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World Class Underpinning Bioscience Rotation Projects 2014-2015

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

Supervisor: Professor Andrea Brand, Physiology Development and Neuroscience

Project abstract: 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.     

Students can choose one of the following projects:    

1. Nutritional control of neural stem cell quiescence and reactivation 

2. Investigating the role of non-coding RNAs in neurogenesis

Learning outcomes and skills acquired: Techniques used include genetics, molecular biology, transgenesis, RNA interference, immunohistochemistry, confocal microscopy, live imaging, transcriptomics, genome-wide DNA-protein interaction assays and bioinformatics.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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The role of NAD and calcium signalling in generating circadian rhythms in Arabidopsis

Supervisor: Professor Alex Webb, Plant Sciences

Project abstract: The circadian clock is a 24 h time keeper, which we have shown is essential for plant productivity (Dodd et al 2005 Science 309, 630 – 633). In this project you will investigate a new pathway we have identified in the plant circadian clock that is based on NAD-mediated modulation of calcium signalling (Dodd et al 2007 Science 318 1730-1731; Dalchau et al 2010 PNAS 107, 13171-13176; Dalchau et al 2011 PNAS 108, 5104 - 5109). In investigating this pathway opportunities are provided for traditional “wet bench” lab training or alternatively “dry bench” bioinformatics training.   You will investigate the temporal and spatial regulation of signalling intermediates within the circadian clock. Bioinformatic tools will be used to identify transcripts with specific regulatory behavioural patterns and temporal dynamics characteristic of predicted novel regulatory signalling components involved in generating NAD-derived signalling intermediates. Alternatively cell physiological analyses will be used to study the temporal and spatial regulation of calcium-sensor protein activity and localisation and its role in regulating circadian clock function.  This project provides the opportunity to identify novel components and regulatory behaviours in a newly identified signalling pathway that is central to the ability of plants to measure time and survive stress.

Learning outcomes and skills acquired: Students following either the bioinformatic or cell physiological programme in the laboratory will obtain skills that will enable them to analyse regulatory networks, signalling pathways and circadian rhythms.  Opportunities will be offered for training in network analysis using time series transcriptional datasets in wild types and genetically disrupted lines. Wet bench investigators will receive training in fluorescence and luminescence imaging of GFP, cytosolic free calcium and luciferase fusions. The experiments will be performed using time series image analysis and experimental manipulation using genetic and pharmacological disruption.

Project availability: Michaelmas and Lent Term

Other relevant themes: Food Security

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Identification of novel imprinted genes and analysis of their epigenetic control using mouse cell-specific transcriptomes

Supervisor: Professor Anne Ferguson-Smith,  Genetics

Project abstract: BLUEPRINT is a European Commission-funded project designed to generate reference epigenomes from ex vivo purified homogeneous populations of mammalian cells (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. Over the past year, whole genome epigenetic and transcriptome data have been  generated from purified cells from fully sequenced inbred strains of mice and reciprocal hybrids. BIoinformatic analysis has suggested the presence of previously unidentified imprinted genes with interesting properties suggesting novel epigenetic mechanisms of epigenetic control. The student will contribute to an experimental project on genomic imprinting and the epigenetic control of genome function (Ferguson-Smith, Nat Rev Genet 2011).

Learning outcomes and skills acquired: Quantitative expression analysis and quantitative epigenetic analysis in mouse.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Control of lineage commitment by chromatin remodeling proteins

Supervisor: Dr. Brian Hendrich, Biochemistry

Project abstract: We are interested in how stem cells make decisions during mammalian development. Specifically we are investigating how chromatin remodeling protein complexes, which influence gene expression levels, facilitate lineage commitment in pluripotent cells both in culture (i.e. Embryonic stem cells and epiblast stem cells) and in very early mouse development.     Work in our lab ranges from developmental biology, e.g.  studying how lineages are made during development, to biochemistry, e.g. understanding how chromatin remodelling complexes influece the activity of RNA Polymerase during active transcription. We have also started a collaboration with Microsoft Research to develop mathematical models of cell differentiation, taking into account single cell gene expression data.

Learning outcomes and skills acquired: Students will learn ES cell culture and manipulation, basic biochemical and molecular biology techniques, chromatin immunoprecipitation and quantitative RT-PCR.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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In vivo mechanisms of morphogenesis in early Drosophila embryos

Supervisor: Dr. Bénédicte Sanson, Physiology Development and Neuroscience

Project abstract: A key frontier in developmental biology is to understand the mechanisms of morphogenesis, that is the shaping of tissue and organs into  functional tridimensional structures. This requires understanding the molecular mechanisms controlling cell behaviours such as cell shape  changes, cell rearrangements, programmed cell death and cell division orientation, in the context of a tissue. Also, at the tissue scale, we  need to understand how individual cell behaviours coordinate and integrate mechanically to change the shape of a tissue. We address  these questions in early Drosophila embryos, during gastrulation and early segmentation. The advantage of this model is the powerful  genetics, but also the extensive knowledge that we have of the early patterning events and the ease of imaging live Drosophila embryos.  We are currently exploring how cell behaviours are controlled during axis extension and tissue boundary formation, processes which are  conserved in all bilateral animals (See Monier et al, 2010, Nature Cell Biology, 2: 60-5; Butler et al, 2009, Nature Cell Biology 11: 859-64;  http://www.pdn.cam.ac.uk/staff/sanson/). We use a range of approaches, including cell biology, confocal microscopy (including light sheet  imaging) and computational analysis of cell behaviours. Many possible projects are available in the lab; please contact bs251@cam.ac.uk if  interested.

Learning outcomes and skills acquired: We are biologists who collaborate with scientists in other fields such as computational sciences, mathematics and physics. So during a PhD, the student will gain the following skills working on a genetically tractable embryo: Drosophila genetics, molecular biology, immunochemistry, live imaging using transgenic fluorescent proteins on a variety of confocal microscopes, including cutting-edge microscopy techniques  such as light sheet, 2 photon and super-resolution. The student will also be exposed to interdisciplinary science in the field of biomechanics. Depending on previous background and individual interest, the student will be able to develop computational and modeling skills applied to image analysis.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Placing new neurons in the Drosophila odor discrimination circuitry using novel targeted expression tools

Supervisor: Dr. Cahir O'Kane,  Genetics

Second supervisor: Dr. Liria Masuda-Nakagawa, Genetics

Project abstract: 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 among a large palette of odors. We focus on the circuitry and its function in the input region of the mushroom bodies, the MB calyx. Here, MB neurons, the KCs, receive input from projection neurons (PNs), the secondary olfactory neurons. We previously developed a map of calyx glomeruli, which shows that PN input to the calyx is stereotypic, whereas KCs receive these inputs in a random and combinatorial manner. This model allows the discrimination of a large number of odors. To understand how the specificity of odor discrimination is achieved, a comprehensive analysis of the different neurons in the calyx circuitry is necessary. New Drosophila transgenic resources make this possible. Large collections of transgenic lines that can target expression of reporter genes to many sets of neurons in the fly brain are becoming available, along with large image datasets describing their brain expression patterns. This project aims firstly to use these resources to identify novel neurons in the calyx circuitry, and secondly, to study their connectivity in the calyx circuitry and how they integrate with previously characterized neurons, using targeted expression of markers for synapses (both pre- and post-synapses) and synaptic contacts.

Background References:

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

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

3. LM Masuda-Nakagawa, NK Tanaka, CJ O’Kane (2005) Stereotypic and random patterns of connectivity in the larval mushroom body calyx of Drosophila. Proc Natl Acad Sci USA 102:19027- 32.

Learning outcomes and skills acquired: Familiarity with genetic tools for marking specific neurons, their axonal and dendritic projections, and their synaptic partners; confocal microscopy

Project availability: Michaelmas and Lent Term

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A microscopy-based functional genomics genomic screen for genes that coordinate cell polarity and the cell cycle

Supervisor: Dr. Rafael Edgardo Carazo Salas, Genetics

Project abstract: Cell polarity and the cell cycle are two key aspects of cell life whose proper coordinated regulation is essential for healthy cellular function. Despite the fact that the molecular gearboxes for each process are well characterized, we essentially ignore the genes or protein networks that coordinate both processes.     Our group has pioneered the development of 3D image-based high-throughput/high-content microscopy pipelines for functional genomics screening. Capitalizing on that technology, we designed a biosensor that reports on the cell polarity and cell cycle state of live yeast cells and are using the biosensor to carry out a genomic screen for genes involved in coordinating both processes, which we will test and validate in human cell lines.     A student in our lab could join this exciting interdisciplinary project, which well underway, either helping us: 1) finish collecting the screens’ data using high-throughput cell biology and microscopy, and/or 2) help us mining the data using quantitative image analysis pipelines and big data statistical/bioinformatics strategies, and/or 3) seeking to validate preliminary candidate hits.    

References:   

Graml, Studera, Lawson, Chessel et al, accepted in Dev Cell   Dodgson J, Chessel A, et al. 2013  Nat Comm 4:1834. doi: 10.1038/ncomms2813.  

Vaggi F, et al.  PLoS Comp Biol (2012) 8(10):e1002732.   Kim DU et al., Nat Biotechnol. 2010 Jun;28(6):617-23.    

Keywords:   Cell polarity, cell cycle, genome-wide knockouts, functional genomics, quantitative image analysis, high-throughput/high-content microscopy, yeast, human cells, big data.

Learning outcomes and skills acquired: Learning outcomes: A student participating in this project will acquire foundations and working knowledge of yeast genetics as well as potentially human cell work, cell polarity and cell cycle control, and systems biology/functional genomics approaches to cell biological questions.     Skills acquired: This project involves the development and use of interdisciplinary methodologies at the interface between cell biology, systems biology, functional genomics bioinformatics and big data analysis. Hence, a student participating in this project will acquire diverse skills such as high-throughput/high-content microscopy, genome-wide knockout screening, large-scale image analysis, and computational modelling and bioinformatics.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Indole signalling and antibiotic resistance

Supervisor: Dr. David Summers, Genetics

Project abstract: 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". We intend to combine biological and physical approaches to further investigate this new mode of signalling and to assess the extent of its involvement in bacterial responses to antibiotic treatment.

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.

Learning outcomes and skills acquired: Basic microbiology and cell biology techniques. An understanding of bacterial signalling mechanisms and their role is bacterial resistance to antibiotics.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Clonality in relation to histiocytoma, a canine tumour that regresses following T cell invasion

Supervisor: Dr. David Sargan, Veterinary Medicine

Project abstract: Histiocytoma is a common neoplasm of the dermis in young dogs in which the major neoplastic cell is a dendritic cell with Langerhans cell characteristics. In most dogs this tumour appears as an isolated growth or a small number of growths, grows rapidly over a period of several weeks, but then regresses after an inflammatory response with T-cell invasion. Occasionally the tumour evades the immune response and goes on to metastasise to visceral sites. The large majority of cancers result from clonal expansion of a single transformed cell. To understand whether this tumour is caused by proliferation that is reactive to an immune challenge or is a result of oncogenic tranformation this project will examine clonality of several tumour biopsies from female dogs, by looking at X inactivation in the tumour. Tumour hosts will be checked for heterozygosity of specific microsatellites on the X chromosome. Semi-pure histiocyte populations will be prepared from the tumour mass by dissection, dispersing and FACS sorting. Methylation state of the X chromosomes in the tumour cells will then be checked by methylation sensitive restriction enzyme cutting followed by PCR amplification of targets on X that have microsatellites within CpG islands containing restriction sites. This assay will give rise to 50% cutting of each X in polyclonal cell populations so that amplified heterozygous microsatellites remain heterozygous, but preferential loss of one or the other allele occurs in clonal cell populations. This assay was developed in humans but has not previously been applied to veterinary species.

Learning outcomes and skills acquired: The student will learn a variety of cell and molecular techniques as outlined above. They will also gain a considerable understanding of tumour formation, and of the biology of X inactivation.

