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PhD Projects for World Class Underpinning Bioscience

Viji Draviam, Dept. of Genetics

Chromosome capture mechanisms

PhD Project Description: Chromosomal stability relies on the proper attachment of chromosomes to microtubule-ends. We reported the first visualisation of how human chromosomes that are first captured along microtubule walls become ultimately tethered to the ends of microtubules. We termed this multi-step process as the end-on conversion process. We have performed an RNAi screen to identify proteins crucial for the various steps of the end-on conversion process. By studying the role of these candidates using high-resolution cell biology and biochemistry tools the student will elucidate how the end-on conversion process is accomplished. The ultimate goal is to demonstrate how the end-on conversion process prevents chromosomal instability (CIN) and thus understand the cause for CIN in cancers.

Referees:

1. Lateral to End-on conversion of chromosome-microtubule attachment requires kinesins CENP-E and MCAK. Shrestha R R L and Draviam V M. (2013) Current Biology. 23: 1-13

2. Microtubule plus-ends within a mitotic cell are ‘moving platforms’ with anchoring, signaling and force-coupling roles. Tamura N and Draviam V M. (2012) Open Biol. 2: 120132

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://sitka.gen.cam.ac.uk/~vmd20/

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Florian Hollfelder, Dept. of Biochemistry

Functional Metagenomics in Picodroplets: Exploring Sequence Space Using Chemical Baits

PhD Project Description: A much broader range of enzyme catalysts than currently known is needed to address a number of challenges – from the implementation of green, sustainable processes in white biotechnology to a fundamental understanding of the evolutionary origin and mechanistic basis of biocatalysis. Microbial ecosystems are viewed as enormous reservoirs of genetic diversity, but 99% of environmental microorganisms are not culturable. Given how difficult it is to read the function of a gene from its sequence, experimental characterisation is the preferred basis of functional annotation of proteins, but expensive and cumbersome. The Figure (if it can be seen in this entry) shows how we are now exploring the hidden diversity of unknown functional proteins, using a high throughput (HT) system of unprecedented capacity. We screen libraries of environmental DNA with a throughput of >106. This system is much cheaper and more convenient than e.g. robotic screening with expensive liquid handling systems. The throughput is also large enough that we beat the odds and find rare catalytic activities. We have just demonstrated this for one specific activity, phosphotriester hydrolysis, and almost doubled the number of known triesterases. Most new triesterases could not have been predicted by bioinformatic methods, because they go by sequence similarity: since we use functional screening the hits are not limited to close sequence neighbours of enzymes with known functions. Indeed, our hits spanned four protein superfamilies and yielded mechanistic arrangements that have not been seen before. Based on this work (just being completed by PhD student Pierre-Yves Colin) we now want to use a range of chemistries – reactions with fluorescence (or absorbance) readouts that can be monitored optically in droplets – to systematically understand where in sequence space chemical potential is located. The choice of the bait substrate alone determines which catalytic processes are identified and systematic exploration of libraries with assays for new substrates will be useful to exhaustively harvest many different classes of enzymes. Of particular interest will be to explore whether enzymes have multiple activities (i.e. are promiscuous): this ‘promiscuome’ (the collection of promiscuous activities from complex meta-proteomes) is completely unpredictable by bioinformatics, but constitutes the chemical ‘raw material’ for evolution. The systematic detection of such catalytic starting points can form the basis of evolutionary models that account for the adaptive potential of a microbial community to degrade foreign compounds, such as pesticides and antibiotics and our hits may also constitute alternative starting points for adaptive evolution. Training. The project involves some well-established microfluidic techniques that can be learned in the first half year. For the design of the bait chemistries and the mechanistic and structural analysis of the identified hits, training in chemistry, chemical biology, bioinformatics and biochemistry will be provided (and any previous experience is useful).

Referees:

• Kintses, B.; Hein, C.; Mohamed, M. F.; Fischlechner, M.; Courtois, F.; Laine, C.; Hollfelder, F., Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. Chem Biol 2012, 19 (8), 1001-9. • Fischlechner, M.; Schaerli, Y.; Mohamed, M. F.; Patil, S.; Abell, C.; Hollfelder, F., Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nature Chem 2014, 6 (9), 791-6.

• Babtie, A. C.; Tokuriki, N.; Hollfelder, F., What makes an enzyme promiscuous? Curr. Op. Chem. Biol 2010, 14 (2), 200-7. • van Loo, B.; Jonas, S.; Babtie, A. C.; Benjdia, A.; Berteau, O.; Hyvönen, M.; Hollfelder, F., An efficient, multiply promiscuous hydrolase in the alkaline phosphatase superfamily. Proc. Natl Acad. Sci USA 2010, 107 (7), 2740-5.

• P.-Y Colin, B. Kintses, F Gielen, C Miton, M Mohamed, D P. Morgavi, D B. Janssen and F Hollfelder., Ultrahigh-throughput Discovery of Promiscuous Enzymes by Picodroplet Functional Metagenomics, submitted.

Other relevant themes: Basic Biosciences Underpinning Health & Bioenergy and Industrial Biotechnology

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

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Karen Lipkow, Babraham Institute

Mechanisms of 3D Genome Dynamics

PhD Project Description: The questions of how genes are regulated remains fundamental even after decades of intense study. Rather than just investigating the linear, one-dimensional sequence of DNA to understand regulatory mechanisms, we can now generate genome-wide data to investigate the complex 3-dimensional organisation of whole genomes [1]. Correlating this 3D organisation with gene expression has resulted in significant advances in understanding gene regulation, in healthy, diseased and aged cells [2]. The next level of complexity derives from the fact that genome organisation is not static, but highly dynamic. These dynamics have been observed to coincide with gene regulatory events [3], and their study is thus fundamental to fully understanding gene regulation. Yet, the molecular mechanisms that drive these changes in genome architecture remain largely unknown. To elucidate the mechanisms that drive the dynamics of chromosome structure and occupancy, we are using a systems biology approach, combining cutting-edge experimental and computational methods. A number of projects are available, depending on the interests and backgrounds of the student. You will receive training in HiC, ChIP-seq, quantitative live-cell microscopy, microfluidics, bioinformatics and/or detailed simulations, and have the opportunity to collaborate on the development of a novel method with a local microfluidics company. You will be part of the Babraham Institute, a BBSRC-funded research institute just outside Cambridge, which houses world-leaders in Nuclear Dynamics, Epigenetics, Signalling and Computational Biology in a highly interactive setting, and of the Cambridge Systems Biology Centre. Please contact Karen Lipkow for further information: KL280@cam.ac.uk.

Referees:

1. Lieberman-Aiden, E. et al. Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome. Science 326, 289–293 (2009).

2. Chandra, T. et al., Global Reorganization of the Nuclear Landscape in Senescent Cells. Cell Reports (2015)

3. Noordermeer, D. et al. The dynamic architecture of Hox gene clusters. Science 334, 222–225 (2011).

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.babraham.ac.uk/our-research/nuclear-dynamics/karen-lipkow; http://lipkow.sysbiol.cam.ac.uk

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Professor Eric Miska, Dept. of Genetics

Second Supervisor/Collaborator: Dr Martin Hemberg - Wellcome Trust Sanger Institute

Transgenerational epigenetic inheritance in C. elegans

PhD Project Description: A central aspect of neo-Darwinian evolutionary theory is that only the information that is stored as nucleotide sequences in the germline DNA is inherited by the offspring. In recent years, however, this dogma has been challenged as there is increasing evidence of epigenetically stored information being inherited, both in humans, animals and plants. The nematode C. elegans is an ideal model system for studying transgenerational epigenetic effects: powerful genetic tools (e.g. CRISPR), a generation time of only three days and a genome 1/30 the size of the human genome. The goal of this project is to investigate how epigenetic properties (e.g. gene expression levels and histone modification patterns) are determined by the environmental conditions of a worm's ancestors (e.g. food levels and stress conditions) at the level of individuals. This project will involve both experimental work to collect genomic data from C. elegans, as well as computational work in developing algorithms and statistical frameworks that will allow us to identify inherited epigenetic features.

Referees:

Ashe, A., Sapetschnig, A., Weick, E.-M., Mitchell, J., Bagijn, M. P., Cording, A. C. & Miska, E. A. (2012). piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans. Cell, 150(1), 88–99.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://ericmiskalab.org

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Catherine Lindon, Dept. of Pharmacology - will be taking up appointment in Sept 2015

PhD Project Description: The Lindon group studies regulation of the cell cycle, focusing on processes required to rebuild interphase after mitosis. For example each daughter cell must adhere to its surroundings, assemble a nucleus around the chromatids it has inherited, and construct networks of organelles (mitochondria, Golgi etc) that fragmented during mitosis. Many events at this time are controlled by ubiquitin-mediated proteolysis orchestrated by the ubiquitin ligase known as Anaphase Promoting Complex (APC/C). Recently we have described the mitotic exit ‘ubiquitome’, a high confidence dataset of proteins targeted by ubiquitination at this transition in the cell cycle. Some of the most interesting hits in our ubiquitome dataset are proteins not previously known to be regulated by specific ubiquitination during mitotic exit, including the nuclear lamins (Lamin A, Lamin B1, Lamin B2) that assemble in networks under the nuclear envelope and influence chromatin organization within the interphase nucleus. We are offering a PhD project that will use a combination of biochemical approaches (including identifying ubiquitination sites on lamins by mass specrometry) and cutting-edge fluorescence imaging approaches (including FRET) to study ubiquitination of lamins in human cells. The project will aim to (1) elucidate APC/C-dependent or –independent pathways controlling ubiquitination of lamins during mitotic exit (2) establish the localization and fate of the ubiquitinated lamins and (3) understand their contribution to the interphase cell. We will be moving to the Department of Pharmacology during 2015.

Referees:

Min M., Mayor U., Dittmar G. and Lindon C. (2014). Using in Vivo Biotinylated Ubiquitin to Describe a Mitotic Exit Ubiquitome from Human Cells. Molecular & Cellular Proteomics 13 2411–2425

Martin C., Chen S. and Jackson D. (2010). Inheriting nuclear organization: can nuclear lamins impart spatial memory during post-mitotic nuclear assembly? Chromosome Res 18:525–541

Other relevant themes: Basic Biosciences Underpinning Health

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

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Professor George Salmond, Dept. of Biochemistry

Bacterial toxin-antitoxin systems, anti-viral abortive infection, "altruistic suicide", and viral host range in a plant pathogen

PhD Project Description: We are working on Gram-negative bacterial plant pathogens (Pectobacterium and Dickeya) the former of which carries a plasmid encoding a Type III toxin-antitoxin (TA) system. This bacterial TA system is activated by bacteriophage infection leading to precocious death of the infected host in a process akin to a prokaryotic "apoptosis". In effect, the infected bacterium is driven into a suicidal response that could be viewed as altruistic because suicide also prevents productive viral replication and release of progeny virus that could go on to kill the bacterial clonal population. Why should a bacterium carry such a Type III TA system, other than for this altruistic model? We aim to test the notion of altruistic suicide. We are interested in the fitness impacts of retaining the Type III TA system both when bacteria are infected by phages and when they are not. We are also investigating how viral mutants evolve spontaneously to escape the abortive infection systems and studying which viral gene products interact (directly or indirectly) with the TA systems, post-infection. We are also interested in the nature of particular bacterial surface receptors for specific phages (viunalikevirsues) and how such receptors and their cognate viral proteins may be manipulated for viral host range extension; with possible phage therapy and synthetic biology implications. This project links various aspects of evolutionary biology, through genetics and molecular biology and environmental microbiology, to phage therapy, synthetic biology, ecology and plant pathology! (see URL link for more information on this and other projects).

Referees:

1. Short, F. et al (2013) Selectivity and self-assembly in the control of a bacterial toxin by an antitoxic noncoding RNA pseudoknot. PNAS, 110 (3) E241–E249. First on-line December 2012, doi:10.1073/pnas.1216039110

2. Blower, TR., et al (2012) Viral evasion of a bacterial altruistic suicide system through RNA-based molecular mimicry enables infectious altruism. PloS Genetics, 8(10): e1003023. doi:10.1371/journal.pgen.1003023

3. Matilla, M., Fang, X. and Salmond, GPC (2014) Viunalikeviruses are environmentally common agents of horizontal gene transfer in pathogens and biocontrol bacteria. The ISME Journal. 8, 2143-2147 doi:10.1038/ismej.2014.150

Other relevant themes: Food Security

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

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Peter Fraser, Babraham Institute

Analysing 3D organisation of the genome using statistical machine learning

PhD Project Description: Recent advances in next generation sequencing technology and Hi-C, a chromosome conformation capture technique [1], has spawned a promising tool to reveal contacts between DNA/chromatin segments genome-wide. In a population Hi-C experiment, a contact map is obtained that reflects the probability of contacts in a population of millions of cells. Such a contact map can be used to construct probabilistic 3D models of chromatin and chromosomes. As chromosome conformation shows remarkable cell-to-cell variability, population Hi-C contact maps have been used to generate an ensemble of 3D structures by inferring an underlying physics-based chromatin model using machine learning techniques. In dynamic processes, though, where a constant physical model cannot be assumed, single cell Hi-C experiments, developed in our lab [2], provide contact information that reflect actual contacts in individual single cells. Single cell contact maps can be used to construct 3D models of chromatin conformation using constrained optimisation techniques, enabling further analysis of the chromatin structure and function. The aim of this project is to analyse 3D organisation of the genome, using statistical machine learning techniques for Hi-C-data-driven chromatin modelling. Potential projects include 3D modelling of single-cell Hi-C contact data, and the exploration of folding landscapes of selected, interesting chromatin domains (Mb-size building blocks of chromatin) using nested sampling, a novel Bayesian algorithm previously used in protein folding [3].

