutrition and zapostnatal leptin treatment, but masked in the presence of both: implications for models of developmental programming. BMC Genomics. Jan 21;15:49. doi: 10.1186/1471-2164-15-49.

3. Gluckman PD, Hanson MA. Living with the past: evolution, development, and patterns of disease. Science. 2004 Sep 17;305(5691):1733-6. Review. PubMed PMID: 15375258.

Other relevant themes: World Class Underpinning Bioscience

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Ben Luisi, Dept. of Biochemistry

Structural and functional studies of membrane transport

PhD Project Description: Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. The project will involve structural and biochemical studies of tripartite complexes involved in drug and protein transport in bacteria, using X-ray crystallography and electron microscopy.

Referees:

Du, D., Wang, Z., James, N.R., Voss, J.E., Klimont, E., Ohene-Agyei, T., Venter, H. Chiu, W. and Luisi, B.F. (2014). Structure of the AcrAB-TolC multidrug efflux pump. Nature. 509, 512-515. Doi:10.1038/nature13205.

Bavro, V.N., Pietras, Z., Furnham, N., Perez-Cano, L., Fernandez-Recio, J., Pei, X.Y., Misra, R. and Luisi, B.F. (2008) Channel-opening and assembly in a bacterial drug efflux machine. Molecular Cell 30, 114-121.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.bioc.cam.ac.uk/people/uto/luisi

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Professor Clare Bryant, Dept. of Veterinary Medicine

Second Supervisor/Collaborator: Dr Pietro Cicuta (Department of Physics) and Dr Gillian Fraser (Department of Pathology)

Salmonella-macrophage interactions: exploring diverse host cell responses uses high resolution single-cell imaging

PhD Project Description: Salmonella enterica causes food-borne infections in humans that range in severity from self-limiting gastroenteritis to systemic typhoid fever. During infection, Salmonella are taken up by macrophages and are either killed in the phago-lysosome or survive by injecting bacterial effectors into the macrophage to subvert its function. At the same time Pattern Recognition Receptors in the macrophage detects the presence of the Salmonella effector proteins and other bacterial molecules to initiate the formation of innate immune signaling platforms that lead to cell death and pro-inflammatory cytokine production. This helps to recruit other immune cells to the infected cell to try and control the infection. This view is an over-simplification because our single cell imaging analysis shows that the majority of cells do not get infected, not all those that are infected die and complex interactions occur between infected and uninfected cells. Using a combination of traditional cell culture techniques, molecular genetics and high-resolution single cell imaging, we aim to characterize key molecular events to determine whether this diversity in response is driven by the bacteria and/or the host. This project will use a range of techniques from measuring changes in bacterial gene expression through to assembly of host immune signaling platforms, to explore why there is such heterogeneity in Salmonella-macrophage interactions.

Referees:

Gog, JR., et al., J. R. Soc. 2012; 9, 2696-707 Man SM et al., Proc Natl Acad Sci. 2014; 111:17588-93

Man, SM et al., Proc Natl Acad Sci 2014; 111(20):7403-8

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.vet.cam.ac.uk/directory/ceb27@cam.ac.uk

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Richard W. Farndale, Dept. of Biochemistry

Plasma protein–collagen interactions and the regulation of haemostasis

PhD Project Description: Plasma glycoproteins, such as von Willebrand factor (VWF), play crucial and pleiotropic roles in the regulation of haemostasis after injury to the blood vessel wall. Interaction between the A domains of VWF and exposed collagen (via A3) and platelet glycoprotein (Gp) Ib (via A1) mediates the initial capture of circulating platelets; the first stage of sealing a damaged blood vessel. We have shown that VWF A3 binds to a specific site in collagen III, using our synthetic peptide libraries (Toolkits); recently we have found a second site in collagens I and II. This will be characterized with a peptide–A3 co-crystal a possible objective. VWF A1 also binds our collagenous peptides. We will express the three A domains, A1, A2 and A3, as a contiguous polypeptide, and discover whether they co-operate, positively or negatively, to support VWF binding to collagen, and the effect of the VWF–collagen interaction in regulating the binding of platelets to GpIb. We will investigate at a structural level, by crystallography and perhaps other techniques, atomic force microscopy or cryoelectron-microscopy. Mutations (R1306/I1309V) in VWF A1 are reported to increase its affinity for GpIb. Do these increase the affinity of VWF for collagen, at rest and under blood flow conditions? VWF binds Factor XIII, prolonging its plasma half-life; does this influence collagen binding? Other Clotting Factors, VII and XII, are reported to bind collagen; we will map these interactions onto the Toolkits, and investigate the haemostatic consequences of Clotting Factor–collagen binding.

Referees:

Lisman et al., (2006). A single high-affinity binding site for von Willebrand factor in collagen III, identified suing synthetic triple-helical peptides. Blood, 108, 3753-6

Farndale et al, (2008) Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions. Biochem Soc Trans 36, 241-50

Brondijk et al. (2012). Implications for collagen I chain registry from the structure of the collage von Willebrand A3 domain complex. Proc Natnl Acad Sci 109, 5235-8

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://collagentoolkit.bio.cam.ac.uk/ http://www.bioc.cam.ac.uk/people/uto/farndale

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Dr Jonathon Pines, Dept. of Zoology

How is the Spindle Assembly Checkpoint turned off?

PhD Project Description: The Spindle Assembly Checkpoint (SAC) is crucial to the control of genomic stability. The SAC is activated by unattached kinetochores and prevents the Anaphase Promoting Complex/Cyclosome (APC/C) from targeting securin and Cyclin B1 for destruction. The SAC achieves this by generating an APC/C inhibitor called the Mitotic Checkpoint Complex (MCC). The MCC is a potent APC/C inhibitor but is itself rapidly disassembled to allow the SAC to respond rapidly to the attachment state of the kinetochores. The aim of this project is to determine how the MCC is disassembled by two different pathways: one depending on the APC/C, specifically the APC15 protein. and the other on the p31Comet protein. We have recently knocked out both alleles of the p31Comet gene and this affords us the means to provide a definitive answer to this crucial problem. The project will investigate both the biochemistry of the pathways, and the dynamics or protein complex assembly and disassembly in vivo using Fluorescence Cross Correlation Spectroscopy our endogenously tagged cell lines. Our specific aims are: i) To determine how p31Comet promotes MCC disassembly. ii) To determine the mechanisms by which the Mitotic Checkpoint Complex disassembles in the absence of p31Comet. iii) To determine the physiological significance of the two pathways in cells and incorporate the results of these studies into a computational model of the SAC.

Referees:

Izawa, D. and Pines, J. (2015) ‘The Mitotic Checkpoint Complex binds a second CDC20 to inhibit active APC/C’ Nature 517, 631-634.

Collin, P., Nashchekina, O., Walker, R. and Pines, J. (2013) ‘The Spindle Assembly Checkpoint works like a rheostat not a toggle-switch’ Nat. Cell Biol. 11, 1378 -1385.

Mansfeld, J., Collin, P., Collins, M.O., Choudhary, J. and Pines, J. (2011) ‘APC15 drives the turnover of MCC-Cdc20 to make the Spindle Assembly Checkpoint responsive to kinetochore attachment’ Nat. Cell Biol. 13, 1234-1244.

Link: http://www2.gurdon.cam.ac.uk/~pineslab/New_Web_Site/Site/Welcome.html

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Austin Smith, Dept. of Biochemistry

METABOLIC REGULATION OF PLURIPOTENT STEM CELL STATES

PhD Project Description: Cells constantly make decisions about their fate. Each choice, life or death, self-renewal or differentiation, is made according to the ever changing environment they sensed. Apart from signals such as growth factors, hormones and cell-cell interactions, nutrient and their downstream metabolites have been increasingly recognized as important post-translational and epigenetic regulators of cell fate. However, the underlying mechanism is still unclear. Our lab focuses on the cues which lead to pluripotent stem cells homeostasis or lineage commitment. Previous observation suggested a correlation between cell state resetting and metabolic resetting. The project aims to investigate this correlation and understand whether metabolites, especially those related to glucose metabolism and cell redox state, can determine cell fate. With the relative ease of carrying out genetic manipulation to pluripotent stem cells, we can potentially identify key mediators of this process. These studies may revolutionize our understanding of the impact of daily nutrient uptake on stem cell fate control, and ultimately aging and disease. These findings may even shed lights on improving conditions for early human embryo development, with potential benefit for IVF.

Referees:

1. Nichols, J. and A. Smith, Naive and primed pluripotent states. Cell Stem Cell, 2009. 4(6): p. 487-92.

2. Zhou, W., et al., HIF1alpha induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC transition. EMBO J, 2012. 31(9): p. 2103-16.

3. Carey, B.W., et al., Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature, 2014

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.stemcells.cam.ac.uk/researchers/principal-investigators/pressor-austin-smith

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Christoph Steinbeck, European Bioinformatics Institute (EBI)

Second Supervisor/Collaborator: Steve Russel/Julian Griffin

Towards a better understanding of lipid metabolism through studies of Drosophila Lipidomics

PhD Project Description: Lipid metabolism is central to normal cellular physiology, including cell division, organelle biogenesis, apoptosis and necrosis. The excess consumption of lipids in the diet has led to the development of obesity epidemic which threatens health by increasing the relative risk of developing diseases such as type 2 diabetes and fatty liver disease. This has led to the term lipotoxicity and it may be a major contributing factor to the disruption of healthy ageing throughout the lifecourse. Despite this, it is still poorly understood how the composition of the lipidome is regulated at the global level and within specific lipid classes, and lipids remain poorly annotated on many network reconstructions of metabolism. It was recently demonstrated that Drosophila melanogaster is an excellent model system for the study of aspects of the fatty acid metabolism in general [1]. This project will bring together Drosophila experts at the University of Cambridge (Prof. Steve Russell, Dept of Genetics and Cambridge Systems Biology Centre), the Lipidomics lab led by Julian Griffin at the MRC Unit for Human Nutrition Research/Biochemistry, University of Cambridge and the Computational Metabolomics Group led by Christoph Steinbeck at the European Bioinformatics Institute in Hinxton to extensively study the lipidomics of Drosophila. The student will grow up a number of Drosophila mutant models related to lipid metabolism, isolate fat pads and perform analysis using high resolution mass spectrometry. The data will be modelled within the Computational Metabolomics Group to identify and document [2] differences between the mutants, and explore how the Drosophila lipidome is regulated. This PhD will improve our understanding of lipid metabolism and how lipotoxicity interacts with the healthy organism throughout the life course and comes under the Basic Biosciences Underpinning Health remit. The year1 rotations in Steve Russell's lab and Julian Griffin's lab to collect the material and perform the lipidomics work. After initial bioinformatics analysis at the Steinbeck group at the EBI, details for the coming rotations will be decided in discussions amongst all parties.

Referees:

[1] Tortoriello, G., Rhodes, B.P., Takacs, S.M., et al. (2013) Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2Monoacylglycerols and Nacyl Amides.. PLoS ONE, 8, e67865.

[2] Haug K, Salek RM, Conesa P, et al. MetaboLights--an open-access general-purpose repository for metabolomics studies and associated meta-data. Nucleic Acids Research. 2013;41(Database issue):D781–6. doi:10.1093/nar/gks1004.

Other relevant themes: Food Security

Link: http://www.ebi.ac.uk/about/people/christoph-steinbeck

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Dr Ross Waller, Dept. of Biochemistry

The function of novel RNA binding proteins in mitochondria of human pathogen Toxoplasma gondii.

PhD Project Description: Apicomplexan parasites comprise over 5000 obligate intracellular parasites of animals, and include human parasites Plasmodium, the causative agent of malaria, and Toxoplasma. During their evolution from free living cells to parasites, apicomplexans uniquely expanded a family of RNA binding proteins (known as RAPs). These proteins are predicted to target to the mitochondrion, suggesting that regulation of mitochondrial processes might have been important to adaptation to parasitism within metazoans, and that RAP proteins are central to these processes. Apicomplexan RAP proteins, however, are completely uncharacterised. This project will examine the roles of RAP proteins in apicomplexan Toxoplasma gondii. RAP protein subcellular locations will be tested by GFP tagging. These tagged versions will also be used to immunoprecipitate individual RAP proteins and test for interacting proteins partners, by mass spectrometry, or bound RNA molecules, by RIP-seq. RAP genes will be knocked out either individually, or in combinations, using CRISPR/CAS9 genome engineering, and the phenotypes of these mutants examined, including transcriptional responses by RNA-Seq. This project will provide the first insights into a new family of proteins whose expansion correlates with the evolution of a major group of pathogens.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.bioc.cam.ac.uk/people/uto/waller

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Dr Melinda J Duer, Dept. of Chemistry

Second Supervisor/Collaborator: Professor Richard W Farndale (Biochemistry)

The role of poly(ADP ribose) in calcification processes

PhD Project Description: The development and turnover of bone tissue is a complicated process – and highly prone to disruption in disease. We have recently shown that the extracellular matrix of bone acquires substantial quantities of poly(ADP ribose) as it prepares to calcify. Poly(ADP ribose) is commonly associated with DNA repair machinery in cell nuclei, so this discovery begs the question of what part poly(ADP ribose) plays in calcifying matrix. The molecular structure of poly(ADP ribose) lends itself to acting as a scaffold to support forming mineral particles, and we have increasing evidence that this is indeed the case. This project will identify the possible binding sites of poly(ADP ribose) in the extracellular matrix and will identify its role in calcification. The Collagen Toolkits developed in Prof Richard Farndale’s laboratory will be used to identify specific collagen sequences that poly(ADP ribose) can bind to, and the relative affinities for different binding sites. NMR spectroscopy methods developed in Dr Duer’s laboratory will be used to assess the relative abundance of the possible binding sites identified on the surfaces of collagen fibrils in vitro and ex vivo. We will then use a combination of X-ray diffraction and solid-state NMR spectroscopy to characterise the atomic structure and detailed chemical composition of extracellular matrix mineral particles formed in the presence of different concentrations of poly(ADP ribose) in vitro, so as to develop hypotheses as to the detailed role of poly(ADP ribose) in calcification. Understanding gained here will have significant impact on our understanding of bone development and disease.

Referees:

Citrate bridges between mineral platelets in bone E. Davies, K.H. Muller, W.C. Wong, C. J. Pickard, D.G. Reid, J.N. Skepper, M.J. Duer*, Proc. Nat. Acad. Sci. USA 111 (2014) E1354–E1363, www.pnas.org/cgi/doi/10.1073/pnas.1315080111

NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization W.Y. Chow, R. Rajan, K.H. Muller, D.G. Reid, J.N. Skepper, W. C. Wong, R.A. Brooks, M. Green, D. Bihan, R.W. Farndale, D.A. Slatter, C.M. Shanahan, M.J. Duer*, Science 344 (2014) 742-746.

Applications of NMR crystallography to problems in biomineralisation: a refinement of the crystal structure and 31P solid-state NMR spectral assignment of octacalcium phosphate. E. Davies, M.J. Duer*, S.E. Ashbrook and J.M Griffin, J. Am. Chem. Soc. 134 (2012) 12508 – 12515

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.ch.cam.ac.uk/person/mjd13 http://www.bioc.cam.ac.uk/people/uto/farndale

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Kathryn Lilley, Dept. of Biochemistry

Second Supervisor/Collaborator: Department of Genetics

The study of the dynamic rearrangement of Golgi Apparatus sub-structures during lineage commitment of mouse embryonic stem cells

PhD Project Description: With advances in genome sequencing and mass spectrometry (MS) it is now possible to generate quantitative information for a biological system at many levels; transcriptome, proteome, metabolome and regulatory interactions via nucleotide binding proteins and protein interaction networks. Despite the advances that genomic and post-genomic science has brought, much of the full complexity of biological function encoded in genomes is yet to be uncovered. A significant amount of functional complexity is brought about by dynamic behaviour of the proteome, as a translated protein may be differentially and dynamically modified, may associate with different binding partners or traffic to different parts of a cell depending on the status of the cell. In this project we will use state of the art high throughput quantitative proteomics methods coupled with fluorescence and electron microscopy, and molecular biology approaches to dissect changes in the sub-structure of the Golgi Apparatus and its fate in cell division upon lineage commitment of mouse embryonic stem cells. In this short rotation project we will look at the subcellular location of important sets of Golgi proteins during transition from an epistem cell like state to that of efficiently differentiated neuroectodermal and mesendodermal cells. The project will involve stem cell culture, plus quantitative mass spectrometry, cell imaging and bioinformatic analysis. The project will fit at the core of the group research activity of both the Lilley and Martinez Arias groups. The student will be supported and assisted by other members of these groups, including bioinformaticians, cell biologists and proteomics experts.

Referees:

Tan, D.J., Dvinge, H., Christoforou, A., Bertone, P., Martinez Arias, A., and Lilley, K.S. (2009). Mapping organelle proteins and protein complexes in Drosophila melanogaster.

J. Proteome Res. 8, 2667-2678. Faunes, F., Hayward, P., Descalzo-Munoz, S., Chatterjee, S.S., Balayo, T., Trott, J., Christoforou, A., Ferrer-Vaquer, A., Hadjantonakis, A-K., Dasgupta, R. et al. (2013). A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells. Development 140, 1171-1183.

Breckels, L.M., Gatto, L., Christoforou, A., Groen, A.J., Lilley, K.S., and Trotter, M.W. (2013). The effect of organelle discovery upon sub-cellular protein localisation. J Proteomics 88, 129-140.

Other relevant themes: World Class Underpinning Bioscience

Link: https://www.bioc.cam.ac.uk/people/uto/lilley http://www.gen.cam.ac.uk/research-groups/martinez-arias

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Steven Lee, Dept. of Chemistry

Second Supervisor/Collaborator: Prof Andres Floto

Mapping hydrophobicity in macrophage membranes with 40nm super-resolution.

