Theme 1

Molecules, Cells and the Basis

for Disease

2017/2018

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1.1 Dissecting antigen presentation to T lymphocytes using dendritic cells derived from genetically modified induced-pluripotent stem cells ....................................................................................... 5 2.1 Mapping the PTPN22 interactome. ......................................................................................... 6 3.1 MicroRNAs as biomarkers of liver regeneration and their link to tumour biology ......................... 7 5.1 Applying single-cell RNA sequencing technologies to establish cardiac stem/progenitor cell heterogeneity ............................................................................................................................ 9 6.1 Developing nanomedicines for immunosuppression in liver transplantation .............................. 10 7.1 Manipulating the stem cell state in vivo .................................................................................. 11 8.1 Mechanisms of bacterial inhibition by interferon-stimulated genes .......................................... 12 9.1 The role of post-translational modification of self proteins in breaking immune tolerance in type 1 diabetes ................................................................................................................................... 13 10.1 Gestational Diabetes, the epigenome and the health of the next generation ........................... 14 11.1 Tackling hearing loss: development, regeneration and reconstruction of the ear ...................... 15 12.1 Mechanism of membrane remodelling in cell biology and replication of HIV-1 and Ebola virus .. 16 14.1 E Gene expression in skin in subjects with reduced biological ageing compared to their chronological ageing ................................................................................................................. 18 15.1 Adhesion receptor signalling regulating lung cancer progression ........................................... 19 16.1 Developing novel, conventional and nanoparticle, inhibitors of the Wnt signaling pathway as a treatment for prostate and breast cancers ................................................................................... 20 17.1 Roles of the RNA-binding proteins LARP4A and LARP4B in cancer cell migration and invasion . 21 18.1 Membrane remodelling during cell division ........................................................................... 22 19.1 Identifying effective drugs to treat oral squamous cell carcinoma. .......................................... 23 20.1 Interventions to improve maternal metabolic profile in obese pregnancy and prevent cardio-metabolic and behavioural deficits in future generations .............................................................. 24 21.1 Molecular dissection of epigenetic signals controlling muscle stem cell behaviour during regeneration ............................................................................................................................. 25 22.1 A Bioengineering Approach To Polarise Glioma Tumour Initiating Cells and Induce Asymmetric Cell Division In Order To Limit Their Tumorigenic Potential .......................................................... 26 24.1 HIV-1 mediated reprogramming of T cell gene expression networks ....................................... 28 25.1 The role of the pro-FLG Ca2+-binding domain in the formation of the skin barrier and pathogenesis of atopic dermatitis .................................................................................................................. 29 26.1 Exploring the general mechanisms of mammalian neural crest migration in vivo using live cell imaging and how it is altered in neuroblastoma cancer cells ......................................................... 30 27.1 Functional interrogation and therapeutic intervention of the molecular pathways driving cutaneous T-cell lymphoma ........................................................................................................ 31 28.1 In-silico design and identification of novel antiviral drugs targeting vector borne viruses (Zika, Dengue & Chikungunya)............................................................................................................. 32 29.1 Defining the role of circulating fibrocytes in the pathogenesis of renal fibrosis; a study of cell-signalling crosstalk .................................................................................................................... 33 30.1 Structural characterisation of a novel cytolytic peptide toxin .................................................. 34 31.1 MS1 - a cardiac regulator of development and stress .............................................................. 35 32.1 Identifying novel molecular mechanisms involved in the generation of new insulin-producing beta cells from adult stem/ progenitor cells………………………………………………………………………………36 33.1 Therapeutic use of regulatory T cells in liver disease: Immunoregulation and tissue regeneration ................................................................................................................................................ 37 34.1 Understanding regulation of cellular energy metabolism ...................................................... 38 35.1 Mechanism of action of a cancer-selective protein toxin .........................................................39 36.1 Mitochondrial microRNA (MitomiRs) and their relationship to mitochondrial bioenergetics ..... 40

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37.1 Using chemical modification of anticancer drug scaffolds to study efflux mediated chemoresistance in cancer stem cells .......................................................................................... 41 38.1 Role of sphingosine-1-phosphate (S1P) signalling in cochlear homeostasis and progressive hearing loss .......................................................................................................................................... 42 39.1 Characterising the epigenome in mice after exposure to environmental carcinogens ............... 43 40.1 Understanding immune responses in intestinal homeostasis and disease ............................... 44 41.1 Broadly neutralizing antibody responses against HIV ............................................................. 45 42.1 The CXCL8-producing T cell: function in health and carcinogenesis ........................................ 46 43.1 Elucidating the crosstalk between lymphocytes and intestinal epithelial cells using human mini-guts .......................................................................................................................................... 47 44.1 Elucidating the molecular mechanisms of peanut allergies .................................................... 48 45.1 Targeting PSK kinases for breast cancer therapy .................................................................. 49 46.1 How do mutations in MYO18B lead to muscle diseases? ........................................................ 50 47.1 Developmental basis of skin diversity .................................................................................. 51 48.1Molecular mechanisms underlying the contractile dysfunction in ageing-related muscle weakness ................................................................................................................................................ 52 49.1 Linking Genotype to Phenotype in Psoriatic Arthritis: determining the role of disease-associated genes in Tc17 cell function and development................................................................................ 55 50.1 TRPV1 and TRPA1 ligands as P-gp substrates: a wide-reaching investigation from basic molecule through to animal models of disease ........................................................................................... 57 51.1 Role of genetic regulation of gene expression in Type 2 Diabetes and Obesity………………………59 52.1 Identifying novel therapeutics to target pancreatic cancer metastasis………………………………..60

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Molecules, Cells and the Basis for Disease

This theme brings together stem cells and regenerative medicine (inc. cellular therapies), immunology, genetics, cellular biology (particularly relating to cancer), and biophysics. These areas – and particularly the interfaces between them – are current strengths and priorities for King’s.

Lead: Professor Rebecca Oakey

When choosing a project from this catalogue in the funding section of the online

application form please enter MRCDTP2017_Theme1

Deadline for application: Sunday 7th May 2017

Shortlisted candidates will be contacted in early June and invited to an interview on June

15th.

Interviews: 15 June 2017

The 2017/18 studentships will commence in September 2017.

For further Information or queries relating to the application process please contact

mrc-dtp@kcl.ac.uk

Projects listed in this catalogue are subject to amendments, candidates invited to

interview will have the opportunity to discuss projects in further detail.

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1.1 Dissecting antigen presentation to T lymphocytes using dendritic cells derived from genetically modified induced-pluripotent stem cells

Co-supervisor 1: Pierre GUERMONPREZ Research Division or CAG: DIIID E-mail: pierre.guermonprez@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/diiid/departments/immunobiology/research/guermonprez/index.aspx Co-supervisor 2: Timothy TREE Research Division or CAG: DIIID Email: timothy.tree@kcl.ac.uk Website:http://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/immunobiology/research/Tree/index.aspx Project Description: Depending on environmental cues, dendritic cells (DCs) can induce the functional inactivation of T cells (i.e. tolerance) or their differentiation in effector/memory T cells (i.e. immunity). Regulatory “immune checkpoints” molecules (PDL1/PDL2, e.g.) or co-stimulatory molecules (CD80/CD86/OX40-L/CD40, e.g.) expressed by DCs determine T cell fate after DC-T cell interactions. DC-based immunotherapies are currently limited by the difficulty to obtain large amounts of DCs of immunogenic phenotype to infuse in patients. Human Induced Pluripotent Stem Cells (iPSCs) represent an inexhaustible source of immunologically compatible cells opening novel iPSCs (termed as iPSCDCs) by recapitulating the physiological differentiation pathway of DCs from hematopoietic stem cells. We intend to apply this technology to iPSCs, genetically modified using CRISPR/Cas technology, in which inhibitory signalling molecules are inactivated and/or activator pathways activated. The ultimate goal of this approach is to generate iPSCDCs with increased potential to activate T cells. iPSCDCs might serve as a proof-of-concept for the development of new cellular immunotherapies in cancer patients. Two representative publications from supervisors: Cross-Presentation of Cell-Associated Antigens by MHC Class I in Dendritic Cell Subsets. Gutiérrez-Martínez E, Planès R, Anselmi G, Reynolds M, Menezes S, Adiko AC, Saveanu L, Guermonprez P. Front Immunol. 2015 Jul 17;6:363. Inflammatory Flt3l is essential to mobilize dendritic cells and for T cell responses during Plasmodium infection. Guermonprez P*, Helft J, Claser C, Deroubaix S, Karanje H, Gazumyan A, Darasse-Jèze G, Telerman SB, Breton G, Schreiber HA, Frias-Staheli N, Billerbeck E, Dorner M, Rice CM, Ploss A, Klein F, Swiecki M, Colonna M, Kamphorst AO, Meredith M, Niec R, Takacs C, Mikhail F, Hari A, Bosque D, Eisenreich T, Merad M, Shi Y, Ginhoux F, Rénia L, Urban BC, Nussenzweig MC. Nat Med. 2013 Jun;19(6):730-8. * corresponding author