Referees:

[1] Lieberman-Aiden et al., Science 326:289-293 (2009)

[2] Nagano et al., Nature 502:59-64 (2013)

[3] Burkoff et al., Biophys J. 102:878-886 (2012)

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.babraham.ac.uk/our-research/nuclear-dynamics/peter-fraser

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Bénédicte Sanson, Dept. of Physiology, Development and Neuroscience

Investigating the orientation of cell divisions in developing epithelia

PhD Project Description: During the embryonic growth and development of animals, thousands of cells must take up their correct position in the embryo and organise themselves into tissues and organs with complex shapes, a process called morphogenesis. Our group investigates the cellular and molecular mechanisms underlying the morphogenesis of epithelial tissues. We use early Drosophila embryos as a model because this multicellular organism is excellent both for genetics and in vivo imaging. In the past years, we have focused on investigating how cells move and change shape during axis extension at gastrulation (Butler et al 2009) and how cells are partitioned into compartments that do not mix during segmentation (Monier et al., 2010). Our approaches include genetics, in vivo imaging, cell tracking and computational analysis. We are particularily interested to understand how the genetic programs interact with the physical context of the embryo to shape cells and tissues. Several projects are available in the lab, including one on the orientation of cell division in the epithelium. The orientation of the cell divisions along the plane of the epithelium can influence the morphogenesis of the tissue (Morin and Bellaiche, 2011). Both polarity cues and tensile forces in tissues have been implicated in the control of oriented cell division, however it is still unclear how these factors integrate in multicellular organisms. We have discovered opposite biases in cell division orientation in the developing epithelium and the investigation of the origin of these biases can form the basis for a PhD project.

Referees:

1) Butler, L. C., Blanchard, G. B., Kabla, A. J., Lawrence, N. J., Welchman, D. P., Mahadevan, L., Adams, R. and B. Sanson (2009). Cell shape changes indicate a role for extrinsic tensile forces in Drosophila germ-band extension. Nature Cell Biology, 11(7), 859–864. doi:10.1038/ncb1894

2) Monier B, Pélissier-Monier A, Brand AH, Sanson B (2010). An actomyosin-based barrier inhibits cell mixing at compartmental boundaries in Drosophila embryos. Nature Cell Biology; 12: pp60-65. doi:10.1038/ncb2005.

3) Morin, X., & Bellaïche, Y. (2011). Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development. Developmental cell, 21(1), 102–119. doi:10.1016/j.devcel.2011.06.012

Other relevant themes: Basic Biosciences Underpinning Health

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

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Martin Hemberg, Wellcome Trust Sanger Institute

Inference of gene regulatory networks from single-cell RNA-seq data

PhD Project Description: Since Watson and Crick first derived the structure of DNA, our knowledge of the genomes has expanded immensly, and today we have identified most, if not all protein coding genes in the human genome. Moreover, molecular biologists have been able to characterize the function of many of these genes, allowing us to understand what happens when a gene is active. However, one aspect of the genome which remains poorly understood is *how* a gene is activated. High-throughput techniques for studying the transcriptome provide us with a method for studying these questions in a global manner. By measuring expression levels at several consecutive time-points, it is possible to infer regulatory relationships. The underlying assumption being that if two genes change their expression in a similar manner, then they are likely to have similar regulatory mechanisms. To date, there are several methods which can be used to infer regulatory relationships from bulk RNA-seq data. However, single-cell RNA-seq [1] provides additional information about the relationship between different genes, since we can estimate distributions of expression levels and not just the means. Furthermore, many established methods rely on results from information theory [2], and these techniques are likely to be even more powerful when combined with single-cell data. The goal of this project is to take full advantage of the data provided by single-cell RNA-seq experiments in order to infer regulatory relationships. Starting from a stochastic model of gene expression, the aim is to develop a statistical procedure and a computational framework to identify regulatory relationships.

Referees:

[1] Junker and van Oudenaarden, Cell, 157 (1), p8-11, 2014.

[2] Margolin et al, BMC Bioinformatics, 7:57, 2006.

Other relevant themes: Basic Biosciences Underpinning Health

Link: https://www.sanger.ac.uk/research/faculty/mhemberg/

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Professor Geoffrey L Smith FRS, Dept. of Pathology

Rapid spreading and immune evasion strategies of vaccinia virus

PhD Project Description: Vaccinia virus (VACV) is a poxvirus that replicates in the cell cytoplasm, encodes about 200 genes and is an excellent model for studying virus-host interactions. Its study has provided fundamental information about cell biology, immunology and how viruses cause disease. Two PhD project options are offered. The first concerns how vaccinia virus exploits microtubule- and actin-based motility to spread rapidly within and between cells (see Doceul et al Science 2010). Newly formed VACV particles use microtubules and the motor complex kinesin-1 to reach the cell surface. Recently, we reported that the VACV proteins E2 and F12 form a complex to engage the kinesin-1 motor via interaction with kinesin light chain isoform 2 (Carpentier et al., PLoS Pathogens, 2015). The project will be to further characterise the roles of these virus proteins in virus egress and determine the order in which they are recruited and how they subvert the function of kinesin-1 to transport virions. The project will be cell biological in nature and make extensive use of recombinant viruses and electron, video and fluorescent microscopy. The second concerns how VACV inhibits innate immunity (review, Smith et al., JGV 2013). VACV expresses scores of inhibitors of innate immune signalling and their study is contributing to knowledge of innate immunity and how this interfaces with the adaptive immune response. The project will focus on a family of VACV proteins that resemble Bcl-2 and determine the structure, binding partners, mechanism of action and contribution to immune evasion of these proteins.

Referees:

1. Doceul, V., Hollinshead, M., van der Linden, L. & Smith, G.L. (2010). Repulsion of superinfecting virions: a mechanism for rapid virus spread. Science, 327, 873-7.

2. Ferguson, B.J., Mansur, D., Peters, N.E., Ren, H. & Smith, G.L (2012). DNA-PK is a DNA sensor for IRF-3 dependent innate immunity. eLife 1, 0047. [56]

3. Carpentier, D.C.J., Gao, W.N.D., Ewles, H., Morgan, G.W. & Smith, G.L. (2015). Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex. PLoS Pathogens. In press.

Other relevant themes: Basic Biosciences Underpinning Health

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

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Ykä Helariutta, Sainsbury Laboratory

Cell patterns matter in plant vascular development

PhD Project Description: Helariutta group studies molecular control of plant vascular development with specific aim at understanding how proper cell patterning is established. Our model system is Arabidopsis primary root, where differentiated vascular tissues are composed of a central xylem axis and two phloem poles. These tissues are separated by procambium, the undifferentiated vascular stem cells. Vascular system is crucial for long-distance transport of water, nutrients and various other components; this specialized tubing also provides support to the plant. Earlier, we have shown that phytohormones cytokinin and auxin regulate vascular patterning in an interactive manner. Along these lines, we use genetic and molecular approaches to search for novel components that may participate in cytokinin and auxin signaling pathways and thus modify vascular patterning. One of the components in cytokinin signaling pathway is AHP6, an inhibitory pseudophosphotransfer protein, that is expressed in specific xylem cell type in the primary root and positively regulated by auxin. To further elucidate regulatory systems underlying vascular development, we perform genetic screens in cytokinin signaling sensitized backgrounds to look for AHP6 misexpression in Arabidopsis primary root. Similarly, we utilize another molecular fluorescent marker for phloem cells in a separate forward genetics approach. Novel mutants with potentially informative misexpression patterns are identified and characterized using a wide range of molecular and histological techniques, bright field and fluorescence/confocal microscopy. Additional approaches involving big data processing, such as genome sequencing, RNAseq and ChIP-seq, are utilized for mutant characterization when applicable.

Referees:

Furuta, K.M., Yadav,S.R., Lehesranta,S., Belevich, I., Miyashima, S., Heo, J., Vatén, A., Lindgren, O., De Rybel, B., Van Isterdael, G., Somervuo, P., Lichtenberger, R., Rocha, R., Thitamadee, S., Tähtiharju, S., Auvinen, P., Beeckman, T., Jokitalo, E., Helariutta, Y. (2014) Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation. Science 345:933-937.

Furuta KM, Hellmann E, Helariutta Y. (2014) Molecular control of cell specification and cell differentiation during procambial development. Annu Rev Plant Biol. 65:607-38.

Bishopp A, Help H, El-Showk S, Weijers D, Scheres B, Friml J, Benková E, Mähönen AP,Helariutta Y. (2011) A mutually inhibitory interaction between auxin and cytokinin specifies vascular pattern in roots. Current Biology 21:917-26.

Link: http://www.slcu.cam.ac.uk/research/helariutta-group

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Michael Akam, Dept. of Zoology

Second Supervisor/Collaborator: Second supervisor - Dr. John Marioni, EMBL-EBI & WT Sanger Institute; Collaborator - Dr. P.D. Evans, Babraham Institute

Spatial transcriptomics of a myriapod brain

PhD Project Description: To understand the origin, evolution and functional diversification of animal nervous systems, it is essential to be able to compare the organisation of the brain in different animal lineages. Until recently, to do this in systems that are not well developed lab models has been very difficult, severely limiting the range of possible comparisons. For example, while the brain is well described in Drosophila and a number of other insects, far less is known about the organisation of the brain in the three other ancient arthropod lineages - crustaceans, myriapods and chelicerates. Our recent work (Hunnekuhl et al 2014) has shown that in at least some respects the brain of a centipede (a representative of the myriapods) retains characteristics of ancestral brain organisation that have been lost in the insects. In this project, building on the recent sequencing of the first myriapod genome (Chipman et al 2014), we will use single cell transcriptomics to characterise the diversity of cell types in the brain of this centipede, and determine the distribution of these cell types in the brain by mapping the expression of key transcription factors and neuropeptide hormones that contribute to the molecular fingerprint of each cell type. This project will be carried out in the Dept of Zoology at Cambridge, and at the European Bioinformatics Institute in Hinxton, in the laboratory of Dr. John Marioni who pioneered the use of single cell transcriptomics. Expertise on neuropeptide hormones will be provided by Dr. Peter Evans (Babraham Insititute) who annotated genes encoding neuropeptide hormones in the centipede genome.

Referees:

Chipman, A. D., Ferrier, D. E. K., Brena, C., Qu, J., Hughes, D. S. T., [other members of the Strigamia genome consortium], Akam, M. and Richards, S. (2014) The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima. PLOS Biology 12(11): e1002005. doi: 10.1371/journal.pbio.1002005

Hunnekuhl, V. S. and Akam, M. (2014) An anterior medial cell population with an apical-organ-like transcriptional profile that pioneers the central nervous system in the centipede Strigamia maritima. Dev. Biol. 396:136-149. doi: 10.1016/j.ydbio.2014.09.020

Achim, K., Pettit J.B., Saraiva L.R., Gavriouchkina D., Larsson T., Arendt D., Marioni J.C. (2015) Single-cell expression profiling and spatial mapping into tissue of origin. Nat Biotechnol. (accepted)

Other relevant themes: Basic Biosciences Underpinning Health

Link: Michael Akam Web Page: www.zoo.cam.ac.uk/directory/michael-akam Marioni lab web page: www.ebi.ac.uk/marioni

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Martin Turner, Babraham Institute

Second Supervisor/Collaborator: Professor Christopher Smith (Dept of Biochemistry)

Identifying Master Splicing Regulators of T Lymphocytes

PhD Project Description: Lymphocyte development and function is dictated by dynamic changes in gene expression. These changes result from the abundance and availability of mRNA in the cell to be translated at a particular time point. RNA abundance and availability are regulated by transcriptional and post-transcriptional mechanisms. Not only is it important how much RNA is transcribed, but also how this is spliced, degraded, localized and translated in the cell. However, while transcription regulation in the immune system has been widely studied our knowledge of post-transcriptional regulation is very limited. This PhD project will focus in studying the functions of PTB proteins in the immune system. PTB proteins regulate several aspects of mRNA metabolism such as alternative splicing, mRNA stability, IRES-driven translation, mRNA localization and polyadenylation. There are several PTB paralogs: Ptbp1 is ubiquitously expressed, Ptbp2 is expressed at high levels in brain and testis and Ptbp3 is highly expressed in the hematopoietic system. Interestingly, the relative levels of Ptbp1 and Ptbp2 have been found to dictate neuronal and muscular development by regulating the splicing patterns of genes crucial for proper development of the brain and muscle. Nonetheless, the functions of these proteins, and in particular of Ptbp3, have not yet been addressed in the hematopoietic system. It remains to be investigated whether their relative expression levels also drive lymphocyte development and function. The roles of PTBs will be investigated in lymphocytes using cell-specific conditional knockout mouse models and genome-wide analyses, such as RNAseq, iCLIP and ribosome profiling.

Referees:

Noncoding RNA and its associated proteins as regulatory elements of the immune system. http://www.ncbi.nlm.nih.gov/pubmed/24840979

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.babraham.ac.uk/our-research/lymphocyte/martin-turner

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Sarah Bray, Dept. of Physiology, Development and Neuroscience

Decoding the Notch response

PhD Project Description: The Notch pathway is one of a small handful of cell signalling pathways that coordinate development, regulating the types and numbers of cells formed in many developmental contexts. Its functions include maintenance of stem/progenitor cells, regulation of cell fates, organizing patterns of growth, and many others. In addition, aberrant Notch activity is implicated in diseases including cancers. A major focus of the work in the lab is on understanding how the pathway operates and what enables its different functional outcomes. We are particularly interested in the nuclear events including how the genome organization, epigenetic context, transcription factor dynamics and target site selection are regulated. To investigate, we are primarily working with the Drosophila model, because of its simplicity and we use a combination of strategies, ranging from genome-wide approaches to in vivo imaging techniques, for investigating core mechanisms. In several projects we are working with computational biologists to analyze and model the data from different perspectives. PhD project could pursue any of these different avenues, depending on the interests and experience of the student.