PhD Project Description: Super-resolution microscopy is an advanced, interdisciplinary, optical imaging technique currently attracting immense interest as a new and exciting way to break the theoretical diffraction limit of visible light (~250nm). Using single molecule control, super-resolution microscopy retains the non-invasive advantages of fluorescence imaging but has the ability to resolve biological structures more compatible with the spatial scale that these events actually take place on, typically attaining resolutions of ~15nm or better. We introduce a new spectrally resolved super-resolution fluorescence microscopy method, which allows simultaneous spectral imaging of single fluorophores with sub-diffraction spatial resolution (>40nm) in condensed phase biological samples . Our method termed sPAINT, exploits the exquisite fluorogenic and solvatochromic properties of hydrophobicity-sensitive fluorophores. The technique works by passing the fluorescence from individual fluorophores through a transmission diffraction grating, in doing so collecting both the positional and spectral information from individual dye molecules. The spatial positions can be used to build up a super-resolved image of the membrane in live cells and the spectral information extracted allows sub-diffraction sampling of hydrophobicity changes in membranes with time . This is achieved using, Nile red, a phenoxazone dye that fluoresces intensely, and with varying emission wavelength based upon the hydrophobicity of its nano environment . We have already combined these in sPAINT successfully in a variety of applications including both: demonstrating the presence of cholesterol-dependant microdomains with different hydrophobicity within the plasma membrane of live SHSY5Y neuron-like cells and to discriminate normal from toxic forms of amyloidogenic Parkinson-associated α-synuclein protein oligomers. The PhD project will be to develop method to generate functional information and enriched nanoscale image and would suit someone with a background in biophysics/photonics.

Referees:

Horrocks, M.H., Palayret, M., Klenerman, D., Lee, S.F. (2014) Histochemistry and Cell Biology, 141 (6), pp. 577-585.

Sharonov, A., Hochstrasser, R.M. (2006) Proc. Natl. Acad. Sci. USA, 103 (50), pp. 18911-18916.

Link: http://www.ch.cam.ac.uk/person/sl591

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William Matthews, Dept. of Psychology

Biological influences on human decision-making

PhD Project Description: Would you rather have a 50% chance of winning £10 or a 5% chance of winning £100? Is it better to have a blueberry muffin right now, or 10 minutes of extra life expectancy? Would you prefer a boring PhD with an OK supervisor or a fascinating one with a potential lunatic? These questions are examples of multi-attribute choices, where people must choose between options which differ on several dimensions. Such decisions are vital to well-being and health, and are affected by macroscopic biological variables including hormone levels, diet, and aging. However, the specific effects of these factors on the information-processing that underlies decision-making is poorly understood. In particular, we are interested in how these variables affect people's sampling, evaluation, and integration of information from multiple sources when they weigh-up competing options. This project will use new techniques to gain deeper insight into these questions. The project will involve human behavioural studies in which key decision variables are systematically varied (e.g., the size, probability, and timing of rewards and losses). Gaze-tracking will monitor how people sample and integrate information prior to choosing, and we will relate these data to formal models of the decision process (Stewart et al., 2015). Within this paradigm, students can choose between studying the effects of (a) steroid sex hormones, (b) dietary tryptophan intake, and (c) cognitive aging. By examining the effects of these macroscopic variables on the microstructure of the decision-process, we will gain new insights into the biological basis of human decision-making.

Referees:

Stewart, N., Hermens, F., & Matthews, W.J. (in press). Eye movements in risky choice. Journal of Behavioral Decision Making. doi: 10.1002/bdm.1854

Coates, J.M., Gurnell, M., & Sarnyai, Z. (2010). From molecule to market: steroid hormones and financial risk-taking. Philosophical Transactions of the Royal Society B 365(1538). 332-343. doi: 10.1098/rstb.2009.0193

Rogers, R.D. et al. (2003), “Tryptophan depletion alters the decision-making of healthy volunteers through altered processing of reward cues.” Neuropsychopharmacology 28(1):153-62. doi: 10.1038/sj.npp.1300001

Link: www.scienceclarity.com

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Dr Brian Ferguson, Dept. of Pathology

Regulation of innate immune nucleic acid sensing by mitokinesis

PhD Project Description: The mitochondria as well as being powerhouses of the cell, responsible for generating energy and regulating metabolism, are also critical regulators of innate immunity and cell death. Many proteins that are essential components of the signalling pathways that drive these stress responses reside in the mitochondrial membrane. Quite beyond their image as static organelles, mitochondria are highly dynamic, sometimes forming large networked structures inside the cell and sometimes residing as small individual units. Fission and fusion processes are therefore required to regulate their subcellular organisation. Mitokinesis is a name given to a proposed mechanism by which the numbers and distribution of mitochondria exist in the cell. Our hypothesis based on descriptions of this process and our own preliminary data is that mitokinesis can regulate innate immune and cell death signalling. This project will use modern cellular and molecular biology techniques to test our hypothesis.

Referees:

J Cell Sci. 2014 Nov 1;127(21):4549-60. 'Novel roles for actin in mitochondrial fission'

Hatch AL, Gurel PS, Higgs HN. 'Mitochondria in innate immune responses' Nature Reviews Immunology 11, 389-402 (June 2011)

A. Phillip West, Gerald S. Shadel & Sankar Ghosh

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.fergusonlab.path.cam.ac.uk/

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Elizabeth Murchison, Dept. of Veterinary Medicine

Evolution of a transmissible cancer in Tasmanian devils

PhD Project Description: The Tasmanian devil facial tumour disease (DFTD) is a transmissible cancer that is spread between Tasmanian devils by the transfer of living cancer cells by biting. Thus DFTD is a single clonal cell lineage that first arose as a cancer in one individual, and subsequently survived beyond the death this original host by “metastasising” through the devil population. DFTD causes the appearance of aggressive facial tumours in affected Tasmanian devils, and the disease is incurable and invariably fatal. Since its emergence in the 1990s, DFTD has caused massive declines in the Tasmanian devil population and there is concern that it could cause extinction of wild devils. The evolutionary trajectory of DFTD can be reconstructed using the somatic genetic changes that have accumulated in its genome. Our laboratory is sequencing DNA from hundreds of DFTD samples collected throughout the epidemic from diverse geographical locations. This project will involve developing methods for analysing genetic variation in DFTD tumours, and building a phylogenetic tree of the DFTD lineage. This tree will be used to analyse the phylogeography and epidemiology of the disease, as well as the evolutionary and mutational processes that have driven and accompanied its spread. This project promises not only to illuminate mechanisms of DFTD spread, but also provides a model with which to study the evolution of cancer more generally. This project would be most suitable for someone with an interest and some background in bioinformatics.

Referees:

Murchison EP , Wedge DC , Alexandrov LB, Fu, B, Martincorena I, Ning Z, Tubio JMC, Werner EI, Allen J, Barboza de Nardi A, Donelan EM, Marino G, Fassati A, Campbell PJ, Yang F, Burt A, Weiss RA, Stratton MR 2014 Transmissible dog cancer genome reveals the origin and history of an ancient cell lineage. Science. Jan 24;3436169:437-40

Murchison EP, Schulz-Trieglaff OB, Ning Z, Alexandrov LB, Bauer MJ, Fu B, Hims M, Ding Z, Ivakhno S, Stewart C, Ng BL, Wong W, Aken B, White S, Alsop A, Becq J, Bignell GR, Cheetham RK, Cheng W, Connor TR, Cox AJ, Feng ZP, Gu Y, Grocock RJ, Harris SR, Khrebtukova I, Kingsbury Z, Kowarsky M, Kreiss A, Luo S, Marshall J, McBride DJ, Murray L, Pearse AM, Raine K, Rasolonjatova I, Shaw R, Tedder P, Tregidgo C, Vilella AJ, Wedge DC, Woods GM, Gormley N, Humphray S, Schroth G, Smith G, Hall K, Searle SM, Carter NP, Papenfuss AJ, Futreal PA, Campbell PJ, Yang F, Bentley DR, Evers DJ, Stratton MR 2012 Genome sequencing and analysis of the Tasmanian devil and its transmissible cancer. Cell. Feb 17;1484:780-91

Murchison EP 2009 Clonally transmissible cancers in dogs and Tasmanian devils. Oncogene 27: S19 – S30

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.vet.cam.ac.uk/directory/murchison

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Professor Michael Wakelam, Babraham Institute

Regulation of autophagy by fatty acids and phospholipases

PhD Project Description: It is now clear that there are a number of mechanistically and functionally distinct forms of autophagy including lipophagy which degrades lipids stored in lipid droplets generating fatty acids which are subsequently metabolised. In contrast to the role of autophagy in generating fatty acids there is now evidence from our and other labs that exogenous free fatty acids can additionally stimulate autophagy. Intriguingly a recent work has suggested that saturated and unsaturated fatty acids regulate autophagy differently by activating distinct pathways, only one of which is Golgi-dependent. Autophagy is a complex multi-factorial process. The signaling pathways that promote stress-induced autophagy involve activation of type III PI3kinase, mTOR and ULK1, there is also evidence that phospholipase D (PLD), both PLD1 and PLD2, is involved in an ill-defined manner. In addition our recent work has pointed to a role for the Golgi located PLD3, a member of the PLD family with no published function or substrate, in regulating autophagy. One autophagy-stimulating lipid is oleate (C18:1) and our previous work has characterized the signaling activated when promoting lipid droplet formation as being mediated by the FFAR4 receptor with activation of Class I PI3kinase, PLD1 and PLD2. This project will aim to characterize the signaling regulating the different forms of autophagy and put the role(s) of fatty acids and their signaling in this regulation into a physiological context, both in terms of lipid storage and metabolism.

Referees:

Schug, Z.T., Peck, B., Zhang, Q., Jones, D.T., Grosskurth, S., Alam, I.S., Smethurst, E., Mason, S., Blyth, K., McGarry, L., James, D., Shanks, E., Kalna, G., Saunders, B., Jiang, M., Howell, M., Lassailly, F., Thin, M. Z., Spencer-Dene, B., Stamp, G., Tardito, S., Harris, A.L., Aboagye, E.O., Critchlow, S.E., Wakelam, M.J.O., Schulze, A. & Gottlieb, E. (2015) “Acetyl-coA synthetase 2 promotes acetate utilization and maintains cancer cell growth under nutrient stress.” Cancer Cell in press

Rohwedder, A., Zhang, Q., Rudge, S.A. & Wakelam, M.J.O. (2014) “Lipid droplet formation in response to oleic acid in Huh-7 cells is a fatty acid receptor mediated event.” J Cell Sci 127, 3104-3115

Bensaad, K., Favaro, E., Lewis, C.A., Peck, B., Lord, S., Collins, J.M., Pinnick, K.E., Wigfield, S., Buffa, F.M., Li, J.-L., Zhang, Q., Wakelam, M.J.O., Karpe, F., Schulze, A. & Harris, A.L. (2014) “Fatty acid uptake and lipid storage induced by HIF-1a contribute to cell growth and survival after hypoxia-regeneration.” Cell Reports 9, 349-365

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.babraham.ac.uk/our-research/signalling/michael-wakelam

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Timothy Weil, Dept. of Zoology

Rapid coordinated cell response through calcium-binding mRNA kink-turn motifs

PhD Project Description: Translational regulation, comprising initiation and transcript degradation, is an important post-transcriptional mechanism for targeting proteins in time and space. Key examples include regulation of pattern formation in oocytes, cell migration and translation at the synapse. In many cases the mechanisms by which directed cues are transduced to regulate translation are not well understood. In cells and tissues an increase in the cytoplasmic [Ca2+] has been shown to coordinate many cellular processes. Ca2+ signaling operates at key transitions where translational regulation has a major role. Currently, however, there is no clear mechanism that directly links Ca2+ signaling to mRNA translation. Kink-turn (k-turn) motifs in RNA dramatically bend the RNA backbone when bound by Ca2+. They are a currently unexplored mechanism that would link translation to physiological and developmental cues. Through deciphering the in vivo function of k-turn motifs, and their role in the regulation of mRNA translation, this project will provide important insight into the coordination of cellular change in many biological processes. This project will exploit the Drosophila egg and early embryo to determine how Ca2+ binding to k-turn motifs affects RNA function, and specifically whether it regulates mRNA translation. Through bioinformatic identification, CRISPR/Cas mutagenesis and CLIP isolation of mRNA associated factors, it is likely a novel mechanism by which mRNA translational control is coordinated will be found. Time permitting, k-turn motifs are a general mechanism for translational regulation in other other tissues and model organisms will be tested.

Referees:

Klein, D. J., Schmeing, T. M., Moore, P. B. & Steitz, T. A. The kink-turn: a new RNA secondary structure motif. EMBO J. 20, 4214–4221 (2001).

Weil, T. T. et al. Drosophila patterning is established by differential association of mRNAs with P bodies. Nat. Cell Biol. 14, 1305–1313 (2012).

York-Andersen, A. H., Parton, R. M., Bi, C. J., Bromley, C. L., Davis, I., Weil, T. T. A single and rapid calcium wave at egg activation in Drosophila. Biology Open. In Press (2015).

Other relevant themes: World Class Underpinning Bioscience

Link: https://sites.google.com/site/tweillab/

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Professor J D Mollon, Dept. of Psychology

A polymorphism of human perception

PhD Project Description: Normal people differ dramatically in the relative numbers of different types of cone cell in their retinas. The ratio of red-sensitive (long-wave, L) and green-sensitive (middle-wave, M) cones has recently been estimated to range from 1:1 to 15:1 within the normal population. These variations reveal themselves perceptually in spatial and temporal sensitivity to different wavelengths. We have developed two perceptual tests that appear to measure the L:M ratio; and in a whole-genome association study have identified a single-nucleotide polymorphism (SNP) that is correlated with these variations. What makes this SNP of great interest is that it lies in the promoter region of RXRG, a gene that encodes the retinoid X gamma receptor. The latter is a nuclear transcription factor that is already known, in mice, to control the differentiation of short-wavelength cones from long-wavelength cones (mice have only two types of cone). The planned project will aim first, to confirm the association between cone ratio and the polymorphism of the promoter region of RXRG; and second, to understand the mechanism by which the individual differences emerge. Two psychophysical measures, and one objective measure (retinal densitometry) will be used, and will be related to genetic sequences of RXRG. The ease with which the cone ratios of the retina can be characterised make this an attractive model system in which to study variation in gene expression in Man. Moreover, knowledge of the molecular control of the differentiation of primordial retinal cells will be of value in future gene therapy for retinal degenerations.

Referees:

Deeb, S. (2006) Genetics of variation in human color vision and the retinal cone mosaic. Curr. Opin. Genet. Devel. 16, 301

Lawrance-Owen, A. J., Bosten, J. M., Hogg, R. E., Bargary, G., Goodbourn, P. T., and Mollon, J. D. (2014) Counterphase modulation flicker photometry: Phenotypic and genotypic associations Journal of the Optical Society of America A 31, A226-231

Hofer, H. J. and Williams, D. R. (2014) Color vision and the retinal mosaic. In Werner, J. S. and Chalupa, L. M. 'The New Visual Neurosciences', MIT Press.

Other relevant themes: World Class Underpinning Bioscience

Link: http://vision.psychol.cam.ac.uk/jdmollon/publications.html

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Pietro Cicuta, Dept. of Physics

Second Supervisor/Collaborator: Julian Rayner, Wellcome Trust Sanger Institute

A quantitative and complete understanding of malaria cell-host interactions

PhD Project Description: In the blood stage of malaria, Plasmodium merozoites must invade human erythrocytes in order to replicate. This is a fascinating cell biological process during which one eukaryotic cell forces its way inside another. To date our understanding of this process has been limited by technology, because the rapid nature of the process (under two minutes) has prevented us from imaging large numbers of individual invasion events. To overcome this technological gap, Dr. Pietro Cicuta’s group in Physics have been developing robotic video microscopy approaches to allow the high throughput capture of individual invasion events (Crick et al., Biophys J 2013, PMID: 23473482, Biophys J 2014, PMID: 25640445, Methods Cell Biol. 2015 PMID: 25640445), working with T.Tiffert and V.Lew in PDN and Dr. Julian Rayner at the Sanger Institute. The Rayner group uses a variety of approaches including experimental genetics and high throughput protein-protein interaction screens to understand the receptor-ligand interactions that allow invasion to occur (Crosnier et al., Nature 2011, PMID: 22080952). The precise nature of this project can be adapted based on the skills and interests of the student. Combining cutting edge genetic approaches with further developments in video microscopy technology, such as using laser tweezers to hold either merozoites or erythrocytes and quantitate attachment forces, or using microfluidics to observe invasion under flow conditions that mimic those of the microvasculature where invasion actually occurs in vivo, the student will be uniquely empowered to understand the function of specific receptor-ligand interactions, and how they come into play during the sequence of processes from initial contact to complete invasion.

Referees:

Crick et al., Biophys J 2013, PMID: 23473482

Crick et al., Biophys J 2014, PMID: 25640445

Crosnier et al., Nature 2011, PMID: 22080952

Other relevant themes: World Class Underpinning Bioscience

Link: http://people.bss.phy.cam.ac.uk/~pc245/

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Christine Farr, Dept. of Genetics

Topoisomerase II (topo2), DNA replication termination and vertebrate centromeres.