Project availability: Michaelmas and Lent Term

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MHC allotype in relation to histiocytoma, a canine tumour that regresses following T cell invasion

Supervisor: Dr. David Sargan, Veterinary Medicine

Second supervisor: Fernando Constantino-Casas, Veterinary Medicine

Project abstract: Histiocytoma is a common neoplasm of the dermis in dogs in which the major neoplastic cell is a dendritic cell. The tumour has similar characteristics to the rare self-healing “Hashimoto–Pritzker” tumour in humans. In most dogs this tumour is an isolated growth, growing rapidly over a period of several weeks, but then regresses after an inflammatory response with increased MHC class II expression and T-cell invasion. Occasionally the tumour evades the immune response and spreads to visceral sites.     Dog Leucocyte Antigen (DLA) genes are highly polymorphic across all breeds but display much lower intra-breed variation. The student will compare MHC Class II genotypes of histiocytoma cases and age matched controls within a flatcoated retriever (FCR) to see whether particular DLA types show association with the disease.     Second exons of DLA-DRB1, DQA1 and DQB1 from DNA of FCR histiocytoma cases will be sequenced and DLA genotypes and haplotypes determined, and compared with pre-existing control data. The experiment size will give power to detect disease associated alleles with genotypic risk/protection >3. We shall also look at metastatic cases if available.      One hazard with assigning odds ratios at single loci is the possibility that a particular line of dogs suffers cancer because of a founder mutation and shares many loci including DLA through identity by descent. To detect the presence of population structure, microsatellites from cases and controls will be analysed using the program Structure.  Cases and controls will then be assigned to sub-populations and DLA frequencies compared within them.   

Learning outcomes and skills acquired: The student will learn to recognise the pathological processes involved in tumour formation and in infalmmation and regression for this tumour. They  will learn multiplex PCR skills. Sequencing will be performed elsewhere, but contig assembly, alignments, heterozygosity checking and odds calculations will be performed in house. The student will also gain an understanding of population stratification and the limitations of OR calculations.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Calculation of chromosome structure and genome architecture in haploid mES cells

Supervisor: Professor Ernest Laue, Biochemistry

Second supervisor: Dr. David Lando, Biochemistry

Project abstract: In order to gain a more system-wide view of chromatin structure within the cell we are exploiting similar computational approaches (to those used to determine structural models of protein complexes) to studies of the global structure of chromatin in single cells. A chromatin conformation capture (3C) experiment, called Hi-C, is being used to provide spatial restraints on the structure. In these experiments restriction fragment digestion of DNA in intact nuclei is followed by the ligation of free DNA ends that are in close 3D spatial proximity. These ligated DNA junctions are then identified by high-throughput sequencing to provide distance restraints for structure calculations of chromatin architecture in the nucleus.    Our initial studies have focussed on calculating the 3D structure of the X chromosome in stably differentiated cells, but in unpublished experiments we have been able to calculate 3D models of the complete haploid genome in ES cell nuclei. Our aim now is to work out how best to use and exploit multicolour super-resolution imaging of NuRD complexes in future work where we combine single molecule imaging with Hi-C experiments carried out on single fixed haploid mESCs. This would allow us to study the association of particular regions of the genome with the NuRD complex, and to ask how this correlates with gene expression during differentiation?   

Recent references:  

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature. 2013 Oct 3;502(7469):59-64. doi: 10.1038/nature12593. Epub 2013 Sep 25. 

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast. Open Biol. 2012 Jul;2(7):120078. doi: 10.1098/rsob.120078.

Learning outcomes and skills acquired: The student would learn about and become familiar with:    •      mES cell culture, cell sorting and single cell studies  •  Next generation high-throughput DNA sequencing and analysis  •            Computational methods to calculate genome structures  •    Bioinformatics studies to analyse e.g. enhancer/promoter interactions

Project availability: Michaelmas and Lent Term

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Single molecule studies of chromatin assembly using super-resolution fluorescence microscopy

Supervisor: Professor Ernest Laue, Biochemistry

Second supervisor: Dr. Srinjan Basu, Biochemistry

Project abstract: In recent years a number of techniques have been developed for studying single molecules using fluorescence microscopy at a higher resolution than that limited by the diffraction of light (so called super-resolution methods). Several research groups have been able to apply these methods to studies in live bacteria, or on the surface of mammalian cells, but we would like to exploit these methods to study nuclear processes in higher eukaryotic organisms.  Our overall objective is to visualize how different Nucleosome Remodelling and Deacetylase (NuRD)/transcription factor complexes assemble and function at particular genomic loci in single mouse embryonic stem cells (mESCs), and to ask how this affects gene expression during ES cell differentiation. We are developing new 3D multicolour single-molecule fluorescence microscopy techniques and plan to carry out three types of project:  1) Multicolour super-resolution imaging of different NuRD/chromatin protein/transcription factor complexes in fixed mouse ES cells during differentiation  2) 3D single-molecule tracking of CHD4 and NuRD complexes in live mESCs to understand how CHD4 functions  3) In-vitro single-molecule binding of CHD4 and purified NuRD complexes to DNA/ nucleosome arrays  In unpublished experiments we have been able to show that distance restraints obtained from single cell Hi-C experiments (one of the chromosome conformation capture, or 3C experiments), will allow us to calculate 3D models of the complete haploid genome in ES cell nuclei. Our long-term aim is to work out how best to use and exploit multicolour super-resolution imaging of NuRD complexes in future work where we combine single molecule imaging with Hi-C experiments carried out on single fixed haploid mESCs. This would allow us to study the association of particular regions of the genome with the NuRD complex, and to ask how this correlates with gene expression during differentiation?   

Recent references: 

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast. Open Biol. 2012 Jul;2(7):120078. doi: 10.1098/rsob.120078. 

Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature. 2013 Oct 3;502(7469):59-64. doi: 10.1038/nature12593. Epub 2013 Sep 25.

Learning outcomes and skills acquired: The student would learn about and become familiar with:    •      Using molecular and cell biological techniques to tag particular proteins with photo-activatable fluorophores  •     Super-resolution microscopy to study the assembly of protein complexes in fixed cells  •  The study of protein dynamics in live cells at the single molecule level

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Genetics and evolution of Drosophila and its viruses

Supervisor: Dr. Frank Jiggins, Genetics

Project abstract: Why do individuals within populations vary in their susceptibility to infection? Answering this question can provide insights into the coevolution of hosts and pathogens, and the molecular interactions that determine the outcome of infection. We are addressing these questions by identifying the genes that cause variation in susceptibility in Drosophila and mosquitoes. This project would involve using techniques in Drosophila genetics and molecular biology. Depending on the interests of the student, we can also offer projects involving bioinformatics and evolutionary genetic analyses. Take a look at the lab website for details of what we work on.

Learning outcomes and skills acquired: This work will provide training in Drosophila genetics, molecular biology and data analysis. Projects can also include an element of bioinformatics

Project availability: Michaelmas and Lent Term

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Profiling DNA methylation in single cells

Supervisor: Dr. Gavin Kelsey, The Babraham Institute

Project abstract: DNA methylation is the classic epigenetic modification in the vertebrate genome, yet it is only recently, with the development of whole-genome bisulphite sequencing (WGBS), that we have been able to assess the methylation landscape across the entire genome.  Because WGBS provides single-base resolution and accurate quantitation, it has rapidly become a gold standard for methylation analysis; however, until very recently, WGBS could only be applied to DNA from thousands of cells, or more, to provide a population average of methylation.  This limitation obscures functionally important cell-to-cell variation in DNA methylation that could have impacts on cell phenotype or potential; for example, in cell lineage decisions or disease progression.  We have recently developed a method capable of profiling DNA methylation of a substantial fraction of the genome in single cells (scBS-seq).  However, although a tremendous advance, our current method is relatively cumbersome, so further development is needed to realise the full potential of single-cell methylation analysis, or to allow it to be adapted to robotic or commercial platforms.  In addition, it would be a huge advantage to combine scBS-seq with single-cell transcription analysis by RNA-seq.  The aim of this project is to develop a truly high-throughput, genome-wide DNA methylation profiling method.  Ref: Smallwood, Lee et al. (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat. Methods doi:10.1038/nmeth.3035.

Learning outcomes and skills acquired: The student will take an existing, cutting-edge method and deconstruct it, examining all aspects in order to find gains that will substantially improve efficiency, robustness and coverage.  Thus, the student will be encouraged to go back to first principles and challenge all steps in a method.  It is also anticipated that part of the work will be with a commercial collaborator, which will give the student exposure to different drivers for success.  Overall, the student will develop skills in molecular biology techniques, generation of next generation sequencing libraries, analysis of sequence data.

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

Other relevant themes: Basic bioscience underpinning health

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Building synthetic carbonyl metalloproteins for CO delivery in vivo

Supervisor: Dr. Gonçalo Bernardes, Chemistry

Second supervisor: Dr. Omar Boutureira, Chemistry

Project abstract: CO-releasing molecules (CORMs) have been proposed as pharmaceutical agents[2] and have the potential to constitute safe treatments if CO release in vivo can be controlled in a spatial and temporal manner.[Angew Chem Int Ed 2014, in press] CORM-3, [RuCl-glycinato(CO)3], has demonstrated beneficial activity in animal models of disease with major clinical indications. Recently, we have detailed the interactions of CORM-3 with proteins which revealed that this ruthenium carbonyl reacts rapidly with plasma proteins leading to Ru species bounded to these proteins.[J Am Chem Soc 2011, 1192] The study showed that CORM-3 reacts with exposed histidine 15 of HEWL to form a stable [Ru(CO)2(H2O)3]2+ adduct after loss of a chloride ion, a glycinate and one CO ligand. This result suggests a rapid reaction of CORM-3 with plasma proteins after entering the blood stream, loses one equivalent of CO in the form of CO2, yielding protein–Ru(CO)2 adducts. It is possible that these adducts, which are carried throughout the body in the circulation, are responsible for CO distribution to different organs and tissues through slow loss of CO, thus preventing rapid CO-Hb elevation. In this project, we will develop a new method for the site-selective modification of exposed histidines on proteins with different metal carbonyls to generate chemically defined carbonyl metalloproteins. These new synthetic carbonyl metalloproteins will allow us to evaluate if carbonyl metalloproteins can act as CO carriers in vivo.

Learning outcomes and skills acquired: The student will acquire key training in organometallic and protein chemistry.

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

Other relevant themes: Bioenergy and industrial biotechnology

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How does vaccinia virus hijack kinesin to move intracellularly on microtubules?

Supervisor: Professor Geoffrey L Smith, Pathology

Second supervisor: Dr David Carpentier, Pathology

Project abstract: Vaccinia virus (VACV) is the vaccine that was used to eradicate smallpox. B VACV is being engineered as a vaccine against other pathogens and as an oncolytic agent against cancers. Our lab is studying how VACV spreads within and between cells and this project concerns how VACV exploits the kinesin-1 microtubule motor complex to transport newly synthesised virions to the cell surface, and what effect VACV has on cellular vesicular traffic.   VACV proteins F12, E2 and A36 have been implicated in IEV transport but their exact roles are unclear. A36 is a transmembrane protein, whereas F12 and E2 lack a transmembrane domain but contain tetratrichopeptide repeats (TPRs) that are also found in kinesin light chains (KLCs) that form part of the kinesin motor. TPRs have been implicated in mediating protein – protein interactions. Kinesin-1 exists as a tetrameric complex containing two copies of the kinesin heavy chain (KHC) and two copies of the kinesin light chain (KLC). Mammalian cells express several isoforms of KLC.  KLC1 and 2 are expressed widely but less is known about KLC3 and 4.   The project will investigate (1) how VACV interacts with the kinesin complex via KLC2, rather than KLC1, and (2) how this interaction affects KLC2 function, such as its role in vesicle trafficking. To do this we will use a cell line expressing GFP-tagged subunit of the Na+,K+ ATPase and monitor the effect of VACV proteins F12/E2/A36 on the ability of these vesicles to traffic.   

Reference: Roberts KL, Smith GL (2008) Trends Microbiol 16: 472-479.