Referees:

Krejci A, Bernard F, Housden BE, Collins S, Bray SJ (2009) Direct Response to Notch Activation: Signaling Crosstalk and Incoherent Logic. Science Signaling 2(55):ra1 doi: 10.1126/scisignal.2000140. 67.

Endo K, Karim MR, Taniguchi H, Krejci K, Kinameri E, Siebert M, Ito K, Bray SJ and Moore AW. (2011) Olfactory receptor neuron identity is diversified by the Drosophila Evi1/Prdm16 Homologue Hamlet that mediates chromatin modification at Notch-target loci. Nature Neuroscience 15(2): 224-33 72.

Terriente-Felix A, Li J, Collins S, Mulligan A, Reekie I, Bernard F, Krejci A and Bray SJ (2013) Notch co-operates with Lozenge/Runx to lock hemocytes into a differentiation programme. Development, 140(4): 926-37.

Other relevant themes: Basic Biosciences Underpinning Health

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

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Jennifer Gallop, Dept. of Biochemistry

Second Supervisor/Collaborator: Steven Lee, Chemistry Department

Step-by-step: single-molecule fluorescence studies of actin regulation

PhD Project Description: The actin cytoskeleton is a dynamic and highly controlled intracellular system of filaments that generates the force needed for cell movement and division, membrane traffic, and maintains cell shape and polarity. It is a critical cell biological effector mechanism in normal morphogenesis and cell function, (e.g. neuronal pathfinding, epithelial maintenance) and also in disease (e.g. cancer metastasis, E coli infection). While actin protein in a test tube recapitulates some aspects of filament assembly and dynamics, actin in the cell is orchestrated by many different proteins that control nucleate, elongate, sever, and bundle in response to cellular signals such as the cell cycle and transcriptional programs and also extracellular growth factors, cytokines and pathogens. Research into cell biology is going through an important change. Monitoring molecular phenomena in live cells at precisely defined locations, as opposed to the entire cell, bulk tissue or in vitro methods has become essential for understanding the cellular mechanisms under study. However the spatial resolution of traditional optical methods is not compatible with the typical spatial regime of cell biological processes. This project will make apply advanced optical single-molecule fluorescence techniques to directly visualize actin in a well-controlled in vitro system that mimics cellular control of actin. Supported lipid bilayers containing PI(4,5)P2 cause the polymerisation of actin into bundled structures that resemble filopodia when cell extracts are added. This project offers unparallel experimental control of biology and the opportunity to follow watch real-time dynamics of a single actin monomer as it assembles into actin bundles.

Referees:

Self-assembly of filopodia-like structures on supported lipid bilayers. Lee K, Gallop JL, Rambani K, Kirschner MW. Science. 2010 329(5997):1341-5.

Super-resolution imaging of the nucleoid-associated protein HU in Caulobacter crescentus SF Lee, MA Thompson, MA Schwartz, L Shapiro, WE Moerner. Biophys J (2011) 100, L31

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www2.gurdon.cam.ac.uk/~galloplab/ http://www.ch.cam.ac.uk/person/sl591

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Jasmin Fisher, Dept. of Biochemistry

PhD Project Description: Our group is interested in the molecular mechanisms controlling cell fate decisions during development, lineage commitment and stem cell maintenance (e.g., the hematopoietic system and germ cell proliferation and apoptosis in C. elegans). Cell fate specification processes must be under strict regulation to ensure life-long homeostasis. A better understanding of the mechanisms controlling cell fate decisions can pave the way to the identification of novel drug targets and improve potential strategies to fight tumorigenesis and cancer. However, due to their enormous complexity and multiple interactions, the comprehension and analysis of these signalling pathways have been a major challenge. We are using program analysis techniques for the construction and analysis of executable models describing cell fate decisions in various model systems. These models are essentially computer programs whose behaviour captures aspects of biological phenomena. Once an executable model has been built, it can be used to get a global dynamic picture of how the system responds to various perturbations. By applying various computational approaches such as Boolean/qualitative networks and state-machines our group devotes itself both to establishing an in-depth understanding of the cell signalling and intercellular communication processes controlling cell fate specification, and to understanding how these processes are orchestrated to establish robust cell fate patterns. There are various possibilities for PhD projects within the group. For example, modelling of glioblastoma development and simulation of whole-organism lineage (C. elegans). For further details, please visit our website: http://www.bioc.cam.ac.uk/people/uto/fisher or email jf416@cam.ac.uk

Referees:

Fisher J. and Henzinger T.A., Executable Cell Biology, in Nature Biotechnology, vol. 25, no. 11, pp. 1239-1249, 2007.

Chuang R., Benque D., Cook B., Hall B.A., Ishtiaq S., Piterman N., Taylor A., Vardi M., Koschmieder S., Gottgens B. and Fisher J. Drug Target Optimization in Chronic Myeloid Leukemia Using an Innovative Computational Platform. Scientific Reports, 2015.

Moignard V., Woodhouse S., Haghverdi L., Lilly J, Tanaka Y, Wilkinson A.C, Buettner F., Nishikawa S.I., Piterman N., Kouskoff V., Theis F.J., Fisher J., Göttgens B. Decoding the Transcriptional Program for Blood Development from Whole Tissue Single Cell Gene Expression Measurements. Nature Biotechnology, 2015.

Other relevant themes: Basic Biosciences Underpinning Health

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

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Dr AP. Jackson, Dept. of Biochemistry

Second Supervisor/Collaborator: Prof. C.L-H Huang (Physiology, Development and Neuroscience); Prof K.S. Lilley (Cambridge Centre for Proteomics)

A role for sodium channel beta-3 subunits in stabilising sodium channel clusters in plasma membranes of neuronal and cardiac cells.

PhD Project Description: Voltage-gated sodium (Nav) channels initiate neuronal and cardiac action potentials. Nav channels co-assemble with other proteins into macromolecular clusters on the neuronal and cardiomyocyte plasma membrane. Yet the wider protein composition of these clusters, how they are stabilised and their functional significance is poorly understood. Nav channels contain an ion-selective alpha subunit with beta subunits. We have shown that beta3 subunits form trimers on the plasma membrane, and can cross-link individual Nav alpha subunits (1). We will test the hypothesis that beta3-induced cross-linking helps stabilise both the Nav channels within these plasma membrane clusters, and their broader protein composition. The project will examine the co-localisation of Nav channel alpha subunits with known cluster components such as potassium and calcium channels on neurones and cadiomyocytes from wild-type mice and genetically modified mice (Scn3b-/-) lacking beta3 expression (2). We will correlate any localisation changes with differences in electrophysiological properties between cells from the wild-type and Scn3b-/- mice. To provide a systematic inventory of proteins within these clusters, and their changes in the absence of beta3, we will exploit a novel proteomic method that can detect proteins in localised plasma membrane clusters (3). Here, a peroxidase is targeted to the Nav channel alpha subunits that will catalyse the biotinylation of proteins within a few nanometres from the channel. Such proteins are isolated by affinity chromatography and identified by mass spectrometry (3). This project provides a strongly interdisciplinary approach that integrates cell-biological, electrophysiological and proteomic approaches to address a major problem in contemporary neurobiology.

Referees:

(1). Namadurai, S., Balasuriya, D., Rajappa, R., Wiemhofer, M., Stott, K., Klingauf, J., Edwardson, J. M., Chirgadze, D. Y., and Jackson, A. P. (2014) Crystal structure and molecular imaging of the Nav channel beta3 subunit indicates a trimeric assembly. The Journal of biological chemistry 289, 10797-10811

(2). Hakim, P., Brice, N., Thresher, R., Lawrence, J., Zhang, Y., Jackson, A. P., Grace, A. A., and Huang, C. L. (2010) Scn3b knockout mice exhibit abnormal sino-atrial and cardiac conduction properties. Acta physiologica 198, 47-59

(3). Li, X. W., Rees, J. S., Xue, P., Zhang, H., Hamaia, S. W., Sanderson, B., Funk, P. E., Farndale, R. W., Lilley, K. S., Perrett, S., and Jackson, A. P. (2014) New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay. The Journal of biological chemistry 289, 14434-14447

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.bioc.cam.ac.uk/people/uto/jacksont http://www.pdn.cam.ac.uk/staff/huang/index.shtml http://www.bioc.cam.ac.uk/people/uto/lilley

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Oliver Stegle, EMBL-EBI

Genetic analysis of molecular traits to uncover downstream molecular consequences of disease loci

PhD Project Description: Gene expression levels and other molecular traits are now being profiled in large cohorts. These efforts provide an unprecedented opportunity to dissect the molecular mechanisms of genotype to phenotype associations that have been identified through GWAS and linkage studies. The most basic approaches to implicate a gene with a GWAS hit test for association of the associated variant with genome‐wide expression levels, thereby identifying colocalized expression quantitative trait loci (eQTL). A critical limitation of such eQTL‐based approached is that i) typically only direct cis regulatory variants are identified and ii) the extent to which a gene is carrying a functional and causal role is not revealed. In this project the aim is to develop new and refined association genetic tools that leverage information from multiple tissues and prior data on regulatory elements to more clearly establish functional relationships between GWAS loci and eQTL studies. We will systematically combine statistical principles based on independence tests with multi‐tissue eQTL datasets and epigenetic information, thereby improving the ability to uncover causal molecular networks downstream of GWAS loci.

Referees:

[1] Genotype-environment interactions reveal causal pathways that mediate genetic effects on phenotype. Gagneur J, Stegle O, Zhu C, Jakob P, Tekkedil MM, Aiyar RS, Schuon AK, Pe'er D, Steinmetz LM. PLoS Genet Volume 9 (2013) p.e1003803

[2] Joint modelling of confounding factors and prominent genetic regulators provides increased accuracy in genetical genomics studies. Fusi N, Stegle O, Lawrence ND. PLoS Comput Biol Volume 8 (2012) p.e1002330 [3] Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells. Buettner F, Natarajan KN, Casale FP, Proserpio V, Scialdone A, Theis FJ, Teichmann SA, Marioni JC, Stegle O. Nat Biotechnol Volume (2015) p.

Link: http://www.ebi.ac.uk/research/stegle

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Andrew Gillis, Dept. of Zoology

Development, growth and repair of cartilage in cartilaginous fishes

PhD Project Description: In mammals, hyaline cartilage is predominantly an embryonic tissue, making up the anlage of the axial and appendicular endoskeleton. The majority of mammalian cartilage is replaced by bone, with hyaline cartilage persisting temporarily in growth plates, and permanently at few sites within the adult skeleton (e.g. in joints, as articular cartilage). Mammalian articular cartilage ceases growth at adolescence, and has very limited capacity for repair (hence the prevalence of joint diseases, such as osteoarthritis). Conversely, cartilaginous fishes (sharks, skate, rays and holocephalans) possess an endoskeleton that is composed entirely of hyaline cartilage, and this skeleton remains cartilaginous throughout adult life. This PhD project will investigate the cellular and molecular mechanisms underlying cartilage development and growth in cartilaginous fishes. The student will characterize differentiation and growth of the endoskeleton of the skate (Leucoraja erinacea), with a particular focus on mode of cartilage growth (e.g. whether cartilage growth occurs by extracellular matrix expansion, re-entry of differentiated chondrocytes into the cell cycle, or recruitment of chondroprogenitor cells from outside the cartilage). The student will test for the presence of persistent chondroprogenitor cells using label-retention assays in juvenile and adult skates, and will characterize the transcriptional profile of such cells by RNAseq and in situ gene expression analysis. Finally, the student will test whether (and how) the skeleton of adult cartilaginous fishes can spontaneously repair damage to hyaline cartilage. This work will establish the skate as a model for hyaline cartilage development, growth, and - perhaps uniquely among vertebrates - spontaneous cartilage repair.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www2.zoo.cam.ac.uk/gillis/

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Andrew Firth, Dept. of Pathology

Novel virus discovery and comparative genomics

PhD Project Description: Research in our lab focuses on viruses with RNA genomes. RNA viruses include influenza A, ebola, rabies, SARS, MERS, Japanese encephalitis, yellow fever, and dengue viruses. While RNA viruses of humans, livestock, crop plants, and some model organisms have been well-studied, RNA viruses of most invertebrate and protist phyla have barely been studied at all. Such viruses tend to be related to known RNA virus groups but are nonetheless typically highly divergent. The identification and in silico characterization of the genome sequences of such viruses is valuable because they provide a broad phylogenetic baseline for studying virus taxonomy and evolution, and for comparative analyses of economically and medically important species. Over the last few years there has been a massive increase in the quantity of relevant sequence data deposited in GenBank; we also have a number of our own datasets. In this project, we will mine RNA sequencing databases from diverse organisms (including human parasites) and environmental samples in order to identify novel RNA virus-derived sequences. These sequences will be used for comparative genomic analyses of novel virus clades and for more distant comparative analyses of medically and economically important viruses . The project may also involve the development of new computational methods for novel virus discovery from high-throughput sequencing data. This is largely a computational project suited to a mathematically orientated student. Some prior experience with programming would be very valuable.

Referees:

1) Li CX, Shi M, Tian JH, Lin XD, Kang YJ, Chen LJ, Qin XC, Xu J, Holmes EC, Zhang YZ (2015) Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. Elife doi: 10.7554/eLife.05378. PMID: 25633976.