PhD Project Description: Topoisomerase 2 is an essential and evolutionarily conserved enzyme that plays a key part in maintaining genome stability. Several lines of evidence suggest that topo2 is involved in the final stages of DNA replication - the fusion of converging replication forks. Moreover, in lower eukaryotes there is evidence that replication forks terminate within centromere domains. Such centromeric TERs would result in DNA entanglements between sister centromeres that need to be resolved to allow chromosome segregation in anaphase. Thus, such linkages, while contributing to the centromere’s structure and function, may also render it especially prone to instability. The multiplicity of processes to which topo2 contributes complicates analysis of cell cycle stage-specific phenotypes. Moreover, the DNA repeat-rich nature of many vertebrate centromeres makes the study of replication fork progression through such domains challenging. To approach this we will focus on chicken DT40 cells because (i) their centromere/kinetochore structure is highly compact; (ii) some centromeres in the chicken genome are made of unique DNA sequences (allowing the mapping of molecular features onto specific DNA sequences) and (iii) a wealth of knockout mutations is available in the hyper-recombinogenic DT40 cell line. Moreover, we have a destabilising domain (degron)-fused version of topo2, that allows topo2 to be rapidly and reversibly depleted from cells upon addition of auxin. This research will lead to a clearer understanding of the role of topo2 in centromere stability and the mechanisms that maintain genome integrity and suppress tumourigenesis.

Referees:

Spence JM, Phua HH, Mills W, Carpenter AJ, Porter ACG, Farr CJ (2007) J. Cell Sci. 120: 3952-3964.

Shang WH, Hori T, Toyoda A et al. (2009) Genome Res. 20: 1219-1228.

Farr, CJ, Antoniou-Kourounioti, M, Mimmack, ML, Volkov, A, and Porter, ACG (2014) The alpha isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells Nucleic. Acids Res. 42(7): 4414-4426

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.gen.cam.ac.uk/research-groups/farr

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J. Michael Edwardson, Dept. of Pharmacology


PhD Project Description: My lab studies the structure and function of proteins at the single-molecule level using atomic force microscopy (AFM). Current projects include investigations of: 1. Activation-induced structural changes in ionotropic glutamate receptors, using fast-scan AFM imaging of the receptors after reconstitution into a lipid bilayer. 2. The interaction of the sigma-1 receptor with ion channels, such as the hERG voltage-gated K+ channel, and the mechanisms by which drugs perturb the interactions between the sigma-1 receptor and its ion channel targets. 3. The mechanism underlying the interaction of urinary exosomes with the primary cilium. Exosomes interact specifically with primary cilia of the tubular epithelial cells. Exosomes from autosomal recessive polycystic kidney disease (ARPKD) mice decorate cilia much more extensively than exosomes from wild type mice. We are interested in how this interaction is perturbed in ARPKD. 4. The structure and behaviour of synaptotagmin, the major Ca2+ sensor for membrane fusion during neurotransmitter release. We have used AFM to investigate the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. We are currently imaging various mutants that affect the behaviour of synaptotagmin, to shed light on the relationship between structure and function. 5. Structure and behaviour of seipin. Disruption of the gene BSCL2 in humans, encoding the protein seipin, causes congenital generalized lipodystrophy. We are currently using AFM to investigate the assembly of seipin and its interactions between seipin and the proteins AGPAT2 and lipin 1, which operate sequentially in the intracellular lipogenesis pathway. I would consider offering a PhD project in any of these areas, or indeed in an entirely new area.

Referees:

Balasuriya, D., D’Sa, L., Talker, R., Dupuis, E., Maurin, F., Martin, P., Borgese, F., Soriani, O., and Edwardson, J.M. (2014) A direct interaction between the sigma-1 receptor and the hERG voltage-gated K+ channel revealed by atomic force microscopy and homogeneous time-resolved fluorescence (HTRF®). J. Biol. Chem. 289, 32353-32363

Liu, H., Bai, H., Xue, R., Takahashi, H., Edwardson, J.M. and Chapman, E.R. (2014) Linker mutations dissociate the function of synaptotagmin I during evoked and spontaneous release and reveal membrane penetration as a step during excitation-secretion coupling. Nat. Neurosci. 17, 670-677

Suzuki, Y., Goetze, T.A., Stroebel, D., Balasuriya, D., Yoshimura, S.H., Henderson, R.M., Paoletti, P., Takeyasu, K. and Edwardson, J.M. (2013) Visualization of structural changes accompanying activation of NMDA receptors using fast-scan AFM imaging. J. Biol. Chem. 288, 778-784

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.phar.cam.ac.uk/research/Edwardson

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Gavin Kelsey, Babraham Institute

Epigenetic coincidence detection for imprint maintenance

PhD Project Description: Epigenetic mechanisms are required to partition our genome into different states of activity and to provide a long-term memory of earlier decisions, such as reinforcing gene expression networks associated with cell lineage determination events. Nowhere are epigenetic states more dynamic, and more critical, than during germ-cell and early embryonic development. However, against this highly dynamic background, some sequences have to retain their epigenetic marks with absolute fidelity, as in the case of imprinted genes. This project will examine the factors that specify the faithful maintenance of imprint marks particularly in early embryos. In particular, it will test how a combination of DNA methylation and histone marks is used as a coincidence detection to confer epigenetic stability. It will examine the ontogeny of these marks, using highly sensitive chromatin and single-cell DNA methylation profiling methods, and their interdependence, using knock-outs for the candidate modifying factors. The results will provide new insights into the possible origin of epigenetic errors in imprinted gene syndromes or associated with adverse environmental factors or assisted reproduction technologies.

Referees:

Smallwood et al. (2011) Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat. Genetics 43, 811–814.

Smallwood and Kelsey (2012) De novo DNA methylation: a germ cell perspective. Trends Genet. 28, 33-42.

Smallwood et al. (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat. Methods 11, 817-820.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.babraham.ac.uk/our-research/epigenetics/gavin-kelsey

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Robert Glen, Dept. of Chemistry

Second Supervisor/Collaborator: Anthony Davenport (Clinical Pharmacology, Addenbrookes Hospital)

Understanding Baised Agonism at GPCRs - designing drugs without de-sensitisation

PhD Project Description: We have discovered remarkable properties of novel peptides [1] at the recently de-orphanised GPCR, Apelin. In an exciting development they were clinically tested in man, and show Apelin control of vasodilation [2]. An ongoing study shows that in cancer, apelin antagonists arrest or eliminate glioblastoma tumours (Nature, submitted) and they also reduce glucose absorption in the gut, by control of transporter expression [3]. Apelin is also vital in early heart development, with a newly discovered peptide Elabella/Toddler activating Apelin. Our peptide receptor probes are giving invaluable insights into the biological effects of Apelin in the development of disease. However peptides have limitations and we now want to discover non-peptides receptor probes. Using molecular dynamics, modelling and medicinal chemistry we will discover new active small molecules. In collaboration with synthetic chemists, we will synthesize and evaluate their pharmacology with clinical partners. The outcome will be novel metabolically stable compounds to investigate the pharmacology of Apelin. The student will receive training in molecular dynamics, computer-aided molecular design, pharmacology and medicinal chemistry. A developing background and interest in these areas as well as software/simulation is desirable.

Referees: 1. Characterization and first-in-human study of the vascular actions of a novel 'biased' apelin receptor agonist. Davenport et al. Hypertension (accepted). 2. Biased ligands at G-protein-coupled receptors: promise and progress. Violin JD et al. Trends in Pharmacological Sciences, July 2014, Vol. 35, No. 7, 308-316. 3. Yang P. Et al. Pharmacology of the Apelin Receptor and Relevance to Pulmonary Arterial Hypertension. http://www.pa2online.org/abstract/abstract.jsp?abid=30008.

Link: http://www.ch.cam.ac.uk/person/rcg28

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Simon Frost, Dept. of Veterinary Medicine

Modeling the evolution of antibody responses

PhD Project Description: Neutralising antibodies form a central part of the adaptive immune system. Through a complex process of immunoglobulin gene rearrangement and somatic hypermutation, massive amounts of genetic diversity can be generated in order to recognise different pathogen structures. Advances in sequencing allow us to obtain a detailed picture of antibody diversity at the sequence level, yet the evolutionary dynamics of antibody responses have yet to be fully characterised. This project will involve the development and application of phylogenetic approaches to characterise how immunoglobulin repertoires evolve, both in the presence and the absence of infection. This will involve mining of large sequence datasets, as well as the development of realistic models of repertoire dynamics. In addition, a comparative approach will be taken to compare the evolution of immunoglobulin genes in different jawed vertebrate species. Skills to be gained: * Understanding of the use of phylogenetics in evolution of immunoglobulins, both germline and rearranged genes * Informatics skills, dealing with large sequence and annotation datasets * Mathematical model of within-host dynamics of antibody responses * Computer programming in languages such as R and Python

Referees:

Frost, S.D.W. et al. (2005) Proc Natl Acad Sci 102:18514-9

Benichou, J. et al. (2012) Immunology. 35:183-9

Georgiou et al. (2014) Nature Biotechnology 32:158-168

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.vet.cam.ac.uk/directory/sdf22@cam.ac.uk http://antibodyo.me

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Simon Frost, Dept. of Veterinary Medicine

Phylodynamics of within-host viral evolution

PhD Project Description: Rapidly evolving RNA viruses such as HIV and hepatitis C virus leave imprints of past transmission and movement in their phylogenetic history. Even within an infected individual, there can be significant viral diversity, reflecting factors such as selection pressure from the immune system and adaptation to target cells. While viral sequence data are rich in information, it is often highly challenging to extract this information from sequence data, necessitating the use of a so-called 'phylodynamic' approach that combines immunological processes with evolutionary models of viral mutation, selection, and recombination. This project will focus on developing within-host models of viral evolution, with a view to improving our understanding of how viruses transmit from one individual to another, and how immune escape occurs following infection. This project will also involve the development and assessment of approaches that incorporate recombination, potentially leading to further insights into the dynamics of multiple infections with the same pathogen, and the selective advantages and disadvantages of recombinant variants. This project will draw on sequence data, both publicly available, and from collaborative projects on viruses that cause chronic infection, such as HIV, and acute infection, such as influenza A. Skills to be gained: * Understanding of the use of phylogenetics in viral evolution * Informatics skills, dealing with large sequence and annotation datasets * Mathematical model of within-host dynamics * Computer programming in languages such as R and Python

Referees:

Frost, S.D.W. et al. (2005) Proc Natl Acad Sci 102:18514-9

Frost, S.D.W. and Volz, E.M. (2010) Philos Trans R Soc Lond B Biol Sci. 2010 365:1879-90

Volz, E. et al. PLoS Comput Biol 2013;9(3):e1002947

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.vet.cam.ac.uk/directory/sdf22@cam.ac.uk

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David Glover, Dept. of Genetics

Second Supervisor/Collaborator: Collaborator - Fiona Watt, King's, London

Effects of supernumerary centrioles upon keratinocytes, hair follicles, and melanocytes

PhD Project Description: Polo-like-kinase 4 (PLK4) is the master regulator of centriole duplication. Centrioles have dual roles – i) core components of the centrosomes essential for the fidelity of cell division ii) as basal bodies required to generate primary cilia essential for many signalling pathways. We have built a mouse line that expresses elevated Plk4 following doxycycline induction. These develop malignant tumours and show cyclical baldness and whitening of the hair. The objective of this project is to determine how these skin phenotypes relate to centriole functions that can be perturbed by Plk4 overexpression. To this end we will apply our knowledge of the molecular cell biology of centriole duplication to study the effects of Plk4 upon the embryonic development of skin and upon the biology of keratinocytes cultured in vitro. This will entail studying the regulation of the symmetry of the cell divisions that generate the stratified epidermis, and signalling events critical for the development of hair follicles and melanocytes. We will complement studies of the effects of Plk4 overexpression by experiments in which we eliminate other components of the centriole duplication pathway. All of the phenotypes we observe are strongly modified by loss of the p53 tumour suppressor. We will therefore study the relationships between cellular events regulated by centrioles in different cell types and p53 status.

Referees:

1. Dzhindzhev NS, Tzolovsky G, Lipinszki Z, Schneider S, Lattao R, Fu J, Debski J, Dadlez M, Glover DM. (2014) Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation. Curr Biol. 24:2526-32. doi: 10.1016/j.cub.2014.08.061. Epub 2014 Sep 25.PMID:25264260

2. Coelho, P.A., Bury, L., Fu, J., Sharif, B., Riparbelli, M.G., Callaini, G., Glover, D.M. and Zernicka-Goetz, M. (2013) Acentriolar spindle formation in the early mouse embryo requires the “centriolar” protein kinase, Plk4 Dev. Cell 27:586-97

3. Dzhindzhev, N., Yu, Q.D., Weiskopf, K., Cunha-Ferreira, I., Riparbelli, M., Rodrigues-Martins, A., Bettencourt-Dias, M., Callaini, G., and Glover, D.M. (2010) Asterless provides a molecular platform for centriole assembly – Nature 467, 714-718

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.gen.cam.ac.uk/research-groups/glover

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Robert Glen, Dept. of Chemistry

Second Supervisor/Collaborator: Anthony Davenport

Discovery of Apelin receptor small molecule agonists and antagonists in cardiovascular disease and cancer

PhD Project Description: We have discovered remarkable properties of novel peptide agonists and antagonists [1] of the recently deorphanised GPCR target Apelin. In an exciting development, our peptides were recently clinically tested in man, and show promise in pulmonary hypertension and heart failure[2]. A recent study shows that in cancer, our peptide apelin antagonists arrest or eliminate glioblastoma tumours (submitted to Nature). These antagonists also reduce absorption of glucose in the gut, leading to a possible treatment in diabetes[3]. Using molecular dynamics, molecular modelling and medicinal chemistry we will discover new chemical entities that are Apelin agonists and antagonists. In collaboration with synthetic chemists, these will be synthesized and evaluated by our pharmacology and clinical partners. The outcome should be novel series of lead compounds (peptido-mimetics) for advancement to pre-clinical studies. The student will receive training in molecular dynamics, computer-aided molecular design, pharmacology and medicinal chemistry. A developing background and interest in these areas as well as software/simulation is desirable.

Referees:

1. Discovery of a Competitive Apelin Receptor (APJ) Antagonist. Macaluso et al. ChemMedChem (2011) 6, 1017-1023.

2. Characterization and first-in-human study of the vascular actions of a novel 'biased' apelin receptor agonist. Davenport et al. Hypertension (accepted).

3. The Intestinal Glucose–Apelin Cycle Controls Carbohydrate Absorption in Mice. Dray et al. Gastroenterology, Volume 144, Issue 4, Pages 771–780.

Link: http://www.ch.cam.ac.uk/person/rcg28

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Eugenia Piddini, Dept. of Zoology

Investigating the molecular mechanisms of cell competition in Drosophila

PhD Project Description: Cell competition is a form of cell-cell interaction that selects the best cells within tissues, acting as a cellular quality control and likely contributing to preserve tissue health. It occurs when cells of differing fitness levels come into contact, causing weaker ‘loser’ cells - which would be perfectly viable on their own – to be eliminated by fitter ‘winner’ cells through the induction of cell death. The mechanisms of cell competition are not well understood and it remains unknown how cell fitness is sensed and how it induces the elimination of loser cells. Our group has identified 217 genes that are specifically expressed in ‘loser’ cells compared to wild-type and has already identified from this list a number of signalling pathways that are specifically activated in losers. The list contains a number of promising genes which could be involved in cell competition. Our next step is to carry out a secondary screen of these genes and investigate their role in cell competition by RNAi mediated silencing of these genes. The student joining on this project would carry out the screen together with a PhD student currently working on this project and would then focus on characterising 2-3 interesting genes emerging from the screen.

Referees:

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

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

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.gurdon.cam.ac.uk/research/piddini

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Eugenia PIddini, Dept. of Zoology

The mechanisms of mechanical cell competition in epithelial cells

PhD Project Description: It is well established that in tissues quality control mechanisms exist to eliminate cells that are not fit. For example fitter cells are known to eliminate less fit cells via a process known as cell competition, and it is likely that this constant selection helps to maintain tissues healthy. Therefore it is important to understand how this process is controlled, so that it can be used to improve organisms’ health. The mechanisms of cell competition are not well understood, however our lab has recently discovered a new mechanism of cell competition in which cells can compete using mechanical compression forces, to induce stress in their neighbours and kill them. We have identified a number of molecules involved in this process such as cell adhesion molecules, stress signalling kinases and the activation of p53, which ultimately leads to cell death. 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? And what mutations in cells make them susceptible to mechanical cell competition? This PhD project will aim to address those questions, using live cell imaging, genetics and cell biological approaches, and mammalian epithelial cells in culture as a model system.

Referees:

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

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

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.gurdon.cam.ac.uk/research/piddini

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Zoe Kourtzi, Dept. of Psychology

Learning and brain plasticity across the lifespan

PhD Project Description: 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. Investigating the mechanisms of this meta-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 meta-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 meta-learning in the context of temporal regularities (i.e. learning sequences of probabilistic events) and categorical structures based on inferences about conceptual similarities. We ask whether young and older learners use the same or different strategies to achieve meta-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 and why some learners are better and faster than others.

Referees:

1. Chang DH, Mevorach C, Kourtzi Z, Welchman AE. (2014) Training transfers the limits on perception from parietal to ventral cortex. Current Biology, 24, 2445-50.

2. Mayhew SD, Li S, Storrar JK, Tsvetanov KA, Kourtzi Z (2010) Learning Shapes the Representation of Visual Categories in the Aging Human Brain. J Cognitive Neuroscience, 22, 2899-912.

3. Kourtzi Z, DiCarlo JJ (2006) Learning and neural plasticity in visual object recognition. Current Opinion in Neurobiology, 16, 152-8.