Learning outcomes and skills acquired: The student will work as a part of a team of postdoctoral and PhD students studying related topics in a well-equipped laboratory and funded laboratory. There will be a broad training in a range of techniques including mammalian cell culture, virology, protein expression in mammalian cells via transfection or viral infection, microscopy using our state of the art confocal, video and electron microscopes, and analysis of protein-protein interactions via immunoprecipitation and immunoblotting. These techniques will be applicable to a broad range of research topics in biological science.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Enterobacterial quorum sensing, plant pathogenesis, bioactive secondary metabolites, and bacteriophage-host Interactions

Supervisor: Professor George Salmond, Biochemistry

Project abstract: We work on the molecular biology of enterobacterial pathogens of plants and animals, namely Erwinia, Dickeya and Serratia. We are studying the role of quorum sensing (QS) in pathogen virulence and examining signal transduction routes and the nature of the corresponding target genes and processes controlled by QS. One such process is the regulation of carbapenem antibiotic production in Erwinia and Serratia and the regulation and biosynthesis of the antimicrobial, immunosuppressant and anti-cancer agent, prodigiosin, in Serratia species. QS also affects production of novel and potentially biotechnologically useful antifungal molecules by rhizosphere bacteria which we have made genetically amenable. QS also regulates production of Serratia gas vesicles – flotation organelles that enable migration to air-liquid interfaces in static cultures. We are also investigating the roles of new phages that infect enterobacterial pathogens, in terms of phage conversion and by exploiting them for functional genomics and synthetic biology. Some of these bacterial pathogens have evolved defensive responses to phage infection (abortive infection systems that act in altruistic suicide, post-infection). We investigate the role of abortive infection mechanisms in these phage-host interactions from evolutionary, physiological, structural and ecological perspectives. Diverse techniques are used in the lab, including bacterial and phage genetics, genomics, proteomics, protein expression, gene fusions, mRNA assays, and assays of antibiotics, quorum sensing molecules and virulence assays.     For more details, see:  http://www.bioc.cam.ac.uk/people/uto/salmond

Learning outcomes and skills acquired: Depending on the particular project chosen, the student will acquire a broad skill set in bacterial genetics, phage biology and viral evolution, gene fusion techniques, genomics and mRNA work, diverse bioassays (including quorum sensing assays, virulence, enzyme assays, buoyancy assays, antibiotic assays and assessment of other bioactives), phase contrast microcopy and EM work - among others.

Project availability: Michaelmas and Lent Term

Other relevant themes: Food security & Bioenergy and industrial biotechnology

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Regulation of the RNAi response in C. elegans by cell signalling

Supervisor: Dr. Howard Baylis, Zoology

Project abstract: RNAi is one of number of small non-coding RNA (sncRNA) mediated processes. In C. elegans and other organisms RNAi is a systemic response. We have discovered that mutations in the IP3/calcium signalling pathway enhance or suppress the RNAi response in C. elegans. IP3 (inositol 1,4,5-trisphosphate) signalling is a core signalling pathway downstream of both RTKs and GPCRs, that transduces a wide range of signals and thus controls many aspects of an animal’s physiology and behaviour. Thus, the RNAi response may be regulated by an animal’s physiological state and the environment in which it finds itself. This has implication for our understanding of RNAi and its application to the manipulation and control of living organisms.

In this rotation you will address one of two questions:

(A) In order to understand the global influence of IP3 signalling on sncRNAs in C. elegans you will use RNAseq or qPCR to identify changes in other sncRNA pathways

(B) One possible environmental modulator of RNAi is starvation, you will therefore test whether genes which mimic starvation and play key roles in nutrient sensing (eg AMPK) alter RNAi.

Learning outcomes and skills acquired: You will work at the interface of molecular, cellular and whole animal biology. You will use a variety of molecular and transgenic approaches. Quantitative analysis of phenotypes will use fluorescence and microscopical techniques. In addition you will learn techniques related to C. elegans.

Project availability: Michaelmas and Lent Term

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Genome editing in C. elegans using CRISPR/Cas9 to test the function of a predicted CpG binding protein Zn-finger

Supervisor: Professor Julie Ahringer, Genetics

Project abstract: Most vertebrate promoters lie in unmethylated CpG-dense islands, while methylation of the more sparsely distributed CpGs in the remainder of the genome is thought to contribute to transcriptional repression. Non-methylated CG dinucleotides at promoters are recognized by CXXC finger protein 1 (Cfp1).  Cfp1 is part of the COMPASS complex, which generates histone H3 lysine 4 trimethylation, an active promoter mark.  Genomic regions enriched for CpGs were thought to be absent or irrelevant in invertebrates that lack DNA methylation, such as C. elegans, however, a Cfp1 ortholog is present.  We recently showed that C. elegans CFP-1 targets promoters with high CpG density and that these promoters are marked by high levels of H3K4me3, as in mammals. Our results indicate that unmethylated CpG-dense sequence is a conserved genomic signal that promotes an open chromatin state and marking by Cfp1 occupancy and H3K4me3 modification in both C. elegans and human genomes.  How COMPASS is recruited to CpG dense sequence is not well understood.  This project is to test whether the CXXC Zn finger of C. elegans CFP-1 is required for its function. The newly developed method of CRISPR/Cas9 genome editing will be used to mutate the conserved cysteines of the CFP-1 Zn finger in vivo and then the function of this mutant CFP-1 will be assessed by analyzing the phenotype of the resulting mutant strain. 

Chen, A.-J., Stempor, P., Down, T.A., Zeiser, E., Feuer, S., and Ahringer, J. (2014) Extreme HOT regions are CpG dense promoters in C. elegans and human, Genome Research, 24: 1138-1146.

Learning outcomes and skills acquired: Experimental design to address biological research questions, CRISPR/Cas9 genome editing, molecular biology, C. elegans handling, microscopy, western blotting

Project availability: Michaelmas and Lent Term

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Executable Programs of Cell Fate Decisions

Supervisor: Dr Jasmin Fisher, Biochemistry

Project abstract: Cell fate determination is a key question in biology. Cell fate specification processes must be under strict regulation to ensure life-long homeostasis. The signalling pathways that are known to play a key role in development - such as Ras, Notch and Wnt - are often deregulated during various diseases due to mutations in key elements of these pathways. 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 disease. However, due to their enormous complexity and multiple interactions, the comprehension and analysis of these signalling pathways is a major challenge.  To study such mechanisms we have been using program analysis techniques for the construction and analysis of executable models describing cell fate decisions in various model systems (e.g., C. elegans, Drosophila, S. cerevisiae, mammalian epidermis, and blood cells). These models are essentially computer programs whose behaviour captures aspects of biological phenomena. Over the years, these efforts have demonstrated successfully how the use of formal methods can be beneficial for gaining new biological insights and directing new experimental avenues. 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. In addition, preliminary studies (in silico) can be quickly performed on a computational model, saving valuable laboratory time and resources for only the most promising avenues. We are currently developing a novel computational platform for the simulation of lineage development. The simulations evolve according to causal rules that determine the behavior of signalling pathways and cell decisions. Creating such programs will help us better understand the orchestration of cell decision processes.   This project aims to create the program that guides the simulation of the lineage of C. elegans and to investigate the robustness and the behavior of the developing embryo. Interacting with C. elegans experts in our group and with computer scientists who are developing the novel platform, the student will summarize the information regarding C. elegans development in the form of a program that corresponds to the causal chains that govern the worm development.

Learning outcomes and skills acquired: In the course of this rotation the student will learn to apply computational modelling methods to gain new biological insights and generate new hypotheses to be tested experimentally. Working in our lab, the student will also get a flavor of the different modelling and analysis tools we have been developing in order to deepen our understanding of complex biological phenomena.

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

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Study of the structure and function of proteins using atomic force microscopy (AFM)

Supervisor: Prof. J. Michael Edwardson, Pharmacology

Project abstract: We study the structure and function of proteins at the single-molecule level using atomic force microscopy (AFM). A number of projects are currently ongoing in the lab, as detailed below, and new recruits to work on any of these projects would be welcomed.    1. The subunit arrangement in ionotropic receptors.  2. Activation-induced structural changes in ionotropic receptors.  3. The interaction of the sigma-1 receptor with ionotropic receptors and ion channels.  4. The structure of proteins involved in autosomal dominant polycystic kidney disease (ADPKD).  5. Investigation of the mechanism underlying the interaction of urinary exosomes with the primary cilium.  6. The structure and behaviour of synaptotagmin.  7. Structure and behaviour of seipin.    Further details are available on my website:    http://www.phar.cam.ac.uk/research/Edwardson   

The following references provide a picture of what we have been doing recently:   

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

2. Namadurai, S., Balasuriya, D., Rajappa, R., Wiemhöfer, 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 β3 subunit indicates a trimeric assembly. J. Biol. Chem. 289, 10797-10811   

3. Balasuriya, D., Goetze, T.A., Barrera, N.P., Stewart, A.P., Suzuki, Y. and Edwardson, J.M. (2013) AMPA and NMDA receptors adopt different subunit arrangements. J. Biol. Chem. 288, 21987-21998   

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

5. Balasuriya, D., Stewart, A.P. and Edwardson, J.M. (2013) The sigma-1 receptor interacts directly with GluN1 but not GluN2A in the GluN1/GluN2A NMDA receptor. J. Neurosci. 33, 18219-18224   

6. Balasuriya, D., Stewart, A.P., Crottès, D., Borgese, F., Soriani O. and Edwardson, J.M. (2012) The sigma-1 receptor binds to the Nav1.5 voltage-gated Na+ channel with four-fold symmetry. J. Biol. Chem. 287, 37021-37019    

7. Sim, M.F.M., Talukder, M.M.U., Dennis, R.J., O’Rahilly, S., Edwardson, J.M. and Rochford, J.J. (2013) Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms. Diabetologia 56, 2498-2506   

8. Oatley, P., Talukder, M.M.U., Stewart, A.P., Sandford, R. and Edwardson, J.M. (2013) Polycystin-2 induces a conformational change in polycystin-1. Biochemistry 52, 5280-5287   

9. Oatley, P., Stewart, A.P., Sandford, R. and Edwardson, J.M. (2012) Atomic force microscopy imaging reveals the domain structure of polycystin-1. Biochemistry 51, 2879-2888

Learning outcomes and skills acquired: Students will be trained in the following skills:    1. Cell culture.  2. Cell transfection.  3. Gel electrophoresis/immunoblotting.  4. Protein isolation.  5. AFM imaging.

Project availability: Michaelmas and Lent Term

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The study of the dynamics of protein interactions during lineage commitment of mouse embryonic stem cells

Supervisor: Professor Kathryn Lilley, Biochemistry

Second supervisor: Professor Alfonso Martinez-Arias, Genetics

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

Learning outcomes and skills acquired: The student will become familiar with the field of stem cell biology, proteomics and mass spectrometry.    The skills acquired will be cell culture, basic protein biochemistry, mass spectrometry, quantitative proteomics approaches including SILAC and isobaric tagging, bioinformatics and data interpretation.     The student will also be expected to give a talk about their short project and will be given training in presentation skills, both oral and written.    The student will be working in two vibrant research groups and will be given the opportunity to work as part of large teams of PhD students and post doctoral research workers.

Project availability: Michaelmas and Lent Term

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Cellular Systems Biology of 3D Chromosome and Chromatin Dynamics in Embryonic Stem Cells and Yeast

Supervisor: Dr Karen Lipkow, The Babraham Institute

Project abstract: The DNA of all eukaryotes is present in the nucleus as chromatin, in which DNA is closely associated with hundreds of different proteins. The precise combination at a given location determines how a gene is regulated and can be captured as a defined ‘chromatin state’ [1]. In all organisms analysed, these states differ in relevant biological properties, such as gene expression and enrichment of gene ontologies. We and others have shown that they are finely scattered in 1D along the chromosomes. In yeast, we could demonstrate that they co-localise in 3D, and that, to our surprise, much of the chromatin is poised, ready for change, under both standard and heat stressed conditions [2].   We are offering two independent computational projects:  1) Bioinformatics: The aim is to advance our computational method to determine chromatin states and apply it to the developmental pathway of stem cell biology. This requires a working knowledge of R, and Python or Perl. The student will be introduced to the data analysis of RNA-Seq and Chip-Seq datasets, and to representing genome wide DNA-DNA contact maps in 3D.    Computational modelling: The aim is to build 3D models of the nucleus and run dynamic simulations to investigate the effects of 1D and 3D genome organisation with respect to chromatin states. This will use different modelling and data analysis approaches, building on our experience in this field [3,4,5], and on structural (Hi-C) and live-cell microscopy experiments in our group. While beneficial, no previous programming experience is required. 

Please see http://lipkow.sysbiol.cam.ac.uk and contact Karen Lipkow to discuss further details.  

[1] Filion GJ, et al. Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 2010;143:212–24. 

[2] Sewitz S, Bancroft J, Brustolini OJB, Kruse K, Stevens T, Babu MM & Lipkow K. Chromatin states in S. cerevisiae reveal that the genome is consistently poised for change. In preparation. 

[3]  Sewitz S & Lipkow K. Simulating bacterial chemotaxis at high spatio-temporal detail. Current Chemical Biology 2013;7:214-23. 

[4]  Claeys Bouuaert C*, Lipkow K*, Andrews SS, Liu D & Chalmers R. The autoregulation of a eukaryotic DNA transposon. eLife 2013;2:e00668. 