2) Smits SL, Bodewes R, Ruiz-Gonzalez A, Baumgartner W, Koopmans MP, Osterhaus AD, Schurch AC (2014) Assembly of viral genomes from metagenomes. Front Microbiol 5:714. PMID: 25566226.

3) Cook S, Chung BY, Bass D, Moureau G, Tang S, McAlister E, Culverwell CL, Glucksman E, Wang H, Brown TD, Gould EA, Harbach RE, de Lamballerie X, Firth AE (2013) Novel virus discovery and genome reconstruction from field RNA samples reveals highly divergent viruses in dipteran hosts. PLoS One 8:e80720. PMID: 24260463.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.firthlab.path.cam.ac.uk/index.html

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Nick Mundy, Dept. of Zoology

Evolutionary genetics of avian coloration - hormonal mechanisms of sexual colour differences

PhD Project Description: Colour differences between the sexes are very common in birds and include iconic examples such as the peacock. Remarkably little is known about how this variation is regulated at the level of the feather and beak. The project will identify changes in gene expression that underlie the different colours of males and females, in galliform and passerine birds. Key questions that will be addressed are the role of hormone receptors in mediating colour changes, and how hormonal control interacts with the gene networks responsible for deposition of pigments (melanins and carotenoids). The main methodology to be employed is RNA-Seq, giving excellent experience in this modern technology. The results will be of fundamental importance for understanding how sexual dimorphism is achieved and also for the molecular mechanisms underlying sexual selection.

Referees:

Walsh, N., Dale, J., McGraw, K. J., Pointer, M. A. and N. I. Mundy (2012) Candidate genes for carotenoid colouration in vertebrates and their expression profiles in the carotenoid-containing plumage and bill of a wild bird. Proceedings of the Royal Society Series B 279, 58-66. DOI: 10.1098/rspb.2011.0765

Link: http://www.zoo.cam.ac.uk/directory/nicholas-mundy

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Andrew Firth, Dept. of Pathology

Mining RNA virus genomes for new gene expression mechanisms

PhD Project Description: Viruses fall into several major groupings but at the highest level they may be divided into those with RNA genomes and those with DNA genomes. RNA viruses include influenza A, ebola, rabies, SARS, MERS, Japanese encephalitis, yellow fever, and dengue viruses, besides many viruses of livestock and most viruses of crop plants. RNA viruses have very compact genomes (2 to 32 kb). Unlike cellular gene expression, which is often controlled at the level of transcription, RNA virus gene expression is very often controlled at the level of translation. RNA viruses have evolved a huge diversity of non-canonical translation mechanisms. Because RNA viruses rely on host-cell ribosomes and other components of the host-cell translational machinery, most of these non-canonical mechanisms can function outside of the context of virus infection and many have potential applications in biotechnology or as tools for molecular biological research. In this project we aim to identify and characterize novel translation mechanisms. We will mine sequence databases for virus sequences highly divergent from previously characterized viruses, identify the protein-coding regions, filter out genes that are likely (based on sequence analysis) to use previously characterized gene expression mechanisms, and, where possible, experimentally characterize cases where genes appear likely to use completely novel expression mechanisms. Biotechnological applications will be investigated as appropriate. This project could be purely computational, or could involve a mixture of experimental and computational work. Proficiency in mathematics and some prior experience with programming would be very valuable.

Referees:

1) Firth AE, Brierley I (2012) Non-canonical translation in RNA viruses. J Gen Virol 93:1385-1409. PMID: 22535777.

2) Minskaia E, Nicholson J, Ryan MD (2013) Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications. BMC Biotechnol 13:67. PMID: 23968294.

3) Fernández IS, Bai XC, Murshudov G, Scheres SH, Ramakrishnan V (2014) Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome. Cell 157:823-831. PMID: 24792965.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.firthlab.path.cam.ac.uk/index.html

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Pedro Beltrao, European Bioinformatics Institute (EBI)

Evolution of protein kinase specificity

PhD Project Description: Protein function is often regulated by protein phosphorylation and over one third of the human proteome has been found to be phosphorylated in-vivo. In humans, this modification is catalysed by approximately 500 protein kinases that recognize specific sequence 'motifs' surrounding the target site. Recent progress in proteomics is rapidly advancing our knowledge of which positions are targeted by protein kinases. However, very little is known about the evolution of the specificity of these crucial regulatory enzymes. This project aims to tackle this question by taking advantage of the increased knowledge of the specificity of extant enzymes and in-vivo determined phosphosites for a growing number of species. To address this the applicant will combine phylogenetic based methods with structural bioinformatics and statistical analysis of mass-spectrometry data.

Referees:

Beltrao P, Bork P, Krogan NJ, van Noort V.Evolution and functional cross-talk of protein post-translational modifications. Mol Syst Biol. 2013 Dec 22;9:714.

Beltrao P, Albanèse V, Kenner LR, Swaney DL, Burlingame A, Villén J, Lim WA, Fraser JS, Frydman J, Krogan NJ. Systematic functional prioritization of protein posttranslational modifications. Cell. 2012 Jul 20;150(2):413-25. doi: 10.1016/j.cell.2012.05.036.

Studer RA, Christin PA, Williams MA, Orengo CA.Stability-activity tradeoffs constrain the adaptive evolution of RubisCO. Proc Natl Acad Sci U S A. 2014 Feb 11;111(6):2223-8.

Link: www.ebi.ac.uk/research/beltrao

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Andrew Firth, Dept. of Pathology

Comparative genomics of RNA viruses

PhD Project Description: The majority of viruses with the potential to cause acute fatal disease in healthy adult humans have RNA genomes. Such viruses include influenza A, ebola, rabies, SARS, MERS, Japanese encephalitis, yellow fever, and dengue viruses. Identification of the full complement of genes and other functional elements in any virus genome is crucial to fully understand its molecular biology and guide the development of effective control strategies. However, RNA viruses have compact multifunctional genomes that frequently contain overlapping genes and non-coding functional elements (e.g. essential replication, translation, and genome packaging signals) embedded within protein-coding sequences. Such features are difficult to detect using conventional experimental or bioinformatic approaches. Our research involves the development and utilization of new comparative genomic techniques to systematically identify hidden features in virus genomes. These analyses can be used to efficiently target experimental work. In this project we will use comparative genomic RNA folding programs to systematically map out functional RNA structural elements in hundreds of virus species. The results will be integrated with previous experimental work to classify functional elements, build search models, and develop software to automatically annotate virus genomes submitted to a webserver. Where appropriate, newly discovered functional elements may be characterized experimentally. This is a computational project suited to a mathematically orientated student. Some prior experience with programming would be very valuable.

Referees:

1) Firth AE (2014) Mapping overlapping functional elements embedded within the protein-coding regions of RNA viruses. Nucleic Acids Res 42:12425-12439. PMID: 25326325.

2) Liu Y, Wimmer E, Paul AV (2009) Cis-acting RNA elements in human and animal plus-strand RNA viruses. Biochim Biophys Acta 1789:495-517. PMID: 19781674.

3) Jagger BW, Wise HM, Kash JC, Walters KA, Wills NM, Xiao YL, Dunfee RL, Schwartzman LM, Ozinsky A, Bell GL, Dalton RM, Lo A, Efstathiou S, Atkins JF, Firth AE, Taubenberger JK, Digard P (2012) An overlapping protein-coding region in influenza A virus segment 3 modulates the host response. Science 337:199-204. PMID: 22745253.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.firthlab.path.cam.ac.uk/index.html

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Jose Silva, Dept. of Biochemistry

Exploring the regulation of gene re-activation during nuclear reprogramming

PhD Project Description: Nuclear reprogramming occurs when a somatic cell nucleus is reverted to a pluripotent state. This can be achieved directly by the use of defined factors. This process was named induced pluripotency and generated cells induced pluripotent stem cells (iPSCs). During nuclear reprogramming cells undergo significant transcriptional and epigenetic changes. However, the interplay and mechanisms underlying these events are not known. An outstanding question in the field concerns the impact of the reprogramming factors on the regulation of reactivation of iPS cell associated genes. Gene expression is known to be controlled by specific regulatory sequences but how these are used during the activation of silenced genes is not known. In particular, we are interested in understanding how initiation of iPS cell gene reactivation relates to the usage of its regulatory sequences. For example, does re-activation occurs as a result of reprogramming factors binding to the regulatory sequences that are involved in the expression of these genes in iPS cells? Or does it occur initially via transiently occupied regulatory sequences which may be more permissible for reprogramming factor binding but are not the same that regulate the expression of these genes in iPSCs? In order to address these questions we will use the double-inducible iNanog-iStat3 system generated in the lab. This system when applied to Epiblast-derived-Stem Cells induces rapid and efficient reprogramming. In conclusion, we expect to be able to relate reprogramming factors with on-going events at target iPS cell associated genes and start deciphering the biology of gene re-activation.

Referees:

1. Stuart HT, van Oosten AL, Radzisheuskaya A, Martello G, Miller A, Dietmann S, Nichols J, Silva JCR. NANOG amplifies STAT3 activation and they synergistically induce the naïve pluripotent program. Current Biology (2014). Feb 3;24(3):340-6. doi: 10.1016/j.cub.2013.12.040.1.

2. Santos R, Tosti L, Radzisheuskaya A, Caballero I, Kaji K, Hendrich B, Silva JCR. Mbd3/NuRD facilitates induced pluripotency in a context dependent manner. Cell Stem Cell (2014). Jul 3;15(1):102-10. doi:10.1016/j.stem.2014.04.019.

3. Costa Y, Ding J, Theunissen TW, Faiola F, Hore TA, Shliaha PV, Fidalgo M, Saunders A, Lawrence M, Dietmann S, Das S ,Levasseur DN, Li Z, Xu M, Reik W, Wang J#, Silva JCR#. Nanog-dependent function of Tet1 and Tet2 in establishment of pluripotency. Nature. (2013) Mar 21;495(7441):370-4. doi: 10.1038/nature11925.

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.stemcells.cam.ac.uk/researchers/principal-investigators/dr-jos-silva http://www.bioc.cam.ac.uk/people/uto/silva http://www.jose-silva-lab.com

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Markus Ralser, Dept. of Biochemistry

The genetic basis of metabolic cooperation in eukaryotic cellular communities

PhD Project Description: Cells that co-exist within the same culture, community or tissue exchange metabolites. This cross-feeding is extensive in bacteria, diversifying communities by metabolic specialisation. Contrastingly, eukaryotes exchange metabolites, yet fail to cross-compensate metabolic deficiencies in pair-wise interactions, which has previously suggested that they might posses lower potential to metabolically cooperate. We have however recently discovered that cooperation is effective in eukaryotes, but limited to complex community structures. We established a synthetic cooperating community in the model eukaryote Saccharomyces cerevisiae that can only survive when cells support each others growth by exchanging essential metabolites. This required eight cell types to cooperate, but together the community successfully entered a state of cooperation, and compensated for metabolic deficiencies to maintain a wild-type like exometabolome, growth parameters, metabolic efficiency and stress resistance. This project is to elaborate the genetic basis of this cooperativity; to use mass spectrometry to identify metabolites shared between cells, to determine the flux exchange rates, and eventually to generate a picture that explains the necessity and advantages of metabolic cooperation. We intend to learn about the processes which drove the evolution of multicellularity, and compare those principles with modern organisms, whose tissues are effectively exchange metabolites to sustain growth. We are a mostly postdoc-driven lab located at the Department of Biochemistry, University of Cambridge, and the MRC National Institute for Medical Research (London) that will transit into the Francis Crick Institute at some point in 2016. The lab is a mixed experimental/computational lab; the ideal applicant for this project comes with a background either in Biochemistry/Analytical Biochemistry, or Cell Biology, Metabolism Research, Microbiology or and/or Functional Genomics. Please visit us at http://ralser.sysbiol.cam.ac.uk/

Referees:

Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in a plausible Archean ocean Keller MA, Turchyn AVT & Ralser M. Molecular Systems Biology 10:725, 2014, Apr 25.

Pyruvate kinase triggers a metabolic feedback loop that controls redox metabolism in respiring cells Gruening NM, Rinnerthaler M, Bluemlein K, Muelleder M, Wamelink MM, Lehrach H, Jakobs C, Breitenbach M, Ralser M Cell Metabolism. 2011 Sep 7 ; 14(3): 415-27

A prototrophic deletion mutant collection for yeast metabolomics and systems biology Michael Mülleder, Floriana Capuano, Pınar Pir, Stefan Christen, Uwe Sauer, Stephen G Oliver & Markus Ralser Nature Biotechnology 2012, pp1176 - 1178, doi:10.1038/nbt.2442

Other relevant themes: Bioenergy and Industrial Biotechnology

Link: http://ralser.sysbiol.cam.ac.uk/

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Rebecca Kilner, Dept. of Zoology

Second Supervisor/Collaborator: Professor Tracey Chapman, School of Biological Sciences, University of East Anglia

A novel mechanism for non-genetic inheritance: telegony in the burying beetle

PhD Project Description: Understanding mechanisms of non-genetic inheritance is key to understanding how quickly organisms can adapt in a rapidly changing world, and can also help solve significant medical problems. Telegony describes a neglected form of non-genetic heredity, in which a mother’s previous mate manages to influence the phenotype of her young, even when he has not sired the offspring. Though telegony has not been studied for many years, recent work on insects suggests a plausible mechanism by which it could work. When a male insect inseminates a female he transfers diverse seminal fluid proteins, some of which influence female physiology, causing her to lay larger eggs for example. In theory, the seminal fluid proteins of an earlier mate might have a lingering effect on female physiology, so changing the resources she allocates to offspring sired by a later male and thus altering the offspring’s phenotype. Our pilot experiments on burying beetles suggest that there may well be inheritance by telegony in this species. In collaboration with Tracey Chapman, you will break new ground by investigating how seminal fluid proteins influence offspring phenotype, and whether they act independently of a male’s genetic contribution. You will combine mating experiments on burying beetles in the lab with genetic and genomic techniques to: a) partition genetic versus non-genetic contributions to offspring size, when the mother has mated with multiple males b) identify proteins in the seminal fluid that might cause telegony c) relate individual variation in seminal fluid proteins to the capacity for telegony.