Link: http://www.abg.psychol.cam.ac.uk

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Dr Torsten Krude, Dept. of Zoology

Second Supervisor/Collaborator: Professor Chris Howe, Department of Biochemistry

Non-coding RNAs as regulators of eukaryotic DNA replication

PhD Project Description: This PhD project brings together molecular cell biology and RNA biochemistry. It will characterise the essential function of novel small non-coding RNAs for the initiation of DNA replication in eukaryotic cells. In vertebrates, non-coding Y RNAs are essential for the initiation of chromosomal DNA replication. We have recently established an experimental approach to isolate RNAs from non-vertebrate organisms that can functionally substitute for vertebrate Y RNAs. This approach is based on a human cell-free DNA replication initiation system, in which endogenous human Y RNAs are replaced by exogenous RNAs to determine their ability to initiate chromosomal DNA replication. Preliminary data support the existence of functionally active RNAs in nematodes, in Drosophila, and in two plant model organisms (Arabidopsis and Chlamydomonas), suggesting that the control of DNA replication by small non-coding RNAs may be a fundamental principle in eukaryotes. The objective of this PhD project is the identification and functional characterisation of novel non-coding RNAs. Using recently generated high-throughput sequencing data from size-purified active RNA fractions, candidate RNAs will be identified, synthesised by in vitro transcription and tested for DNA replication initiation function in the human cell-free system. The RNA motifs and structural elements required for function will be identified by systematic mutagenesis. The physiological function of these active RNAs will be characterised in the animal and/or plant model organisms in vivo, utilising powerful genetic tools and antisense oligonucleotide technologies.

Referees:

Christov, C.P., T.J. Gardiner, D. Szüts, and T. Krude. 2006. Functional requirement of noncoding Y RNAs for human chromosomal DNA replication. Mol Cell Biol. 26, 6993-7004.

Collart, C., C.P. Christov, J.C. Smith, and T. Krude. 2011. The midblastula transition defines the onset of Y RNA-dependent DNA replication in Xenopus laevis. Mol Cell Biol. 31, 3857-3870.

Wang, I., M.P. Kowalski, A.R. Langley, R. Rodriguez, S. Balasubramanian, S.T. Hsu, and T. Krude. 2014. Nucleotide Contributions to the Structural Integrity and DNA Replication Initiation Activity of Noncoding Y RNA. Biochemistry 53, 5848-5863.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.zoo.cam.ac.uk/directory/dr-torsten-krude

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Ruth Murrell-Lagnado, Dept. of Pharmacology

The role of the P2X4 receptor ion channel in the regulation of lysosome function

PhD Project Description: Lysosomes are not simply compartments of degradation but have important functions in cell signalling and energy metabolism and a central role in autophagy. Many diseases, including neurodegenerative disease and cancer, are associated with a disruption in the normal function of lysosomes. Previously we showed that one member of the family of purinergic receptor ion channels, P2X4, is targeted to lysosomes and is activated by a combination of elevated luminal ATP and a reduction in the acidity of the lysosome. This activation promotes calcium flux through the pore and generates a local calcium signal that promotes lysosome fusion with other compartments. We are particularly interested in the regulation of lysosome fusion with the autophagosome. We have recently obtained evidence that activation of lysosomal P2X4 receptors regulates autophagic flux. This activation can be triggered by plasma membrane receptors that generate a cytosolic calcium signal. A particularly interesting example is P2X7, another purinergic receptor, which is highly calcium permeable and expressed at the cell surface. P2X4 and P2X7 are often co-expressed particularly in the immune system and we have shown that they act synergistically to regulate autophagy. This project will aim to elucidate the molecular and cellular mechanisms that underlie this regulation. Our findings will have important implications for the role of these receptors in cancer. There will be the opportunity to utilize a combination of techniques in this project, including molecular biology, protein biochemistry, fluorescence imaging and electrophysiology.

Referees:

Huang P, Zou Y, Zhong XZ, Cao Q, Zhao K, Zhu MX, Murrell-Lagnado R.D, Dong XP (2014) P2X4 Forms Functional ATP-activated Cation Channels on Lysosomal Membranes Regulated by Luminal pH. Journal of Biological Chemistry. 289 (25), 17658-17667

Qureshi, O.S, Paramasivam, A., Yu, J.C.H and Murrell-Lagnado R.D. (2007) Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis Journal of Cell Science. 120, 3838-3849.

Setembre et al., (2013) Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nature Reviews (Molecular Cell Biology). 14, 283-295.

Link: http://www.phar.cam.ac.uk/research/Murrell-Lagnado

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Clemens Kaminski, Dept. of Chemical Engineering and Biotechnology

Second Supervisor/Collaborator: Dr. Gabriele Kaminski Schierle

Development of microfluidic devices and C. elegans protein aggregation models for high throughput screening using multiparametric imaging

PhD Project Description: Aggregation of misfolded proteins is a characteristic hallmark of many neurodegenerative disorders, e.g. Alzheimer’s disease (AD). The ability to observe these aggregation processes and the corresponding structures formed in situ is therefore a key requirement to understand the molecular mechanisms of the disease. We have recently developed a FRET biosensor which permits us to monitor the aggregation kinetics of amyloid proteins and directly correlate it to the disease phenotype in vivo in worm models (1). The student will first develop a fluorescently-tagged worm (C. elegans) model of Amyloid beta (Aβ) aggregation and validate the model using biophysical techniques (2). Then, a microfluidic platform will be developed to permit microscopy imaging of phenotype and aggregation state. The phenotype is typically measured by observing motor behaviour, which can be obtained by image analysis of movies obtained by microscopy. Last, the student will implement the microfluidic platform optimised for automated FLIM imaging that permits a direct readout of the protein aggregation state. With those protocols in place, the student will be able test several small drug molecules, currently developed against Aβ (Chemistry dept). The student will develop skills in manipulating the genotype of C. elegans and in synthesis of recombinant proteins, including in vitro characterisation (AFM, TEM, nanoscopy). She/He will also learn how to design and construct microfluidic devices for high-throughput screening (CAD designs, programming, lithography). The student will further profit from the interdisciplinary environment of the group with regards to state-of-the-art fluorescence microscopy techniques and their background in protein misfolding diseases.

Referees:

(1) Kaminski Schierle GS, Bertoncini CW, Chan FTS, van der Goot AT, Schwedler S, Skepper J, Schlachter S, van Ham T, Esposito A, Kumita JR, Nollen EAA, Dobson CM, Kaminski CF, "A FRET sensor for non-invasive imaging of amyloid formation in vivo", ChemPhysChem, 12(3), 673–680, (2011).

(2) van Ham TJ, Esposito A, Kumita JR, Hsu ST, Kaminski Schierle GS, Kaminski CF, Dobson CM, Nollen EA, Bertoncini CW, "Towards multiparametric fluorescent imaging of amyloid formation: Studies of a YFP model of alpha-synuclein aggregation", J. Mol. Biol. (2010) 395, 627–642.

(3) Pinotsi D, Büll AK, Galvagnion C, Dobson CM, Kaminski-Schierle GS, Kaminski CF, "Direct Observation of Heterogeneous Amyloid Fibril Growth Kinetics via Two-Color Super-Resolution Microscopy," Nano Letters (2013), 14 (1), 339–345.

Other relevant themes:

Link: http://laser.ceb.cam.ac.uk/

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Nicolas Le Novere, Babraham Institute

Computational modelling of pluripotency and cell fate regulation

PhD Project Description: Our group uses bioinformatic methods and mathematical modelling to study the basic processes of life. Biological research now relies on the generation and analyses of large amounts of quantitative data, coming for example from nucleic acid sequencing and mass spectrometry. This data needs to be processed, quantified and put in context. Based on the information acquired from experiments and existing literature one can build mathematical models that can then be simulated under various conditions. The success or failure of reproducing observed behaviours tell us if we adequately understand the mechanisms of life. This activity is an important part of what is now called "systems biology". The systems biology paradigm recognises that the behaviour of any living system emerges from the interactions between many of its components and cannot be fully understood by studying those components in isolation. The main biological focus of the group is to understand how cellular and molecular systems interpret signals from their environment and adapt their behaviour as a consequence. This entails understanding how the various cells receive and transduce the signal, the interplay of different signalling pathways, and the final outcome for cell physiology, including gene expression and cell fate. One of our main interests is the maintenance and differentiation of stem cells. PhD projects will take take place within that landscape, focussing in particular on the cross-talks between signalling pathways and the epigenetics machinery.

Referees:

Le Novère N. Quantitative and logic modelling of gene and molecular networks. Nature Reviews Genetics (2015) online advance publication http://dx.doi.org/110.1038/nrg3885

Lai M., et al. Modulation of calmodulin lobes by different targets: an allosteric model with hemiconcerted conformational transitions. PLoS Computational Biology (2015) 10(1):e0116616

Thiele I. A community-driven global reconstruction of human metabolism. Nature Biotechnology (2013) 31: 419–425

Other relevant themes: World Class Underpinning Bioscience

Link: http://lenoverelab.org

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Dr Frances Henson, Dept. of Veterinary Medicine

Second Supervisor/Collaborator: Dr Mark Birch, Department of Surgery

The role of skeletal stem cells at the osteochondral interface.

PhD Project Description: It has long been viewed that a pool of stem cells that gives rise to the progenitors capable of differentiating into osteoblasts and chondrocytes must reside in bone to allow skeletal maintenance and repair throughout life. Recent studies have identified a skeletal stem cell and illustrated its ability to form bone and cartilage tissue [1,2] but what remains unclear is how progenitor populations are sustained through development into adulthood and how anatomical distribution of these cells is maintained to ensure appropriate regional response. This project will use markers that allow stem cell lineage tracing alongside an osteochondral injury model in mice to follow the cycle of activation, recruitment and repair in bone and cartilage, and investigate why this response diminishes with age. Transcriptomics and bioinformatics studies coupled with in vitro cell culture analysis will contrast the regionally defined progenitor pools and seek to provide insight into the molecular mechanisms that govern the maintenance of skeletal stem cells and the cues that signal their role in bone and cartilage homeostasis.

Referees:

1. Chan CK, Seo EY, Chen JY, Lo D, et al. Identification and specification of the mouse skeletal stem cell. Cell. 2015 Jan 15;160(1-2):285-98.

2. Worthley DL, Churchill M, Compton JT, Tailor Y, et al. Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential. Cell. 2015 Jan 15;160(1-2):269-84.

Link: http://www.orthopaedics.cam.ac.uk http://www.vet.cam.ac.uk/directory/fmdh1@cam.ac.uk

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Kristian Franze, Dept. of Physiology, Development and Neuroscience

Second Supervisor/Collaborator: Prof. James Fawcett, Brain Repair Centre

Neurons feel the force: mechanics in the nervous system

PhD Project Description: Key aspects in the development and regeneration of the nervous system include the formation of neuronal axons and their subsequent growth and guidance through the tissue. These processes involve motion and must thus be driven by forces. However, while our understanding of the biochemical and molecular control of neuronal growth is increasing rapidly, how mechanical signalling contributes remains poorly understood. To tackle this problem, we take an interdisciplinary approach by combining tools from biology, physics and engineering. We use different model organisms, including Xenopus and mouse, and combine standard techniques in biology, such as phase contrast and confocal laser scanning microscopy, genetic manipulation of embryos and cells, qPCR and Western blotting with atomic force microscopy, traction force microscopy and compliant cell culture substrates. Our main goal is to understand what mechanical signals neurons encounter during development and regeneration, how they respond to these signals, and how to manipulate their response to ultimately facilitate regeneration after spinal cord injuries, which in humans can currently not be promoted. For projects, requirements are high levels of enthusiasm and curiosity, a background in physics or engineering is NOT necessary. Possible PhD projects include: - Investigating neuronal mechanosensitivity, focusing on the distribution and activity of mechanosensitive ion channels, calcium dynamics etc. in vitro and in vivo - Investigating mechanical changes in spinal cord tissue following injuries plus mechanical manipulation of injured tissue to facilitate regeneration in vitro and in vivo - Investigating the interaction of mechanical and chemical signals

Referees:

Pagliara et al.: Auxetic nuclei in embryonic stem cells exiting pluripotency. Nature Materials 13:638-644 (2014)

Franze K: The mechanical control of nervous system development. Development 140:3069-3077 (2013)

Hardie RC and Franze K: Photomechanical responses in Drosophila photoreceptors. Science 338(6104):260-263 (2012)

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.pdn.cam.ac.uk/staff/franze/index.shtml

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Roger Keynes, Dept. of Physiology, Development and Neuroscience

The mechanism and function of a contact-repulsive protein that regulates nerve growth

PhD Project Description: Background: Our laboratory is interested in the molecular control of nerve development and regeneration. One of our experimental systems is based on the segmented repeat pattern of spinal nerves in birds and mammals, and how this is achieved in the embryo. Each somite is divided into halves, one of which repels outgrowing motor and sensory axons, forcing them to traverse the other half. We have identified a glycoprotein as the source of this repellent activity. It is expressed selectively by cells in the repellent half-somite, causes collapse of axon growth cones in vitro, and its knock-down in vivo by siRNA causes axons to traverse both somite halves. We have also shown recently that it is expressed on the human astrocyte surface, where it may be co-opted to regulate neural plasticity. Aims: Using the growth cone collapse assay, the project will assess the molecular mechanism of this repellent. We will test whether small molecule inhibitors of its activity block collapse when applied to cultured neurons. We will also identify the cognate receptor on axon growth cones, and assess the expression and function of the repellent during development and in the adult CNS. Outcomes: Training in diverse molecular and cellular techniques will enable the student to make original observations, as the system is cutting new territory. Elucidation of this contact-repellent protein is expected to yield new molecular targets for therapeutic strategies to ameliorate neurodegeneration and promote nerve regeneration.

Referees:

Differing Semaphorin 3A concentrations trigger distinct signaling mechanisms in growth cone collapse. RPC Manns, GMW Cook, CE Holt, RJ Keynes Journal of Neuroscience 32, 8554-8559 (2012)

Protein synthesis dependence of growth cone collapse induced by different Nogo-A-domains. R Manns, A Schmandke, A Schmandke, P Jareonsettasin, G Cook, ME Schwab, C Holt & R Keynes PLoS One, 10.1371/journal.pone.0086820 (2014)

Growth cone collapse assay. GMW Cook, P Jareonsettasin & R Keynes in Axon Growth and Regeneration: Methods and Protocols Methods in Molecular Biology, vol. 1162, Springer Science and Business Media, New York (2014)

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.pdn.cam.ac.uk/staff/keynes/index.shtml

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Nicholas Ktistakis, Babraham Institute

How autophagy is initiated: an approach combining live imaging with super resolution microscopy.

PhD Project Description: Autophagy is a conserved cellular response to a variety of stress conditions, including nutrient limitation and the accumulation of damaged proteins/organelles. Upon induction, a novel double membrane vesicle termed autophagosome is formed in the cytosol which encloses cytosolic material for delivery and degradation in the lysosomes. For starvation-induced autophagy the material to be degraded includes a broad and largely random selection of cytosolic proteins which are used to generate novel aminoacids. For other types of autophagy, the cargo is the material (either protein or organelle) to be eliminated. We have been using live imaging combined with biochemical methods to identify and characterise the earliest structures formed during the induction of autophagy. We have identified in the past one such early membrane structure (which we termed omegasome) that is enriched in the lipid PI3P and dynamically connected to the ER. Other related structures appear to be puncta of some type made up of the earliest regulator of the pathway, the protein kinase ULK and its associated components. These structures also appear to have some functional connection with the endoplasmic reticulum. The aim of this project is to combine live imaging with super resolution microscopy in order to provide a spatiotemporal description of how ULK puncta are formed, and their conversion to downstream autophagy intermediates such as omegasomes. All technologies required (live imaging and SIM and STORM super resolution protocols) are up and running in the lab, and encouraging preliminary data have already been obtained.

Referees:

Karanasios, E and Ktistakis NT 2014. Live-cell imaging for the assessment of the dynamics of autophagosome formation: Focus on early steps. Methods S1046-2023

Karanasios E, Stapleton E, Manifava M, Kaizuka T, Mizushima N, Walker SA, and NT Ktistakis 2013 Dynamic association of the ULK1 complex with omegasomes during autophagy induction. J Cell Sci 126: 5224-38

Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G and Ktistakis NT 2008. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 182: 685-701

Other relevant themes:

Link: http://www.babraham.ac.uk/our-research/signalling/nicholas-ktistakis http://www.metabolism.cam.ac.uk/directory/ktistakis/

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Marisa Segal, Dept. of Genetics

Cell cycle control of spindle pole asymmetry in budding yeast.

PhD Project Description: Polarised spindle orientation directs differential cell fate and features prominently in self-renewing stem cell divisions. S. cerevisiae is a unique paradigm for spindle orientation in a cell dividing asymmetrically. The yeast spindle must intersect the bud neck, a configuration dictated by the asymmetric targeting of the yeast counterpart of the centrosomes - the spindle pole bodies (SPB). After duplication, the old SPB is tethered to the bud by its existing astral microtubules while the newly assembled SPB lacks them and is therefore confined to the mother cell. We have elucidated the structural basis for this outstanding asymmetry - the initial absence of Spc72 (a receptor for microtubule nucleation) at the new SPB outer plaque due to asymmetric phosphorylation of an SPB component by cyclin-dependent kinase (CDK). The aim of this project is to reveal the mechanism linking CDK to Spc72 dynamic asymmetry via this key substrate. We plan genetic and biochemical analyses to establish the mechanism for asymmetric phosphorylation. Moreover, we will monitor SPB dynamic assembly using tandem fluorescent timers, a novel approach to track protein mobility by quantitative microscopy analysis to explore the direct role of phosphorylation in vivo and its significance to asymmetric fate. Spindle pole components are highly conserved, as are the principles for asymmetric function in self-renewing stem cell divisions. Imbalanced self-renewal has been implicated in oncogenesis. Thus, our work will help uncover mechanisms for control of asymmetric centrosome function, assess their importance in normal development and the impact of their derangement in disease.