[5] Schmidt H, Sewitz S, Andrews SS & Lipkow K. An integrated model of transcription factor diffusion shows the importance of intersegmental transfer and quaternary protein structure for target site finding. PLoS ONE, in press.

Learning outcomes and skills acquired: We are welcoming students from a variety of backgrounds, such as Life Sciences, Physics, Mathematics, Engineering or Computer Science. We are a mixed wetlab/computational group, and you will be exposed to state-of-the-art experimental and computational methods, produce and analyse large datasets, and work quantitatively. The project will take place in the Babraham Institute and in close connection to the Cambridge Systems Biology Centre. You will collaborate with world-leading experts in Nuclear Dynamics, Epigenetics, Signalling and Computational Biology.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Neurons feel the force: mechanics in the nervous system

Supervisor: Dr Kristian Franze, Physiology Development and Neuroscience

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

Possible rotation projects include:   

  • Investigating different aspects of neuronal mechanosensitivity,e.g., calcium dynamics or axonal growth velocities as a function of substrate stiffness, etc. 
  • Comparing substrate stiffness-dependent neuronal growth of nervous tissue explants with isolated neurons and illuminate interactions between mechanical and intercellular signalling. 
  • Investigating mechanotaxis, i.e., mechanically guided growth of neuronal axons on custom-built culture substrates.

Learning outcomes and skills acquired: In this project, you will work in an interdisciplinary environment. You will learn how to communicate with scientists with different backgrounds and pick up approaches and tricks from different fields.  You will learn different cutting edge experimental techniques (depending on project choice; e.g., primary neuronal cultures, how to build your own mechanically complex cell culture substrates, confocal laser scanning microscopy, etc.) and get insight into automated, quantitative image processing and data analysis. You will also be able to practice your writing and presentation skills and receive feedback on your presentation and report.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Molecular mechanisms underlying the function of tandem-repeat proteins, dissected using biophysical methods

Supervisor: Dr. Laura Itzhaki, Pharmacology

Project abstract: A major focus of research in our group is a class of proteins with very striking architectures, known as tandem-repeat proteins. Examples are ankyrin repeats, tetratricopeptide repeats, armadillo repeats and HEAT repeats, and they are frequently deregulated in human diseases including cancer and respiratory and cardiovascular diseases. The individual modules of repeat proteins stack in a linear fashion to produce highly elongated, superhelical structures, thereby presenting an extended scaffold for molecular recognition. The term ‘scaffold’ implies a rigid architecture; however, as suggested by their Slinky spring-like shapes, it is thought that repeat arrays utilise much more dynamic and elastic modes of action. For example: stretching and contraction motions to regulate the activity of a bound enzyme; reversible nanosprings to operate ion channels; proteins that wrap around their cargoes to transport them in and out of the nucleus. The modular architecture of repeat proteins makes them uniquely amenable to the dissection of their biophysical properties as well as the rational redesign of these properties. We are interested in understanding how the process of folding and unfolding of this distinctive class of proteins directs their functions in the cell.  We are also looking at small molecule and peptide-based approaches to target these proteins for therapeutic benefit; examples include inhibitors of ankyrin-repeat protein gankyrin for the treatment of liver cancer and ankyrin-repeat protein tankyrase for the treatment of breast cancer. Lastly, we are exploring the design of artificial repeat proteins and the construction of novel self-assembling repeat-protein nanomaterials.    We have a number of projects that would be suitable for a rotation period, all involving site-directed mutagenesis and biophysical analysis.

Learning outcomes and skills acquired: During the project the student will acquire a range of skills including some or all of the following: 

1.  Molecular biology to make mutant variants of a protein. 

2.  Protein expression and purification techniques. 

3.  A range of biophysical methods, such as fluorescence and circular dichroism spectroscopies, thermal and chemical denaturation, stopped-flow and isothermal titration calorimetry, will used to investigate the stability, folding and molecular recognition properties of the proteins.  

4.  For certain projects, additional approaches may be used, for example single-molecule fluorescence and force microscopy

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Modelling the segmentation gene network in Drosophila and other insects

Supervisor: Michael Akam, Zoology

Project abstract: The segmentation gene network of Drosophila is a textbook example of how gene interactions generate pattern during development.  However, many characteristics of this gene network remain poorly understood - for example how the process of spatial patterning is coordinated in time as well as space.  Attempts to model segment patterning already undertaken highlight missing data, and suggest  specific additional experiments that are needed to refine the model.  The project will involve working with Drosophila, and to collect such data, using techniques to manipulate and monitor gene expression in wild type and mutant embryos, including double fluorescent in situ hybridisation and confocal microscopy.

Learning outcomes and skills acquired: During this project you will learn to work with Drosophila embryos, one of the key model systems for developmetnal and genetic studies.  You will apply data generated to an explicit modelling framework, and assess how best to refine the model as a result.

Project availability: Michaelmas and Lent Term

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Novel RNA protein interactions that promote signal transduction in the immune system

Supervisor: Dr. Martin Turner, The Babraham Institute

Project abstract: Synopsis of the project: The experimental approach will begin with the student learning how to optimize immuno-precipitations.  By varying the stringency of the immuno-precipitation conditions a condition will be found that is suitable to recover the protein from cells in which RNA has been covalently cross-linked to protein using U.V. light (Huppertz et. al 2014). From there the student will prepare cDNA libraries for next generation sequencing. Sequence results will be analysed by the student under the guidance of an expert bioinformatician to precisely identify RNA bound to signalling proteins.  Follow up experiments will involve siRNA to knockdown identified lncRNAs and to measure the impact of this upon signalling processes.  If time permits the region of the protein interacting with the RNA will be identified and the functional consequences of its mutation upon signal transduction determined.   

References: 

Huppertz et. al.  iCLIP: protein-RNA interactions at nucleotide resolution.  Methods. 2014.  65:274-87.   

Turner et al. Noncoding RNA and its associated proteins as regulatory elements of the immune system.  Nature Immunology in press  (June 2014 issue).

Learning outcomes and skills acquired: molecular biology and bionformactics in the context of signal transduction in the immune system.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Asymmetric assembly of spindle poles dictating spindle polarity and pole inheritance

Supervisor: Dr. Marisa Segal, Genetics

Project abstract: Chromosomal segregation along an axis of cell polarity is a hallmark of asymmetric cell divisions throughout evolution. S. cerevisiae is a unique model to probe the mechanisms for spindle orientation in a cell dividing asymmetrically. In yeast, astral microtubules position the spindle such that chromosomal segregation occurs across the bud neck. This polarised orientation is dictated by spindle pole body (SPB, the yeast counterpart of the centrosome) asymmetric function by which the old SPB inherited from the preceding cell cycle is invariantly targeted to the bud. Such functional asymmetry has been more recently observed in self-renewing stem-cell divisions. We have previously shown that asymmetric SPB inheritance stems from a structural asymmetry between old and newly duplicated SPBs (Juanes et al. 2013 Curr. Biol. 23:1310). Here we propose an in-depth quantitative analysis to monitor SPB dynamic assembly along the cell cycle underlying this outstanding asymmetry.  We will exploit tandem fluorescent protein timers (TFTs), an approach that can effectively allow us to track individual protein mobility between subcellular compartments in living cells (Khmelinskii et al. 2012 Nat Biotechnol 30: 708). The work will involve the generation of strains (wild type and cell cycle mutants) expressing TFTs of selected SPB components to be used in quantitative live imaging analysis. These studies will enable us to model asymmetric protein recruitment linking SPB history (old versus new) and fate (mother versus bud) and create a framework for further dissecting the underlying cell cycle controls.

Learning outcomes and skills acquired: The project aims to achieve an integrative view of the mechanisms for spindle pole assembly and their cell cycle control. We will also generate a framework to model the impact of phosphorylation on protein dynamic assembly. From the execution of this project, a student will gain a deep understanding of molecular genetics, expertise in recombinant DNA technologies, live imaging microscopy and computational digital analysis. Through the use of our broad-based computational platform for quantitative live imaging analysis a student will also acquire a set of transferable skills to be applied in other areas.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health & World class underpinning bioscience

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Mechanical forces at the onset of pluripotent stem cell organization

Supervisor: Professor. Magdalena Zernicka-Goetz, Physiology Development and Neuroscience

Second supervisor: Dr. AlexanDr.e Kabla, Engineering

Project abstract: Mechanical forces are key drivers of tissue morphogenesis during the development of multicellular organisms. However, how these mechanical forces impact on cell fate decisions in the mammalian embryo is currently unknown.    The laboratory of Magdalena Zernicka-Goetz developed an in vitro culture system that allows mouse embryos to undergo the pre- to post-implantation transition outside body of the mother (Bedzhov and Zernicka-Goetz Cell 2014). This is the time when the platform of the foetus becomes established. Using this system, a previously unknown morphogenetic event that transforms the apolar pluripotent cells of the epiblast into a polarized epithelial structure in the shape of a rosette was discovered. This event can be reproduced in ES cells providing a simpler experimental model.  The aim of this interdisciplinary project is to understand how mechanical forces at this crucial developmental stage impact on the geometry, position, transcriptional regulation and reorganization of the pluripotent epiblast cells.   The transition from an apolar mass of pluripotent cells to an organized epithelial rosette will be analyzed by time-lapse microscopy, and the shape, movement and coordinated behaviour of individual cells will be tracked. This geometric characterization will be the basis to generate a mechanistic model of the forces that drive the morphogenesis of the rosette. This will be done in the laboratory of Alexandre Kabla, who has developed a number of computational tools to analyze morphogenetic reorganization of cells. The predictions from the model will be validated by manipulating the mechanical properties of the cells and analysing gene expression patterns.

Learning outcomes and skills acquired: The student will acquire experience in both classical and modern techniques of developmental and stem cell biology as well as biophysical modelling.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Computational modelling of stem cell pluripotency and differentiation

Supervisor: Dr. Nicolas Le Novere, The Babraham Institute

Second supervisor: Pinar Pir, The Babraham Institute

Project abstract: Pluripotent stem cells have the ability to differentiate into any other body cell type. Stem cells are unrivalled tools to understand cell differentiation and tissue development, offer alternative to animal testing and hold great promise for regenerative medicine. Our lab is building computational models of stem cells to understand the maintenance of their pluripotency and the regulation of their differentiation into other cell types. The models are based on prior knowledge from literature and mining data from public databases and experiments done at the Babraham Insitute. The models contain a list of biochemical reactions involved in signaling pathways, gene regulatory networks and epigenetic machinery. Their simulation allows to predict the temporal evolution of molecule concentrations and other cellular parameters.    The rotation student will join existing projects in the group but develop her/his own activity. Depending on the particulars of the project, the student will be particularly trained in genomics data analysis or dynamical modeling.  The supervision will be ensured by experienced senior post-docs. In addition to the research activity the student will attend a a large diversity of seminars at the institute, including institute-wide lecture and meetings of the Epigenetics department.

Learning outcomes and skills acquired: Mathematical modelling, numerical simulations, bioinformatics of omics datasets.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Evolution of CYP genes and bright yellow carotenoid coloration in birds

Supervisor: Dr. Nick Mundy, Zoology

Project abstract: The CYP genes encode cytochrome P450 enzymes that involved in detoxification reactions and a wide range of other important functions. In birds, we have recently obtained evidence that a CYP is involved in converting dietary yellow carotenoid pigments to red carotenoids, that are crucial for display in some species. In other birds, such as canaries, a different mechanism has evolved to derive bright yellow carotenoids. Making use of the recently available canary genome, this project will investigate whether CYPs are responsible for bright yellow carotenoid production. The project will involve mining the canary and other avian genomes (more than 40) for CYPs, and using molecular evolutionary analysis to determine whether there are any unusual patterns in canary CYP evolution that indicate a role in carotenoid metabolism.    