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2.1 Mapping the PTPN22 interactome Co-supervisor 1: Andrew P. Cope Research Division or CAG: DIIID E-mail: andrew.cope@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/diiid/centres/cmcbi/research/cope/index.aspx Co-supervisor 2: Dylan M. Owen Research Division or CAG: Randall Institute Email: dylan.owen@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/cell/owen/index.aspx Project Description: Scientific background: PTPN22 encodes a protein tyrosine phosphatase that negatively regulates antigen receptor signalling by targeting Src and Syk family kinases. The PTPN22-R620W genetic variant is strongly associated with susceptibility to RA, T1D and lupus through mechanisms that are poorly understood. We have demonstrated that PTPN22 is a negative regulator of adhesion receptor signalling; the disease-associated variant promotes integrin-dependent cell adhesion. We also discovered that PTPN22 is sequestered in large clusters in the steady state that disperse following receptor stimulation, permitting association of the phosphatase with its substrates. In this project we will study mechanisms of PTPN22 clustering and de-clustering in T cells, informed by proteomic profiling and high resolution imaging of PTPN22 and its interacting partners. This will offer new insights into pathways regulated by PTPN22 that underpin susceptibility to autoimmunity. Skills training: Cell signalling and protein purification; generation of mutant proteins for functional analysis; imaging by confocal and super resolution microscopy; Bayesian cluster analysis; functional analysis of T lymphocytes. Objectives: Year 1: The PTPN22 interactome will be defined using the BioID system. PTPN22 constructs will be generated for transfection of T cells, interacting proteins purified using streptavidin beads and identified by mass spectrometry. Year 2: Super resolution microscopy will be used to visualise cluster assembly and disassembly of PTPN22 and key interacting partners; transfecting cells with fluorescent fusion proteins will permit imaging of live cells. Year 3/4: Functional analysis of T cells expressing mutants of PTPN22 and its interacting partners, including cell adhesion and migration, and effector function. Two representative publications from supervisors: Rubin-Delanchy P, Burn GL, Griffié J, Williamson DJ, Heard NA, Cope AP, Owen DM. Bayesian cluster identification in single-molecule localization microscopy data. Nat Methods 2015;12:1072-1076. Burn GL, Cornish GH, Potrzebowska K, Samuelsson M, Griffie J, Minoughan S, Yates M, Ashdown G, Pernodet N, Morrison VL, Sanchez-Blanco C, Purvis H, Clarke F, Brownlie RJ, Vyse TJ, Zamoyska R, Owen DM, Svensson LM, Cope AP. Super-resolution imaging of the cytoplasmic phosphatase PTPN22 links integrin-mediated adhesion with autoimmunity. Sci Signaling 2016;9:ra99.

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3.1 MicroRNAs as biomarkers of liver regeneration and their link to tumour biology Co-supervisor 1: Dr. Varuna R. Aluvihare Research Division or CAG: Transplantation Immunology & Mucosal Biology E-mail: varuna.aluvihare@kcl.ac.uk Co-Supervisor 2: Dr Siamak Salehi Research Division or CAG: Transplantation Immunology & Mucosal Biology Email: Siamak.salehi@kcl.ac.uk Project Description:

Regenerative competence is species and tissue specific. In higher eukaryotes efficient solid organ regeneration is restricted to the liver. Regeneration competence encompasses regulated cell proliferation, differentiation and remodelling to enable replacement of tissue or whole complex body parts. In our previous studies we identified microRNAs (miRNA) that regulate successful and failed human liver regeneration in an auxiliary transplant model. We also demonstrate that miRNA associated with successful regeneration drive more aggressive tumour behaviour. Conversely miRNA linked to failed regeneration can inhibit tumour growth.

Our goal is to understand the processes that regulate liver regeneration or prevent it. We will use a variety of clinical settings which involves regeneration such as transplantation or acute liver failure to identify the responsible miRNAs and investigate their link to tumour behaviour and its aggressiveness. It may lead to the development of novel biomarkers that better predict both the spontaneous liver regeneration/recovery and potential novel treatment strategy for human cancers by deploying regulatory miRNA inhibitors of regeneration thereby improving patient survival.

Y1: Establish experience in lab techniques (sample collections, RNA and DNA extraction protocols, molecular biology techniques PCR, qPCR, micro- arrays and vector design and construction) Y2: Analyse and interpret data obtained from a tissue samples and invitro validation of clinical observations Y3: In vivo experiments to validate invitro findings using animal models for regeneration and cancer Y4: Explore putative miRNA clinical applications + Write up Techniques to be utilized will range from basic molecular biology techniques to FACS,

microRNA arrays, NGS, transfection of microRNA mimics and inhibitors by construction of Lentiviral and Adenoviral vectors. Tissue culture techniques and primary human hepatocyte isolation and culture. In vivo techniques such as partial hepatectomy and xenograft tumor models, In vivo imaging techniques … Liver Studies at King’s College Hospital provides a unique combination of clinicians and scientists working together allowing strong collaboration on translational research. The clinical material available for analysis will be obtained from adult patients according to human tissue act guidelines. Two representative publications from supervisors: Human Liver Regeneration Is Characterized by the Coordinated Expression of Distinct MicroRNA Governing Cell Cycle Fate S. Salehi, †, H. C. Brereton, †, M. J. Arno, D. Darling, A. Quaglia, J. O’Grady, N. Heaton and V. R. Aluvihare †Contributed equally American Journal of Transplantation 2013; 13: 1282–1295093 The microRNA Expression Profile in Donation after Cardiac Death (DCD) Livers and Its Ability to Identify Primary Non Function

Shirin Elizabeth Khorsandi☯, Alberto Quaglia☯, Siamak Salehi☯, Wayel Jassem‡, Hector Vilca-

Melendez‡, Andreas Prachalias‡, Parthi Srinivasan‡, Nigel Heaton*☯☯ = These authors contributed equally to this work. ‡ = These authors also contributed equally to this work.

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Published: May 15, 2015 DOI: 10.1371/journal.pone.0127073 PLOS ONE

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5.1 Applying single-cell RNA sequencing technologies to establish cardiac stem/progenitor cell heterogeneity Co-Supervisor 1: Dr. Georgina M. Ellison-Hughes Research Division or CAG: Centre of Human and Aerospace Physiological Sciences, Centre for Stem Cells & Regenerative Medicine E-mail: Georgina.ellison@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/georgina.ellison.html Co-Supervisor 2: Prof. Juha Kere Research Division or CAG: Genetics and Molecular Medicine Email: juha.kere@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/juha.kere.html Project Description: We and others have shown that the adult myocardium, including human, harbours a population of resident (endogenous) multi-potent cardiac stem cells (eCSCs). A variety of markers (i.e. c-kit, Sca-1, MDR1/2, PDGFrα, Mesp1) have been used to identify and delineate eCSCs in different species and throughout development, causing conceptual uncertainty because of heterogeneous definition. In this project you will apply innovative and state-of-the art technologies, such as laser capture microdissection (LCM), enzymatic digestion and micromanipulation, to isolate single cells from the heart. Then you will apply single-cell RNA sequencing utilising the single-cell tagged RT (STRT) protocol, with unique molecular identifiers (UMIs), to explore transcriptome heterogeneity of the eCSCs, and their relationship between each other. You will utilise computational methods to leverage the full complexity within single-cell transcriptome data, and will mine the eCSC transcriptome data for specific, novel marker genes in order to better characterise the eCSCs. You will determine multi-lineage priming, which reflects the cells competency (based on the presence of multi-lineage affiliated genes) to adopt one of several fates and will employ computational prediction to determine their relationship to each other, multipotency and cardiomyogenic commitment. Objective 1: Optimising single-cell isolation techniques from heart tissue. Objective 2: Single-cell transcription profiling of eCSCs utilising single-cell tagged RT (STRT) protocol. Objective 3: Addressing the heterogeneity inherent in the Sca-1pos/c-kitpos eCSCs. Two representative publications from supervisors: Ellison GM, Vicinanza C, Smith AJ, Aquila I, Leone A, Waring CD, Henning BJ, Stirparo GG, Papait R, Scarfò M, et al. Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell. 2013, 154:827-842. Smith AJ, Lewis FC, Aquila I, Waring CD, Nocera A, Agosti V, Nadal-Ginard B, Torella D, Ellison GM. Isolation and characterization of resident endogenous c-Kit(+) cardiac stem cells from the adult mouse and rat heart. Nat Protoc. 2014, 9:1662-1681.

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6.1 Developing nanomedicines for immunosuppression in liver transplantation Co-Supervisor 1: Dr Khuloud T Al-Jamal Research Division or CAG: IPS E-mail: Khuloud.al-jamal@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/en/persons/khuloud-aljamal(4e743c3a-574c-4ef2-b205-12211b409e05)/projects.html Co-Supervisor 2: Prof. Giovanna Lombardi Research Division or CAG: DTIMB Email: giovanna.lombardi@kcl.ac.uk www.kcl.ac.uk/medicine/research/divisions/timb/about/people/profiles/giovannalombardi.aspx Project Description:

Immunosuppression is an important strategy used in the medical field in cases of organ transplantation and auto-immune diseases. Current approaches to control unwanted immune responses involve administration of immunosuppressant drugs of known side-effects. Cell-based therapy including the use of pluripotent stem cells and allogenic stem cells is an emerging new field used in tissue regeneration and/or tissue transplantation. These suffer from poor survival or rejection following implantation. Potential solutions involve the use of cell-substitutes (e.g. exosomes) or the use of immuno-modulators. Immuno-modulators are generally biological molecules, relatively large in size molecules with poor kinetics in vivo.

Nanomedicine has emerged as a new field utilises nano-sized carriers that can deliver small drug molecules (immunosuppressants) or larger proteins (immuno-modulators) in a more controlled and targeted manner. This overall reduces the dose, improve efficiency and reduce side effects of the delivered drugs. Nanomedicines accumulate passively in liver tissues due to presence of sinusoids able to tarp these nanoparticles.