Referees:

1. Juanes MA, Twyman H, Tunnacliffe E, Guo Z, ten Hoopen R and Segal M (2013) Spindle pole body history intrinsically links pole identity with asymmetric fate in budding yeast. Curr. Biol. 23: 1310-1319

2. ten Hoopen R, Cepeda-García C, Fernández-Arruti R, Juanes MA, Delgehyr N and Segal M (2012) Mechanism for astral microtubule capture by cortical Bud6p priming spindle polarity in S. cerevisiae. Curr. Biol. 22: 1075-1083

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.gen.cam.ac.uk/research-groups/segal

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Pier Paolo D'Avino, Dept. of Pathology

Cross-talk between microtubules and actin filaments in animal cells

PhD Project Description: The cytoskeleton controls shape, polarity, movement, and division in animal cells. Deregulation of all these processes has been implicated in various human diseases, including cancer. Cytoskeleton dynamics relies on two types of filamentous structures: microtubules and actin filaments. Although these two types of filaments regulate distinct aspects of cell dynamics, they often need to co-operate in many cellular processes. How this co-operation is established and regulated, however, is still largely unknown. This project aims at identifying the signalling pathways and molecules that control this cross-talk between microtubules and actin filaments. Depolymerisation of actin filaments at the cell cortex in Drosophila cells causes the formation of strong microtubules bundles, indicating that a cortical actin mesh inhibits microtubule polymerisation (D'Avino et al., 2008; Eggert et al., 2004; Kiger et al., 2003). Large-scale RNAi screens have identified several proteins whose depletion causes a similar phenotype and not surprisingly most of these proteins are known actin regulators (Eggert et al., 2004; Kiger et al., 2003). However these screens could neither identify the proteins necessary for the polymerisation and stabilisation of the microtubule arrays nor the signalling molecules required to transmit the signal from the cortex to the cytoplasm. We will use differently designed RNAi screens to identify these molecules in Drosophila cells. We will then investigate if these molecules and their roles have been conserved in human cells. This project will unravel key mechanisms and signalling pathways regulating cytoskeletal dynamics in animal cells.

Referees:

D'Avino, P.P., Takeda, T., Capalbo, L., Zhang, W., Lilley, K., Laue, E., and Glover, D.M. (2008). Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site. J Cell Sci 121, 1151-1158.

Eggert, U.S., Kiger, A.A., Richter, C., Perlman, Z.E., Perrimon, N., Mitchison, T.J., and Field, C.M. (2004). Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. PLoS Biol 2, e379.

Kiger, A., Baum, B., Jones, S., Jones, M., Coulson, A., Echeverri, C., and Perrimon, N. (2003). A functional genomic analysis of cell morphology using RNA interference. J Biol 2, 27.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.path.cam.ac.uk/research/investigators/davino/

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Prof Graham J Burton, Dept. of Physiology, Development and Neuroscience

Oxygen and early placental development

PhD Project Description: Establishment of the placenta is fundamental to a successful pregnancy and to life-long health. Early development of the human placenta takes place in a low oxygen environment, supported by secretions from the endometrial glands rather than the maternal circulation. The principal substrate for the placenta is glucose, and evidence suggests a high percentage is metabolised to lactate rather than to carbon dioxide through oxidative phosphorylation. This is reminiscent of Warburg metabolism that occurs in tumours, allowing cancer cells to survive in low oxygen environments. In the placenta, it may reduce oxygen consumption, so maintaining the supply to fetus, as well as providing precursors for nucleotide synthesis required for cell proliferation. Low oxygen environments are also important for maintenance of stem cells, and there is increasing evidence that stress-response pathways are involved in stem cell differentiation. We recently reported placental dysmorphogenesis in a mouse model with high levels of endogenous endoplasmic reticulum stress. The aim of this project is to explore the significance of the unique low oxygen environment for early placental development. Expression and activity of key enzymes in the aerobic glycolytic pathway in human placentas will be assessed in order to gauge the extent of Warburg metabolism. The effects of oxygen, and of the allied endoplasmic reticulum stress, on lineage differentiation of trophoblast stem cells will be investigated and the signaling pathways identified. Finally, the protective effects of antioxidants and ER chaperones will be tested, as these may represent beneficial dietary supplements for women in the periconceptional period.

Referees:

Yung HW, Hemberger M, Watson ED, et al. Endoplasmic reticulum stress disrupts placental morphogenesis: implications for human intrauterine growth restriction. J Pathol 2012;228:554-64.

Coller HA. Is cancer a metabolic disease? Am J Pathol 2014;184:4-17.

Burton GJ, Jauniaux E, Charnock-Jones DS. The influence of the intrauterine environment on human placental development. Int J Dev Biol 2010;54:303-12.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.pdn.cam.ac.uk/staff/burton/index.shtml

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Trevor W Robbins, Dept. of Psychology

Second Supervisor/Collaborator: Johan Alsio

Functions of dorsal raphé serotonin projections

PhD Project Description: The proposed project aims to elucidate the functional role in cognition of the neuronal input from the dorsal raphé nucleus (DRN) to the forebrain, with a focus on serotonin (5-HT) release into nuclei within the prefrontal cortex (PFC), striatum, and amygdala. Changes in mood hypothetically mediated by 5-HT may impair the executive control over cognitive performance exerted by the PFC. Thus, PFC 5-HT is important for reversal learning and its depletion in the orbitofrontal sector of the PFC of rodents and marmosets impairs such performance[1]. In outbred rats, low levels within the OFC of 5-HT metabolites accordingly predict maladaptive perseverative responding in reversal tasks[2]. Signalling at the 5-HT(2C) receptor seems particularly important, as manipulation of this receptor within the OFC improves cognitive flexibility. Modulation of 5-HT transmission in rodents also biases sensitivity to reinforcing outcomes, decreases in 5-HT activity enhancing responses to negative feedback and vice versa[4]. Such effects are hypothetically mediated by DRN 5-HT projections to the PFC and amygdala. These functional hypotheses will be tested using a combination of neuropharmacological and genetic techniques focusing on Cre-recombinase driving transgenic rats and mice, which will allow the selective expression of DREADDs and other transgenes in DRN neurons or in their target cells in the PFC, striatum, or amygdala. 5-HT release measured by in-vivo microdialysis or in-vivo electrochemistry and immunohistochemistry for 5-HT markers will provide validation of the manipulations. The project will enable students to combine training in computer-controlled measurement of cognitive behaviour in rodents, with surgical and neurobiological techniques.

Referees:

1. Clarke, H.F., Dalley, J.W., Crofts, H.S., Robbins, T.W. & Roberts, A.C. (2004) Cognitive inflexibility after prefrontal serotonin depletion. Science, 304, 878-880.

2. Barlow, R.L., Alsiö, J., Jupp, B., et al. (2015) Markers of Serotonergic Function in the Orbitofrontal Cortex and Dorsal Raphé Nucleus Predict Individual Variation in Spatial-Discrimination Serial Reversal Learning. Neuropsychopharmacology, in press.

3. Bari, A., Theobald, D.E., Capriolo, D., et al (2010) Serotonin modulates sensitivity to reward and negative feedback in a probabilistic reversal learning task in rats. Neuropsychopharmacology, 35, 1290-1301

Link: http://www.neuroscience.cam.ac.uk/directory/profile.php?Trevor

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Jon Houseley, Babraham Institute

Second Supervisor/Collaborator: Nicolas Le Novère

Ageing in a changing world

PhD Project Description: Ageing is a major risk factor in many serious medical conditions and therefore goes hand-in-hand with decreasing health. Ageing is almost universal in eukaryotes and primitive ageing has even been argued in bacteria. Dissecting pathways that control, suppress and even promote ageing in simple tractable organisms should provide clues as to the underlying drivers of ageing in humans and how the ageing process could be modulated. We recently found that ageing in yeast is closely connected with the pathways underlying cellular adaptation to new environments. By studying mutants responding better or worse to environmental change, we aim to understand how yeast turn ageing to their advantage, and to what extent cells can manipulate their rate of ageing. The results of existing studies are complex and biochemical networks underlying ageing remain incomplete. Part of the problem comes from the difficulty of using high-throughput methods to assay lifespan and healthspan in yeast in the unbiased manner required to produce large datasets for computational biology. In this project, we will study growth and gene expression responses to environmental change. We will integrate this data with existing knowledge regarding the biochemical pathways underlying ageing and construct networks to model environmental adaptation and ageing in yeast. We will analyse the structure and function of these networks to predict new factors which modulate ageing and test these predictions in the laboratory. This project lies at the interface of laboratory and computational methods, and will provide the successful candidate with an excellent grounding in both areas.

Referees:

Buchel, F. et al. (2013). Path2Models: large-scale generation of computational models from biochemical pathway maps. BMC systems biology 7, 116.

Houseley, J. et al. (2008). A ncRNA Modulates Histone Modification and mRNA Induction in the Yeast GAL Gene Cluster. Mol Cell 32, 685-695.

Denoth Lippuner, A. et al. (2014). Budding yeast as a model organism to study the effects of age. FEMS microbiology reviews 38, 300-325.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.babraham.ac.uk/our-research/epigenetics/jon-houseley http://www.babraham.ac.uk/our-research/signalling/nicolas-le-novere

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Nianshu Zhang, Dept. of Biochemistry

Second Supervisor/Collaborator: Prof Steve Oliver

Regulation of quiescence entry and exit in yeast

PhD Project Description: The vast majority of cells, whether unicellular or part of a multicellular organism, exist in a non-proliferating state known as G0. However, it is poorly understood how the transition into, and survival during, the G0 state is regulated. Yet many human disorders arise when the G0 state is dysregulated, leading to diseases such as Alzheimer’s or cancer. Using the budding yeast as a model, we have revealed a number of TOR-/PKA-regulated signaling pathways which are vital to quiescence entry and chronological life span. The objective of this Project is to reveal the molecular mechanisms by which these signaling pathways coordinate the transition from exponential growth to quiescence, including cell cycle arrest. The methodologies used will range from biochemical and molecular biological techniques to systems approaches, such as synthetic genetic arrays, transcriptomics, proteomics and modelling.

Referees:

1. Zhang N, Quan Z, Rash B, Oliver SG. 2013 Synergistic effects of TOR and proteasome pathways on the yeast transcriptome and cell growth. Open Biology. 3(5):120137

2. Pir P, Gutteridge A, Wu J, Rash B, Kell DB, Zhang N, Oliver SG. 2012 The genetic control of growth rate: a systems biology study in yeast. BMC Syst Biol. 6:4.

3. Zhang N, Oliver SG (2010) The transcription activity of Gis1 is modulated by proteasome-mediated limited proteolysis. J. Biol. Chem. 285, 6465-6476.

Other relevant themes: Bioenergy and Industrial Biotechnology

Link: http://www.sysbiol.cam.ac.uk/Investigators/Zhang

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Randall Johnson, Dept. of Physiology, Development and Neuroscience

Role of hypoxic response in metastasis

PhD Project Description: The project will be focussed on determining how metastatic cancer cells are influenced by the low oxygen levels found in primary tumors and in turn how this causes them to migrate and proliferate at new sites. A particular focus will be on models of human breast cancer and metastatic progression to the lung, including how inflammation in the primary site and the metastatic site influence metastatic success.

Referees:

Stockmann C, Doedens A, Weidemann A, Zhang N, Greenberg J, Cheresh D, and Johnson RS. (2008) Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis. Nature 456:814-818

Branco-Price C, Zhang N, Schnelle M, Evans C, Katschinski DM, Liao D, Ellies L, and Johnson RS. (2012) Endothelial cell HIF-1α and HIF-2α differentially regulate metastatic success. Cancer Cell 21:52-65

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.pdn.cam.ac.uk/staff/rjohnson/index.shtml

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Pier Paolo D'Avino, Dept. of Pathology

Investigating the role of the midbody in determining stemness and cell fate

PhD Project Description: Cell division controls growth, development and reproduction in all organisms. This process plays a key role not only in the transmission of the genetic information, but also in determining the fate of the two daughter cells. This is particular relevant in the division of stem cells, which generate two cells with distinct properties: one cell maintains stemness while the other differentiates. Recent studies have proposed that an organelle that forms between the two daughter cells, dubbed the midbody, could play a role on determining cell fate in stem cell division (Chen et al., 2013), but this hypothesis has not been confirmed by studies in animal models (Salzmann et al., 2014). This project aims at investigating the contribution of the midbody to stemness and cell fate by analysing the effect of mutation in genes involved the formation and organisation of the midbdoy in the fruitfly Drosophila melanogaster. We will use these mutants to investigate whether abnormal midbody formation affects cell fate in the division of both somatic and germline stem cells. This project could lead to the identification of important mechanisms governing stem cell identity.

Referees:

D'Avino, P.P., Takeda, T., Capalbo, L., Zhang, W., Lilley, K., Laue, E., and Glover, D.M. (2008). Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site. J Cell Sci 121, 1151-1158.

Eggert, U.S., Kiger, A.A., Richter, C., Perlman, Z.E., Perrimon, N., Mitchison, T.J., and Field, C.M. (2004). Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. PLoS Biol 2, e379.

Kiger, A., Baum, B., Jones, S., Jones, M., Coulson, A., Echeverri, C., and Perrimon, N. (2003). A functional genomic analysis of cell morphology using RNA interference. J Biol 2, 27.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.path.cam.ac.uk/research/investigators/davino/

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Amanda Sferruzzi-Perri, Dept. of Physiology, Development and Neuroscience

Resource allocation and intrauterine development

PhD Project Description: My laboratory is focussed on understanding the aetiology of pregnancy complications and the regulation of developmental processes in a wider context. We focus our studies on the molecular control placenta as it is the main determinant of fetal growth, responsible for materno-fetal nutrient and oxygen transfer and secretes hormones and growth factors thought to adapt maternal physiology to support pregnancy and lactation. We employ genetic and environmental manipulations of the insulin-like growth factor, phosphoinositide-3 kinase (IGF-PI3K) system and in vivo functional assays and cellular, histological and molecular techniques in vitro. Therefore, a PhD could involve: 1) Deciphering the molecular mechanisms governing placental differentiation and function. 2) Examining how the placenta may influence the metabolic or cardiovascular systems of the mother and thus, her ability to allocate resources (nutrients and oxygen) to the developing fetus. 3) Determining how the placenta senses and responds to the environment of the mother and thereby regulates fetal growth. 4) Identifying the significance of changes in maternal-fetal resource allocation due to altered placental function, on the development and health of the offspring later in life.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.pdn.cam.ac.uk/staff/sferruzzi-perri/index.shtml

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Silvia Vignolini, Dept. of Chemistry

Second Supervisor/Collaborator: Braybrook, Siobhan Ariel

Smart Cellulose composites for cell culture and experimentation

PhD Project Description: Cellulose is the earth’s major biopolymer and is of tremendous economic importance globally. Cellulose is found in the cell wall of plants but can be also synthetized by animals and bacteria. Among the bacteria, the Glucanacetobactera can procure glucose, sugar, glycerol or other organic substrates and convert them into pure cellulose [1]. Bacterial cellulose can simply be isolated and it is organized into a micro-fibrillated highly porous network (hydrogel). Due to its high purity, hydrophilicity, this cellulose hydrogel offers a wide range of special applications, e.g. as a food matrix, as an acoustic or filter membrane, as ultra-strength paper and as a good substrate for tissue regeneration and growth [2]. Within this project we aim to produce smart composite for cell culture made of bacterial cellulose. The project will enable the Ph.D student to work in a highly interdisciplinary environment to produce new platform for cell culture where growth and experimental manipulation are enhanced. The adhesion to the culture substrate can be improved by nano-patterning of the surface of the produced film. Morphologically similar cells, generally difficult to distinguish by conventional methods will be grown on gold nanoparticle templates to improve surface-enhanced Raman spectroscopy (SERS) from the cellulose substrate [3]. We expect that the student will develop a multi-functional substrate that provides label-free and non-invasive characterization, and that can play a vital role in identifying cell types in biomedical stem cell research.

Referees:

[1] Bacterial cellulose' a masterpiece of nature's arts J. Materials Science (2000) 35, 261–270

[2] Bacterial Cellulose Production and its Industrial Applications, J. Bioprocess Biotech (2014) 4, 150 doi: 10.4172/2155-9821.1000150

[3] Intracellular SERS Nanoprobes For Distinction Of Different Neuronal Cell Types, Nano Lett. (2013), 13, 2463−2470

Link: http://www.ch.cam.ac.uk/group/vignolini

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Ragnhildur Thora Karadottir, Dept. of Veterinary Medicine

Activity dependency of myelination

PhD Project Description: 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. 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. We now wish to address whether, and by what mechanisms, neural activity regulates OPCs differentiation and myelination, by: (1) expressing Ch2R in parallel fibres of the molecular layer of the mouse cerebellum, axons that normally are not myelinated and have at a low rate (0.5Hz) in-vivo, and light stimulate the neurons in vivo at high frequencies (5Hz) and quantify myelination. This will show if the frequency of neuronal activity is sufficient to initiate myelination. (2) Express Ch2R in DRG neurons in-vitro, and culture with OPCs, stimulate the neurons at different frequencies to identify a frequency code for myelination. OPCs will then be sorted afterwards and qPCR and western blot used to identify intracellular mechanisms for activity dependent myelination. The project will involve optogenetics, cell culture work, electrophysiology, surgery, immunohistochemistry and imaging.

Referees:

Lundgaard I, Luzhynskaya A, Stockley JH, Wang Z, Evans KA, Swire M, Volbracht K, Gautier HO, Franklin RJM., ffrench-Constant C, Attwell D & Káradóttir R (2013). Neuregulin and BDNF induce a switch to NMDA receptor dependent myelination by oligodendrocytes. PLoS Biol 11(12):e1001743.