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   

Watanabe KP et al (2013) Avian cytochrome P450 (CYP) 1-3 family genes: isoforms, evolutionary relationships and mrNA expression in chicken liver. PLoS ONE 8:e75689. DOI:10.1371/journal.pone.0075689

Learning outcomes and skills acquired: The project will provide training in a strongly interdisciplinary area at the intersection between molecular biology, biochemistry and evolutionary biology. The skills acquired include bioinformatics relating to nucleotide databases, molecular evolutionary genetic analysis, and analysis of adaptive evolution.        modern bioinformatic skills

Project availability: Michaelmas and Lent Term

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Modelling the dynamics of bacteria in vivo

Supervisor: Dr. Olivier Restif, Veterinary Medicine

Project abstract: Salmonella enterica is a leading source of food poisoning and severe opportunistic or systemic infection. For several years our group has pioneered data-driven mathematical modelling of within-host bacterial infection dynamics. We have acquired data from experiments in mice using tagged strains of Salmonella that have given us detailed insight into the spatiotemporal dynamics of infection and the effects of vaccination and antibiotics in vivo. More recently we have started investigating the environmental stage of foodborne bacteria using the nematode Caenorhabditis elegans as a host for Salmonella enterica. Mathematical models are essential tools for both the design and analysis of experiments in order to maximise the information that can be retrieved. This project will build upon existing models that have been recently developed for these systems and apply them to new data. Ongoing work will provide ample opportunities to expand this project into a PhD research project that could include lab work.

Learning outcomes and skills acquired: We have extensive experience in training biology students into computational modelling methods. Some familiarity with mathematical models (differential equations) and programming (especially using R) would be preferable, but the scope of the project will be tailored to the student's background. Learning outcomes will be: improved quantitative and programming skills, statistical techniques and experimental design.

Project availability: Michaelmas and Lent Term

Other relevant themes: Food security

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Whole genome screens for novel DNA replication factors using C.elegans

Supervisor: Dr. Philip Zegerman, Biochemistry

Project abstract: The genome must be copied in its entirety before every cell division. Importantly, factors required  for accurate genome duplication are frequently mutated in diseases, most notably in cancer cells,  and DNA replication is a key target of many current and emerging chemotherapies. Despite this,  our understanding of the control of genome duplication in the context of a whole animal is  extremely poor. In this proposal we utilise the advantages of the nematode Caenorhabditis elegans  for both mechanistic cell biology and genome wide genetic screens to drive replication research  beyond what is currently achievable in unicellular and in vitro systems. To investigate the  molecular mechanisms of metazoan replication initiation we will combine C.elegans genetics with  the development of a unique assay to visualise the recruitment of replication factors to origins in  single cells in real-time. To further our understanding of genome duplication in animals we will use  the synergistic methods of protein-interaction and whole genome synthetic lethality screens to  generate an unprecedented resource of novel metazoan regulators of DNA replication in  development. Capitalising on our expertise in multiple systems, we will test the significance of new  discoveries in vertebrate models. Together the innovations in this proposal will provide unique  insight into the developmental mechanisms of replication initiation regulation and through screens  for factors controlling genome duplication we have the potential to discover new drug targets and  bio-markers of relevance to human diseases such as cancer.

Learning outcomes and skills acquired: Learning outcomes  Through this course, students should gain  

• An understanding of the molecular mechanisms that control several related processes essential for cell division and how these controls help maintain the integrity of the genome as it is transmitted from one cell to its progeny

• An awareness of gaps and uncertainties in our current knowledge and how these may be addressed by future research

• An awareness of the techniques available to study these processes and their limitations 

• A knowledge of the relevance of these processes to the generation and possible treatment of disease 

• An ability to think critically about current molecular cell biological approaches

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Global evolutionary reconstruction of the septin family using phylogenetic methods

Supervisor: Dr. PeDr.o Beltrao, European Bioinformatics Institute

Second supervisor: Dr. Romain Studer, European Bioinformatics Institute

Project abstract: The septin gene family (GTP binding proteins) was discovered forty years ago and is now well studied in mammals and yeasts. In each organism, multiple duplicated genes (paralogs) have been identified. Septins have the ability to form long heteropolymers, composed of multiple paralogs. In yeast, they participate in the robustness of the membrane during cell division. In human, they may also participate in the prevention of bacterial invasion by the formation of “cages” around bacterial pathogens. While this gene family has not been found in higher plants, it does exist in green and brown algae. Our collaborators Prof. Richard Garratt and Dr. Ana Paula Araújo have recently crystallised one structure from the green algae Chlamydomonas. While the biochemical function of this gene family is quite well understood, very few studies have investigated the evolutionary history of these genes. Either they focused on one particular clade (mostly vertebrates or fungi) or they were realised before the massive increase of complete sequenced genomes.    The proposed project would identify homologous of septins in as many genomes as possible. The next step will be the alignment and the construction of phylogenetic trees to infer the evolutionary history of the septin family. This will allow identifying the divergence timepoint of paralogs, which genes are strict orthologs and their importance in major evolutionary events, such as multicellularity in animals. The availability of 3D structures and modern evolutionary tools could help to identify key residues responsible for functional divergence or participating in protein-protein interactions between septin homologs.

Learning outcomes and skills acquired: The student will have the opportunity to work on an evolutionary project, using various computational tools. He/She will first learn how to find homologous sequences in the relevant database (Genbank, Ensembl) using Blast searches. He/She will then learn how to cluster these sequences in different proteins families and align them using state-of-the-art tools (MAFFT, PRANK, ClustalOmega). Finally, he/she will learn how to produce a phylogenetic tree (PhyML, RAxML). Depending on the advance of the project, he/she will also have the opportunity to identify and visualise key residues on 3D protein structures (PyMOL). This will also introduce command-line Unix basic skills.

Project availability: Michaelmas and Lent Term

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Physical properties of the host/pathogen interaction in blood stage malaria

Supervisor: Dr. Pietro Cicuta, Physiology Development and Neuroscience

Second supervisor: Julian Rayner, The Wellcome Trust Sanger Institute

Project abstract: Plasmodium merozoites must invade human erythrocytes in order to replicate – a fascinating cell biological process during which one eukaryotic cell forces its way inside another in under two minutes. To date our understanding of this process has been limited by technology, because the rapid nature of the process has prevented us from imaging large numbers of individual invasion events. To overcome this technological gap, Dr. Pietro Cicuta’s group in Physics have been developing robotic video microscopy approaches to allow the high throughput capture of individual invasion events (Crick et al., Biophys J 2013, PMID: 23473482, and Biophys J 2014, in press), working with T.Tiffert in Dept. of PDN.   Dr. Cicuta is also collaborating on this work with Dr. Julian Rayner at the Sanger Institute, who uses a variety of approaches including experimental genetics and high throughput protein-protein interaction screens to understand the receptor-ligand interactions that allow invasion to occur (Crosnier et al., Nature 2011, PMID: 22080952).   In this project the student will learn state of the art video microscopy techniques in Dr. Cicuta’s lab, and become experienced with in vitro culture and genetic manipulation of Plasmodium parasites in Dr. Rayner’s lab. The overall goal is to combine cutting edge genetic approaches with further developments in video microscopy technology, such as using laser tweezers to hold either merozoites or erythrocytes and quantitate attachment forces, or using microfluidics to observe invasion under flow conditions that mimic those of the microvasculature where invasion actually occurs in vivo, in order to understand the function of specific receptor-ligand interactions during invasion.

Learning outcomes and skills acquired: Automated imaging; optical trapping; cell culture of P.Falciparum (malaria); genetics and proteomics screens.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Phototransduction and TRP channels in Drosophila

Supervisor: Professor Roger Hardie, Physiology Development and Neuroscience

Project abstract: Drosophila photoreceptors represent an important genetic model for sensory transduction and G-protein coupled signaling. The light response is mediated by phospholipase C (PLC), which leads to the activation of Ca2+ permeable “TRP” channels. First discovered in the Drosophila eye, TRP channels are now recognized as a major cation channel family playing vital roles in Ca2+ signaling throughout the body. In the photoreceptors TRP-mediated Ca2+ influx acts at multiple molecular targets, shaping the kinetics of the light response and mediating light adaptation, and regulating a range of cellular responses. A range of projects are available to study the cellular and molecular machinery underlying phototransduction in Drosophila.  Techniques available include: electrophysiology; imaging of optical probes (e.g. GFP-tagged constructs); molecular biology; classical and molecular genetics; cell culture; immunocytochemistry; confocal and electron microscopy.  

For lab details and references see   http://www.pdn.cam.ac.uk/staff/hardie/index.shtml

Learning outcomes and skills acquired: Sensory transduction and ion channel physiology, Drosophila genetics, electrophysiology, in vivo optophysiology techniques

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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How do transcription factors find their targets in the genome?

Supervisor: Dr. Rob White, Physiology Development and Neuroscience

Project abstract: 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 in experiments to identify the key determinants of functional Hox specificity. 

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.

Learning outcomes and skills acquired: Understanding transcription factor binding and the issues, chromatin-immunoprecipitation with quantitative PCR, cell culture and transfection, opportunity also for bioinformatic analysis of ChIP-Seq datasets

Project availability: Michaelmas and Lent Term

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Transcription factor specificity during drosophila embryonic development

Supervisor: Professor Steven Russell, Genetics

Second supervisor: Dr. Boris Adryan, Genetics

Project abstract: 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.

Learning outcomes and skills acquired: The project will provide experience in generating and analysing genome-wide datasets, training in molecular cloning techniques, experience in analysing Drosophila embryos and basic training in dealing with genome sequence.

Project availability: Michaelmas and Lent Term

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Information coding in cerebellar input systems

Supervisor: Dr. Steve Edgley, Physiology Development and Neuroscience

Project abstract: As you will know, the nervous system carries information via "digital" action potential (Spike) signalling. This requires information to be represented within the spikes generated by neurons. Although we've known this for many years, the fundamentals of how information is represented are still very unclear.     Many laboratories work on the general problem of how the spike code of neurons represents information. In my laboratory we work on the cerebellum, a major part of the brain involved in controlling movement. We have a very detailed knowledge of the organisation and connectivity in cerebellar circuits, which are one of the best understood circuits in the brain where there is hope of understanding function.     In recent years work in my laboratory has shown some unusual properties of Spike signalling in neurons that carry information into the cerebellum. In particular one of the pathways providing input to the cerebellum (the lateral reticular nucleus - LRN) shows very unusual spike behaviour, suggesting that the timing of peripheral events may be an important feature of it's information content is (Xu et al. 2013).  LRN provides projections to one part of the cerebellum. A different nucleus (NRTP in short) with some anatomical features like the LRN provides input to the lateral, more recently evolved, parts of the cerebellum.  The question of interest is therefore whether NRTP has spike coding properties similar to those of the lateral reticular nucleus.   This is a realistic project on which progress could be made in the lab rotation.

Learning outcomes and skills acquired: The student would learn basic electrophysiological skills in this project. These include:  an understanding of the methods needed to record activity from single nerve cells from the intact brain, including laboratory animal anaesthesia, surgical preparation and physiological maintenance   practical experience of searching for, discriminating and acquiring data from the brain (both mass recordings and single neuron activity)  Methods for activating brain pathways and for manipulating activity with pharmacological manipulation.  basic methods to analyse patterns of neural cell activity, and responses to stimulation of different pathways.  Note though that the Home Office regulations in the UK would limit what a student can do so the work would need to be done in collaboration.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Calcium-dependent regulation of NMDA glutamate receptors in substantia nigra dopaminergic neurons

Supervisor: Dr. Sue Jones, Physiology Development and Neuroscience

Project abstract: N-methyl-D-aspartate (NMDA) receptors are important cell surface ligand-gated ion channel receptors that are ubiquitously expressed in mammalian brain neurons. NMDA receptors have some unique properties, including voltage-dependent Mg2+ block, such that they require both glutamate and depolarization to open the integral ion channel, and permeability to Ca2+ ions. Ca2+ ions are important intracellular signalling molecules and regulate neuronal development, survival, and adaptation. We have previously shown that substantia nigra dopamine neurons are composed of GluN2B and GluN2D protein subunits (e.g. Brothwell et al., 2008), making NMDA receptors in dopamine neurons different from those at mature glutamatergic synapses in other brain neurons. We are currently studying the regulation of NMDA receptor function and expression (Wild et al., 2014). The aim of this project is to use whole-cell patch-clamp recordings from substantia nigra dopamine neurons in brain slices from mice to investigate the regulation of NMDA receptors by calcium-dependent intracellular signalling molecules.   

Brothwell, Barber, Monaghan, Jane, Gibb & Jones (2008). NR2B- and NR2D-containing synaptic NMDA receptors in developing rat substantia nigra pars compacta dopaminergic neurones. Journal of Physiology 586:739-750.    

A.R. Wild, S. Jones, A.J. Gibb (2014). Activity dependent regulation of NMDA receptors in substantia nigra dopaminergic neurones. J Physiology 592:653-668.