Referees:

Chapman, T. 2001 Seminal fluid-mediated fitness traits in Drosophila. Heredity 87:511-521 doi:10.1046/j.1365-2540.2001.00961.x

Crean, A. J. et al. 2014. Revisiting telegony: offspring inherit an acquired characteristic of their mother's previous mate. Ecology Letters 17: 1545-1552 DOI: 10.1111/ele.12373

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://nicrophorus.zoo.cam.ac.uk

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Matthias Landgraf, Dept. of Zoology

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

PhD Project Description: We are interested in how neuronal networks are genetically specified and assembled during development. Using genetics and imaging, we study the formation and subsequent adjustment of synapses. A key element is the ability of neurons to homeostatically tune their connectivity, excitability and morphology in response to changes in activity. This is critical for neuronal networks: during development, for network function to emerge, and to remain within appropriate activity ranges. We discovered that neurons undergo structural homeostatic adjustments of their synaptic terminals upon changes in activity. Wondering how neurons ‘know’ their preferred activity range, we discovered that neurons use a metabolic by-product to obtain a readout of activity: Reactive Oxygen Species (ROS). Briefly, ROS are by-products of mitochondrial ATP production. We find that ROS act as second messengers that are necessary and sufficient for regulating homeostatic synaptic terminal growth. We want to understand how neurons sense increases in ROS levels, and the effector pathways that mediate change at the synapse. For the former we will carry out a biochemical screen, for the latter we harness genetic interactions. Intriguingly, ROS are also known to affect chromatin structure, opening the possibility of metabolically driven epigenetic regulation of neuronal structure and function. We work with the locomotor network of Drosophila. This provides us with uniquely identifiable interconnected cells: body wall muscles, their motoneurons and their pre-motor interneuron. We can specifically manipulate each element of this network and, using high resolution imaging and computer aided 3D reconstructions, quantify changes to complex dendritic and presynaptic arbors.

Referees:

1. Milton, V. J. et al. Oxidative stress induces overgrowth of the Drosophila neuromuscular junction. Proc Natl Acad Sci U S A 108, 17521–17526 (2011).

2. Zwart, M. F., Randlett, O., Evers, J. F. & Landgraf, M. Dendritic growth gated by a steroid hormone receptor underlies increases in activity in the developing Drosophila locomotor system. Proc Natl Acad Sci U S A (2013). doi:10.1073/pnas.1311711110

3. Tripodi, M., Evers, J. F., Mauss, A., Bate, M. & Landgraf, M. Structural homeostasis: compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input. PLoS Biol 6, e260 (2008).

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.zoo.cam.ac.uk/departments/neural-network

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Chris Smith, Dept. of Biochemistry

Analysis of molecular mechanisms of splicing regulation by PTB and co-regulators

PhD Project Description: Alternative pre-mRNA splicing plays a major role in bridging the gap between the relatively small number of genes compared to the much greater complexity of expressed proteomes. To understand the mechanisms by which alternative splicing is regulated between cell-types it is necessary to characterize the regulatory proteins, their interactions with regulatory RNA sequences and how they influence splicing complex assembly. We have investigated the functions of Polypyrimidine Tract Binding protein (PTB) as both a repressor and activator of splicing (Ref 2). PTB has four RNA Recognition Motif (RRM) domains, the second of which is particularly important for function by binding to both RNAs and proteins that contain short linear peptide motifs known as PTB RRM Interacting (PRI) motifs (Ref 3). Recently, we characterized a set of nuclear proteins that interact specifically with the second RNA binding domain of PTB (Ref 1). Among these was the nuclear matrix protein Matrin3, which we found to be an independent regulator of many alternative splicing events. A number of other interesting proteins also contain PRI motifs and are known to be components of spliceosome complexes and/or associated with RNA Polymerase II. This project will focus on characterizing the nature of the interaction of these proteins with PTB and their roles in mediating the splicing repressor and activator activities of PTB. The project will involve a range of cellular, molecular and RNA biology techniques.

Referees:

1. M. Coelho, J. Attig, N. Bellora, J. König, M. Hallegger, M. Kayikci, E. Eyras, J. Ule, C.W.J. Smith. Nuclear Matrix Protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB. EMBO Journal (2015) DOI 10.15252/embj.201489852

2. Llorian, M., Schwartz, S., Clark, T.A., Hollander, D., Tan, L.Y., Spellman, R., Gordon, A., Schweitzer, A.C., la Grange, P., Ast, G., and Smith, C.W.J. (2010). Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB. Nat Struct Mol Biol 17, 1114-1123.

3. A.P. Rideau, C. Gooding, P.J. Simpson, T.P. Monie, M. Lorenz, S. Hüttelmaier, R.H. Singer, S. Matthews, S. Curry & C.W.J. Smith. A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain. Nature Structural and Molecular Biology. 13, 839-848 (2006)

Other relevant themes: Basic Biosciences Underpinning Health

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

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Howard Baylis, Dept. of Zoology

How is RNAi regulated by cell signalling?

PhD Project Description: RNAi is one of a number of small ncRNA mediated processes in animals. We have recently shown that signalling mediated by the second messenger, IP3 regulates the strength of responses to exogenous RNAi in C. elegans. This is the first signalling pathway which has been shown to modulate RNAi in C. elegans. The novelty of this discovery is that it suggests a mechanism by which the physiological state of an animal and thus its environment can regulate the RNAi response. We now wish to dissect the mechanism of this action by identifying further molecules involved in the process and pin-pointing the cells in which they act. By so doing we aim to link external and internal signals to RNAi. To do this we will use a range of molecular (including including small RNA-seq), cellular, genetic and transgenic techniques, in combination with image analysis and the quantification of whole animal physiology and behaviour. In addition to answering fundamental questions about RNAi the results may suggest routes for improving the exploitation of RNAi in other systems.

Referees:

Nagy AI*, Vázquez-Manrique RP*, Lopez M, Christov C, Sequedo MD, Herzog M, Herlihy AE, Bodak M, Gatsi R and Baylis HA (*Joint first authors) (2015) IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans. EMBO Rep. DOI 10.15252/embr.201439585

Baylis H A and Vazquez-Manrique R P (2011) Genetic analysis of IP3 and calcium signalling pathways in C. elegans. Biochim. Biophys. Acta-Gen. Subj. 1820, 1253-1268 DOI: 10.1016/j.bbagen.2011.11.009

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.zoo.cam.ac.uk/departments/baylis/research

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Abigail Fowden, Dept. of Physiology, Development and Neuroscience

Second Supervisor/Collaborator: Professor Susan Ozanne Institute of Metabolic Science

Diet induced obesity and exercise during pregnancy: consequences for maternal and offspring metabolic health

PhD Project Description: In developed countries, women of reproductive age becoming increasing obese. Even those of normal body mass index are gaining excess weight during pregnancy, primarily as fat. Human epidemiological observations and experimental animal studies have shown that enhanced fat deposition and obesity during pregnancy are associated with abnormal birth weight and and an increased risk of the offspring developing obesity and metabolic disorders in later life. However, the mechanisms operating during pregnancy that underlie these detrimental outcomes remain largely unknown. Nor is it clear whether inventions advocated to combat the metabolic dysfunction of obesity, such as increased exercise, are beneficial to the development of the fetus in either normal or obese mothers. The aim of the project is to investigate the effects of diet-induced obesity coupled with exercise on the maternal metabolic adaptation to pregnancy and the allocation of nutrients to conceptus growth in mice using a combination of in vivo and in vitro techiques. The project is designed to answer three specific questions: 1) What effects does diet-induced obesity have on maternal metabolic adaptation to pregnancy? 2) How are these adaptations altered by exercise in normal and obese dams? and 3) What are the immediate and long-term consequences of maternal diet-induced obesity and exercise on the offspring, including the phenotype of the placenta, the organ responsible for metabolic signalling to the mother and supplying nutrients to the fetus? The project will incorporate studies at the whole body (eg glucose tolerance, insulin sensitivity, body composition), tissue (eg placental nutrient transfer, tissue glucose uptake) and molecular (eg insulin signalling pathway and miRNA analyses) levels in the mothers and their offspring. The project, therefore, provides unique training in in vivo physiological measurements (eg three pool tracer methodology, hyperinsulinaemic-euglycaemic clamps, TDNMR), stereological tissue analyses (eg surface area, placental vessel volumes) and in vitro molecular techniques including Western blotting, PT-PRC and ELISA. All the facilities and techniques needed for this interdisciplinary project are available in the participating laboratories (Metabolic Research Laboratories, Physiology, Development and Neuroscience and Centre for Trophoblast Research).

Referees:

Vaughan OR, Sferruzzi-Perrir AN, Coan PM & Fowden AL (2012) Environmental regulation of placental phenotype: Implications for fetal growth Reprod. Fert. Develop. 24:80-96.

Fowden AL & Moore T (2012). Maternal-fetal resource allocation:co-operation and conflict Placenta 33: Suppl 2 e11-e15.

Bouret S, Levin BE & Ozanne SE (2015). Gene-environment interactions in controlling energy and glucose homeostasis and the developmental origins of obesity. Physiol. Rev. 95:47-82.

Other relevant themes: Basic Biosciences Underpinning Health

Link: www.pdn.cam.ac.uk www.mrl.ims.cam.ac.uk

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Liria Masuda-Nakagawa, Dept. of Genetics

Second Supervisor/Collaborator: Cahir O'Kane

Neuronal circuitry of the Drosophila larval mushroom body

PhD Project Description: To understand how the brain discriminates among a large palette of sensory concepts, and how these are used to form memories, we use the fruitfly Drosophila. The mushroom bodies (MBs) of the insect brain are centers for olfactory learning, and provide a substrate to understand how brains can discriminate. We focus on the circuitry of the input region of the MBs, the calyx. Here, MB neurons, the KCs, receive input from projection neurons (PNs), secondary olfactory neurons. We previously showed that PN input to the calyx is stereotypic; that KCs connect to these inputs in a random combinatorial manner; and that a single inhibitory neuron mediates a negative feedback loop from KC outputs to their calyx inputs. This model allows the discrimination of a large number of odors. However, there are additional neurons in the calyx circuit; their high evolutionary conservation implies that they are important to this model discrimination centre. We need to know how they are connected, using both anatomical and functional criteria. New Drosophila transgenic resources facilitate this: large collections of lines to target reporter gene expression to many sets of neurons, along with large image datasets of their expression; markers like split GFP for neuronal contacts and termini; transgenes like GCaMP to monitor neuronal activity; and transgenes like channelrhodopsin or shibire to manipulate neuronal activity. This project will explore the connectivity of novel neurons in the calyx circuitry, and how they integrate with previously characterized neurons; its goal the logic governing selectivity of discrimination.

Referees:

1. LM Masuda-Nakagawa, K Ito, T Awasaki, CJ O’Kane (2014). A single GABAergic neuron mediates feedback of odor-evoked signals in the mushroom body of larval Drosophila. Front Neur Circ 8:35

2. LM Masuda-Nakagawa, N Gendre, CJ O'Kane, RF Stocker (2009) Localized olfactory representation in mushroom bodies of Drosophila larvae. Proc Natl Acad Sci USA 106, 10314-9.

3. L Luo, EM Callaway, K Svoboda (2008) Genetic dissection of neural circuits. Neuron 57, 634-660.

Link: http://www.gen.cam.ac.uk/research-groups/liria-masuda-nakagawa

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Andrea Brand, Dept. of Physiology, Development and Neuroscience

Stem cells to synapses: regulation of self-renewal and differentiation in the nervous system

PhD Project Description: Discovering how stem cells are maintained in a multipotent state and how their progeny differentiate into distinct cellular fates is a key step in the therapeutic use of stem cells to repair tissues after damage or disease. We are investigating the genetic networks that regulate stem cells in the Drosophila nervous system. Stem cells can divide symmetrically to expand the stem cell pool, or asymmetrically to self-renew and generate a daughter cell destined for differentiation. The balance between symmetric and asymmetric division is critical for the generation and repair of tissues, as unregulated stem cell division results in tumourous overgrowth. By comparing the transcriptional profiles of symmetrically and asymmetrically dividing stem cells, we are identifying the molecular switches that regulate stem cell behaviour. Neural stem cells transit through a period of quiescence at the end of embryogenesis. We discovered that insulin signalling is necessary for these stem cells to exit quiescence and reinitiate cell proliferation. We showed that a glial niche secretes the insulin-like peptides that reactivate neural stem cells in vivo. We are investigating the systemic and local signals that regulate stem cell growth and proliferation and the role of glia in inducing neural stem cell exit from quiescence. Techniques used include genetics, molecular biology, transgenesis, RNA interference, immunohistochemistry, confocal microscopy, live imaging, transcriptomics, genome-wide DNA-protein interaction assays and bioinformatics.

Referees:

Spéder, P. and Brand, A.H. (2014). Gap junction proteins in the blood-brain barrier control nutrient-dependent reactivation of Drosophila neural stem cells. Developmental Cell 30, 309-321.

Southall, T.D., Davidson, C.M., Miller, C., Carr, A. and Brand, A.H. (2014). Dedifferentiation of neurons precedes tumour formation in lola mutants. Developmental Cell 28, 685-96.