Káradóttir R, Hamilton N, Bakiri Y & Attwell D (2008). Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter. Nature Neuroscience 11(4): 450-456

McKenzie IA, Ohayon D, Li H, Faria JP de, Emery B, Tohyama K, Richardson WD (2014) Motor skill learning requires active central myelination. Science 346:318–322.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.stemcells.cam.ac.uk/researchers/principal-investigators/dr-ragnhildur-thra-kradttir

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Cahir O'Kane, Dept. of Genetics

Axonal endoplasmic reticulum and axon degeneration in Drosophila

PhD Project Description: Maintaining the structure and function of axons as long as 1m requires a lot of engineering, the importance of which is revealed in diseases where it goes wrong – e.g. hereditary spastic paraplegia (HSP), which shows preferential degeneration of longer spinal cord axons. Identifying the causative disease genes helps to reveal the processes that maintain function of long axons. Many HSP mutations affect proteins that model ER membrane curvature by inserting in one face of the membrane, hinting at an important role for ER in axon function. Axonal ER is principally smooth tubular ER, unlike rough ER sheets in neuronal cell bodies. Its length and continuity, from dendrites through cell body and axons to presynaptic terminals, liken it to a "neuron-within-a-neuron", which might potentially conduct signals through neurons, independent of action potentials, but faster than microtubule-based transport. We use Drosophila to study the mechanisms of organisation of axonal ER. We have developed markers for it, ways to look at it in fixed and live preparations, and at the effects of mutant genotypes on it. At least one Drosophila HSP protein is important for its organisation in longer but not in shorter axons. A number of areas are possible for a PhD, including (1) ultrastructural topology and continuity of ER network organisation in wildtype and HSP mutant axons; (2) comprehensively testing requirements for Drosophila HSP genes, and identifying new genes, in axonal ER organisation; (3) testing the requirement for ER in axonal function and signaling.

Referees:

FM Menzies, M Garcia-Arencibia, S Imarisio, N C O'Sullivan, T Ricketts, BA Kent, MV Rao, W Lam, ZW Green-Thompson, RA Nixon, LM Saksida, TJ Bussey, CJ O'Kane*, DC Rubinsztein* (2014) Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity. Cell Death & Differentiation; doi: 10.1038/cdd.2014.151

NC O’Sullivan, TR Jahn, E Reid, CJ O’Kane* (2012) Reticulon-like-1, the Drosophila orth¬olog of the Hereditary Spastic Paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons. Hum Mol Gen 21:3356-65 C Blackstone, CJ O’Kane, E Reid (2011) Hereditary spastic paraplegias: membrane traffic and the motor pathway. Nat Rev Neurosci 12:31-42

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.gen.cam.ac.uk/research-groups/okane

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Heidi Welch, Babraham Institute

New G protein signalling pathways to regulate Rac GTPases

PhD Project Description: Rac-GTPases control morphology, migration and other crucial cell behaviours (1). They are tightly regulated through several types of proteins, including the GEFs that activate them (2). Our lab discovered P-Rex family Rac-GEFs which control normal Rac function in many cell types, but when deregulated can drive cancer progression (3,4). P-Rex family Rac-GEFs are activated by heterotrimeric G proteins upon cell stimulation through G protein-coupled receptors (GPCRs) (3,4). The fact that this activation occurs through the catalytic domain of P-Rex suggests that G proteins might also be able to regulate other types of Rac-GEFs, as many of these proteins harbour similar domains. This project is to explore the existence of such a new signalling pathway. You will i) investigate the effects of G proteins on the catalytic activity of a range of recombinant Rac-GEFs, and ii) use an unbiased approach by purifying novel G protein dependent regulators through their effects on Rac activity. For promising hits, you will characterise their mechanism of regulation and their functional role in the cell. The identification of a novel G protein-dependent Rac signalling pathway will be a step-increase in our knowledge of the processes that regulate cell shape and migration. Such a pathway could also be exploited for targeting inflammatory conditions and cancer metastasis. (1) Heasman & Ridley A (2008) Nat Rev Mol Cell Biol. (2) Rossman K et al (2005) Nat Rev Mol Cell Biol. (3) Welch HC et al (2002) Cell. (4) Welch HC (2015) Small GTPases. Lab website: http://www.babraham.ac.uk/our-research/signalling/heidi-welch

Referees:

Welch HCE. (2015). Regulation and Function of P-Rex Family Rac-GEFs. Review. Small GTPases. In Press. Pan D, Amison RT, Riffo-Vasquez Y, Spina D, Cleary SJ, Wakelam MJ, Page CP, Pitchford SC, Welch HCE. (2015). P-Rex and Vav Rac-GEFs in Platelets Control Leukocyte Recruitment to Sites of Inflammation. Blood. In Press. (Epub 23 Dec 2014)

Johnsson A-K E, Dai Y, Nobis M, Baker MJ, McGhee EJ, Walker S, Schwarz JP, Kadir S, Morton JP, Myant KB, Huels DJ, Segonds-Pichon A, Sansom OJ, Anderson K, Timpson P, Welch HC. (2014). A Rac-FRET Mouse for Spatiotemporal Analysis of Rac Activity in Living Tissue. Cell Rep 6, 1153-1164.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.babraham.ac.uk/our-research/signalling/heidi-welch

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David Klenerman, Dept. of Chemistry

PhD Project Description: Our research is based on exploiting cutting edge single molecule fluorescence, single molecule tracking and super resolution imaging to directly image individual biological molecules on or in live cells. Our work is highly collaborative and our group contains physically trained scientists to help maintain and adapt the instrumentation. We would offer a PhD project in three main areas: 1. The molecular basis of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, aiming to identify the key protein aggregates that lead to the onset and spreading of disease. 2. The molecular basis of T cell triggering, which underpins the adaptive immune response, focusing on following the initial changes in the spatial organisation of the signalling protein in the T-cell membrane when encountering a model lipid bilayer or cell surface . 3. The molecular basis of Toll-like receptor signalling, part of the innate immune response, following the oligomerisation of these proteins in the cell membrane and the assembly of large macromolecular signalling complexes in real-time.

Referees:

1.Direct Observation of the Interconversion of Normal and Toxic Forms of alpha-Synuclein”, N. Cremades, S.I.A Cohen, E. Deas, A.Y. Abramov, A.Y. Chen, A. Orte, M. Sandal, R.W. Clarke, P. Dunne, F.A. Aprile, C.W. Bertoncini, N.W. Wood, T.P.J. Knowles, C.M. Dobson, D. Klenerman. Cell, 149, 1048, (2012).

2. “The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β 1-40 peptide”, P Narayan, A Orte, RW Clarke, B Bolognesi, S Hook, KA Ganzinger, S Meehan, MR Wilson, CM Dobson, D Klenerman – Nat. Struct. Molec. Biol., 19, (2012).

3. “Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells”, J.R. James, S.S. White, R.W. Clarke, A.M. Johansen, P.D. Dunne, D.L. Sleep, W.J. Fitzgerald, S.J. Davis, & D. Klenerman, PNAS,104, 17662-17667, (2007).

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.ch.cam.ac.uk/person/dk10012

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Andrew Waller, Animal Health Trust

Second Supervisor/Collaborator: Duncan Maskell

Identification of better vaccine targets for the prevention of Strangles.

PhD Project Description: This project will exploit transposon directed insertion-site sequencing (TraDIS) to provide an unprecedented insight into the virulence mechanisms employed by, and immune responses against S. equi, the causative agent of Strangles in horses. This project will identify new vaccine targets with which to improve the health of horses around the World. During her MSc project at the AHT, Amelia Charbonneau successfully developed a TraDIS method for S. equi by adapting a technique that was originally developed by Professor Maskell’s group for the study of Salmonella Typhi [1]. A pool of 115,951 S. equi mutants was generated by the random insertion of the ISS1 transposon, which is equivalent to approximately one insertion every 18 bp across the S. equi genome [2]. The identification of viable insertion sites by TraDIS identified 253 genes that are essential or important to the in vitro growth of S. equi. In part 1 of this project, TraDIS will be used to identify mutants less able to attach to monolayers of equine cells, acquire metal ions or resist oxidative stress in the presence of hydrogen peroxide. In part 2 of the project, TraDIS will be used to identify novel protective antigens by comparing the viability of mutants in whole equine blood in vitro following pre-treatment with sera from naïve, vaccinated [3] or convalescent horses. This project fits within the BBSRC’s theme of Bioscience for Health and exploits a data driven biology approach to significantly enhance our knowledge of S. equi and our ability to develop effective vaccines.

Referees:

1. Langridge, G.C., Phan, M.D., Turner, D.J., Perkins, T.T., Parts, L., Haase, J., Charles, I., Maskell, D.J., Peters, S.E., Dougan, G., Wain, J., Parkhill, J. & Turner, A.K. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res 19, 2308-16 (2009).

2. Holden, M.T., Heather, Z., Paillot, R., Steward, K.F., Webb, K., Ainslie, F., Jourdan, T., Bason, N.C., Holroyd, N.E., Mungall, K., Quail, M.A., Sanders, M., Simmonds, M., Willey, D., Brooks, K., Aanensen, D.M., Spratt, B.G., Jolley, K.A., Maiden, M.C., Kehoe, M., Chanter, N., Bentley, S.D., Robinson, C., Maskell, D.J., Parkhill, J. & Waller, A.S. Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens. PLoS Pathogens 5, e1000346 (2009).

3. Guss, B., Flock, M., Frykberg, L., Waller, A.S., Robinson, C., Smith, K.C. & Flock, J.I. Getting to grips with strangles: an effective multi-component recombinant vaccine for the protection of horses from Streptococcus equi infection. PLoS Pathog 5, e1000584 (2009).

Link: http://www.aht.org.uk/

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Ewan St. John Smith, Dept. of Pharmacology

Neuronal activation by acid: pain and breathing.

PhD Project Description: 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.

Referees:

Schuhmacher, L. N., Srivats, S. and Smith, E. S. (2015) Structural domains underlying the activation of acid-sensing ion channel 2a. Mol Pharmacol in press, doi 10.1124/mol.114.096909

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

Link: http://www.phar.cam.ac.uk/research/Smith

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Alfonso Martinez Arias, Dept. of Genetics

PhD Project Description: We have recently developed a method to recapitulate early mammalian development in tissue culture from aggregates of Embryonic Stem cells (van den Brink et al. 2014 Development 141, 4231-4242 doi: 10.1242/dev.113001). This system opens up the possibility of exploring how cellular processes impinge in pattern formation in mammalian embryos, The projects involve combinations of computational and experimental techniques.

Referees:

van den Brink et al. 2014 Development 141, 4231-4242 doi: 10.1242/dev.113001

Turner et al. 2014 Development 141, 4243-4253 doi: 10.1242/dev.112979.

Other relevant themes: World Class Underpinning Bioscience

Link: http://amapress.gen.cam.ac.uk/ http://www.gen.cam.ac.uk/research-groups/martinez-arias

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Stephen Graham, Dept. of Pathology

Structural basis of lysosomal fusion

PhD Project Description: Eukaryotic cells are defined by their compartmentalisation, different membrane-bound organelles containing distinct chemical environments. Cells have evolved an elaborate system of membrane trafficking to move macromolecules between such organelles, the movement, docking and subsequent fusion of organelles being tightly regulated by a number of protein complexes. Nowhere is the tight regulation of membrane fusion more important than at the lysosome, a low-pH compartment that lies at the end of the endocytic pathway and contains acid hydrolases that digest its luminal contents (i.e. the cell’s recycling centre). We are interested in defining the molecular basis, at atomic resolution, of how cells regulate the docking and fusion of lysosomes with organelles such as late endosomes or autophagosomes. Understanding this process will help us understand a range of processes important for health, from antigen presentation and processing of the Parkinson’s disease protein α-synuclein to infection of cells by filoviruses like Ebola virus. This project will combine macromolecular crystallography, biophysics and cell biology to investigate the structure and function of the protein complexes that modulate lysosome fusion, key amongst these being the six-protein HOPS complex. We have already identified a number HOPS components and cellular binding partners that can be recombinantly expressed and purified in quantities suitable for crystallisation experiments. Structural and biophysical characterisation of these complexes will provide unique insights to guide functional assays to that probe the regulation of lysosomal fusion in cells.

Referees:

SC Graham, L Wartosch, SR Gray, EJ Scourfield, JE Deane, JP Luzio, DJ Owens (2013) Structural basis of Vps33A recruitment to the human HOPS complex by Vps16. Proceedings of the National Academy of Sciences of the USA, 110: 13345-13350. doi: 10.1073/pnas.1307074110

JE Carette, M Raaben, AC Wong, AS Herbert, G Obernosterer, N Mulherkar, AI Kuehne, PJ Kranzusch, AM Griffin, G Ruthel, P Dal Cin, JM Dye, SP Whelan, K Chandran, TR Brummelkamp (2011) Ebola virus entry requires the cholesterol transporter Niemann-Pick C1. Nature 477: 340-343. doi: 10.1038/nature10348

H Balderhaar and C Ungermann (2013) CORVET and HOPS tethering complexes – coordinators of endosome and lysosome fusion. Journal of Cell Science 126: 1307-1316. doi: 10.1242/jcs.107805

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.path.cam.ac.uk/research/investigators/graham/

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Colin W Taylor, Dept. of Pharmacology

Superresolution analyses of IP3-evoked calcium signals

PhD Project Description: How does calcium, the simplest of all intracellular messengers, effectively and specifically regulate so many cellular activities? Calcium signals result from regulated opening of ion channels. The distribution of these channels within cellular membranes plays an important part in defining the spatial organization of calcium signals, and thereby their ability to mediate selective communication between specific extracellular stimuli and cellular responses. In most cells, calcium signals are initiated by receptors that stimulate formation of IP3, which then stimulates calcium release from ER through a family of calcium channels, IP3 receptors (IP3R). With superresolution microscopy, we can resolve the tiny calcium signals that result from opening of single IP3R in intact cells. These ‘optical patch-clamp’ analyses reveal that as stimulus intensities increase, single IP3R respond first, then small clusters of IP3R respond together, and finally calcium signals propagate regeneratively across cells as waves. Hence, the nature of the calcium signal changes with stimulus intensity. We have only a rudimentary understanding of how this recruitment of the fundamental building blocks of IP3-evoked calcium signals occurs. What are the roles of IP3-evoked clustering of IP3R, IP3 and ER mobility, Ca2+-regulation of IP3R, and inactivation mechanisms in the genesis of calcium signals? Our recent (unpublished) progress with fluorescent labelling of endogenous IP3R (via gene-editing) and with high-resolution analyses of single-channel openings of lone and clustered IP3R in living cells (optical patch-clamp) provide the tools needed to address with unrivalled resolution the fundamental mechanisms that govern the organization of IP3-evoked calcium signals.

Referees:

Rahman, T-U, Skupin, A, Falcke, M & Taylor CW (2009) Clustering of IP3 receptors by IP3 retunes their regulation by IP3 and Ca2+. Nature. 458, 655-659. PMID 19348050.

Seo, M-D, Velamakanni, S, Ishiyama, N, Stathopulos, PB, Rossi, AM, Khan, SA, Dale, P, Li, C, Ames, JB, Ikura, M & Taylor, CW (2012) Structural and functional conservation of key domains in InsP3 and ryanodine receptors. Nature 483, 108-112. PMID 22286060.

Thurley, K, Tovey, SC, Moenke, G, Prince, VL, Meena, A, Thomas, AP, Skupin, A, Taylor, CW* & Falcke, M* (2014) Reliable encoding of extracellular stimuli within random sequences of Ca2+ spikes. Sci Signal. 7, ra59. PMID 24962706

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.phar.cam.ac.uk/research/taylor/Summary

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Guy Brown, Dept. of Biochemistry

PhD Project Description: Eaten alive: does phagocytosis of neurons or synapses by microglia contribute to ageing and inflammatory neurodegeneration? 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) stressed-but-live neurons, and thereby cause neuronal death and loss. This project will investigate the signals and receptors involved in this process, and whether phagocytosis is beneficial or detrimental in particular pathologies. By identifying the signals and receptors involved we hope to develop drugs and other treatments for the neuronal loss occurring in diseases such as Alzheimer’s and Parkinson’s disease. The project will use cell culture, transgenic animals, fluorescence microscopy and molecular cell biology.

Referees:

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

Neher JJ, Neniskyte U, Hornik T, Brown GC (2014) Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo. Glia 62:1463-75.

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.

Link: www.guybrown.net

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Dr Gonçalo Bernardes, Dept. of Chemistry

Second Supervisor/Collaborator: Prof Chris Schofield, University of Oxford

PhD Project Description: Histones are key structural and functional proteins in chromatin. Through post-translational modification (PTM) processes, histone proteins undergo modification at multiple sites and residue types. Methylation, acetylation, ubiquitination, and several other acylations (including succinylation and crotonylation) of lysine (Lys) are examples of PTMs common to histones. Many of these are dynamic and regulate chromatin structure, transcription, and DNA replication and repair. A biochemical understanding of how histone PTMs regulates such function is a current frontier in biology. Site-selective protein-modification chemistry promises to allow the facile preparation of proteins with defined PTMs from full-length recombinantly produced precursors. A common approach to achieve histones bearing defined PTMs is through the generation of thioether mimics of Lys methylation and acetylation. Although, the thioether moiety is prone to oxidation yielding sulfoxide and/or sulfone-like structures that hamper their use for in vivo studies. In this project, we will explore a new chemical conjugation method based on Aza-Michael addition of amine nucleophiles to Dha that produces a natural-like –NH– bond for the installation of PTMs on histone proteins. In addition, we will also explore the use of hydroxylamine derivatives that would allow for a “switch-on” mechanism based on pH instability of the bond formed upon reaction of the hydroxylamine with Dha. This project will provide advanced training in synthetic & protein chemistry as well as in molecular and cellular biology with the potential to unravel the biological roles that histones bearing defined PTMs have on genetic regulation.