This 4-year project will focus on developing nanomaterials to induce immunosuppression in liver transplant animal model, with the clinical translation to man in mind. A biodegradable nanoparticle (NP) (polymer or lipid-based) will be loaded with therapeutic agent(s) and used as an implant or in an injectable form. During this project, therapeutic strategies include using NP encapsulating immunosuppressant agents (e.g. rapamycin) which is hypothesised to increase drug half-life, tissue specificity which reduce the side effect of the proposed drug(s). The second strategy to be exploited involves using nanocarriers encapsulating therapeutic agents (immune-modulators) to be used alone or in combination with implanted cells (or tissues) with the aim of improving cell survival or reduce their immunogenicity.

The project consists of the following stages: (i) preparation and characterisation of the nanocarriers containing the immunosuppressant/immunomodulatory drug(s); (ii) in vitro studies (cytotoxicity assays, intra-cellular uptake and immune suppression); (iii) in vivo imaging and therapy studies in relevant mouse model. Two representative publications from supervisors: Safinia N, Vaikunthanathan T, Fraser H, Thirkell S, Lowe K, Blackmore L, Whitehouse G, Martinez-Llordella M, Jassem W, Sanchez-Fueyo A, Lechler RI, Lombardi G. Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation. Oncotarget. 2016 Feb 16;7(7):7563-77. doi: 10.18632/oncotarget.6927. El-Gogary R, Rubio N, Wang JT, Al-Jamal WT, Bourgognon M, Kafa H, Naeem M, Klippstein R, Abbate V, Leroux F, Bals S, Van Tendeloo G, Kamel AO, Awad GA, Mortada ND, Al-Jamal KT. Polyethylene glycol conjugated polymeric nanocapsules for targeted delivery of quercetin to folate-expressing

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cancer cells in vitro and in vivo. ACS Nano. 2014 Feb 25;8(2):1384-401. doi: 10.1021/nn405155b. Epub 2014 Jan 23. 7.1 Manipulating the stem cell state in vivo Co-Supervisor 1: Cynthia Andoniadou Research Division or CAG: Dental Institute E-mail: cynthia.andoniadou@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/cynthia.andoniadou.html Co-Supervisor 2: Isabelle Miletich Research Division or CAG: Dental Institute Email: isabelle.miletich@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/isabelle.miletich.html Project Description: The pituitary gland is essential for the regulation of key physiological processes such as growth, metabolism, fertility and the stress response. This organ can fail to produce hormones at any stage of life, or develop tumours, both with devastating consequences. Our lab focuses on the stem cell involvement in both of these processes. The project will study the function of novel determinants that regulate stem cell potential and homeostasis. We have identified the Hippo kinase cascade to be active in the pituitary gland and in the stem cells, but its function in this organ is currently unknown. This pathway regulates organ size in other tissues and when downregulated, promotes stem cell potential, increases proliferation and prevents apoptosis. We will manipulate this pathway to address the impact on stem cells, if their potential can be reactivated after loss and if it can be inhibited when overactive such as during neoplastic disease. This is applicable to regenerative medicine approaches with the ultimate goal to open new avenues for safer and better treatments for human conditions. Objectives 1. Through use of genetic tools, to establish the function of individual pathway components in pituitary stem cell regulation. 2. To establish interactions between Hippo and other signalling pathways in the gland that influence proliferation. Laboratory skills training provided Mouse genetics, developmental biology, dissection, immunofluorescence, ex vivo organ culture, in vitro culture, confocal microscopy, molecular biology, RNAscope in situ hybridisation. Two representative publications from supervisors: Lodge EJ, Russell JP, Patist AL, Francis-West P and Andoniadou CL. (2016) Expression analysis of the Hippo cascade indicates a role in pituitary stem cell development. Front. Physiol. Mar 31;7:114. Andoniadou CL et al, (2013) Sox2+ stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell, Oct;13(4):433-45.

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8.1 Mechanisms of bacterial inhibition by interferon-stimulated genes Co-Supervisor 1: Charlotte Odendall Research Division or CAG: DIIID Email: charlotte.odendall@kcl.ac.uk Co-Supervisor 2: Stuart Neil Research Division or CAG: DIIID E-mail: stuart.neil@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/infectious/research/Neil/index.aspx

Project Description:

Type I and III interferons (IFNs) are produced in response to non-self by the innate immune system. Although better described as inhibitors of viral infections, we found that IFNs block the growth of Salmonella within host cells. IFNs function by inducing a large family of 300 proteins termed Interferon stimulated genes (ISGs), with a wide range of different functions. This project seeks to determine which ISG(s) are responsible for IFN-mediated inhibition of bacterial growth, using Salmonella Typhimurium and Shigella sonnei as models. These pathogenic bacteria cause disease using type III secretion systems (T3SS), molecular needles that enable the transport of virulence proteins into host cells.

We will first perform an expression screen using an ISG library. Individual ISGs will be expressed and their ability to block bacterial replication will be assessed. In parallel we will perform siRNA screens of known ISGs. ISGs will be knocked down and infection assays will be carried out in the presence of IFNs. Positive hits will be cells that no longer control bacterial infection in the presence of IFN (Year 1 and 2). In parallel, we will test well-characterised ISGs that could have effects on the known lifecycles of bacteria (Year 1 and 2). For example, IFITM proteins affect membrane fluidity, and could potentially affect the lifecycle of Salmonella (that replicates in membrane-bound vacuoles) or Shigella (that requires vacuolar lysis and escape). Identified ISGs will be further studied to determine which step of the bacterial intracellular lifecycle is affected (Year 3). The roles of ISGs in inhibition of bacterial virulence will potentially then be studied in vivo in well-established models (Year 3). This project will investigate both aspects of host pathogen interactions, as well as bridge the fields of virology and bacteriology as IFNs and ISGs are normally considered as antiviral factors. Work involving bacterial virulence, replication and pathogenesis assays will be done with Charlotte Odendall while Stuart Neil will be mostly involved with the ISG screens. Techniques involved will be: - Molecular Biology - Tissue culture - Flow cytometry - Microbiology - Infection assays with different bacterial pathogens - Gene knockdowns and knockouts by RNAi and CRISPR/Cas9 - Biochemistry (western immunoblotting, immunoprecipitation) - Microscopy - In vivo infection models Two representative publications from supervisors: Odendall, C. et al. Diverse intracellular pathogens activate type III interferon expression from peroxisomes. Nat. Immunol. 15, 717–726 (2014). Foster et al. Resistance of Transmitted Founder HIV-1 to IFITM-mediated restriction. Cell Host and Microbe 2016 Oct 12;20(4):429-442. doi: 10.1016/j.chom.2016.08.006

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9.1 The role of post-translational modification of self proteins in breaking immune tolerance in type 1 diabetes Co-Supervisor 1: MARK PEAKMAN Research Division or CAG: DIIID E-mail: mark.peakman@kcl.ac.uk Website: www.kcl.ac.uk/lsm/research/divisions/diiid/.../peakman/index.aspx Co-Supervisor 2: YUK-FUN LIU Research Division or CAG: DNS Email: fun.liu@kcl.ac.uk Website: n/a Project Description: Approximately 80 human diseases come under the umbrella term “autoimmune”, and around 1 in 20 of the population will suffer from one of these conditions during their life. Although there is a great deal known about the genes that predispose to autoimmunity, there is still a very poor understanding as to how and why the immune system turns on self, and initiates the processes that cause tissue damage. Recently, a potentially important pathway through which immune tolerance to self can be broken has been suggested. “Post-translational modifications” (PTMs) of self proteins can alter them in a way that renders them highly antigenic and circumvents thymic tolerance induction. In the autoimmune disease Type 1 diabetes, there are new models proposed through which this can occur, and this PhD project will focus on these pathways. Initial studies will use new techniques we have developed (cell and molecular) to identify PTMs. In the middle of the PhD the studies will examine how PTMs can be presented as antigens to the immune system. The final studies will examine how this is relevant to the autoimmune responses we can detect in patients with Type 1 diabetes. This highly translational project will include studying patient samples, a range of skill sets (cell biology, molecular techniques, flow cytometry), and strong interaction (including an exchange visit) with the Technische Universitat Dresden as part of our KCL: Dresden TransCampus (http://www.kcl.ac.uk/lsm/research/transcampus/index.aspx). Two representative publications from supervisors: Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity. Cole DK, Bulek AM, Dolton G, Schauenberg AJ, Szomolay B, Rittase W, Trimby A, Jothikumar P, Fuller A, Skowera A, Rossjohn J, Zhu C, Miles JJ, Peakman M, Wooldridge L, Rizkallah PJ, Sewell AK. J Clin Invest. 2016 Jun 1;126(6):2191-204. Hydrophobic CDR3 residues promote the development of self-reactive T cells. Stadinski BD, Shekhar K, Gómez-Touriño I, Jung J, Sasaki K, Sewell AK, Peakman M, Chakraborty AK, Huseby ES. Nat Immunol. 2016 Aug;17(8):946-55.