Southall, T.D., Gold, K.S., Egger, B., Davidson, C.M., Caygill, E.E., Marshall, O.J. and Brand, A.H. (2013). Cell type-specific profiling of gene expression and chromatin binding without cell isolation: Assaying RNA Pol II occupancy in neural stem cells. Developmental Cell 26, 101-112.

Other relevant themes: Basic Biosciences Underpinning Health

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

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Dr Andreas Bender, Dept. of Chemistry

Using Gene Expression Data for Stem Cell Differentiation and Treating Cancer and Other Diseases

PhD Project Description: The Bender Group at at Department of Chemistry currently comprises about 20 members from all areas of science, and focuses on the integration of chemical and biological data for the analysis of mechanisms of action of drugs, and the design and discovery of potential novel treatments. In recent years, we have successfully integrated bioactivity data of compounds with biological readouts, in particular gene expression data, for the selection of compounds to induce directed stem-cell differentiation into eg cardiac myocytes (of significant relevance for regenerative diseases in the pharmaceutical context), as well as to select selectively cytotoxic compounds for more targeted cancer treatment approaches. The project we now propose within the context of this DTP aims to capitalize on those advances even further, and to improve the computational algorithms we develop in our group to analyze information from chemical and biological sources in collaboration with various external collaborators who are able to perform experimental testing in a variety of settings (from stem cells to various disease models). Students working on this project will either have a background in the biological or chemical natural sciences (biology, pharmacology, chemistry, biochemistry, etc.) or in computer science/programming, and be willing to acquire skills in the respective other discipline. Given that the amount of chemical and biological data available is increasing steadily, skills to handle the information available will be very sought after in the near future, and hence this project will equip the graduate student working on it with excellent subsequent career perspectives.

Referees:

Using transcriptomics to guide lead optimization in drug discovery projects. B Verbist, G Klambauer, L Vervoort, W Talloen, QSTAR Consortium, Z Shkedy, O Thas, A Bender, HW Göhlmann, S Hochreiter – Drug Discov Today (2015, in press) http://dx.doi.org/10.1016/j.drudis.2014.12.014

Polypharmacology modelling using proteochemometrics (PCM): Recent methodological developments, applications to target families, and future prospects I Cortés-Ciriano, QU Ain, V Subramanian, EB Lenselink, O Méndez-Lucio, AP Ijzerman, G Wohlfahrt, P Prusis, TE Malliavin, GJP Van Westen, A Bender – MedChemComm (2015) 6, 24 http://dx.doi.org/10.1039/c4md00216d

Extending in silico mechanism-of-action analysis by annotating targets with pathways: application to cellular cytotoxicity readouts. S Liggi, G Drakakis, A Koutsoukas, I Cortes-Ciriano, P Martínez-Alonso, TE Malliavin, A Velazquez-Campoy, SC Brewerton, MJ Bodkin, DA Evans, RC Glen, JA Carrodeguas, A Bender – Future Med Chem (2014) 6, 2029 http://dx.doi.org/10.4155/fmc.14.137

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.ch.cam.ac.uk/group/bender/index http://www.andreasbender.de/

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Frank Jiggins, Dept. of Genetics

Genetic variation in the susceptibility of Drosophila to infection

PhD Project Description: In most species it is normal to find extensive genetic variation in susceptibility to infectious disease. Identifying the genes that are causing this variation allows us to understand both the evolutionary processes that maintain this variation in populations, and how hosts evolve resistance to infection at a molecular level. This project will combine large-scale whole genome sequencing with the powerful tools of Drosophila genetics to identify and characterize genes that cause variation in susceptibility to infection. A combination of experiments and analysis of patterns of genome sequence variation can then be used to understand why this variation is maintained in populations. We can also offer projects on other related projects in the lab.

Referees:

Magwire, MM, Fabian, DK, Schweyen, H, Cao, C, Longdon B, Bayer, F and Jiggins, FM 2012 Genome-wide association studies reveal a simple genetic basis of resistance to naturally coevolving viruses in Drosophila. PLoS Genetics. 8: e1003057.

Magwire, MM, Bayer, F, Webster, CL, Cao, C and Jiggins, FM 2011 Successive Increases in the Resistance of Drosophila to Viral Infection through a Transposon Insertion Followed by a Duplication. PLoS Genetics. 7: e1002337.

Martinez, J, Longdon, B, Bauer, S, Chan, Y-S, Miller, WJ, Bourtzis, K, Teixeira, L and Jiggins, FM 2014 Symbionts commonly provide broad spectrum resistance to viruses in insects: a comparative analysis of Wolbachia strains. PLoS Pathogens. 9: e1004369

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www.jiggins.gen.cam.ac.uk

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Tuomas Knowles, Dept. of Chemistry

Second Supervisor/Collaborator: Martin Welch, Department of Biochemistry, mv240@cam.ac.uk

Tuning functionality – using natural amyloids as a functional biological material in micro-gels

PhD Project Description: Amyloid fibrils are nanoscale protein filaments, initially discovered and characterized in relation to a number of disease states. Amyloid fibrils are however not only associated with amyloid diseases. They can also be exploited by living systems in which case they are known as functional amyloids. Escherichia coli and various Pseudomonas strains are known to utilize amyloid fibrils to change the external environment in a beneficial manner (for them!) by using amyloid fibrils to aid in the colonization of surfaces through the formation of biofilms. Micro-gels have been proposed as carriers of potential drug molecules by encapsulation of the drug inside the gel and thereby facilitate a slow release of the drug through diffusion out of the gel material. By controlled aggregation of the proteins from bacterial amyloids inside micro-droplets it is possible to spatially restrict the aggregates formed while also controlling the overall shape of the resulting aggregate. The purpose of the present project is to use a material designed to self-associate by Nature as building blocks for a new “designer” material. Since the functional bacterial amyloids have evolved to aggregate, the gel material can be formed from pure protein under physiological conditions. Consequently, it is possible to “re-tune” the characteristics of the functional amyloids from being beneficial to the bacteria to being a functional nanomaterial. This is a completely novel approach to making micro-gels and the project will significantly aid in our current understanding of the formation of and release of bioactive reagents captured within micro-gels.

Referees:

Shimanovich, U., et al., Protein microgels from amyloid fibril networks. ACS Nano, 2015. 9(1): p. 43-51.

Otzen, D., Functional amyloid: turning swords into plowshares. Prion, 2010. 4(4): p. 256-64

Other relevant themes: Basic Biosciences Underpinning Health

Link: http://www-knowles.ch.cam.ac.uk/

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Andrew Gillis, Dept. of Zoology

The development and evolution of gill arch appendages in cartilaginous fishes

PhD Project Description: Appendages provide excellent models with which to investigate the molecular basis of pattern formation. Indeed, decades worth of investigation into the molecular basis of tetrapod limb development have contributed substantially to our understanding of fundamental processes of organogenesis (e.g. axis establishment, developmental signalling centres, coordinated cell proliferation and differentiation). Cartilaginous fishes (sharks, skates, rays and holocephalans) possess endoskeletal appendages (branchial rays) that project laterally from their gill arches, and it was suggested over a century ago that these appendages are the evolutionary antecedent to paired fins and limbs. However, this controversial hypothesis of transformational homology remains effectively untested, owing to a lack of data on branchial ray development in cartilaginous fishes. I have established the skate (Leucoraja erinacea) as a developmental model system for the investigation of skeletal patterning in cartilaginous fishes. Previous work in the lab has discovered an epithelial signalling centre (the distal epithelial ridge, DER) that coordinates outgrowth and patterning of branchial rays, as well as evidence that the sonic hedghog, fibroblast growth factor and retinoic acid signalling pathways may act as molecular effectors of DER function. This PhD project will investigate the role of these developmental signalling pathways in patterning branchial rays, by employing experimental embryological manipulations, experimental dissection of signalling pathway function in vivo, fate mapping techniques and gene expression analysis (in situ and RNAseq) in skate embryos. This research will yield insight into basic mechanisms of vertebrate appendage patterning, and may also shed light on the evolutionary origin of paired fins and limbs.

Referees:

Gillis JA, Modrell MS, Baker CVH (2013) Developmental evidence for serial homology of the vertebrate jaw and gill arch endoskeleton. Nat. Commun. 4: 1436.

Gillis JA, Rawlinson KA, Bell J, Lyon WS, Baker CVH, Shubin NH (2011) Holocephalan embryos provide evidence for gill arch appendage reduction and opercular evolution in cartilaginous fishes. Proc. Nat’l Acad. Sci. U.S.A. 108: 1507-1512.

Gillis JA, Dahn RD, Shubin NH (2009) Shared developmental mechanisms pattern the gill arch and paired fin skeletons in vertebrates. Proc. Nat’l Acad. Sci. U.S.A. 106: 5720-5724.

Other relevant themes:

Link: http://www2.zoo.cam.ac.uk/gillis/Gillis_Zoology/Home.html

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Clare Baker, Dept. of Physiology, Development and Neuroscience

The development and evolution of vertebrate electroreceptors

PhD Project Description: Fundamental biological questions include how different cell types/organs are specified during development, and how novel cell types/organs evolve. An excellent model for both questions is the vertebrate lateral line system. Ancestrally, this comprises lines of 'neuromasts' on the head and trunk, containing mechanosensory hair cells (essentially identical to vestibular hair cells in our inner ear) that respond to local water movement, flanked on the head by fields of electrosensory 'ampullary organs' that respond to weak electric fields in water, allowing prey/predator detection. Both cranial neuromasts and ampullary organs develop from elongating cranial lateral line placodes. Electroreception was independently lost in the lineages leading to teleosts, frogs and amniotes. In a fascinating example of convergent evolution, electroreceptors that detect low-frequency environmental electric fields evolved independently at least twice within the teleosts. Within these teleost lineages, the 'weakly electric' teleost groups further independently evolved an electric organ generating high-frequency electric fields, and electroreceptors that respond to electric organ discharges. The main models for lateral line development, the zebrafish and the frog Xenopus, lack electroreceptors. To date, the lab has primarily studied electroreceptor development in the Mississippi paddlefish (a non-teleost ray-finned fish), with some work in the axolotl (a salamander), an experimentally tractable amphibian, and the little skate, a cartilaginous fish. The PhD project aims to understand further the development and evolution of vertebrate electroreceptors by investigating the cellular and molecular mechanisms of electroreceptor development in the axolotl or in weakly electric teleosts (gymnotiforms and/or mormyrids), using imaging, comparative transcriptomic and functional approaches.

Referees:

Modrell, M. S., Bemis, W. E., Northcutt, R. G., Davis, M. C. and Baker, C. V. H. (2011) Electrosensory ampullary organs are derived from lateral line placodes in bony fishes. Nature Communications 2: 496, DOI:10.1038/ncomms1502 (http://www.ncbi.nlm.nih.gov/pubmed/21988912)

Gillis, J. A., Modrell, M. S., Northcutt, R. G., Catania, K. C., Luer, C. A. and Baker, C. V. H. (2012) Electrosensory ampullary organs are derived from lateral line placodes in cartilaginous fishes. Development 139, 3142-3146 (http://www.ncbi.nlm.nih.gov/pubmed/22833123)

Modrell, M. S. and Baker, C. V. H. (2012) Evolution of electrosensory ampullary organs: conservation of Eya4 expression during lateral line development in jawed vertebrates. Evol. Dev. 14, 277-285 (http://www.ncbi.nlm.nih.gov/pubmed/23017075)

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

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Boris Adryan, Dept. of Genetics

Second Supervisor/Collaborator: Steve Russell

Transcription factor specificity during Drosophila embryonic development

PhD Project Description: Metazoan development can be considered as the progressive elaboration of a series of gene regulatory networks driven by the combinatorial interaction of transcription factors (TFs) at the cis- regulatory modules controlling the expression of target genes. In many cases different tissues utilise overlapping sets of TFs to direct radically different programmes of cellular differentiation and morphogenesis. Drosophila has served as an excellent model for understanding general principles in development due to well established genetics and genomics: this project will take advantage of experience in fly genome and developmental biology to explore aspects of TF specificity in the development of the embryonic tracheal system and CNS ventral midline. Both of these organ systems rely on the activity of an overlapping set TFs: The POU-domain protein Ventral veins lacking (Vvl) is required for the specification of both tissues. Vvl interacts with bHLH-PAS proteins, Trachealess (Trh) in trachea and Single-minded (Sim) in the midline, which both form heterodimers with the Tango (Tgo) protein. In the midline an additional factor, the Sox-domain protein Dichaete, is known to be required in partnership with Sim-Tgo-Vvl to activate target genes. The project will explore aspects of these regulatory networks by: characterising Dichaete binding in midline cells via ChIP-on-chip or ChIP-seq analysis; generate a GFP-tagged version of Vvl for similar studies; explore the phenotypic consequences of expressing dominant negative forms of Dichaete in the tracheal system; carry out a bioinformatics analysis of defined tracheal and midline target genes to identify binding motifs enriched in the regulatory regions of target genes.

Link: http://logic.sysbiol.cam.ac.uk http://flypress.gen.cam.ac.uk

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Chris Jiggins, Dept. of Zoology

Evolution and speciation in the tropics

PhD Project Description: Our research studies evolution at the population and species level in new world tropical butterflies. In particular, we are interested in the predictability of evolution – to what extent do different populations follow the same evolutionary trajectories. Convergent evolution, such as mimicry, offers the opportunity to ask whether the same genes, or the same kinds of genetic changes are involved repeatedly when different populations undergo similar evolutionary changes. By studying the genetic basis of adaptive traits we can answer questions regarding the origins of genetic variation needed for evolution (from novel mutation, hybridization, or standing variation), the kinds of mutations involved in evolutionary change (cis-regulatory versus structural protein changes) and the genetic architecture of genes involved in adaptation and speciation (many genes or few?). The huge diversity of divergent populations and species in Heliconius offers a wealth of natural variation with which to address these questions. Possible projects can range from developmental studies of wing pattern formation through to genetic and genomic studies of behaviour and other evolutionarily relevant traits.