Referees:

T. Kouzarides, Cell 2007, 128, 693. M. D. Simon, F. Chu, L. R. Racki, C. C. de La Cruz, A. L. Burlingame, B. Panning, G. J. Narlikar, K. M. Shokat, Cell 2007, 128, 1003.

J. M. Chalker, L. Lercher, N. R. Rose, C. J. Schofield, B. G. Davis Angew. Chem. Int. Ed. 2012, 51, 1835 .

Link: http://www.ch.cam.ac.uk/person/gb453

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Wolfram Schultz, Dept. of Physiology, Development and Neuroscience

PhD Project Description: Rewards are crucial objects that induce learning, approach behavior, choices and emotions. Whereas emotions are difficult to investigate in animals, the learning function is mediated by neuronal reward prediction error signals which implement basic constructs of reinforcement learning theory. These signals are found in dopamine neurons, which emit a global reward signal to striatum and frontal cortex, and in specific neurons in striatum, amygdala and frontal cortex projecting to select neuronal populations. The approach and choice functions involve subjective value, which is objectively assessed by behavioral choices eliciting internal, subjective reward preferences. Utility as prime value decision variable of economic choice theory incorporates various influences, including risk, delay, effort and social interaction. Appropriate for formal decision mechanisms, this variable concerns object value, action value, difference value and chosen value, which are coded by specific reward neurons. Non-value influences on choices including superstition, prejudice, tradition, strategy and heuristics are not considered here. Although all reward, reinforcement and decision variables are theoretical constructs, their neuronal signals constitute measurable physical implementations and as such confirm the validity of these concepts. The neuronal reward signals provide guidance for behavior while constraining the free will to act.

Referees:

Tobler PN, Christopoulos GI, O'Doherty JP, Dolan RJ, Schultz W. Risk-dependent reward value signal in human prefrontal cortex. Proc. Natl. Acad. Sci. (USA) 106: 7185-7190, 2009.

Burke CJ, Tobler PN, Baddeley M, Schultz W. Neuronal mechanisms of observational learning. Proc Natl Acad Sci (USA) 107, 14431-14436, 2010.

Medic N, Ziauddeen H, Vestergaard MD, Henning E, Schultz W, Farooqi IS, Fletcher PC. Dopamine modulates the neural representation of subjective value of food in hungry subjects. J Neurosci 34: 16877-16889, 2014.

Link: http://www.pdn.cam.ac.uk/staff/schultz/index.shtml

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Anne Ferguson-Smith, Dept. of Genetics

PhD Project Description: Our team focuses on the epigenetic control of genome function and of developmental processes. Research activities fall within three themes (a) Stem cells and the epigenetic programme in vivo and in vitro; (b) The relationship between genome, epigenome and phenotype and (c) Environment, development and disease. We are a multidisciplinary team of experimental researchers and bioinformaticians working on key questions in mammalian developmental genetics using mouse models.

Referees:

Radford EJ, Ito M*, Shi H*, Corish JA, Yamazawa K, Ignanaitis E, Seisenberger S, Hore TA, Reik W, Erkek S, Peters A, Patti ME*, Ferguson-Smith AC*. (2014) In utero undernourishment perturbs the adult sperm methylome, and is linked to metabolic disease transmission. Science 345(6198):1255903

Charalambous M, Teixeira da Rocha S, Rowland T, Ferron S, Ito M, Radford E, Schuster Gossler K, Hernandez A, Ferguson-Smith AC. (2012) Imprinted gene dosage is critical for the transition to independent life. Cell Metabolism 15(2) 209-221

Ferron S, Charalambous M, Radford E, McEwen K, Wildner H, Hind E, Morante-Redolat J, Laborda J, Guillemot F, Bauer S, Farinas I. Ferguson-Smith AC (2011). Postnatal loss of Dlk1 imprinting in stem cells and niche-astrocytes regulates neurogenesis. Nature 475 7355 381-385. Review: Ferguson-Smith AC. (2011).Genomic imprinting: the emergence of an epigenetic paradigm. Nature Reviews Genetics 12(8) 565-575

Link: http://www.gen.cam.ac.uk/research-groups/ferguson-smith

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Dr Debbie Guest, Animal Health Trust

PhD Project Description: We have two main areas of research. First, the development of stem cell therapies to aid tendon regeneration in horses. Horses frequently suffer from tendon injuries and these heal through the formation of scar tissue which leads to a high rate of re-injury. Autologous mesenchymal stem cells (MSCs) are being used clinically to aid tendon regeneration but little is known about their mechanism of action. We demonstrated poor long-term tissue integration of MSCs suggesting that they do not make a direct, physical contribution to tissue repair through differentiation. Instead, our evidence suggests that MSCs may function through modulation of injury-induced inflammation. In contrast, pluripotent equine embryonic stem cells (ESCs) have a high survival in the injured tendon and appear to undergo differentiation to tenocytes. We are now working to define the pathways underpinning the different mechanism of stem cell function in tendon repair. Second, we have derived induced pluripotent stem cells (iPSCs) from horses to allow the in vitro modelling of inherited diseases. Fractures occur frequently in racing Thoroughbreds and although we have demonstrated that fracture risk has a genetic component, it is a complex disorder with multiple genes involved. We are beginning to identify these genes using whole genome sequencing and 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.

Referees:

1. Paterson Y. Z., Rash, N., Garvican, E. R., Paillot, R., & Guest, D.J. (2014) Equine mesenchymal stromal cells and embryo-derived stem cells are immune privileged in vitro. Stem Cell Research and Therapy. 5, 90.

2. Barsby, T., Bavin, E. & Guest D. J. (2014) 3-Dimensional Culture and Transforming Growth Factor Beta3 Synergistically Promote Tenogenic Differentiation of Equine Embryo-Derived Stem Cells. Tissue Engineering Part A. 20, 2604-2613.

3. Guest, D. J., Smith, M. R. W. & Allen, W. R. (2010) Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendons. Equine Veterinary Journal. 42(7), 636-642.

Other relevant themes:

Link: http://www.aht.org.uk/cms-display/profiledg.html

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Christopher Huang, Dept. of Biochemistry

Second Supervisor/Collaborator: Dr. Andrew Grace

PhD Project Description: Ryanodine receptor (RyR2)-mediated release of sarcoplasmic reticular (SR) Ca2+ following electrical activation is essential to initiation of the heartbeat, and return of such released Ca2+ into the SR store is essential in maintainence of membrane electrical stability during the supervening diastole. Beta-adrenergic stimulation is strategic in the capacity of the heart to adjust its strength and frequency of contraction in response to exercise challenge and is thus central to human wellbeing particularly in the ageing population with blunting of this response. The classical scheme for beta-adrenoreceptor stimulation results in activation of the Gs G-protein. This results in an adenylate cyclase activation that in turn activates protein kinase A and phosphorylation of its targets including sarcolemmal ion channels, the ryanodine receptor (RyR2), sarcoplasmic reticular (SR) ATPase (SERCA) and calcium/calmodulin-dependent protein kinase II (CaMKII). These changes lead to the necessary adjustments in Ca2+ handling. Recent reports have suggested an existence of an alternative pathway contributing to the Ca2+ control network. Thus, the recently discovered exchange protein directly activated by cAMP (Epac) is also thought to mediate adrenergic effects upon Ca2+ release and membrane excitability to influence ventricular arrhythmic tendency. The extent to which this novel system contributes to normal cardiac adaptation and properties is unknown. This project will manipulate Epac activation in both normal hearts, as well as RyR2-P2328S hearts with an increased predisposition to RyR2-mediated SR Ca2+ leak. Expeeriments will be perfomred in the absence and in the presence of the RyR2-blocker Dantrolene Na, to assess the relative importance of this novel activation pathway in order to develop novel quantitative network models describing the regulatory pathways for RyR2-mediated Ca2+ release both during systolic activation and during diastole, as well as the implications for the resulting cytosolic Ca2+ levels on the remaining Ca2+ homestatic processes. This will lead to a more complete description of cellular Ca2+ regulation that had hitherto omitted this novel control mechanism. This has eventual implications for human health in view of alterations in these Ca2+ release processes with age.

Referees:

De Rooij J, Zwartkruis FJT, Verheijen MHG, et al. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 1998;396(6710):474–7.

Hothi SS, Gurung IS, Heathcote JC, Zhang Y, Booth SW, Skepper JN, Grace AA, Huang CL. Epac activation, altered calcium homeostasis and ventricular arrhythmogenesis in the murine heart. Pflugers Arch. 2008 Nov;457(2):253-70.

Zhang Y, Wu J, Jeevaratnam K, King JH, Guzadhur L, Ren X, Grace AA, Lei M, Huang CL, Fraser JA. Conduction slowing contributes to spontaneous ventricular arrhythmias in intrinsically active murine RyR2-P2328S hearts. J Cardiovasc Electrophysiol. 2013 Feb;24(2):210-8.

Link: http://www.murrayedwards.cam.ac.uk/contacts/contactdetails/personal_pages/prof_huang

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Patrick Varga-Weisz, Babraham Institute

Second Supervisor/Collaborator: Marc Veldhoen Babraham Institute

PhD Project Description: Epigenetic stability in intestinal stem cells during ageing and in inflammation Patrick Varga-Weisz (with the group of Marc Veldhoen, Babraham Institute) 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 named Smarcad1 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. The gastrointestinal tract contains the largest surface of the body with specialised epithelial cells that separate mammalian hosts from the environment. Intestinal epithelial cells (IECs) are made up of several distinct lineages derived from a multi-potent progenitor, continually self-renewing, form an active part of the immune system and are adapted to selectively host bacteria. The luminal content influences cell differentiation and gene regulation of IECs. We will investigate how the intestinal content affects the transcriptome and epigenome of intestinal cells. One focus is on the role of the aryl hydrocarbon receptor (AhR), a transcriptional regulator activated by diet- and microbial-derived ligands, and the chromatin remodelling factor Smarcad1. Phenotypic analysis of mice deficient in AhR or Smarcad1 indicated potential crosstalk between these two factors. 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).

Referees:

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).

Link: http://www.babraham.ac.uk/our-research/nuclear-dynamics/patrick-varga-weisz

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Mikhail Spivakov, Babraham Institute

Second Supervisor/Collaborator: Helle Jørgensen, Cambridge University Department of Medicine

PhD Project Description: 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 vital for the maintenance of healthy vasculature throughout the lifespan. Intriguingly, VSMCs originate from two different embryonic tissues, and tissue-specific differences in their ‘eagerness’ to undergo phenotypic switching in response to injury have been observed. 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 this response, which can be reflected by their global gene expression profiles. We are studying VMSC heterogeneity by profiling gene expression genome-wide in individual cells using the state-of-the-art method of 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. Although this project necessarily involves a bioinformatics component, the balance between the “wet-lab” and computational work can be adjusted to suit the candidate’s objectives. The candidate will join our friendly and motivated team (www.regulatorygenomicsgroup.org), while working in close collaboration with our partner laboratory at Cambridge University. Specific bioinformatics training will be provided; some experience in computational data analyses and/or programming is desirable, but not essential.

Referees:

1. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004; 84:767-801. PMID: 15269336.

2. Baslan T, Hicks J. Single cell sequencing approaches for complex biological systems. Curr Opin Genet Dev 2014; 26C:59-65. PMID: 25016438.

Link: http://www.babraham.ac.uk http://www.regulatorygenomicsgroup.org

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Barbara Blacklaws, Dept. of Veterinary Medicine

PhD Project Description: Fibrotic changes in tissue are one of the problems of ageing and although the aetiology is often unknown infections are implicated in many cases. We have a model of fibrosis in persistently murine norovirus infected Stat1-/- mice. These animals show fibrosis in both the spleen and liver that is not present in persistently infected wild-type mice and yet similar levels of virus are found in both mouse strains. This model poses many questions on how the same virus infection in two different host backgrounds may lead to different chronic tissue responses. The project will address both the virus and host response to persistent infection. Initially, changes in the virus populations from both wild-type and Stat1-/- mice will be analysed by next generation sequencing methods. Changes in the viral genome will be analysed for evidence of immune evasion or mutation in non-structural proteins that could lead to change in function that could contribute to fibrosis. Secondly the response of Stat1-/- compared with wild-type mice during the fibrotic changes will be analysed by transcriptomic analysis by next generation sequencing. Evidence from ageing animals suggests that fibrosis accumulates because myofibroblasts that initiate fibrosis to start tissue repair become resistant to apoptosis and so continue to secrete collagen. In younger animals these cells become senescent and are cleared to allow resolution of the scar. Transcriptomic results will be analysed to determine if genes involved in cell cycle control or proliferative signalling pathways are altered in persistently infected Stat1-/- mice in comparison to wild-type mice. Results will be confirmed by qRT-PCR, cell purification and immunofluorescent staining of infected tissues; this is to determine which particular cell type(s) contribute to the altered gene expression profile. Staining for viral products will be analysed for localisation with cells involved with the fibrotic lesions to determine if direct viral stimulation is necessary for pathology. Fibrotic diseases are increasingly important in our ageing population with many having no known aetiology. Using the two different mouse strains for comparison, the project above allows us to study the same injury signal in different host backgrounds to determine pathways to the fibrotic changes.

Link: http://www.vet.cam.ac.uk/directory/bab2@cam.ac.uk

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Colin Russell, Dept. of Veterinary Medicine

Mapping the risk of cross species influenza virus transmission

PhD Project Description: Assessing the pandemic risk posed by non-human influenza A viruses remains a complex challenge. The substantial diversity amongst influenza viruses in animals makes creating pandemic preparedness measures against all viruses infeasible. Thus there is a need to prioritize viruses of concern. Currently, influenza pandemic risk assessment is driven by a simple idea: non-human influenza viruses that cause sporadic human infections pose a greater risk than viruses that have not infected humans. However, this intuitive idea has little empirical support. Detecting influenza viruses of concern relies on systematic characterization of influenza viruses circulating in wild and domesticated animal populations. However, existing surveillance networks are largely ad hoc, varying substantially by host and geographical region, and with only a small proportion of the data entering the public domain. This project seeks to design non-human influenza surveillance strategies by using statistical analyses to determine levels of coverage by geographic region, host species, and human-animal interface risk factors. The first phase of the project will involve assembling a global database of known animal influenza virus occurrence, surveillance activities, and information on potential for human-animal interactions. The second phase of the project will systematically identify critical gaps in animal influenza virus surveillance by geographically mapping the distribution of animal influenza viruses, the surveillance activities that monitor them, and factors that could affect human to animal transmission. The project will involve the assembly and manipulation of large data sets, mapping using Geographic Information Systems (GIS) tools such as ArcGIS, and statistical modeling analyses using boosted regression trees and generalized linear models. Depending on the progress of the project there may also be opportunities to explore the use of mathematical models of influenza virus population dynamics.

Other relevant themes: Food Security

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Luca Pellegrini, Dept. of Biochemistry

PhD Project Description: The faithful inheritance of genetic information is essential to cellular life. Successful transmission of the genetic material requires the accurate replication of the genome and the repair of any DNA lesion that might block replication or alter the encoded message. Failure of the complex molecular systems that preserve the integrity of our DNA is a major contributing factor in human disease. The object of PhD projects in our laboratory is to understand structure and function of the macromolecular assemblies responsible for the maintenance and replication of our genome. We use the tools of structural biology to recover three-dimensional information of proteins and protein complexes important to DNA replication and repair, and we combine the structural evidence with biochemical, biophysical and computational analysis to reveal molecular mechanisms.

Referees:

1: Perera RL, Torella R, Klinge S, Kilkenny ML, Maman JD, Pellegrini L. Mechanism for priming DNA synthesis by yeast DNA Polymerase α. Elife. 2013 Apr 2;2:e00482. doi: 10.7554/eLife.00482.

2: Blackwood JK, Rzechorzek NJ, Abrams AS, Maman JD, Pellegrini L, Robinson NP. Structural and functional insights into DNA-end processing by the archaeal HerA helicase-NurA nuclease complex. Nucleic Acids Res. 2012 Apr;40(7):3183-96. doi: 10.1093/nar/gkr1157.

3: Klinge S, Hirst J, Maman JD, Krude T, Pellegrini L. An iron-sulfur domain of the eukaryotic primase is essential for RNA primer synthesis. Nat Struct Mol Biol. 2007 Sep;14(9):875-7.

Link: http://www.bioc.cam.ac.uk/people/uto/pellegrini

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Ole Paulsen, Dept. of Physiology, Development and Neuroscience

Second Supervisor/Collaborator: Dr Olivia Shipton, PDN

Lateralisation of hippocampal memory function in mice

PhD Project Description: The hippocampus is a cortical structure of importance for certain forms of memory. In humans, hippocampal function is lateralised, often assumed to be linked to the location of the language centre in the left hemisphere. However, it was recently discovered that there is a difference in synaptic plasticity between the left and right hippocampus in mice, raising the possibility of more fundamental functional differences between the two sides. The aim of the proposed project is to use optogenetics to unravel these differences. Optogenetics is a technique that enables cell type- and location-specific activation and silencing of neurons. Using this technique, we recently demonstrated that the magnitude of long-term potentiation of synaptic transmission (LTP) depends on whether the presynaptic input originates in the left or right hippocampus. In fact, whereas left input shows robust LTP, there is no LTP in the right input (Kohl et al., 2011). Moreover, when we silence left or right input during a spatial memory task in awake behaving mice, long-term memory is impaired following left silencing, but intact following right silencing (Shipton et al., unpublished). The aim of the PhD project is to use optogenetics to investigate further this left-right asymmetry in hippocampal function. Depending on the interests of the student, the research question addressed could be at any level from the molecular mechanisms of this synaptic left-right asymmetry to its neural circuit consequences and relation to behaviour.