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10.1 Gestational Diabetes, the epigenome and the health of the next generation Co-Supervisor 1: Professor Lucilla Poston Research Division or CAG: Division of Women’s Health E-mail: lucilla.poston@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/wh/index.aspx Co-Supervisor 2: Dr Jordana Bell Research Division or CAG: Division of Genetics and Molecular Medicine Email: Jordana.bell@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/gmm/departments/twin/research/bell/index.aspx Project Description: The incidence of gestational diabetes mellitus (GDM) is increasing with the rising prevalence of maternal obesity. Children born to mothers with GDM have heightened risk of obesity and metabolic disease in adulthood. The purpose of this studentship is to improve understanding of the mechanisms through which GDM exposure in utero translates to the development of obesity in the children. Increasing evidence suggests that maternal GDM induces stable modifications of the offspring epigenome, resulting in persistently changed gene expression and a lifelong increased risk of obesity and associated disorders. By utilising the biobank of the largest RCT of a lifestyle intervention (low glycaemic index diet and increased physical activity) in obese pregnant women (UK Pregnancy Better Eating and Activity Trial, UPBEAT), an RCT which lowered maternal dietary glycaemic index, increased physical activity) and reduced infant fat mass, this studentship will directly assess the role of epigenetic processes in the association between maternal GDM and offspring adiposity. Specifically, the student will address the interaction between the maternal metabolic profile (metabolome) the neonatal epigenome and childhood adiposity. The student will benefit from an established collaboration between KCL and the University of Southampton. Dr Karen Lillycrop (Southampton) an expert in epigenetics will play an important supervisory role in addition to the KCL supervisors. The student will join a vibrant research group in the KCL Division of Women’s Health. The student will acquire skills in the fields of women’s health, epigenetics, statistical modelling and gestational diabetes and benefit from the excellent KCL graduate training. Two representative publications from supervisors: Poston L, Bell R, Croker H et al. (2015) Effect of a behavioural intervention in obese pregnant women (the UPBEAT study): a multicentre, randomised controlled trial. The Lancet Diabetes & Endocrinology 3, 767-777. Tsai P-C, van Dongen J, Tan Q, Willemsen G, Christiansen L, Boomsma DI, Spector TD, Valdes AM, Bell JT. (2015) DNA methylation changes in the IGF1R gene in birth weight discordant adult monozygotic twins. Twin Research and Human Genetics 13, 1-12.

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11.1 Tackling hearing loss: development, regeneration and reconstruction of the ear Co-Supervisor 1: Prof Abigail Tucker Research Division or CAG: Craniofacial Development & Stem Cell Biology, KCL E-mail: Abigail.tucker@kcl.ac.uk http://www.kcl.ac.uk/dentistry/research/divisions/craniofac/ResearchGroups/TuckerLab/TuckerLab.aspx Co-Supervisor 2: Mr Dan Jiang PhD FRCSI(Otol) FRCS(ORL-HNS) Research Division or CAG: Otolaryngology, Head & Neck Surgery, Guys and St Thomas’s Hospital Honorary Prof: Craniofacial Development & Stem Cell Biology, KCL Email: dan.jiang@kcl.ac.uk https://kclpure.kcl.ac.uk/portal/en/persons/dan-jiang(cd25e784-010c-4321-a3ac-7189d23ea839)/publications.html Project Description: Birth defects associated with the middle and external ear lead to conductive hearing loss where sound fails to pass to the inner ear. Our knowledge of how these birth defects arise is limited, hampering our ability to correct defects. We aim to study the development of the ear taking advantage of mouse mutants with aberrant ear development and data from patients attending the Ear Clinic at St Thomas’ Hospital. The project is a collaboration between an expert in ear development in mice (Prof Tucker) and a clinician specialising in ear surgery (Prof Jiang). Overall we aim to understand how the ear forms and integrates so that ear defects can be more successfully repaired and the ear reconstructed during surgery. Aim 1 (year 1): To investigate the normal process of external ear formation during mouse embryonic development. Aim 2 (year 1 & 2): To understand the mechanisms behind ear defects using mouse models of human syndromes associated with external ear defects. These will include 22q11.2 deletion syndrome (Tbx1 mice), Branchio-oto-renal syndrome (Eya1 mice), LADD syndrome (Fgf10 mice), and holoprosencephaly (Gas1 mice). Aim 3 (year 2 & 3): To analyse CT scans from patients with ear defects to correlate the findings from the mouse with humans, and to assess the success of reconstructive surgery. Skills training: The student will be trained in a range of molecular biology techniques, anatomy and regenerative biology, while having access to clinical data. In addition critical thinking, presentation and writing skills will be taught. Two representative publications from supervisors: Thompson, H. Tucker, A.S. (2013). Dual origin of the epithelium of the middle ear. Science 339, 1453-1456.

Eze N, Jiang D, O'Connor AF. (2014) The atretic plate – a conduit for drill vibration to the inner ear. Acta Otolaryngol. 134(1):14-8.

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12.1 Mechanism of membrane remodelling in cell biology and replication of HIV-1 and Ebola virus Co-Supervisor 1: Prof. Juan Martin-Serrano Research Division or CAG: DIIID E-mail: juan.martin_serrano@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/diiid/departments/infectious/research/Martin-Serrano/indexJD.aspx Co-Supervisor 2: Dr. Jorge Bernardino de la Serna Research Division or CAG: Department of Physics Email: jorge.bernardino_de_la_serna@kcl.ac.uk Project Description: The Endosomal Sorting Complex Required for Transport (ESCRT) pathway catalyses the scission of thin membranous stalks found in diverse cellular processes, including multivesicular body (MVB) formation, cytokinetic abscission and nuclear envelope reformation. These fundamental events share a common topology and they all require ESCRT-III, a filament forming multi-protein complex that promotes membrane deformation and scission. In other words, the ESCRT complexes form the only cellular machinery capable of inducing membrane invaginations that protrude away from the cytoplasm, and this unique property explains why these proteins are hijacked by enveloped viruses, such as HIV-1 and Ebola virus, to facilitate their budding. In this project the student will take advantage of existing cell lines that express physiological levels of fluorescently tagged ESCRT-III subunits. These cells will be used to visualise ESCRT-III filament formation by live-cell microscopy under strict physiological conditions. The combination of this experimental setting with bespoke biophysical analysis will allow the study of the dynamics of ESCRT-III polymerization, and how the biogenesis of these polymers facilitates membrane remodeling. A complementary approach will take advantage of super-resolution microscopy (SIM an STED) that are available in the host labs. Advanced quantitative fluorescence microscopy techniques and imaging analysis tools will be employed to reveal the role of ESCRTs in membrane remodelling during viral budding at the spatial and temporal nanoscale, including the tracking of single viral budding events. Two representative publications from supervisors: Host factors involved in retroviral budding and release. Martin-Serrano J, Neil SJ. Nature Reviews in Microbiology. 2011 Jun 16;9(7):519-31. doi: 10.1038/nrmicro2596 ESCRT machinery: Damage control at the nuclear membrane. Ventimiglia LN, Martin-Serrano J. Cell Res. 2016 Jun;26(6):641-2. doi: 10.1038/cr.2016.52.

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14.1 Gene expression in skin in subjects with reduced biological ageing compared to their chronological ageing Co-Supervisor 1: Dr Mario Falchi, PhD Research Division or CAG: Genetics Division, Kings College, Twin Research Unit, St Thomas Hospital London E-mail: mario.falchi@kcl.ac.uk Co-Supervisor 2: Dr Veronique Bataille, MD PhD FRCP Research Division or CAG: Genetics Division, Kings College, Twin Research Unit, St Thomas Hospital London Email: bataille@doctors.org.uk Project Description: The TwinsUK cohort includes 12,000 twins used to study the genetic and environmental aetiology of age-related complex traits. Measures of biological ageing comprise cataract score, strength, muscle mass on DEXA scan, lung function, telomere length, bone mineral density, naevus count, creatinine and others, as well as measures derived from epigenetics and glycomics data. The final aim of this PhD is to identify novel gene and pathways involved in skin ageing and skin cancer (one of the most common cancers in the world). 1st year: Integrate phenotypic and –omics data already available in the TwinsUK cohort to define reliable biological ageing scores. Gene expression data in skin are available on more than 700 twins: the student will learn how to analyse the RNA sequencing data. Differential expression analysis will be used to identify genes associated with reduced and accelerated biological ageing. 2nd: Gene network analysis will be used to identify modules of co-expressed genes correlated with the observed gap between biological and chronological age, to identify pathways associated with decelerated biological ageing. Modules and hub genes (genes exerting the main control within each module) will be investigated for their role in risk to skin cancer, skin, and systemic ageing. 3rd: The biological function of identified genes and modules will be investigated through whole-genome sequencing, metabolomics, metagenomics, and epigenetics data. 4th: During the fourth year, the student will write their thesis. The student is expected to write manuscripts for peer review as well as presenting his work at national and international meetings. Two representative publications from supervisors: Falchi M et al Genome-wide association study identifies variants at 9p21 and 22q13 associated with development of cutaneous nevi. Nat Genet. 2009 41:915-9 V Bataille et al Nevus size and number are associated with telomere length and represent potential markers of a decreased senescence in vivo Cancer Epidemiol Biomark Prev 2007 16:1499-1502