Learning outcomes and skills acquired: As part of this project you would learn (i) how to prepare brain slices from mice. (ii) To make patch-clamp recordings from dopamine neurons in brain slices, and how to use the basic electrophysiolgical equipment needed to do this. (iii) Basic neurophysiological and neuropharmacological approaches. (iv) How to collect and analyse synaptic responses and interpret your data in light of the existing literature. (v) How to present your findings to other lab members. By the end of the project you would have some preliminary data on the regulation of NMDA receptors in dopamine neurons.

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

Other relevant themes: Basic bioscience underpinning health

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Diversity-oriented synthesis of bioactive small molecules

Supervisor: Prof David Spring, Chemistry

Project abstract: 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.

Learning outcomes and skills acquired: Synthetic Chemistry, Chemical Biology, Biophysical Analysis skills.

Project availability: Michaelmas and Lent Term

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Functional characterisation of novel non-coding RNAs from Drosophila melanogaster that promote DNA replication

Supervisor: Dr. Torsten Krude, Zoology

Project abstract: Small non-coding RNAs are essential for the initiation of chromosomal DNA replication in vertebrate organisms. They are known as Y RNAs and interact with the evolutionarily conserved protein machinery for DNA replication. To date, no functional Y RNA homologues have been identified outside of vertebrates and therefore, the question arises whether other non-coding RNAs fulfil similar essential functions in non-vertebrate organisms such as insects or plants.   We have recently isolated candidate RNAs from the fruit fly Drosophila melanogaster, that can functionally substitute for vertebrate Y RNAs in a biochemically defined cell-free system for the initiation of chromosomal DNA replication. This rotation project will focus on the identification and initial characterisation of these RNAs. It can thus be developed into a full PhD project on the full characterisation of these RNAs, using the full power of the model system Drosophila melanogaster including biochemical, cellular, genetic and developmental techniques.  This rotation project initially aims to identify the novel RNAs isolated from Drosophila melanogaster by cDNA sequencing and computational analysis. It will then move on to synthesise the individual RNAs in vitro from defined DNA templates, which will be designed and generated as part of the rotation project. Individual candidate RNAs will be purified by ion exchange chromatography and gel filtration. Finally, the ability of the purified candidate RNAs to initiate DNA replication will be tested in the biochemically defined cell-free system.  All of the technology and expertise required for the success of this project is established in the host lab.

Learning outcomes and skills acquired: The student will benefit from learning a wide array of biochemical and molecular cell biology techniques, including several original assays that were developed in our laboratory. Initial training will include recombinant DNA technology, preparatory and quantitative polymerase chain reaction (PCR), RNA synthesis in vitro and RNA purification by ion exchange chromatography. Importantly, the student will also learn to use an original human cell-free DNA replication initiation assay, including cultivation of human cell lines, preparation of cell extracts and nuclei, and confocal immunofluorescence microscopy. The student will also be trained and supervised in data analysis, writing and oral presentation skills.

Project availability: Michaelmas and Lent Term

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Supervisor: Professor Vassilis Koronakis, Pathology

Project abstract: 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.

Learning outcomes and skills acquired: 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.

Project availability: Michaelmas and Lent Term

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Occurrence of resilin in insect attachment structures

Supervisor: Dr. Walter Federle, Zoology

Project abstract: Resilin is a widespread protein in arthropod cuticle that has unique rubber-like properties. It can recover almost 100% of the elastic energy put into it during deformation, and it is therefore the most resilient biological material known to date. Resilin is typically found in parts of the insects’ cuticular exoskeleton which undergo frequent and repeated deformation or store elastic energy for locomotion. It contains infrequent molecular cross-links formed of di- and tri-tyrosines, which emit a characteristic blue autofluorescence when excited under ultraviolet light. The presence of resilin has been inferred from UV-autofluorescence for various body parts of insects. However, the existing evidence is in most cases inconclusive, as cuticle may contain other fluorescing proteins and the characteristic emission spectrum and pH-dependence of resilin have rarely been used as identification criteria.  We will verify claims that resilin is present in smooth and hairy adhesive structures of insects. As the function of conventional adhesives normally requires absorbing rather than returning energy, the presence of resilin would be unusual, and would suggest that resilience is a defining feature of biological adhesives. Specimens of insect adhesive structures will be cryo-sectioned and examined using confocal microscopy with UV-excitation, in comparison with other well-characterized test samples of resilin. Analyzing the cuticular fluorescence emission spectrum and its pH-dependence will allow us to distinguish resilin from other autofluorescent types of cuticle.

Learning outcomes and skills acquired: The rotation will provide the student with an introduction to the running of an experimental biology project. This will include project planning, time management, working unaided and the effective presentation of data in workgroup meetings.   The student will acquire a range of technical skills over the course of the project, including cryo-sectioning, specimen preparation, confocal microscopy, and spectral analysis. The student will also learn to perform statistical analysis of the data obtained.

Project availability: Michaelmas and Lent Term

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Ontogeny and scaling of adhesive hairs and attachment forces in spiders

Supervisor: Dr Walter Federle, Zoology

Project abstract: The body mass of animals climbing with adhesive pads varies over seven orders of magnitude from the smallest mites to the largest geckos. Surface attachment is generally more difficult for larger animals, as the area available for adhesive structures increases more slowly with size than body mass. Many spiders possess footpads covered with dense arrays of tiny hairs which allow them to climb on vertical and inverted substrates. Some spiders undergo extreme changes in body size from the smallest spiderlings to adult spiders, yet all stages possess pads with adhesive hairs. How do their adhesive systems change with body size?  We will study the ontogeny of adhesive pad morphology and attachment performance in tarantulas (Theraphosidae), from small spiderlings to fully grown adults. Morphological parameters will be measured using light and electron microscopy, and attachment forces of footpads will be quantified using a custom-made force measurement set-up. We will test whether morphological characters scale isometrically with body size and whether the spiders’ attachment performance is consistent with the morphological changes. The data will test the recently proposed hypothesis that larger climbing animals increase their adhesive efficiency by developing denser arrays of finer adhesive hairs. The results of this project will be essential for understanding the scaling and function of animal attachment devices and for the development of bio-inspired adhesives.

Learning outcomes and skills acquired: The rotation will provide the student with an introduction to experimental biology and biomechanics, including project planning, time management, independent laboratory work, data analysis and the presentation of data in workgroup meetings.   The student will acquire a range of technical skills over the course of the project, including specimen preparation, light and electron microscopy, as well as computer-controlled force measurements. The student will learn how to perform statistical analysis of the data obtained, and compare the results to predictions from theory.

Project availability: Michaelmas and Lent Term

Other relevant themes: Food security

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Modelling the exit from the cell cycle

Supervisor: Dr. Yuu Kimata, Genetics

Second supervisor: Philip Greulich, Cavendish Laboratory

Project abstract: How do cells decide whether to continue or cease proliferation in vivo? To gain insight into this crucial decision-making process, we aim to generate a mathematical model of the molecular event that regulates cell cycle exit. In the rotation project, we will focus on the protein interactions occurring between four key cell cycle regulators during the developmentally programmed cell cycle exit in Drosophila embryonic epidermis. By taking advantage of the vast amount of experimental data published on this tissue, we will create an initial computable model, which will be later refined and validated by experiments.  The student who will apply for this project is expected to have the following skills:  - mathematical skills for a physics/mathematics graduate   - basic programming skills  - some knowledge in numerically solving differential equations (e.g. in Matlab, Maple, Mathematica, or in C)   - basic understanding of cellular processes + deep interest in biological problems

Learning outcomes and skills acquired: From this project the student will be able to learn the theoretical approach to a fundamental biological question, increase the knowledge of cell cycle/developmental biology, develop their programming/mathematical skills.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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How to strigolactones modulate auxin transport?

Supervisor: Prof Ottoline Leyser, Sainsbury Laboratory

Project abstract: Strigolactones are a relatively recently discovered class of plant hormone. Among other things, they play a central role in regulating shoot branching. The mechanism of strigolactone signaling is still unclear. One rapid response is the removal of auxin transporters of the PIN family from the plasma membrane. This correlates with reduced auxin transport, with implications for the degree of shoot branching. We are adopting a range of approaches to investigate how strigolactones trigger PIN depletion from the plasma membrane. This include transient assays tracking the localization of fluorescently tagged strigolactone signaling components in tobacco leaf epidermal cells, and in Arabidopsis stems. In addition, we are investigating which motifs in the PIN protein family are necessary for strigolactone responsiveness. A range of projects contributing to this ongoing work are available. 

Project availability: Michaelmas and Lent Term

Other relevant themes: Food Security

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Genetic code expansion

Supervisor: Dr Jason Chin, Department of Chemistry

Project abstract: The project will develop genetic code expansion approaches for studying post translational modification.  The application of these approaches will be followed up to address problems of biological significance.  The student will learn a range of approaches in molecular biology and chemistry and directed evolution that are relevant to genetic code expansion and synthetic biology.

Project availability: Michaelmas and Lent Term

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Genetic markers for cell dynamics in Marchantia

Supervisor: Dr Jim Haseloff, Department of Plant Sciences

Project abstract:

The liverworts (or Marchantiophyta) are descendants of the earliest terrestrial plants. The group is characterised by morphological simplicity, likely matched by simple underlying genome structures. Marchantia polymorpha is the best characterised of the liverworts. It is a thalloid liverwort, growing with flat sheet-like tissues which possess distinct upper and lower surfaces. The lower surface of the plant has specialised root-like cells, or rhizoids. The body of the thallus contains oil bodies, present as scattered differentiated cells. The upper surface has a modular structure, with repeated formation of units that form primitive cell complexes adapted for photosynthesis and gas exchange. Each morphological unit is marked by a single, permanently open central pore.

The liverworts have alternate haploid and diploid generations. Like other bryophytes, the gametophyte or haploid generation is dominant phase of the life cycle. M. polymorpha has a global distribution, and is often found as a weed in horticulture. The plant produces vegetative propagules. These form vegetatively inside conical splash cups. Superficial cells on the inside of a cup undergo cell proliferation to form a group of cells carried on a short stalk, the cells continue to proliferate in regular fashion to form a bilobed gemma. This is eventually detached from the stalk, and can be dispersed from the cup, typically by water splash. Gametogenesis is under environmental control, and can be induced by exposure to far-red light. Antheriophores and archegoniophores grow upward from the Marchantia thallus, with gamete-forming structures supported by pedestals. Antheridia form on the upper surface of a disc, and archegonia form underneath an arrangement of spokes. Genetic crosses can be performed by transfer of sperm from mature antheridiophores to archegonia by pipette. After fertilisation and zygote formation, the diploid phase of the life cycle continues with cell proliferation, meiosis and spore formation. The spores and elaters are packaged within yellow sporangiophores, suspended under archegoniphores. Both gemma and spores can be used for transformation and propagation of plantlets during experiments. 

Similar to other lower plants, M. polymorpha regenerates easily, and was used in early plant tissue culture studies. A number of plastid and nuclear transformation techniques have been developed for M. polymorpha, including a recent method for Agrobacterium tumefaciens mediated transformation using germinating spores. This offers simple high throughput production of transformed plants (Ishizaki et al. 2008). We have a draft genome for the Cambridge 1 (male) and 2 (female) isolates of M. polymorpha. The project will be based on the construction and use of new cell state markers by transformation and imaging of promoter and gene fluorescent gene fusions. Confocal microscopy techniques will be used for quantitative imaging of cell states, subcellular and tissue geometries to produce anatomical cell fate maps. This will aid the construction of computational models for early Marchantia development.

References:

Ishizaki K, Chiyoda S, Yamato KT, Kohchi T. Plant Cell Physiol.49:1084-91, 2008.

Project availability: Michaelmas and Lent Term

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Functions of polypyrimidine tract binding protein co-regulators

Supervisor: Dr. Chris Smith

Project abstract: Polypyrimidine tract binding protein (PTB) is an RNA binding protein that regulates alternative splicing, pre-mRNA 3’ end processing, translation and mRNA stability1. RNA binding is mediated by four RNA Recognition Motif (RRM) domains; the inter-RRM linkers are also important for function and the second RRM interacts with linear peptide motifs of the form [S/G][I/L]LGxxP2. Structural characterization indicated the crucial role of PTB tyrosine 247 in the interaction with an SLLGEPP motif from the splicing co-regulator Raver13. Analysis of proteins that were pulled down with GST-RRM2, but not the Y247Q mutant revealed numerous interactors, many with [S/G][I/L]LGxxP like motifs. Some of the other factors are involved in functions with which PTB has been associated, such as pre-mRNA 3’ end processing (WDR33 and CSTF2), but for which a molecular connection has not previously been established. Others suggest previously unexpected potential roles for PTB. For example, the KIAA1967 protein contains a SLLGPPP motif and is a component of the DBIRD complex, which connects RNA Pol II and alternative splicing4. This project aims to validate, and characterize the functional roles of, one or more of the potentially interesting PTB interactors.   1. Kafasla, P. et al.,  Biochem Soc Trans 40, 815 (2012).  2. Rideau, A. P. et al., Nat Struct Mol Biol 13, 839 (2006).  3. Joshi, A. et al., Structure 19, 1816 (2012).  4. Close, P. et al. Nature 484, 386 (2012).