Referees:

The Heliconius Genome Consortium Butterfly genome reveals promiscuous exchange of mimicry adaptations among species. Nature 487, 94–98 (2012). Martin, S. H. et al.

Genome-wide evidence for speciation with gene flow in Heliconius butterflies. Genome Res. (2013). Nadeau, N. et al.

Population genomics of parallel hybrid zones in the mimetic butterflies, H. melpomene and H. erato. Genome Res. gr.169292.113 (2014).

Link: www.heliconius.cam.ac.uk

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Juan Mata, Dept. of Biochemistry

Exploring the small proteome: a global approach

PhD Project Description: The prediction and experimental identification of small proteins (900 translated sORFs of 20 codons or longer, which correspond to ~ 20% of the known proteome of this well-studied organism. Very little is known about the expression and function of SEPs. The project will use S. pombe as model to study the expression and functions of SEPs. The project may involve some of the following approaches: ribosome profiling to predict additional SEPs, proteomics to experimentally validate SEP expression, and genetic and cell biological methods to investigate the function of SEPs. References: [1] Duncan C and Mata J (2014) The translational landscape of fission yeast meiosis and sporulation. Nat Mol Struct Biol doi:10.1038/nsmb.2843

Referees:

1. Hasan A, Cotobal C, Duncan C and Mata J (2014) Systematic analysis of the role of RNA-binding proteins in the regulation of RNA stability. PLoS Genet 10 (11): e1004684

2. Duncan C and Mata J (2014) The translational landscape of fission yeast meiosis and sporulation. Nat Mol Struct Biol doi:10.1038/nsmb.2843

3. Mata J (2013) Genome-wide mapping of polyadenylation sites in fission yeast reveals widespread alternative polyadenylation. RNA Biology 10 (8) 1-8

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

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Dr Olivier Restif, Dept. of Veterinary Medicine

Modelling the dynamics of bacterial infection in vivo using tagged strains.

PhD Project Description: Animal models retain a crucial role in biomedical research on bacterial infections. Over the last few years, the use of so-called tagged strains has initiated a major change in the way animal experiments can be designed and exploited, in line with the 3Rs policy (replacement, reduction and refinement of animal use). Tagged strains are genetically engineered clones of bacteria that contain a short DNA sequence (or barcode) inserted in a non-coding region of the chromosome. By infecting an animal with a mixture of tagged strains, it becomes possible to gain much more detailed insight into the dynamics of infection, effectively obtaining multiple replicates from a single animal. In order to fully exploit the data from such experiments, it is necessary to use mathematical and statistical models borrowed from population dynamics or population genetics. To date, however, these analytical tools have been developed on a case-by-case basis for each experimental system, and their usefulness for experimental design has not been fully exploited. The Department of Veterinary Medicine has been at the forefront of research in this area for more than ten years, with a unique synergy between microbiologists and modellers. The aim of this proposed PhD project is to develop unified modelling techniques that will be applicable to multiple experimental systems for both data analysis and experimental design. The student will have access to extensive datasets (published and unpublished) and in-house expertise on state-of-the-art techniques for both bacterial genetics and computational modelling.

Referees:

C Coward, O Restif, R Dybowski, AJ Grant, DJ Maskell, P Mastroeni 2014. The Effects of Vaccination and Immunity on Bacterial Infection Dynamics In Vivo. PLoS pathogens 10 (9), e1004359

Dybowski R, TJ McKinley, P Mastroeni and O Restif 2013. Nested Sampling for Bayesian Model Comparison in the Context of Salmonella Disease Dynamics. PLoS One 8:e82317.

Mastroeni P, A Grant, O Restif and DJ Maskell. 2009. A dynamic view of the spread and intracellular distribution of Salmonella enterica. Nature Reviews Microbiology 7:73-80.

Link: http://www.vet.cam.ac.uk/directory/or226@cam.ac.uk

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David Summers, Dept. of Genetics

Indole signalling in bacteria

PhD Project Description: Bacteria communicate by the exchange of chemical signalling molecules that regulate key processes such as pathogenicity, stress responses and co-operation within mixed communities. We have recently proposed a novel mechanism whereby a short-lived, high concentration of a signalling molecule, indole, regulates bacterial growth and cell division. We call this the "pulse signalling hypothesis". In this project we will combine of biological and physical approaches to further investigate this new mode of signalling and to assess the extent of its involvement in bacterial responses to a range of environmental stresses. A key objective of this proposal is to discover whether there is indeed a single mechanism underpinning all indole signalling. The project will include: (i) An investigation of the mode of indole signalling in response to stress induced by a range of conditions including antibiotic challenge and oxidative stress. (ii) A biophysical investigation of the effect of a wide range of indole concentrations on membrane conductivity, combined with a genetic investigation of the mechanism by which changes in conductivity are linked to modification of bacterial metabolism and changes in gene expression in response to stress.

Referees:

1. Pinero-Fernandez, S., Chimerel, C., Keyser, U.F., and Summers, D.K. (2011). Indole transport across Escherichia coli membranes. Journal of Bacteriology 193, 1793-1798.

2. Chimerel, C., Field, C. M., Piñero-Fernandez, S., Keyser, U. F., & Summers, D. K. (2012). Indole prevents Escherichia coli cell division by modulating membrane potential. Biochimica et Biophysica Acta - Biomembranes, 1818(7), 1590-1594.

3. Gaimster H, Cama J, Hernandez-Ainsa S, Keyser UF, Summers DK. The Indole Pulse: A New Perspective on Indole Signalling in Escherichia coli. PLoS One. 2014;9(4):e93168.

Other relevant themes: Basic Biosciences Underpinning Health

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

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Guy Brown, Dept. of Biochemistry

Eaten alive - cell death by phagocytosis

PhD Project Description: 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. Cells are phagocytosed as a result of: i) expressing eat-me signals on their surface, ii) losing don’t-eat-me signals, and/or iii) binding of opsonins. It is now clear that otherwise-viable cells can expose/bind such phagocytosis-promoting signals as a result of cell stress, activation or senescence. Phagoptosis is probably the most common form of cell death in the body as it is responsible for erythrocyte turnover. And there is increasing evidence that it mediates physiological death of neutrophils, T cells, platelets and stem cells, and thereby regulates inflammation, immunity, clotting and neurogenesis. Phagoptosis is a major form of host defence against pathogens and cancer cells. However, recent evidence indicates that excessive phagoptosis may kill host cells in inflammatory conditions, contributing to haemophagic conditions, and neuronal loss in the inflamed brain.

Other relevant themes: Basic Biosciences Underpinning Health

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Pentao Liu, Wellcome Trust Sanger Institute

Second Supervisor/Collaborator: Sarah Teichmann. European Bioinformatics Institute

PhD Project Description: Totipotent Stem Cells of Mammals Pentao Liu, Ph.D. Human pluripotent stem cells hold great promise for both basic research and regenerative medicine. The current human embryonic stem cells (ESCs), though extensively used in the stem cell community, are still difficult to manipulate and not considered to be the “truly” or naïve pluripotent stem cells. Intensive efforts have been made to capture and to maintain new or better human stem cells. We have established cultures of new human stem cells. These new cells, unlike the standard pluripotent ES cells or induced pluripotent stem cells (iPSCs), are totipotent. They can differentiate not only to embryonic lineages but also to trophoblasts, and are thus similar to the blastomeres in early preimplantation embryos. The mouse totipotent stem cells can efficiently differentiate to cells in both the embryo proper and the placenta. Totipotent stem cells therefore have broad applications in basic research and in translational research. This project will be first on functional and genomic (including epigenomics) studies of totipotent stem cells. The knowledge from these studies will aim establishment of similar stem cells from medically and agriculturally important mammalian species including pig and cow. Totipotent stem cells from these farm animal species should facilitate genetically modification of their genomes and reproduction including cloning. The Sanger Institute and the Wellcome Trust Genome Campus provide a collaborative academic environment for this study. Reference Ryan, D. J., Wang, W., Tsang, J. C-H., Yang, J., Lan, G., Gao, X., Antunes, L., Yu, Y., Kolodziejczyk, A., Campos, L., Wang, J., Wang, C., Yang, F., Bussell, J., Ramirez-Solis, R., Teichmann, S., Zou, X., Lu, L. and Liu, P. (2015). Establishment in Culture of Mouse Expanded-Potential Stem Cells. Under review.

Referees:

Ryan, D. J., Wang, W., Tsang, J. C-H., Yang, J., Lan, G., Gao, X., Antunes, L., Yu, Y., Kolodziejczyk, A., Campos, L., Wang, J., Wang, C., Yang, F., Bussell, J., Ramirez-Solis, R., Teichmann, S., Zou, X., Lu, L. and Liu, P. (2015). Establishment in Culture of Mouse Expanded-Potential Stem Cells. Under review.

Tsang, C. J., X. Gao, and Liu, P. (2014). Cellular reprogramming by transcription factor engineering. Current Opinion in Genetics & Development 28C, 1-9.

Gao, X., Yang, J., Tsang, J., Ooi, J, and Liu, P. (2013) Reprogramming to Pluripotency Using Designer TALE Transcription Factors Targeting Enhancers. Stem Cell Reports. 1,183-197.

Link: http://www.sanger.ac.uk/research/faculty/pliu/

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David Spring, Dept. of Chemistry

PhD Project Description: Our research is focussed on combining synthetic organic chemistry with biology and medicine, inventing new technology where necessary. This project will primarily give experience in organic synthetic skills, and some prior experience would be desirable. In the project we would make small molecules that have (or should have) interesting biological activities. They can be used as chemical probes of biological processes, and be potential chemotherapeutic agents. Specific projects will be designed taking into account the rotation students preferences, but some possible areas of biology that could be explored in this way are: Antibacterial discovery: http://www-spring.ch.cam.ac.uk/research/antibiotics.shtml Quorum sensing pathways: http://www-spring.ch.cam.ac.uk/research/qs.shtml And protein-protein interaction (PPI) modulation with small molecules. http://www-spring.ch.cam.ac.uk/research/ppi.shtml If you are interested in any of these topics and the application of organic synthesis to address them, then we hope you would be interested in our groups research.

Referees:

Synthesis of a novel polycyclic ring scaffold with antimitotic properties via a selective domino Heck-Suzuki reaction, E. Alza, L. Laraia, B. M. Ibbeson, S. Collins, W. R. J. D. Galloway, J. E. Stokes, A. R. Venkitaraman, D. R. Spring, Chem. Sci. 2015, 6, 390-396.

Diversity-Oriented Synthesis of Drug-Like Macrocyclic Scaffolds Using an Orthogonal Organo- and Metal Catalysis Strategy, A. Grossmann, S. Bartlett, M. Janecek, J. T. Hodgkinson, D. R. Spring, Angew. Chem. Int. Ed. 2014, 53, 13093-13097.

Functionalised staple linkages for modulating the cellular activity of stapled peptides, Y. H. Lau, P. de Andrade, S.-T. Quah, M. Rossmann, L. Laraia, N. Skold, T. J. Sum, P. J. E. Rowling, T. L. Joseph, C. Verma, M. Hyvonen, L. S. Itzhaki, A. R. Venkitaraman, C. J. Brown, D. P. Lane, D. R. Spring, Chem. Sci. 2014, 5, 1804-1809.

Link: http://www-spring.ch.cam.ac.uk

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Prof. Vasillis Koronakis, Dept. of Pathology

PhD Project Description: The control of numerous bacterial diseases and infections through the use of antibiotics has arguably been one of the greatest achievements of modern medical science. However, the inexorable rise in antibiotic resistance among pathogens poses an increasing threat, with the very real possibility that previously easily-controlled infections might again become untreatable diseases. One key mechanism through which bacteria may become resistant to multiple antibiotics involves the up-regulation of multidrug (MDR) efflux pumps in the bacterial cell envelope which eject drugs and other noxious molecules, including antibiotics, out of bacterial cells. Our laboratory studies the structure and operation of these important biological machines at the molecular level, opening the possibility that we might eventually develop countermeasures to antimicrobial drug resistance. This project is of a molecular/biochemical nature. You will seek to purify and structurally characterise a novel family of efflux pumps identified in Clostridium difficile, the cause of pseudomembranous colitis and a prominent hospital acquired infection. The ultimate aim is to determine the atomic structure of MDR pumps by X-ray crystallography. Working on this project, you will gain laboratory experience in cloning, protein expression and purification, ITC (isothermal titration calorimetry) and X-ray crystallography - all established techniques within our lab.

Referees:

Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C (2000) Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405: 914 - 919

Andersen C, Koronakis E, Bokma E, Eswaran J, Humphreys D, Hughes C, Koronakis V (2002). Transition to the open state of the TolC periplasmic tunnel entrance. Proceedings of the National Academy of Sciences (USA) 99: 11103 - 11108

Higgins M, Boekma E, Koronakis E, Hughes C, Koronakis V (2004) Structure of the periplasmic component of a bacterial antibiotic efflux pump. Proceedings of the National Academy of Sciences (USA) 101: 9994-99

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

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Anne Ferguson-Smith, Dept. of Genetics

PhD Project Description: Epigenetic control of genome function in purified mammalian cells. BLUEPRINT is a European Commission-funded project designed to generate reference epigenomes from purified homogeneous populations of mammalian cells ex vivo (Adams et al., Nature Biotech. 2012). Dr David Adams (Wellcome Trust Sanger Institute) and Prof Anne Ferguson-Smith (Department of Genetics, University of Cambridge) direct the only mouse model work-package on this programme – a hypothesis driven project designed to quantify the functional relationship between genome and epigenome. Over the past year, whole genome epigenetic and transcriptome data have been generated from purified cells from different inbred strains of mice and hybrids. These high quality datasets allow us to explore fundamental questions around the relationships between genotype, epigenotype and transcriptome. The student will select one of many projects associated with the BLUEPRINT mouse epigenome programme.