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15.1 Adhesion receptor signalling regulating lung cancer progression Co-Supervisor 1: Professor Maddy Parsons Research Division or CAG: Randall Division E-mail: maddy.parsons@kcl.ac.uk Website: www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/motility/parsons/index.aspx Co-Supervisor 2: Professor George Santis Research Division or CAG: AALB Division/Cancer CAG Email: George.santis@kcl.ac.uk Website: www.kcl.ac.uk/lsm/research/divisions/aalb/about/people/profiles/santisg.aspx Project Description: The Coxsackie and Adenovirus Receptor (CAR) is a transmembrane receptor that plays a key role in controlling adhesion between adjacent epithelial cells. CAR is expressed in normal epithelial cells, but has also been shown to be upregulated in a number of human tumours including lung carcinomas. However, the potential mechanisms by which CAR contributes to cancer cell growth remain unclear. Our recent data has shown that CAR increases recruitment of immune cells during lung inflammation. Further unpublished data demonstrated an important role for CAR in promoting lung cancer cell proliferation in vitro and in vivo. In this context, CAR is also important for maintaining junctions between proliferating human lung cancer cells and this promotes optimal signaling downstream of the epidermal growth factor receptor (EGFR) to perpetuate cell division. However, whether CAR plays an additional role in immunomodulation within tumours to promote cancer cell growth remains unknown. CAR has never before been studied as a potential therapeutic target for inflammation, and as the CAR knockout mouse is embryonic lethal, this receptor has also not previously been studied in the context of the adult lung. The aim of this project is to use biochemistry, cell biology, advanced imaging and in vivo analysis to define how CAR depletion from tumour cells in non-immunocompromised mice impacts tumour growth and stromal organization. This data will provide novel insight into the role of CAR in homeostasis and disease, and provide a solid foundation for future in vivo studies of this receptor as a potential therapeutic target. The two supervisors have collaborated closely for many years and their complementary expertise in basic and translational research will provide the student with an excellent training opportunity. Two representative publications from supervisors: Morton PE et al. TNF promotes CAR-dependent migrations of leukocytes across epithelial monolayers. Sci Reps. 2016. 19;6:26321 Kiuchi T et al. The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility. Sci Signal. 2014 Aug 19;7(339):ra78. doi: 10.1126/scisignal.2005157.

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16.1 Developing novel, conventional and nanoparticle, inhibitors of the Wnt signaling pathway as a treatment for prostate and breast cancers Co-Supervisor 1: Aamir Ahmed Research Division/Department or CAG: Molecular Medicine and Genetics E-mail: aamir.ahmed@kcl.ac.uk Website: www.kcl.ac.uk/lsm/research/divisions/gmm/departments/stemcells/people/Dr-Aamir-Ahmed.aspx Co-Supervisor 2: Prokar Dasgupta, , Professor of Urological Innovation, Hon. Consultant Urologist, KCL Research Division/Department or CAG: DTIMB Liver, Renal, Urology, Transplant, Gastro/Gastro Intestinal Surgery Clinical Academic Group Email: Lynne.Saker@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/prokar.dasgupta.html Project Description: The Wnt signalling pathway plays a key role in carcinogenesis of prostate, penile, colon and breast cancers. Wnt signalling is transduced by two key transducers, intracellular free calcium ([Ca2+]i) and ß-catenin, a potent transcription factor co-activator. We have shown that the activation of Wnt signalling first increases [Ca2+]i that depolarizes the cell and nuclear electrical potential to facilitate ß-catenin translocation into the nucleus to activate gene (including numerous proto-oncogenes) transcription. We have a described a novel mechanism for the Wnt signaling pathway that involves cell membrane potential and inhibition of free [Ca2+]i. Very recently we have identified inhibitors that can reduce Wnt induced: 1. free [Ca2+]i 2. ß-catenin translocation into the nucleus where it activates gene transcription which is implicated in carcinogenesis. A patent (GB2014/053138) for targeting the Wnt pathway as a treatment for cancer was awarded recently. The purpose of this project will be to develop these novel Wnt pathway inhibitors, organic compounds and specifically designed nanopraticles (with London Centre for Nanotechnology), and to identify the mechanisms by which these may act to inhibit Wnt signaling in vitro and tumorigenicity in vivo. The following techniques, established in our laboratory, will be used: 1. Design and synthesis of specific nanoparticles using chemical techniques 2. [Ca2+]i release and cell membrane currents by simultaneous patch clamp electrophysiology and live calcium imaging using confocal microscopy 3. ß-catenin translocation into the nucleus using immunocytochemistry 4. Cell growth ± Wnt ligands and ±Wnt inhibitors using live cell imaging 5. Using human models of cancer in mice Two representative publications from supervisors: Arya M, Thrasivoulou C, Henrique R, Millar M, Hamblin R, Davda R, Aare K, Masters JR, Thomson C, Muneer A, Patel HR, Ahmed A. Targets of wnt/ß-catenin transcription in penile carcinoma. PLoS One 10:e0124395, 2015

Thrasivoulou, C, Millar, M and Ahmed, A. Activation of intracellular calcium by multiple Wnt ligands and translocation of ß-catenin into the nucleus: a convergent model of Wnt/Ca2+ and Wnt/ß-catenin pathways. J. Biol.Chem. 288: 35651–35659, 2013

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17.1 Roles of the RNA-binding proteins LARP4A and LARP4B in cancer cell migration and invasion Co-Supervisor 1: Agi Grigoriadis Research Division or CAG: Craniofacial Development & Stem Cell Biology E-mail: agi.grigoriadis@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/agi.grigoriadis.html Co-Supervisor 2: Sasi Conte Research Division or CAG: Randall Email: sasi.conte@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/structural/conte/index.aspx Project Description: Cancer metastasis is associated with poor prognosis and is difficult to treat. To metastasize, cancer cells invade surrounding tissues, then enter blood vessels and spread to other sites. Using RNAi screens we have identified novel regulators of cancer cell migration and invasion, including the RNA-binding protein LARP4A. LARP4A and its close relative LARP4B are mutated in several cancers, and our recent data identified mutations that affect LARP4A function (Seetharaman et al., 2016). LARP4A/B are believed to bind to mRNA poly(A) tails and thereby regulate translation, although their mRNA targets are not known. They also interact with Poly(A)-binding protein (PABP) and RACK1, both of which are involved in mRNA translation. This project aims to determine how LARP4A/B regulate cancer invasion, and whether they could be targets for cancer therapy. Skills training: cell culture, siRNA/plasmid transfection, CRISPR/Cas9, site-directed mutagenesis, confocal microscopy, biochemical/biophysical analysis of protein-protein interactions, in vitro and in vivo cancer migration/invasion. Year 1: Roles of LARP4A/LARP4B mutants found in cancers in regulating their interactions with PABP, RACK1 and poly(A), using biochemical and biophysical approaches. Effects of these mutants on intracellular localization of LARP4A/B and cancer cell migration. Year 2: Effects of LARP4A/B depletion and overexpression on translation of Rho GTPase signalling network proteins that have a similar phenotype to LARP4A/B in our RNAi screen results. Determine if LARP4A/B cancer-associated mutants alter expression or localization of these Rho network proteins. Year 3: Test selected LARP4A/B mutations for effects on cancer cell-cell adhesion, invasion and metastasis in cells and in animal models. Two representative publications from supervisors: Seetharaman, S., Flemyng, E., Shen, J., Conte, M.R., Ridley, A.J. (2016) The RNA-binding protein LARP4 regulates cancer cell migration and invasion. Cytoskeleton, doi: 10.1002/cm.21336. Reymond, N., Im, J.H., Garg, R., Cox, S., Soyer, M., Riou, P., Colomba, A., Muschel, R.J., Ridley, A.J. (2015) RhoC and ROCKs regulate cancer cell interaction with endothelial cells. Mol. Oncol. 9, 1043-1055.

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18.1 Membrane remodelling during cell division Co-Supervisor 1: Dr Jeremy Carlton Research Division/Department or CAG: Cancer E-mail: Jeremy.Carlton@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/cancer/research/sections/cellbio/mtcd.aspx Co-Supervisor 2: Prof Riki Eggert Research Division/Department or CAG: Randall and Chemistry Email: Ulrike.Eggert@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/motility/eggert/index.aspx Project Description: During division, cells undergo dramatic remodelling of their membranes and cytoskeleton. Membrane-bound organelles, including the nuclear envelope, the endoplasmic reticulum, the Golgi apparatus and mitochondria have to be equally partitioned between dividing daughter cells, which requires extensive organelle rearrangement. Whilst we have varying amounts of information about the proteins involved in different organelles’ remodelling, our understanding of the role of lipids in these process remains severely underappreciated. Faithful cell division is essential for the maintenance of genome integrity and failures in this process are thought to underlie a variety of human malignancies. The goal of this project is to investigate the roles lipids and membrane associated proteins play in the remodelling of organelles during cell division. We will focus on a number of organelles, and by removing enzymes necessary for production of various lipid species or proteins that can shape membranes, you will test whether these organelles are remodelled and separated correctly during division and will analyse the consequences of perturbing this remodelling on cell division. You will use genome-editing to introduce epitope tags into organelle markers and will extract the tagged markers and will perform mass spectrometry and lipidomic analysis to identify lipid species bound. Once candidate lipids have been identified, you will verify interactions using liposome-based binding assays. You will join laboratories examining organelle remodelling during cell division (Carlton laboratory – ESCRTs; Eggert laboratory - lipids and the cytoskeleton) and will be trained in techniques including molecular biology, advanced imaging, protein biochemistry, lipidomics and lipid biochemistry. Two representative publications from supervisors: ESCRT-III controls nuclear envelope reformation. Olmos Y, Hodgson L, Mantell J, Verkade P, Carlton JG. Nature (2015) 522:236-9 Dividing cells regulate their lipid composition and localization. Atilla-Gokcumen GE, Muro E, Relat-Goberna J, Sasse S, Bedigian A, Coughlin ML, Garcia-Manyes S, Eggert US. Cell (2014) 156:428-39.