Learning outcomes and skills acquired: Analysis of protein-protein and protein-RNA interactions. Cell culture analysis of alternative pre-mRNA splicing and/or 3' end processing.

Project availability: Lent Term (January - March 2015)

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Decoding the Notch signal

Supervisor: Professor Sarah Bray

Project abstract: 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. Rotation projects could pursue any of these different avenues, depending on the interests and experience of the student.

Project availability: Lent Term (January - March 2015)

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Cross species transmission of influenza viruses

Supervisor: Dr Debra Elton

Project abstract: The natural hosts of most influenza A viruses are wild aquatic birds, which usually show minimal clinical signs of infection. There are many different subtypes, named according to their surface proteins haemagglutinin (HA) and neuraminidase (NA).  Although a relatively rare event, influenza can be transmitted across the species barrier to new hosts, including horses, humans, pigs, poultry and seals. A major hurdle in this process is the ability of the virus to bind to and infect cells of a new species, a process that is largely controlled by HA.    HA binds to sialic acid residues on the surface of cells and viruses adapted to different species are usually adapted to bind preferentially to certain types of sialic acid. Avian influenza viruses bind to sialic acid with an α2-3 linkage whereas human viruses bind to those with α2-6. Equine influenza H3N8 viruses also bind to α2-3 but have a preference for glycolyl rather than acetyl side chains. We have identified two naturally occurring mutations in clinical isolates of equine influenza that have changes in the receptor-binding pocket of HA. One of these is found in both avian and equine viruses that have adapted to transmit in dogs. The other is associated with the switch in binding between avian and human receptors, but was previously thought to require a second substitution for full effect. This project will continue work in our laboratory to investigate the effect of these changes on receptor binding specificity, using molecular and cell-based approaches.

Learning outcomes and skills acquired: The project will be based in the Virology group at the Animal Health Trust where the student will benefit from wide-ranging expertise with an emphasis on veterinary research with a clear benefit to animal health. The student will acquire and utilise a variety of molecular, cell and virology techniques including reverse genetics & RT-PCR and through participation in laboratory meetings have the opportunity to develop oral presentation skills

Project availability: Lent Term (January - March 2015)

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CRISPR-mediated modulation of APC/C activity

Supervisor: Dr Jonathon Pines

Project abstract: We have developed live cell and biochemical analyses of the Anaphase Promoting Complex/Cyclosome that have allowed us to develop ideas for how mitosis is regulated by ubiquitin-mediated proteolysis. We are now in a position to test these ideas by exploiting CRISPR-mediated genomic engineering to knockout components of the pathway and asses how this changes APC/C activity and mitosis. We have a number of CRISPR constructs ready to go and will begin by knocking out the Cdh1 protein to test how this changes substrate selection, exit from mitosis and the timing of DNA replication in the next cell cycle.

Learning outcomes and skills acquired: This project will involve CRISPR-mediated gene targeting, fluorescence cell sorting, genomic analysis by PCR and time-lapse microscopy. Correct experimental design and data interpretation will be taught.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Estimating molecular diversification rates in biodiversity hotspots

Supervisor: Dr Andrew Tanentzap

Project abstract: Conservation biologists often try to protect species-rich areas that are threatened by habitat loss.  However, the rate at which regions produce biodiversity is seldom considered.  New advances in molecular phylogenetics are now making it possible to estimate rates of species diversification across the world’s bioclimatic regions (Madriñán et al. 2013).  This project would aim to test how diversification rates vary among the world’s biodiversity hotspots and whether they should influence the prioritization of conservation actions.     The project will involve searching the literature and biodiversity databases for species endemic to different regions.  The student will compile sequence data for monophyletic clades in each region from published databases (i.e. Genbank), and identify suitable fossils to use for calibrating evolutionary relationships in time.  New phylogenies will be derived using Bayesian evolutionary analysis by sampling trees (BEAST) and used to estimate diversification rates..    A major bias in existing analyses is that high net diversification may arise because extinction rates are low rather than there being many new species produced.  There is potential to use a new method for recovering estimates of speciation and extinction rates from time-calibrated phylogenetic trees (Heath et al. 2014), and we can use simulated trees to test the sensitivity of the analyses to different extinction scenarios.    Heath, T.A. et al. 2014. The fossilized birth-death process: a coherent model of fossil calibration for divergence time estimation. PNAS E2957–E2966.  Madriñán, S. et al. 2013. Páramo is the world's fastest evolving and coolest biodiversity hotspot. Frontiers in Genetics article 192.

Learning outcomes and skills acquired: During this project, the student will learn how to use molecular sequences to build time-calibrated phylogenetic trees.  They will gain skills in data mining, sequence alignment, tree estimation, and statistical/simulation modelling, as well as experience with a range of  computational tools (e.g. BEAST and R).

Project availability: Lent Term (January - March 2015)

Other relevant themes: World class underpinning bioscience

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Site-selective chemical modification of proteins

Supervisor: Professor Matthew Gaunt

Project abstract: This project involves the development of chemical methods designed towards the selective functionalization of proteins. Central to this project is the deployment of a novel reagent with multiple reactivity modes such that not only can the protein undergo selective functionalization but can be further elaborated through conjugation with another biomolecule.

Learning outcomes and skills acquired: this project will provide the development of basic chemical synthesis and chemical biology techniques such as small molecule purification, microscale reactions, mass spec analysis of proteins, and conjugation reactions

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Investigating molecular interactions important for herpesvirus tegument assembly

Supervisor: Dr Colin Crump

Project abstract: Herpesviruses cause a wide range of diseases in animals and humans, many of which have significant economic impact on livestock industries and human health. They are structurally complex viruses composed of an icosahedral capsid, a tegument layer and a lipid envelope. The tegument is vital for the assembly of virions and also contains numerous enzymes that are delivered into the cytoplasm of cells during virus entry to modulate many cellular pathways. This project will investigate the protein-protein interactions involved in the assembly of herpesviruses, building upon our recent studies on the interaction between the essential herpes simplex virus type-1 (HSV-1) proteins VP16 and VP1/2. Examples of important tegument enzymes that modulate the host cell includes the virion host shutoff protein (vhs, an RNAse), two protein kinases (pUS3 and pUL13), and an E3 ubiquitin ligase (ICP0). Vhs is of particular interest: we have recently demonstrated this tegument protein is important for HSV-1 to evade the antiviral factor tetherin, and the delivery of vhs as part of the viral tegument is likely to be key for the virus to subdue early cellular defences. Therefore a parallel aim of this project will be to define and inhibit the interactions required for virion incorporation of host modulatory proteins such as vhs, and then generate viruses that either do or don’t package these factors into viruses. This will allow the contribution of specific tegument proteins for establishing productive infection to be investigated both in vitro and in vivo.

Learning outcomes and skills acquired: This project will provide experience in mammalian cell culture, handling containment level 2 pathogens, protein-protein interactions assays (e.g. co-precipitation assays), molecular biology techniques (e.g. generation of recombinant viruses using large (>150kb) viral genomes cloned as bacterial artificial chromosomes), fluorescence microscopy, potentially electron microscopy and super-resolution microscopy, and classical virology techniques.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Investigation of the molecular mechanism of Oct4 in nuclear reprogramming

Supervisor: Dr Jose Silva

Project abstract: The lab research is centered on the biology of nuclear reprogramming using the process of induced pluripotency, that is, the conversion of a differentiated cell, such as neural and skin cells, back into a pluripotent cell. This was first described by Takahashi and Yamanaka in 2006[1]. Oct4 is the key master regulator of this process, however, its molecular mechanisms are not known. Recently, our lab demonstrated that a defined dose of Oct4 controls the reprogramming of differentiated cells and then the differentiation of these into all the cell lineages of an organism[2,3]. We are now dissecting the molecular mechanisms of how Oct4 mediates these cell state transitions and the rotation project on offer will focus on aspects of this.    Visit our website to learn more about our lab: http://www.jose-silva-lab.com    References:  1. Takahashi K, Yamanaka S (2006) Cell.  2. Radzisheuskaya A, Le Bin Chia G, Dos Santos RL, Theunissen TW et al. (2013) Nat Cell Biol.  3. Radzisheuskaya A, Silva JC (2014) Trends Cell Biol.

Learning outcomes and skills acquired: Cell culture (ESCs, EpiSCs, iPSCs), including maintenance, expansion, genetic manipulation, reprogramming, timecourses, flow cytometry. RNA extraction for RNAseq and RT-qPCR. ChIP.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Identifying gaps in animal influenza virus surveillance

Supervisor: Dr. Colin Russell

Project abstract: Assessing the pandemic risk posed by non-human influenza A viruses remains a complex challenge. The substantial diversity amongst influenza viruses in animals makes creating pandemic preparedness measures against all viruses infeasible. Thus there is a need to prioritize viruses of concern. Currently, influenza pandemic risk assessment is driven by a simple idea: non-human influenza viruses that cause sporadic human infections pose a greater risk than viruses that have not infected humans. However, this intuitive idea has little empirical support.  Detecting influenza viruses of concern relies on systematic characterization of influenza viruses circulating in wild and domesticated animal populations. However, existing surveillance networks are largely ad hoc, varying substantially by host and geographical region, and with only a small proportion of the data entering the public domain. This rotation project is part of a substantially larger project that seeks to design non-human influenza surveillance strategies by using statistical analyses to determine levels of coverage by geographic region, host species, and human-animal interface risk factors.  In this rotation project, you will use influenza virus sequence data from domesticated animals to create an influenza occurrence map. You will then work on combining this information with ongoing surveillance and data collection efforts to identify substantive gaps in genetic data collection and sharing.

Learning outcomes and skills acquired: You will gain experience working with and analysing large datasets, methods for analysing genetic data, and tools for visualising geographic data (such as ArcGIS). This project is entirely computational and will help develop quantitative skills.

Project availability: Lent Term (January - March 2015)

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Is hearing impaired in white-eyed mutant circkets? A behavioural, biophysical and neurophysiological study

Supervisor: Dr Berthold Hedwig

Project abstract: Hearing in insect is based on highly sensitive mechanoreceptors, which are integrated into auditory organs for sound perception and frequency detection. In Drosophila a molecular analysis of proteins and channels involved in the mechano-electrical transduction process revealed evidence that rhodopsins known from the visual processing cascade may also play a role in the mechanical transduction process (Senthilan et al. (2012) Cell 150, 1042–1054). We keep a line of white-eyed crickets which lacks the photoreceptor screening pigments but may have other defects of the visual system as well. Behavioural experiments have demonstrated that this mutant cricket has a poor acoustic orientation.  We speculate that the poor auditory behaviour may be related to a defect in the insect’s hearing organ. The project aims at (i) demonstrating the altered auditory behaviour in the mutants, (ii) recording and comparing the biomechanical sensitivity of the tympanic membrane with a laser vibrometer in wild type and mutant crickets and (iii) at characterising the threshold and response of the auditory afferents using extracellular recordings from the auditory nerve. These measurements should indicate if the hearing organ in these white eyed mutants is impaired or if the observed behaviour is due to higher processing.

Learning outcomes and skills acquired: In this project you will approach an interesting question at three different levels: behaviour, biophysics and neurophysiology. This will provide very different training opportunities for your research skills. You will work in the environment of an active and cooperative research group with the aim to make you as independent and critical as possible. You will also be able to practice your writing and presentation skills and will receive feedback on your presentation and report.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health, Food Security

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Modeling the within-host dynamics of antibody escape

Supervisor: Dr. Simon Frost

Project abstract: RNA viruses such as HIV and hepatitis C virus can cause chronic infection, despite the presence of potent, ongoing antibody responses. Viral sequence data, coupled with ex vivo data on the sensitivity of viral clones to neutralisation can provide insights into the pathways of immune escape. The goal of this project is to link mathematical models of antibody escape to the patterns of viral evolution, and compare the model results with data.