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

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Professor Roger Hardie, Dept. of Physiology, Development and Neuroscience

Ca2+ dependent feedback in Drosophila phototransduction

PhD Project Description: Phototransduction in microvillar photoreceptors is mediated by a canonical G-protein coupled phospholipase C (PLC) signalling cascade. In Drosophila this results in the activation of two distinct cation channels, TRP and TRPL, which are the prototypical members of the TRP ion channel superfamily[1]. Phototransduction in Drosophila is notable for its sensitivity and speed, generating large responses to single photons (quantum bumps), with a temporal resolution ~10-100 fold faster than in vertebrate rods. The TRP channel in particular is highly Ca2+ selective and Ca2+ influx via TRP channels has critical roles in regulating the gain and kinetics of phototransduction. In particular both activation and termination of the electrical response are accelerated ca 10-fold by Ca2+ influx indicating both positive and negative feedback effects of Ca2+ [review 2]. Negative feedback acts at multiple sites – including rhodopsin and PLC. In addition the channels themselves are both facilitated and inhibited by Ca2+, but the molecular basis is unknown. TRP is also incorporated into a multimolecular signalling complex via the a PDZ-domain scaffolding protein (INAD), but the functional significance of this interaction is unresolved. This project will use a combination of genetics, electrophysiology (whole-cell patch clamp), optophysiology (indicator dyes and GFP tagged probes) and molecular biology to investigate mechanisms underlying Ca2+ dependent regulation. Molecular genetic techniques will be used to generate flies expressing TRP channels and scaffolding proteins in which potential regulatory domains (such as calmodulin binding sites and PDZ interaction sites) are systematically mutated, and quantitative, state-of-the art physiological techniques used to analyse their consequences in vivo. This project should provide substantial and important new information on how TRP channels are regulated. Since phototransduction utilises the ubiquitous PLC signalling cascade, the mechanisms discovered are likely to be of more general significance. The project should provide excellent training in a range of disciplines including sensory physiology, ion channels, electrophysiology, imaging and molecular genetics.

Referees:

1. Hardie RC, Minke B (1992) The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Neuron 8:643-651.

2. Yau KW, Hardie RC (2009) Phototransduction motifs and variations. Cell 139:246-264.

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Dr Alex Webb, Dept. of Plant Sciences

PhD Project Description: We perform cutting edge research to understand the biology of plant cells. We are interested in how plants measure time and how this information is integrated with stress signalling. We believe that stress signalling is tightly linked to the daily rhythms of the plants so that the plant can make the appropriate responses to a stress signal, such as cold, dependent on the time of day the signal is perceived. We use experimental approaches and also develop new systems biology approaches. Projects for both experimentalists and theoreticians are available. We focus on the interactions between the internal circadian clock and cell signalling. We study how signalling pathways regulate the circadian clock and how the circadian clock regulates signalling. Projects on offer include the role of a Ca2+ switch in the circadian clock, regulation of circadian systems by NAD, reconstruction of transcriptional hierarchy of circadian Ca2+ signalling using bioinformatic and systems analysis of circadian transcriptomes, reverse genetic dissection of circadian Ca2+ signalling, mathematical modelling of circadian signalling, the role of photosynthetically derived sugars in regulating clock function and analyses of circadian clocks in crop plants. We have specialist facilities for circadian analyses including three automated imaging and photon counting systems for luminescence measurements, two low background PMT systems for aequorin measurements, a single cell FRET imaging workstation, a 16 camera automated leaf movement imaging system and a 6 channel infra red gas analysis system.

Referees:

Haydon, M.J., Mielczarek, O., Robertson, F.C., Hubbard, K.E. and Webb, A.A.R. (2013) Photosynthetic entrainment of the Arabidopsis circadian clock. Nature 502, 689–692.

Dodd, A.N., Gardner, M.J., Hotta, C.T., Hubbard, K.E., Dalchau, N., Love, J., Assie, J.M., Robertson, F.C., Kyed Jakobsen, M., Gonçalves, J., Sanders D. and Webb A.A.R. (2007) A cADPR-based feedback loop modulates the Arabidopsis circadian clock. Science 318, 1789 -1792.

Dodd, A.N., Salathia, N., Hall, A., Kévei, E., Tóth, R., Nagy, F., Hibberd, J.M., Millar, A.J. and Webb, A.A.R. (2005) Plant circadian clocks improve growth, competitive advantage and survival. Science 309, 630 – 633.

Link: http://www.plantsci.cam.ac.uk/research/alexwebb

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Dr Giles Yeo, Dept. of Clinical Biochemistry

Second Supervisor/Collaborator: Dr Darren Logan, Wellcome Trust Sanger Institute

Analysis of individual hypothalamic pro-opiomelanocortin (POMC) neurons

PhD Project Description: Over the past 15 years, insights from genetic studies have revealed that the brain leptin-melanocortin signalling is central to the control of mammalian food intake, with genetic disruption of most components of the pathway resulting in severe obesity in both mouse and man. Pro-opiomelanocortin (POMC) is a prohormone precursor for the melanocortin peptides and is produced in a distinct population of neurones the arcuate nucleus (ARC) of the hypothalamus. These melanocortin peptides are released from POMC neurones upon leptin stimulation and activate downstream melanocortin-4 receptor (MC4R) neurones in the paraventricular nucleus (PVN), resulting in reduced food intake and increased energy expenditure. However, the POMC neurons are not a homogenous group of neurons. Some, for example, are responsive to glucose or insulin, while others are responsive to leptin. To date, no one knows how many different populations of POMC neurons there are, and therefore how many are responsible for the regulation of food intake. To this end, we have specifically isolated POMC neurons by fluorescence activated cell sorting (FACS) from transgenic mice overexpressing EGFP in POMC neurons, and have obtained, using RNAseq in collaboration with the Sanger Institute, the transcriptomes from 96 individual POMC neurons. This project will involve a) analyses of these 96 transcriptomes and identification of genetic markers useful to categorise the POMC neurons; b) validation of these markers at the protein level in primary neuronal culture of FACS sorted POMC-GFP neurons; c) identify and validate in vivo the subset of POMC neurons that play a key role in the control of food intake. The eventual goal is to identify the specific POMC neurons and the underlying molecular mechanisms that play a role in controlling energy balance.

Referees:

Gumy L*, Yeo GSH*, Tung YCL, Zivraj K, Willis D, Coppola G, Lam BYH, Twiss JL, Holt C & Fawcett J. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localisation (2011) RNA. 17(1):85-98. Epub 2010 Nov 23. (*co-first authors.).

Zivraj K, Tung YCL, Piper M, Gumy L, Fawcett J, Yeo GSH† and Holt C†. Subcellular profiling reveals distinct and developmentally regulated repertoire of growth cone mRNAs. (2010)J Neuroscience. 17;30(46):15464-78. († co-corresponding author).

Jovanovic Z, Tung YCL, Lam BYH, O’Rahilly S and Yeo GSH. Identification of the global transcriptomic response of the hypothalamic arcuate nucleus to fasting and leptin. (2010) J Neuroendocrinol. 22(8):915-25.

Link: http://www.mrl.ims.cam.ac.uk/staff/AI/Yeo/index.php

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Dino Giussani, Dept. of Physiology, Development and Neuroscience

PhD Project Description: DEVELOPMENTAL PROGRAMMING OF HEART HEALTH. In addition to genetics and lifestyle risk factors, it is now accepted that the quality of the intrauterine environment also plays a role in shaping cardiovascular function in adult life. In turn, the quality of the intrauterine environment is primarily determined by the available nutrient and oxygen supply to the unborn child. Consequently, several studies have reported that reductions in oxygen and nutrient delivery to the fetus, as occurs in placental insufficiency or preeclampsia, can trigger cardiovascular abnormalities in the adult offspring. However, the mechanisms underlying developmental programming of alterations in cardiovascular health in offspring from complicated pregnancy remain unknown. This PhD project will test the hypothesis that oxidative stress underlies the molecular basis via which developmental programming of alterations in cardiovascular health occur in offspring from complicated pregnancy. If true, maternal treatment with antioxidants of complicated pregnancies may offer plausible clinical intervention to maintain cardiovascular health in the offspring. Using an integrative approach at the whole animal, isolated organ, cellular and molecular level, we have shown in several animal models that prenatal hypoxia promotes aortic and cardiac wall thickening with endothelial and cardiac dysfunction in the adult offspring. Further, these effects can be prevented by maternal treatment with vitamin C. While this provides proof of concept, only extremely high doses of vitamin C proved effective, making translation to human difficult. The PhD will adopt a similar approach to test different antioxidant regimes. http://news.sciencemag.org/sciencenow/2012/02/embryos-starved-of-oxygen-may-be.html

Referees:

Giussani, D.A., Camm, E.J., Niu, Y., Richter, H.G., Blanco, C.E., Gottschalk, R., Blake, E.Z., Horder, K.A., Thakor, A.S., Hansell, J.A., Kane, A.D., Wooding, F.B.P., Cross C.M. & Herrera. E.A. (2012). Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress. PLoS ONE 7(2): e31017. News piece written in Science Magazine about this work entitled: Embryos Starved of Oxygen May Be 'Programmed' for Heart Disease by Jean Friedman-Rudovsky on 13 February 2012. http://news.sciencemag.org/sciencenow/2012/02/embryos-starved-of-oxygen-may-be.html

Richter, H.G., Camm, E.J., Modi, B.N., Naeem, F., Cross, C.M., Cindrova-Davies, T., Spasic-Boskovic, O., Dunster, C., Mudway, I.A., Kelly, F.J., Burton, G.J., Poston, L. & Giussani, D.A. (2012). Ascorbate prevents placental oxidative stress and enhances birth weight in hypoxic pregnancy in rats. Journal of Physiology 590.6, 1377-1387. Paper selected as Editor’s Choice.

Niu, Y., Herrera, E.A., Evans, R.D. & Giussani, D.A. (2013). Antioxidant treatment improves postnatal survival and prevents impaired cardiac function in at adulthood following postnatal glucocorticoid therapy. J. Physiol. 591(Pt 20), 5083-93. Paper selected for Editorial Perspectives.

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

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Rob White, Dept. of Physiology, Development and Neuroscience

PhD Project Description: The function of transcriptional control networks is highly dependent on the ability of transcription factors (TFs) to identify and act on their appropriate specific target genes. However TFs commonly bind to short degenerate sites occurring very frequently in the genome. How then is functional specificity generated? There are two basic models; 1) that TFs bind in complexes with specificity generated by multiple DNA-protein and protein-protein interactions or 2) that chromatin structure plays the key role in controlling accessibility and target availability. The truth may lie somewhere between these extreme positions. We are studying this issue using the family of Hox TFs in Drosophila. They dramatically illustrate the problem of TF specificity as each member of the Hox family exhibits clear functional specificity in vivo and yet they show very similar DNA binding preferences in vitro. With regard to the above models for functional specificity, there is evidence that Hox proteins bind DNA together with cofactors and more recently we have demonstrated, in genome-wide studies of Hox protein binding, that chromatin accessibility also plays a major role. To dissect this further we have established a cell culture system that allows us to study the genome-wide binding and function of different Hox proteins. In this system we can manipulate the availability of cofactors and also modulate the chromatin accessibility. This project will involve studying the binding of Hox proteins using Chromatin-Immunoprecipitation coupled with next generation sequencing (ChIP-Seq) and analysing the datasets to identify the key determinants of functional Hox specificity.

Referees:

Choo SW, White R and S Russell (2011) Genome-wide analysis of the binding of the Hox protein Ultrabithorax and the Hox cofactor Homothorax in Drosophila. PLoS ONE 6: e14778.

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

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Prof. Stephen P. Jackson, Wellcome Trust Sanger Institute

PhD Project Description: DNA damage can block genome replication and transcription, and can yield mutations or wider-scale genome aberrations that threaten cell viability. To counter these threats, all organisms possess a highly evolutionarily conserved “DNA damage response” (DDR) to detect DNA damage, signal its presence and mediate its repair. The DDR impacts on myriad cellular events and prevents various age-related human diseases (1). This project will take advantage of a platform that we have recently established that combines the powerful molecular genetics of yeast with state-of-the-art next-generation sequencing and bio-informatics tools. Building on our recent success with similar work, one aspect of the project will be to identify second-site suppressors of phenotypes caused by defects in a known crucial DDR component, thereby providing insights into the function of that component and potentially also identifying new DDR modulators. The other aspect of the project will be to define DNA-damage induced mutational spectra of wild-type yeast strains and of strains bearing deletions in various known or candidate DDR components. This latter work should provide important insights into mutagenesis and DDR-pathway functions, and could also identify novel DDR components that have hitherto escaped detection by conventional approaches. While work during year 1 and early year 2 will focus on these yeast-based approaches, subsequent work will likely then address – by analyses of DDR functions, advanced microscopy and various molecular approaches – of analogous phenomena and DDR factors in human tissue culture cells. This project will take place within a well resourced cell and molecular biology laboratory but will also benefit from ready access to state-of-the-art DNA sequencing and bioinformatics within the Gurdon Institute and at the Wellcome Trust Sanger Institute.

Referees:

(1) Jackson, S.P., and Bartek, J. (2009). The DNA-damage response in human biology and disease. Nature 461, 1071-1078.

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

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