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19.1 Identifying effective drugs to treat oral squamous cell carcinoma Co-Supervisor 1: Fiona M. Watt Research Division or CAG: GMM E-mail: fiona.watt@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/gmm/departments/stemcells/wattfiona.aspx Co-Supervisor 2: Allessandra Vigilante

Research Division or CAG: GMM

Email:

Website: Project Description: Oral squamous cell carcinoma (OSCC) is a tumour of the multilayered epithelia lining the mouth. It is the sixth most common cancer affecting men in the UK and the long-term survival rate is only 50%. One of the challenges of treating OSCC is that it is highly heterogeneous at both the genetic and cellular levels. To create an in vitro model that accurately reflects this heterogeneity, we have generated a panel of human OSCC cell lines with a mutational spectrum that is representative of primary OSCC in The Cancer Genome Atlas (Hayes et al., 2016). The goal of the proposed PhD project is to collaborate with researchers at the Wellcome Trust Sanger Institute to use the cell lines to identify drugs that prevent OSCC growth in culture (see Iorio et al (2016) Cell 166:740-754) and in xenografts (Benaich et al., 2014). The objective of the rotation project is to develop robust, quantitative assays for cell proliferation, death and differentiation, based on high content imaging. The goal of year 1 is to screen the available drug library for hits. In year 2 the effects of the drugs will be confirmed using additional cell based assays (Hayes et al., 2016) and growth of transplanted cells in NSG mice (see Benaich et al., 2014) In the final year mechanistic experiments will be used to establish how certain drugs or combinations of drugs target cells that are mutant for NOTCH1, CASP8 and FAT1 (see Hayes et al., 2006). Two representative publications from supervisors: Benaich, B., Woodhouse, S., Goldie, S.J., Mishra, A., Quist, S.R., and Watt, F.M. (2014) Rewiring of an epithelial differentiation factor, miR-203, to inhibit human squamous cell carcinoma metastasis. Cell Reports 9:104-117 Hayes, T.F., Benaich, N., Goldie, S.J., Sipilä, K., Ames-Draycott, A., Cai, W., Yin, G. and Watt, F.M. (2016) Integrative genomic and functional analysis of human oral squamous cell carcinoma cell lines reveals synergistic effects of FAT1 and CASP8 inactivation. Cancer Letters 383:106-114.

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20.1 Interventions to improve maternal metabolic profile in obese pregnancy and prevent cardio-metabolic and behavioural deficits in future generations. Co-Supervisor 1: Dr Paul Taylor Research Division or CAG: Women’s Health Academic Centre, KHP E-mail: paul.taylor@kcl.ac.uk Website: www.kcl.ac.uk/wh Co-Supervisor 2:Prof Clive Coen Research Division or CAG: Women’s Health Academic Centre, KHP Email: clive.coen@kcl.ac.uk Website: www.kcl.ac.uk/wh Project Description: Maternal obesity is now the single biggest obstetric risk factor, and it is now widely recognised that maternal obesity is not only a risk factor for pregnancy outcomes (e.g. pre-eclampsia, gestational diabetes and fetal macrosomia) but also for the long term health of the child, with increased risk of obesity and related comorbidities. Diet and nutrition in pregnancy are modifiable risk factors for offspring metabolic health and offer the opportunity for intervention to stem the growing tide of childhood obesity and impact on the cardiovascular and mental health of the next generation. We have identified two candidate compounds, resveratrol and polydextrose which we intend to test for safety and efficacy (therapeutic potential) in a rat model of obesity in pregnancy. Resveratrol is a polyphenolic compound with numerous biological activities and anti-oxidant properties, found in high concentrations in the skins of red grapes. Polydextrose, on the other hand, is a soluble fibre with low glycaemic index and pro-biotic properties. The study will employ rodent models to advance understanding of the mechanism of action of these compounds in improving maternal metabolic profiles in obese pregnancy and their disease-preventive potential for cardiovascular (blood pressure) metabolic (obesity and diabetes) and behavioural deficits (cognitive function and ADHD) in future generations. Interventions with these two promising compounds, conceivably acting through divergent pathways, will also provide insight and mechanistic understanding of how obesity in pregnancy can beget cardio-metabolic disorders in childhood. The studentship focuses on in vivo physiological techniques and is supported by an MRC Project grant. Two representative publications from supervisors: Poston L & Taylor PD. Obesity in Pregnancy and the Legacy for the Next Generation. What Can we Learn from Animal Models? RCOG Obesity Study Group (2007). Taylor PD. Samuelsson AM & Poston L. (2014) Maternal Obesity and the Developmental Programming of Hypertension: A role for leptin. Acta Physiol 2014, 210, 508–523.

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21.1 Molecular dissection of epigenetic signals controlling muscle stem cell behaviour during regeneration Co-Supervisor 1: Robert Knight Research Division or CAG: Craniofacial Development and Stem Cell Biology E-mail: robert.knight@kcl.ac.uk www.kcl.ac.uk/dentistry/research/divisions/craniofac/ResearchGroups/KnightLab/KnightLab.aspx

Co-Supervisor 2: Fiona Wardle Research Division or CAG: Randall Division of Cell and Molecular Biophysics Email: Fiona.wardle@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/signalling/wardle/index.aspx Project Description: Muscle stem cells (muSCs) are critical for maintaining muscle strength in disease, injury and ageing. They are highly migratory, yet almost nothing is known about their behaviour during regeneration in vivo, as it has been problematic to visualise migration to injury sites in mouse or human. We have recently described the use of transparent zebrafish for imaging muSC responses to injury and for identifying regulators in vivo. Epigenetic signals control chromatin packaging and hence gene expression and recent evidence suggests that these signals may influence the migratory ability of many cell types. We find this includes muSCs. This project therefore aims to test the importance of an epigenetic modifying protein, Ezh2, in controlling muSC responses in zebrafish models of muscle injury. This will be achieved by time-lapsed imaging of muSCs responses to small laser injuries using confocal microscopy to measure cell responses. Ezh2 function in muSCs will be tested using a mutant, by addition of small molecule inhibitors or over-expression of functionally altered Ezh2 genes; Ezh2-dependent changes to gene expression and chromatin marks will be evaluated by sorting cells and performing qPCR and ChIP-qPCR in combination with immunolabelling and in situ hybridisation. Training will be given in all these techniques. Objectives for the project are: 1. determine whether loss of Ezh2 function compromises muSC migration and ability to regenerate muscle (year 1) 2. determine whether small molecule inhibitors of Ezh2 affect muSC responses to injury (year 2) 3. identify candidate targets of Ezh2 regulating muSCs during regeneration (year 3-4) Two representative publications from supervisors: Knappe, Stefanie; Zammit, Peter S.; Knight, Robert D. 'A population of Pax7- expressing muscle progenitor cells show differential responses to muscle injury dependent on developmental stage and injury extent'. Frontiers in aging neuroscience, Vol. 7, No. 161, 25.08.2015. Nelson, A.C., Cutty, S.J., Niini, M., Stemple, D.L., Flicek, P., Corinne Houart, C., Bruce, A.E.E., Wardle, F.C. (2014). Global identification of Smad2 and Eomesodermin targets in zebrafish identifies a conserved transcriptional network in mesendoderm and a novel role for Eomesodermin in repression of ectodermal gene expression. BMC Biology, 12(1):81.

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22.1 A Bioengineering Approach To Polarise Glioma Tumour Initiating Cells and Induce Asymmetric Cell Division In Order To Limit Their Tumorigenic Potential Co-Supervisor 1: Dr Shukry James Habib Research Division or CAG: Centre for Stem Cells and Regenerative Medicine E-mail: Shukry.habib@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/gmm/departments/stemcells/people/dr-shukry-j-habib.aspx Co-Supervisor 2: Prof Jeremy Green Research Division or CAG: Craniofacial Development and Stem Cell Biology Email: jeremy.green@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/jeremy.green.html Project Description: Adult gliomas are malignant human brain tumours with no curative therapy available. They can occur via transformation of adult neural progenitor cells (NPCs) into glioma tumour-initiating cells (GTICs). Several studies have shown that NPCs usually divide asymmetrically to produce one NPC and another differentiated cell, however during gliomagenesis the cells mainly divide symmetrically, promoting tumour growth. This project will investigate these cell division processes, and engineer strategies to polarise GTICs in order to direct them to divide asymmetrically. We hypothesise that the Wnt signaling pathway could be employed for controlling the cellular polarity and division of GTICs, as the majority of gliomas do not acquire mutations in the Wnt signalling components. The Habib lab has engineered localised Wnt niches that can induce asymmetric cell division of embryonic and adult stem cells. The combination of our bioengineering approaches with cell polarity expertise (Green lab) provide grounds to explore the mechanistic regulation of GTIC division and opportunities to limit their tumorigenic potential. 1st -2nd year: Culturing and characterising human NPCs and GTICs. Employing 3D microscopy to study the division NPCs and GTICs by establishing a molecular segregation map for cell polarity proteins, Wnt pathway components and cell fate markers. Purification of Wnt proteins will also be done. 3nd- 4th year: Engineering localised Wnt niches and testing their effect on NPCs and GTIC division mode. Flow cytometery to separate between the daughter cells of GTICs and investigating their tumorigenic potential in in vitro and in vivo transplantation assays. Two representative publications from supervisors: Habib SJ et al A localized Wnt signal orients asymmetric stem cell division in vitro. Science 2013 Tabler JM, Yamanaka H, Green JB. PAR-1 promotes primary neurogenesis and asymmetric cell divisions via control of spindle orientation. Development 2010