Learning outcomes and skills acquired: This project will provide training in the handling and processing of high- throughput sequencing data; in computational statistics applied to sequence data; and in mathematical modeling of host-pathogen interactions. The student will also gain knowledge of virology (in particular, in genomic organisation and patterns of molecular adaptation) and antibody-mediated immunity.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health, Food Security

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Design of nanobody-fluorescent protein interactions for use in next generation single-molecule super-resolution microscopy

Supervisor: Dr. Lucy Colwell

Second supervisor: Dr. Steven Lee & Dr. Erwin De Genst

Project abstract: Nanobodies are single-chain antibodies derived from camelids that bind to protein targets with high affinity and specificity [1]. These properties have recently been exploited to produce nanobodies that efficiently bind green fluorescent protein (GFP), thus enabling any GFP-tagged construct to yield nanometer spatial resolution in single-molecule super-resolution microscopy [2]. However, imaging of more complex biological phenomena: such as macromolecular complexes and other bio-interactions requires targeted development of new nanobodies specific for fluorescent protein variants whose spectral emission differ to allow efficient multicolor imaging.     This project brings together theoretical and experimental tools in a synergistic way to better understand how sequence and structure relate to binding affinity in order to improve bio-imaging. Statistical inference techniques, based on amino acid coevolution, will be used to construct a model for the molecular specificity code that dictates nanobody-protein interaction specificity. The student will 1) create a database of known nanobody-protein interactions to characterise the specificity determining nanobody sequence residues, 2) use the resulting model to design nanobodies that interact specifically with the desired fluorescent protein targets and 3) express the designed nanobodies in bacteria and test their efficacy using super-resolution imaging tools in the Lee lab. The project has the potential to further our understanding of rational design of protein interactions in a broad context.       Background references: [1] Muyldermans, Serge. "Nanobodies: natural single-domain antibodies." Annual review of biochemistry 82 (2013): 775-797. [2] Ries, Jonas, et al. "A simple, versatile method for GFP-based super-resolution microscopy via nanobodies." Nature methods 9.6 (2012): 582-584.

Learning outcomes and skills acquired: The student will learn standard molecular biology techniques such as site directed mutagenesis using PCR, molecular cloning, bacterial protein expression and purification. The purified nanobodies will be characterized for binding to the desired target using surface plasmon resonance and Isothermal calorimetry. Advanced microscopy, single-molecule spectroscopy, super-resolution imaging, optics and photonics techniques can also be acquired, in addition to experience in statistical inference, bioinformatic and programming approaches that make use of protein sequence evolution to identify specificity determining residues.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Bioenergy and industrial biotechnology

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Measuring changes in protein stability in living cells

Supervisor: Dr Catherine Lindon

Project abstract: Ubiquitin-mediated proteolysis controls the turnover of many cellular proteins, but is a process that is difficult to study in living cells. We are piloting the use in mammalian cells of tandem fluorescent protein timers (tFTs), first described for use in budding yeast [1]. tFTs provide a readout for the stability of tagged proteins in single cells, through measurement of the ratio of a slow-folding mCherry fluorescent protein to a fast-folding GFP (with which it is linked in tandem). We propose to use tFT tags to study changes in protein turnover during the cell cycle. In preliminary time-lapse imaging experiments of dividing cells we have shown that the mCherry:GFP ratio of a known substrate of ubiquitin-mediated proteolysis varies three-fold across the cell cycle.  A rotation project is available with the following aims:  (i) to optimize the time-lapse imaging protocols we are developing, using different tFT-tagged versions of known substrates of cell cycle proteolysis  (ii) to test the turnover of protein hits from our recent screen which identified proteins specifically polyubiquitinated during exit from mitosis [3].  (iii) to investigate how turnover of these proteins is related to growth conditions    [1] Khmelinskii A, Keller PJ, Bartosik A, Meurer M, Barry JD, Mardin BR, Kaufmann A, Trautmann S, Wachsmuth M, Pereira G, Huber W, Schiebel E, Knop M (2012). Nat Biotechnol 30  708-14  [2] Min M, Lindon C (2012). Semin. Cell Dev. Biol. 23 482-491  [3] Min M, Mayor U, Dittmar G, Lindon C (2014). Mol Cell Proteomics 13 2411-25

Learning outcomes and skills acquired: Students will learn standard molecular biology techniques, mammalian cell culture and electroporation, and fluorescence timelapse imaging on an Olympus widefield platform. They will acquire broadly applicable skills in image acquisition and analysis.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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The neuropeptide hormones of arthropods:  Conservation and divergence in the neuropeptide hormone complement of centipedes

Supervisor: Prof Michael Akam

Second supervisor: Peter Evans

Project abstract: Neuropeptides control many aspects of physiology in arthropods, as they do in all animals.  Some neuropeptides have widely conserved roles, but others have specific functions in a single lineage - for example controlling the moult cycle in arthropods.  Insect and crustacean peptide hormones have been studied extensively, but until recently, other ancient arthropod lineages remained uncharacterised.         We have used the sequenced genome of the centipede Strigamia maritima to characterise the complement of peptide hormones in this myriapod [1], which represents an ancient lineage of arthropods that invaded land independently of the insects. We have also shown that Strigamia retains aspects of ancient brain organisation that have been lost in insects, including an anterior medial neurosecretory brain centre. [2].  The aim of this project will be to identify which neuropeptides are expressed by this anterior medial region, and where and when other neuropeptides are expressed in Strigamia as a first step towards mapping out the evolution of the neuropeptidome in arthropods.      [1]    Chipman, A. D., Ferrier, D. E. K., Brena, C., Qu, J., Hughes, D. S. T., Evans, P.  et al, 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  Published 25 November 2014    [2]   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  Published 26 Sept 2014

Learning outcomes and skills acquired: Planning a research project  Mining genome and transcriptome data to identify genes, confirm annotation and design reagents  Generation of qPCR and in situ hybridisation probes  Quantitation of RNAs in embryo samples  in situ anlysis of neuropeptide RNA and protein expression in embryos  Reconstruction of gene expression in relation to brain morphology by confocal microscopy

Project availability: Lent Term (January - March 2015)

Other relevant themes: Food Security

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Metabolic regulation of stem cell differentiation and reprogramming

Supervisor: Professor Austin Smith

Project abstract: Cells constantly make decisions about their fate. Each choice, life or death, self-renewal or differentiation, is made according to the ever changing environment they sensed. Apart from signals such as growth factors, hormones and cell-cell interactions, nutrient-responsive metabolites have been increasingly recognized as important mediators of crosstalk between extracellular flux, cellular signalling and epigenetic regulation of cell fate. However, the underlying mechanism is still unclear. Our lab focuses on the cues which lead to pluripotent stem cells homeostasis or lineage commitment. Previous observation suggested a correlation between cell fate-changing and metabolic resetting. The project aims to dissect this correlation and investigate whether metabolites, especially those related to glucose metabolism and cell redox state, can determine cell fate. With the relative ease of carrying out genetic manipulation to pluripotent stem cells, we can potentially identify key mediators of this process. These studies may revolutionize our understanding of the impact of daily nutrient uptake on stem cell fate control, and ultimately aging and disease.

Learning outcomes and skills acquired: The student will receive training in a variety of molecular and cellular biology techniques including gene expression analysis, cell culture, cloning, and genome editing. Subsequently, the student will be able to explore the metabolic state of the stem cells using the cutting edge extracellular flux analyser combined with flow cytometry and confocal imaging. In addition to laboratory-based skills, the students will be trained for independent thinking and designing the experiment, data interpretation, communication and presentation skill.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health, Food Security

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Biomimetic substrates for pluripotent stem cell culture

Supervisor: Dr Kevin Chalut

Second supervisor: Jose Silva

Project abstract: In collaboration with Jose Silva’s laboratory, we are interested in how cell-matrix and cell-cell interactions influence the acquisition and maintenance of pluripotency. Our work focuses on how biophysical parameters can be optimised to promote the differentiation of pluripotent cells into adult tissues such as adipose tissue and skeletal muscle. To uncover the biophysical and molecular mechanisms regulating stem cell function, we engineer novel hydrogel matrices of varying Young's moduli (mechanical stiffness) using a range of synthetic biopolymers. We then functionalise these with extracellular matrix proteins and adhesion molecules for an optimal biomimetic substrate for pluripotent cells. The project on offer, which is part of a collaboration with the laboratory of Florian Hollfelder from Biochemistry and Jose Silva from the Stem Cell Institute, will extend our current studies to three dimensions. The aim of the rota project is to optimise 3D matrix conditions – involving the synthesis of the matrices and characterisation of iPS cells within these conditions. Ultimately, the outcome of this project will be part of a greater effort to make a better pluripotent cell through creating a biomimetic environment for pluripotency.

Learning outcomes and skills acquired: Synthesis of 2D and 3D hydrogels, atomic force microscopy iPS and ES cell culture, qPCR and RNA seq

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Investigating Communication between active sites in Thiamine Diphosphate-Dependent Enzymes

Supervisor: Dr Finian J. Leeper

Project abstract: Thiamine diphosphate (TPP) is a coenzyme used by many important enzymes, including pyruvate dehydrogenase, the key enzyme that links glycolysis to the citric acid cycle. All TPP-dependent enzymes are homodimers (or dimers of dimers) and there is evidence of communication between the active sites in the dimer such that they act alternately. One possible means of communication is a water-filled channel between the TPP molecules belonging to the two active sites and it has been suggested that the alternation of activity is effected by injection of a proton into the channel from one active site and withdrawal of a proton by the other active site. In this project we plan to test this hypothesis by producing a mutant of the dimeric enzyme in which there is a mutation in just one of the two active sites. If this results in loss of all activity for the dimer it will show that the activities of the two sites are indeed tightly coupled. This project requires prior experience in gene cloning, site-directed mutagenesis and expression of proteins in E. coli.  Further reading:  R. A. W. Frank, F. J. Leeper and B. F. Luisi (2007) Structure, mechanism and catalytic duality of thiamine-dependent enzymes, Cell. Molec. Life Sci., 64 (7-8), 892-905.

Learning outcomes and skills acquired: Further experience of gene cloning, site-directed mutagenesis and expression of proteins in E. coli. Methods for assaying enzyme activities. Enzyme kinetics. Mechanisms of TPP-dependent enzymes.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Inference of gene regulatory network using single-cell RNA-seq data

Supervisor: Dr Martin Hemberg

Project abstract: 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.      [1] Junker and van Oudenaarden, Cell, 157 (1), p8-11, 2014.  [2] Margolin et al, BMC Bioinformatics, 7:57, 2006.

Learning outcomes and skills acquired: This is a computational project where you will learn how to process and analyze RNA-seq data. Moreover, you will learn about stochastic models of gene expression, as well as graph theory and network inference methods.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Transgenerational epigenetic inheritance in C. elegans

Supervisor: Dr Martin Hemberg

Project abstract: 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 [1] 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.

Learning outcomes and skills acquired: 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 levelsand stress conditions) at the level of individuals. This project will involve both experimental work to collect genomic data from worms, as well as computational work in developing algorithms and statistical frameworks that will allow us to identify inherited epigenetic features.

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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Biophysical model of transcription factor binding

Supervisor: Dr Martin Hemberg

Project abstract: It is widely accepted that a large fraction of the genome serves a regulatory role. Today, one of the major challenges in genomics is to understand the principles and the mechanism by which genes are regulated. Binding by transcription factors (TFs) is one of the most important steps in gene regulation, and one of the most widely studied problems in computational biology is the prediction of where a specific TF will bind to a given DNA sequence. Despite numerous publications on this topic, our ability to predict TF binding sites remains limited. Most existing methods, however, only rely on the information about the underlying DNA sequence, ignoring any information about the physical structure of the protein and the DNA. Structural biologists have obtained atomic-resolution models of hundreds of TFs, and we hypothesize that by taking this information into consideration, it will be possible to improve in vivo binding site predictions. Starting from the detailed x-ray structure, one first has to obtain a lower-dimensional representation of the protein [1] which will make it possible to calculate binding energies efficiently for different DNA sequences [2]. By taking the protein-DNA structure into consideration, the aim is to develop improved heuristics and algorithms for identifying TF binding sites.

Learning outcomes and skills acquired:

Project availability: Lent Term (January - March 2015)

Other relevant themes: Basic bioscience underpinning health

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