Referees:

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, Leitz JR, Dworzak J, Acton CEJ, Tunbridge EM, Denk F, Dawson HN, Vitek MP, Wade-Martins R, Paulsen O and Vargas-Caballero M (2011) Tau protein is required for amyloid β–induced impairment of hippocampal long-term potentiation. J Neurosci 31: 1688-1692.

Shipton OA and Paulsen O (2013) GluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticity. Philos Trans B 369: 20130163.

Link: http://noggin.pdn.cam.ac.uk/

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Heike Laman, Dept. of Pathology

PhD Project Description: Fbxo7/PARK15 is an F-box protein that captures substrates for ubiquitination by SCF-type E3 ligases. We have recently shown that Fbxo7 regulates the autophagy (‘self-eating’) of mitochondria but the molecular basis for this is not known. To gain some insight into this process, we have conducted high-throughput proteome microarrays and yeast two hybrid screens to identify novel, ubiquitinated substrates of Fbxo7 and discovered several protein components that are potential key regulators of this. The student will perform functional validation of these proteins involved in mitochondrial biology, and can take advantage of model systems in the lab including cultured cell lines, differentiation of stem cells, and mouse models and use biochemistry, cell biology and live cell imaging techniques. Our goal is to determine how Fbxo7-mediated ubiquitination of these proteins regulates mitochondrial turnover and dynamics.

Referees:

1- D. E. Nelson, S. J. Randle and H. Laman. 2013. Beyond ubiquitination: The atypical functions of Fbxo7 and other F-box proteins. Open Biol. 3:130131. http://dx.doi.org/10.1098/rsob.130131.

2- V. S. Burchell*, D. E. Nelson*, A. Sanchez-Martinez*, M. Delgado-Camprubi, R. M. Ivatt, J. H. Pogson, S. J. Randle, S. Wray, P. A. Lewis, H. Houlden, A. Y. Abramov, J. Hardy, N. W. Wood, A. J. Whitworth&, H. Laman& and H. Plun-Favreau&. 2013. The Parkinson’s disease genes Fbxo7 and parkin interact to mediate mitophagy. Nat Neurosci. 2013 Aug 11. doi: 10.1038/nn.3489. *Equal first authorship. &Equal senior authorship.

3- D. E. Nelson and H. Laman. 2011. A competitive binding mechanism between Skp1 and Exportin (CRM1) controls the localisation of a subset of F-box proteins. J Biol Chem. 286(22):19804-15.

Link: http://www.path.cam.ac.uk/research/investigators/laman/research.html

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Prof Nicky Clayton, Dept. of Psychology

Are jays' cache protection strategies sensitive to observers' internal states?

PhD Project Description: Both Western scrub jays and Eurasian jays, like many other members of the corvid family (crows), use a variety of different strategies to protect their caches from observers who might later steal their caches. The strategies employed by caching jays appear to be sensitive to the sensory abilities of observers, for example when an observer can see and hear the caching event, cachers place their caches further away from the observer, preferentially cache in shaded areas or in out-of-sight locations, suggesting that the cacher might be sensitive to what the observer can and cannot see. In contrast, when an observer cannot see but can hear the caching event, cachers place caches preferentially in quiet substrate and thus limit the probability that the observer will be alerted to the caching event, suggesting that they might be sensitive to what the observer can and cannot hear. However, it is yet unknown whether cachers can take into account observers' internal states, such as desires and beliefs, when protecting their own caches from being stolen. Recent studies have shown that Eurasian jay males can take into account their female partner's desires for different foods when sharing food with her during their courtship feeding. Thus, these birds might be able to ascribe desires to others. The current project aims to investigate whether this ability is specific to the food-sharing behaviour or can also be shown within the context of food-caching. Even though observers can memorise the location of another jay's cache, they are most likely to pilfer the cache very soon after the caching event. Thus, the observer's desire for different foods might affect their motivation to pilfer caches. If cachers can take into account the observer's desire for different foods, they might be able to protect their caches more effectively, for example by caching food that the observer currently does not desire.

Referees:

Ostojić, L., Shaw, R. C., Cheke, L. G. & Clayton, N. S. (2013). Evidence suggesting that desire-state attribution may govern food sharing in Eurasian jays. Proc. Natl. Acad. Sci 1101, 4123-4128.

Ostojić, L., Legg, E. W., Shaw, R. C., Cheke, L. G. & Clayton, N. S. (2014). Can male Eurasian jays disengage from their current desire to feed the female what she wants. Biology Letters, 10, 20140042.

Clayton, N. S. (2014). EPS Mid Career Award Lecture. Ways of Thinking: From Crows to Children and Back Again. Quarterly Journal of Experimental Psychology.

Link: http://www.memlab.psychol.cam.ac.uk/index.html www.lucycheke.com http://www.psychol.cam.ac.uk/ccl

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Dr Laura Itzhaki, Dept. of Pharmacology

PhD Project Description: Our research focuses on a class of proteins with very distinctive architectures, known as tandem-repeat proteins. Tandem-repeat proteins, which include ankyrin repeats, tetratricopeptide repeats, armadillo repeats and HEAT repeats, are frequently deregulated in human diseases including cancer and respiratory and cardiovascular diseases. The individual modules of repeat proteins stack in a linear fashion to produce highly elongated, superhelical structures, thereby presenting an extended scaffold for molecular recognition. The term ‘scaffold’ implies a rigid architecture; however, as suggested by their Slinky spring-like shapes, it is proposed that repeat arrays utilise much more dynamic and elastic modes of action. For example: stretching and contraction motions to regulate the activity of a bound enzyme; reversible nanosprings to operate ion channels; proteins that wrap around their cargoes to transport them in and out of the nucleus. Thus, repeat proteins have been proposed to be a distinct structural class midway between globular structured proteins and intrinsically disordered proteins. The modular architecture of repeat proteins makes them uniquely amenable to the dissection of their biophysical properties as well as the rational redesign of these properties. We are interested in understanding how the process of folding and unfolding of this distinctive class of proteins directs their functions in the cell. We are also looking at small molecule and peptide-based approaches to target these proteins and their binding partners for therapeutic benefit. Lastly, we are exploring the fabrication of novel self-assembling repeat-protein nanomaterials. PhD projects will combine protein engineering with a range of biophysical and biochemical methods.

Referees:

Tsytlonok, M., Craig, P.O., Sivertsson, E., Serquera, D., Perrett, S., Best, R.B., Wolynes, P., Itzhaki, L.S. Complex energy landscape of a giant repeat protein. Structure 21: 1954-65 (2013). Previewed in “Falling Down: landscape and kinetics of one-dimensional protein folding”. Neira, J.L. Structure 21: 1905-7 (2013).

Javadi Y., Itzhaki L.S. Tandem-repeat proteins: regularity plus modularity equals design-ability. Curr. Opin. Struct. Biol. 23: 622-31 (2013). Werbeck, N.D., Rowling, P.J., Chellamuthu, V.R., Itzhaki, L.S. Shifting transition states in the unfolding of a large ankyrin repeat protein. Proc. Natl. Acad. Sci. USA 105: 9982-7 (2008). See Commentary: 'The capillarity picture and the kinetics of one-dimensional protein folding'.

Ferreiro, D.U. & Wolynes, P.G. Proc. Natl. Acad. Sci. USA 105: 1893-4 (2008).

Link: http://www.phar.cam.ac.uk/research/Itzhaki/research

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Wolf Reik, Babraham Institute

PhD Project Description: 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 at the nearby Sanger Institute (http://www.sanger.ac.uk/research/projects/singlecellcentre). 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.

Referees:

Smallwood SA, Lee HJ, Angermueller C, Krueger F, Saadeh H, Peat J, Andrews SR, Stegle O, Reik W & Kelsey G (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nature Methods 11: 817-820 Lee HJ, Hore, TA & Reik W (2014) Reprogramming the methylome: erasing memory and creating diversity. Cell Stem Cell 14: 710-719

Ficz G, Hore TA, Santos F, Lee HJ, Dean W, Arand J, Krueger F, Oxley D, Paul YL, Walter J, Cook SJ, Andrews S, Branco MR & Reik W (2013) FGF signalling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13: 351-359 Seisenberger S, Andrews S, Krueger F, Arand J, Walter J, Santos F, Popp C, Thienpont B, Dean W & Reik W (2012) The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol Cell 48: 849-862

Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, Marques CJ, Andrews S & Reik W (2011) Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473: 398-402 Popp C, Dean W, Feng S, Cokus SJ, Andrews S, Pellegrini M, Jacobsen SE & Reik W (2010) Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency. Nature 463: 1101-1105

Link: www.babraham.ac.uk/our-research/epigenetics/reik www.sanger.ac.uk/research/projects/singlecellcentre

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Magdalena Zernicka-Goetz, Dept. of Physiology, Development and Neuroscience

PhD Project Description: The development of an embryo takes place through a series of morphogenetic movements and cell fate decisions. Implantation is a crucial event in mammalian pregnancy that generates the basic body plan. How a simple group of pluripotent cells develops to build a body has remained a key question to which an answer is largely unknown. This is because this developmental transition happens when embryo implants and so becomes hidden from observations and manipulations within the body of the mother. We have recently established a culture system that allows us to film this transition as it happens in living embryos, outside the body of the mother (Morris et al., Nature Comm 2012; Bedzhov et al., Nature Protocols, 2014). This has revealed that pluripotent cells of the embryo self-organise into a rosette-like structure from which the future body will emerge (Bedzhov and Zernicka-Goetz, Cell 2014). Moreover, we can instruct ES cells to mimic this process when culture in the 3D system. The aim of this PhD is to discover the mechanism, on cellular and molecular level, of this self-organisation using both embryos and ES cells developing in vitro. Specifically: (1) in order to understand how morphogenesis happens, ES cells carrying fluorescent protein tagged transgenes, that mark different cell types, will be introduced into blastocysts and their fate will be followed by the time-lapse fluorescent microscopy and 4D analyses. (2) in order to understand the role of Wnt, BMP and Nodal signalling pathways at this developmental transition, mutant ES cells in such pathways will be introduced into blastocysts to follow the consequences for morphogenesis and gastrulation. This PhD project will establish the thus far hidden sequence of events leading to gastrulation and the mechanisms of this key developmental transition.

Referees:

Morris et al Nature Communication 2012 (Zernicka-Goetz group)

Bedzhov and Zernicka-Goetz, Cell 2014 (Zernicka-Goetz group)

Bedzhov et al Nature Protocols 2014 (Zernicka-Goetz group)

Link: http://www2.gurdon.cam.ac.uk/~zernickagoetzlab/

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Professor Jonathan L Heeney, Dept. of Veterinary Medicine

Second Supervisor/Collaborator: Richard Leggett, BBSRC The Genome Analysis Centre, Paul Kellum, Sanger Institute.

Viral discovery in complex diseases of humans and animals

PhD Project Description: 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 pathological 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. The PhD student 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.

Referees:

Daly GM, Leggett RM, Rowe W, Stubbs S, Ramirez-Gonzalez R, Caccamo M, Bernal W, Heeney JL. Host subtraction, filtering and assembly validations for novel viral discovery in illumina next generation sequencing data. PLOSone, in press.

Daly, G., Bexfield, N., Heaney, J., Stubbs, S., Mayer, A.P., Palser, A., Kellam, P., Drou, N., Caccamo, M., Tiley, L., Alexander, G.J.M., Bernal, W., Heeney, J.L. (2011). A viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing. PLoS One. Dec; 6(12):e28879.

Varela, M., Landskron, L., Lai, R.P.J., McKinley, T.J., Bogers, W.M.J.M., Verschoor, E.J., Dubbes, R., Barnett, S.W., Frost, S.D.W., Heeney, J.L. (2011). Molecular evolution analysis of the Human Immunodeficiency Virus type 1 envelope in SHIV-infected macaques: implications for challenge dose selection. Journal of Virology. Oct;85(19), pp.10332-45.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.vet.cam.ac.uk/directory/jlh66@cam.ac.uk

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Professor Jonathan L Heeney, Department of Veterinary Medicine

Second Supervisor/Collaborator: Dr Marc Veldhoen, BBSRC's Babraham institute.

Professor of Comparative Pathology and head of the Lab of Viral Zoonotics

PhD Project Description: The gut mucosa is the largest (roughly 400 m2) surface area in the body that is in direct contact with the microbial enteric environment, and importantly, the immune system. Disturbances in the microbiota have consequences for gut immunity and the response to viral infections. The aims of the project are to understand how the microbiota influences enteric immunity to viral infections and how dysregulation may predispose to certain types of viral infections. The specific objectives of the project are to understand how age and disturbances in the bacterial community of the gut influence the innate immune system, and how it affects responds to RNA viral infections. We have found that specific triggering of intra-epithelial lymphocytes (IELs) can afford protection of mice from murine norovirus infection by up-regulating type I, II, & III interferons in IEFs and have demonstrated that mice pre-stimulated are completely protected from RNA viral (MNV) infection. This project will explore the effect of age and microbial changes that occur during antibiotic use to understand the mechanisms of why susceptibility to RNA viruses increases. This project is at the interface of microbial genomics and immune system interaction.The techniques involved will include experimental immunology, deep sequencing, bioinformatics, ex-vivo immune analysis and virus titre analysis from in vivo exposure to different RNA viruses.

Referees:

Veldhoen, M. Heeney, JL. (2014) A helminth-mediated viral awakening. Trends in Immunology Oct;35(10);452-3. * Shortland A, Chettle J, Archer J, Wood K, Bailey D, Goodfellow I, Blacklaws BA, Heeney JL. (2014). Pathology caused by persistent norovirus infection. J Gen Virol. Feb;95(Pt 2):413-22.

Sholukh AM, Watkins JD, Vyas HK, Gupta S, Lakhashe SK, Thorat S, Zhou M, Hemashettar G, Bachler BC, Forthal DN, Villinger F, Sattentau Q, Weiss RA, Agatic G, Corti D, Lanzavecchia A, Heeney JL, Ruprecht RM. Defense-in-depth by mucosally administered anti-HIV dimeric IgA2 and systemic IgG1 mAbs: complete protection of rhesus monkeys from mucosal SHIV challenge. Vaccine, in press.

Other relevant themes: World Class Underpinning Bioscience

Link: http://www.vet.cam.ac.uk/directory/jlh66@cam.ac.uk

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Dr Matthew Harper, Department of Pharmacology

Platelet death signalling in haemostasis

PhD Project Description: The balance between cell death and cell survival is essential to organism homeostasis and both must be tightly regulated. Cell death can be controlled by regulated death programs including apoptosis and several distinct regulated necrosis pathways. Blood platelets are an essential element in the primary haemostatic system that prevents bleeding. Platelet count is a delicate balance between platelet production and platelet death. Circulating platelets live for only ten days in humans, and approximately 100 billion platelets are made and destroyed every day. Platelet lifespan appears to be controlled by intrinsic apoptosis. In contrast, regulated necrosis and phosphatidylserine exposure in activated platelets may contribute to their role in haemostasis. Platelet death pathways must therefore be tightly controlled to maintain a constant platelet count and normal haemostasis. This project aims to understand cell death signalling in platelets. What triggers different platelet death pathways? How are platelet death pathways controlled by intracellular signalling? What are the roles of different regulated death programs in platelet physiology? To address these questions, detailed analyses of molecular and cellular mechanisms of platelet death in vitro will be combined with physiological analysis in vivo. This project will enhance our understanding of haemostasis. Moreover, the conserved nature of these death pathways makes this of relevance to a broad range of cells throughout the body.

Referees:

Procoagulant platelets: are they necrotic? Jackson SP, Schoenwaelder SM. Blood (2010) 116: 2011-8.

Chloride channels are necessary for full platelet phosphatidylserine exposure and procogulant activity. Harper MT, Poole AW. Cell Death & Disease (2013) 4: e969.

Transient receptor potential channels function as a coincidence signal detector mediating phosphatidylserine exposure. Harper MT et al. Science Signaling (2013) 6: ra50

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Guy Brown, Dept. of Biochemistry

How nutrition affects phagocytosis of cells in development, ageing and neurodegeneration

PhD Project Description: Overnutrition results in age-related pathology, including dementia, and may accelerate ageing itself. Understanding how this occurs at the cellular level may enable treatments for overnutrition and related pathologies. This project will investigate how overnutrition affects phagoptosis. Phagoptosis is a type of cell death, caused by phagocytosis of the cell, and thus is prevented by blocking phagocytosis. Phagocytosis of an otherwise-viable cell may occur because the cell is stressed, activated, senescent, damaged, recognised as non-self or misrecognised. And phagoptosis is greatly enhanced during inflammation. This project will investigate how nutrients and nutrient signalling affect phagocytes (including macrophages and microglia) and target cells, to cause aberrant phagocytosis of cells. For example, we will investigate whether: i) overnutrition activates aberrant phagocytosis of neurons and synapses by microglia in part by activating the NADPH oxidase, and ii) overnutrition stresses target cells promoting their phagocytosis in part by modifying surface sugars. The project will use cell culture, transgenic animals, fluorescence microscopy and molecular cell biology.

Referees:

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

Vilalta A & Brown GC (2014) Deoxyglucose prevents neurodegeneration in culture by eliminating microglia. J Neuroinflammation. 11:58.

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.

Other relevant themes: Food Security

Link: www.guybrown.net

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