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24.1 HIV-1 mediated reprogramming of T cell gene expression networks Co-Supervisor 1: Michael MALIM Research Division or CAG: DIIID E-mail: michael.malim@kcl.ac.uk Website: http://www.kcl.ac.uk/malim Co-Supervisor 2: Rebecca OAKEY Research Division or CAG: GMM Email: rebecca.oakey@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/gmm/departments/mmg/researchgroups/OakeyLab/index.aspx Project Description: Virus infection triggers two fundamental types of cellular response: those that inhibit infection (termed immune responses) and those that promote virus production, persistence and/or dissemination (here called reprogramming). The balance between these opposing forms of response dictates the overall outcome of infection and, ultimately, contributes to the pathogenic consequences for the infected host. To date, little is understood regarding the capacity of the pathogenic retrovirus, HIV-1, to reprogramme infected T lymphocytes, or the consequences of such changes for altering cell function. This project will build upon a substantial body of data where we have demonstrated that HIV-1 reorganises the transcriptional landscape of T lymphocytes within the first few hours of infection. We will employ a multi-disciplinary approach including virology, molecular genetics, biochemistry, high-throughput nucleic acid sequencing, single-cell analytics and bioinformatics to tackle the following key questions. One, what are the virus determinants that drive these RNA expression changes and through which signalling pathways do they operate? Two, what genome-wide alterations in chromatin and epigenetic marks underpin changes in RNA levels, and which steps of RNA biogenesis are regulated? Three, do all cells respond to infection equivalently or is there cell-to-cell variation; and if the latter, what cell-specific signatures underpin the differences? Four, how does reprogramming impact the fate of HIV-1 infection, perhaps by altering virus production or persistence (a form of which is called latency)? Together, insight in these areas will yield new information on the dynamic interplay between HIV-1 and its human host. Two representative publications from supervisors: Goujon, C., Moncorgé, O., Bauby, H., Doyle, T., Ward, C.C., Schaller, T., Hué, S., Barclay, W.S., Schulz, R. and Malim, M.H. (2013). Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection. Nature 502, 559-562. This paper employed comparative transcriptomics to identify candidate innate immune inhibitors of HIV infection, that were subsequently functionally screened and validated. Prickett, A.R., Barkas, N., McCole, R.B., Hughes, S., Amante, S.M., Schulz, R., and Oakey, R.J. Genome wide and parental allele specific analysis of CTCF and Cohesin binding sites in mouse brain reveals a tissue-specific binding pattern and an association with differentially methylated regions. Genome Research 2013. 23(10):1624-1635. This paper illustrates the use of genome wide sequencing techniques in understanding gene regulation.

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25.1 The role of the pro-FLG Ca2+-binding domain in the formation of the skin barrier and pathogenesis of atopic dermatitis Co-Supervisor 1: Dusko ILIC Research Division or CAG: Women’s Health E-mail: dusko.ilic@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/dusko.ilic.html Co-Supervisor 2: Carsten FLOHR Research Division or CAG: GMM Email: carsten.flohr@kcl.ac.uk Website: www.kcl.ac.uk/lsm/research/divisions/gmm/departments/dermatology/Groups/flohr/index.aspx Project Description: Atopic dermatitis (AD) affects 20% of children and 5% of adults in the UK and has a profound effect on patients’ quality of life. A genetic predisposition for skin barrier dysfunction, together with environmental factors, such as exposure to hard water (high CaCO3 levels) and the use of protease containing detergents, results in the typical immunological phenotype of AD. Normal epidermis displays a marked Ca2+ gradient, which controls the expression of filaggrin (FLG) and differentiation of the epidermis. Recent genetic studies have found that loss-of-function mutations in the FLG gene are present in up to 50% of AD patients. Pro-FLG has a Ca2+-binding domain of unknown function, which is cleaved off when pro-FLG is proteolytically processed into functional FLG during the biogenesis of the stratum corneum. We will use CRISPR/Cas9 gene-editing technology to delete the FLG Ca2+-binding domain in healthy human embryonic stem cell (hESC) lines. We will then use these isogenic normal and mutated hESC to differentiate various skin cell types and build 3D in vitro models of full thickness skin. The developed model will be used to assess processes related to keratinocyte differentiation and skin barrier integrity to determine the role of the pro-FLG Ca2+-binding domain in the formation of a functional skin barrier and the pathogenesis of AD. We will also expose the outer surface our 3D in vitro model to varying CaCO3 levels to assess the effect on skin barrier integrity. The project will inform novel methods of treatment and disease prevention. Two representative publications from supervisors:

Petrova A, Celli A, Jacquet L, Dafou D, Crumrine D, Hupe M, Arno M, Hobbs C, Cvoro A, Karagiannis P, Devito L, Sun R, Adame LC, Vaughan R, McGrath JA, Mauro TM, Ilic D. 3D In vitro model of a functional epidermal permeability barrier from human embryonic stem cells and induced pluripotent stem cells. Stem Cell Reports 2014;2:675-689.

Perkin MR, Craven J, Logan K, Strachan D, Marrs T, Radulovic S, Campbell LE, MacCallum SF, McLean WH, Lack G, Flohr C. Association between domestic water hardness, chlorine, and atopic dermatitis risk in early life: A population-based cross-sectional study. J Allergy Clin Immunol 2016;138(2):509-516.atopic dermatitis risk in early life: A population-based cross-sectional study. J Allergy Clin Immunol 2016;138(2):509-516.

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26.1 Exploring the general mechanisms of mammalian neural crest migration in vivo using live cell imaging and how it is altered in neuroblastoma cancer cells Co-Supervisor 1: Karen J. Liu Research Division or CAG: Craniofacial Development and Stem Cell Biology E-mail: karen.liu@kcl.ac.uk www.kcl.ac.uk/dentistry/research/divisions/craniofac/ResearchGroups/Liu-Lab/index.aspx Co-Supervisor 2: Matthias Krause Research Division or CAG: Randall Division of Cell and Molecular Biophysics Email: matthias.krause@kcl.ac.uk Project Description: Neural crest cells are multipotent embryonic stem cells that give rise to diverse tissues such as melanocytes, craniofacial skeleton and peripheral nervous system. During embryogenesis the neural crest cells delaminate from the neural tube, undergo an epithelial to mesenchymal transition (EMT), and migrate to populate distant organs. The highly migratory activity of these cells is critical to their in vivo function—not only are their ultimate tissue descendants widespread in the organism, but failures to regulate migration and differentiation in the correct locations leads to cancers such as neuroblastoma. Despite the importance of the neural crest, the specific mechanisms underlying this migratory activity and its control are poorly understood, especially in mammals. In this project we propose a novel signal transduction pathway in which anaplastic lymphoma kinase (ALK), a kinase mutated in neuroblastoma, phosphorylates glycogen synthase kinase (GSK3) to regulate the actin cytoskeleton via Lamellipodin (Lpd), thereby controlling neural crest migration. Overall goals: 1) Identify the cellular behaviours of mammalian neural crest cells as they migrate in their native milieu. Approach: in vivo imaging of migrating neural crest cells. Techniques: in vivo live cell microscopy, embryology, computational image analysis. 2) Pinpoint the requirements for GSK3 and Lpd during neural crest migration using genetic mutants. Approach: Tissue culture, genetic analysis, molecular biology, biochemistry. This project will have implications for understanding head patterning, human congenital anomalies, and more broadly, processes that involve cell migration, such as wound healing and cancer cell metastasis. Two representative publications from supervisors: Tabler et al., Fuz mutant mice reveal shared mechanisms between ciliopathies and FGF-related syndromes. Dev Cell 2013 Jun 24; 25(6):623-35.

Liu et al., Chemical rescue of cleft palate and midline defects in conditional GSK3b mice. Nature 2007 Mar 1; 446(7131):79-82.

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27.1 Functional interrogation and therapeutic intervention of the molecular pathways driving cutaneous T-cell lymphoma Co-Supervisor 1: Dr Tracey Mitchell Research Division or CAG: Genetics and Molecular Medicine/GRIDA E-mail: tracey.mitchell@kcl.ac.uk Website:https://www.kcl.ac.uk/medicine/research/divisions/gmm/departments/dermatology/Groups/WhittakerLab/index.aspx Co-Supervisor 2: Professor Sean Whittaker Research Division or CAG: Genetics and Molecular Medicine/GRIDA Email: sean.whittaker@kcl.ac.uk Website:https://www.kcl.ac.uk/medicine/research/divisions/gmm/departments/dermatology/Groups/WhittakerLab/index.aspx Project Description: All cancers are characterised by the acquisition of gene mutations and structural aberrations that initiate and drive the disease. Mapping the genomic landscape of tumour genomes is a powerful approach to facilitate the identification of potential therapeutic targets. Primary cutaneous T-Cell lymphoma (CTCL) is a heterogeneous malignancy of mature memory type, skin homing T-cells. There are no effective treatments for CTCL patients with advanced stage disease and as a consequence the survival rate is dismal (~3 years). Our research program aims to use the genomic basis of CTCL to identify biomarkers for patient stratification and to design novel therapies. We have recently performed a deep sequencing study that has identified >800 gene mutations and copy number changes in CTCL tumours. Analysis of these data at the gene pathway level has identified TCR signalling, cell survival, genome maintenance, DNA damage repair and epigenetic regulation as the key pathways mediating the pathogenesis of CTCL. The aim of this PhD is to follow up this work by functional validation of identified genes and involved pathways. The main objectives are to: (i) examine the functional role of candidate driver mutations in the malignant transformation of T-cells and will involve complementary experiments to examine gene function in healthy T-cells, cell lines and tumour cells (years 1/2); and (ii) to screen identified mutations in specific pathways against small molecule libraries to identify potential therapeutic targets (years 3/4). A range of cutting-edge molecular genetic and immunological techniques will be used. Work will be undertaken in our state-of-the-art laboratories in the Division of Genetics and Molecular Medicine. Two representative publications from supervisors: Woollard WJ, Pullabhatla V, Lorenc A, Patel VM, Butler RM, Bayega A, Begum N, Bakr F, Dedhia K, Fisher J, Aguilar-Duran S, Flanagan C, Ghasemi AA, Hoffmann RM,Castillo-Mosquera N, Nuttall EA, Paul A, Roberts CA, Solomonidis EG, Tarrant R,Yoxall A, Beyers CZ, Ferreira S, Tosi I, Simpson MA, de Rinaldis E, Mitchell TJ, Whittaker SJ. Candidate driver genes involved in genome maintenance and DNA repair in Sézary syndrome. Blood. 2016 Jun 30;127(26):3387-97. doi:10.1182/blood-2016-02-699843. Epub 2016 Apr 27. PubMed PMID: 27121473.

Woollard WJ, Kalaivani NP, Jones CL, Roper C, Tung L, Lee JJ, Thomas BR, Tosi I, Ferreira S, Beyers CZ, McKenzie RC, Butler RM, Lorenc A, Whittaker SJ, Mitchell TJ. Independent Loss of Methylthioadenosine Phosphorylase (MTAP) inPrimary Cutaneous T-Cell Lymphoma. J Invest Dermatol. 2016 Jun;136(6):1238-46.doi: 10.1016/j.jid.2016.01.028. Epub 2016 Feb 9. PubMed PMID: 26872600.

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28.1 In-silico design and identification of novel antiviral drugs targeting vector borne viruses (Zika, Dengue & Chikungunya) Co-Supervisor 1: Dr Daniele Castagnolo, Lecturer Research Division or CAG: Institute of Pharmaceutical Science E-mail: daniele.castagnolo@kcl.ac.uk Website: https://sites.google.com/site/danielecastagnoloresearchgroup/home Co-Supervisor 2: Dr David Barlow, Lecturer Research Division or CAG: Institute of Pharmaceutical Science E-mail: dave.barlow@kcl.ac.uk Website: www.kcl.ac.uk/lsm/research/divisions/ips/research/pharmabio/Staff/Barlow/index.aspx Project Description: The project aims at developing urgently needed drug candidates targeting mosquito-borne viruses, such as Zika, Dengue and Chikungunya, which are causing an increasing number of outbreaks in humans worldwide and for which there are no current treatments available. •In Year 1, two in-silico approaches will be undertaken in Dr Castagnolo’s Lab to design novel Zika, Dengue and Chikungunya inhibitors. (1) A ligand-based virtual screening approach will be adopted to identify structurally-innovative small molecules targeting vector borne viruses. New hit candidates will be identified from chemical databases (e.g., ZINC) and synthesized at KCL. In parallel, (2) a structure-based approach will be followed to design new inhibitors targeting specific viral proteins (particularly protease and polymerase of Zika and Dengue). • The newly identified hit compounds will then be tested for their antiviral activity. Enzymatic screening on viral proteins will be carried out in the Dr Castagnolo's newly refurbished Lab for biology at IPS. Phenothypic screening will be carried out in the BL3 lab of our collaborator Prof. Johan Neyts at KU Leuven, Belgium (4 months placement). (Year 2/3). •The SAR of the drugs will be evaluated and the hit compounds will be then optimised (Year 3) leading to the identification of Drug Leads active againt Zika, Dengue and Chikungunya viruses (4 Year).

This project is highly multidisciplinary and it will offer a unique combination of expertise and skills ranging from in-silico computational design, virtual screening on viral proteins, chemistry and biology, virology up to commercial and industrial research. Two representative publications from supervisors: Magri, A.; Reilly, R. Scalacci, N.; Radi, M.; Hunter, M.; Ripoll, M.; Patel, A.; Castagnolo, D.* “Rethinking the old antiviral drug moroxydine: discovery of novel analogues as potent anti-hepatitis C virus (HCV) agents” Bioorg. Med. Chem. Lett. 2015, 25, 5372-5376. Vincetti, P.; Caporuscio,F.; Gioiello,A.; Mancino,V.; Suzuki, Y.; Yamamoto, N.; Crespan, E.; Maga, G.; Rastelli,G.; Castagnolo, D.; Kaptein, S.; Leyssen,P.; Neyts, J.; Costantino, G.; Radi, M. “Discovery of multi-target antivirals acting on both the dengue virus NS5-NS3 interaction and the host Src/Fyn kinases” J. Med. Chem.2015, 58, 4964-4975

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29.1 Defining the role of circulating fibrocytes in the pathogenesis of renal fibrosis; a study of cell-signalling crosstalk Co-Supervisor 1: Dr Claire Sharpe Research Division or CAG: Division of Transplantation Immunology & Mucosal Biology Email: Claire.sharpe@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/claire.sharpe.html Co-Supervisor 2: Professor Antony Dorling Research Division/Department or CAG: Division of Transplantation Immunology & Mucosal Biology E-mail: anthony.dorling@kcl.ac.uk Website: www.kcl.ac.uk/lsm/research/divisions/timb/about/people/profiles/anthonydorling.aspx Project Description: Interstitial fibrosis is the process that leads to kidney failure, regardless of the initiating disease (e.g. diabetes, high blood pressure, kidney transplant rejection), yet there is no drug available to stop this. Understanding which cells are responsible for producing scar tissue and the signalling cascades that control this process will help us to design drugs which can protect patients from the need for dialysis or kidney transplantation. We have evidence that, following injury, circulating cells known as fibrocytes are responsible for damage caused to blood vessels within the kidney. These cells may also migrate into kidney tissue and be responsible for kidney scarring. These cells express a molecule called tissue factor, which triggers the coagulation cascade but also signals through pro-fibrotic signalling pathways such as Ras monomeric GTPases, via PAR-1. We have developed a transgenic strain of mice that overexpress the naturally-occurring human tissue factor pathway inhibitor (TFPI) on CD31+ fibrocytes. This project aims to: 1) Discover what percentage of scar-forming cells are derived from circulating fibrocytes in a mouse model of renal fibrosis (induced by aristolochic acid) (year 1) 2) Compare the degree of fibrosis that develops between the transgenic and wild-type mice to see whether TFPI is anti-fibrotic (year 2) 3) To understand the cross-talk between Ras and PAR-1 signalling to highlight new potential targets for drug discovery (year 3) 4) Test the impact of targeting those signalling molecules that have been newly identified as key in the fibrotic pathway in in-vitro models using CRISPR technology. (year 4). Two representative publications from supervisors: Fibrocytes mediate intimal hyperplasia post-vascular injury and are regulated by two tissue factor-dependent mechanisms. Chen D1, Ma L, Tham EL, Maresh S, Lechler RI, McVey JH, Dorling A. J Thromb Haemost. 2013 May;11(5):963-74.

Antisense knockdown of Kirsten­Ras inhibits fibrosis in a rat model of unilateral ureteric obstruction. Jia­Hui Wang, Lucy J. Newbury, A.S. Knisely, Brett Monia, Bruce M. Hendry and Claire C. Sharpe. Am J Pathol. 2012 Jan;180(1):82-90

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30.1 Structural characterisation of a novel cytolytic peptide toxin Co-Supervisor 1: Julian Naglik Research Division or CAG: Mucosal & Salivary Biology Division, Dental Institute E-mail: julian.naglik@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/julian.naglik.html Co-Supervisor 2: Antoni Borysik Research Division or CAG: Department of Chemistry Email: antoni.borysik@kcl.ac.uk Website: http://www.kcl.ac.uk/nms/depts/chemistry/people/core/borysikantoni.aspx Project Description: We have recently identified the first cytolytic peptide toxin (Candidalysin) secreted by any human fungal pathogen. Candidalysin is exclusively expressed by hyphal filaments of Candida albicans and is essential for mucosal pathogenesis, epithelial activation and damage induction1. Candidalysin is an amphipathic 31-mer peptide, and central to damage induction is its ability to intercalate and permeabilise cell membranes. However, the mechanism of cell damage is currently unknown. Therefore, the objectives are to understand how Candidalysin intercalates and causes membrane destabilisation and cell damage. We will investigate the structure, dynamics and assembly of Candidalysin using a broad range of cutting edge techniques to understand how this peptide toxin intercalates and destabilises epithelial membranes. In Year 1&2 the student will use molecular dynamics (MD) simulations to understand the self-assembly properties of the peptide and generate candidate peptide assemblies. The MD simulations will be complimented by native mass spectrometry (MS) and ion-mobility MS experiments, which will be used to determine the respective stoichiometry and shape of the assembled peptides2. In Year 2&3, the structure and dynamics of the peptide pores will be determined by hydrogen-deuterium exchange MS. The student will also capitalise on recent advances in native MS to understand the role of lipids in the structure and dynamics of the pore-forming properties of Candidalysin. This multidisciplinary project combines structural biology, infection, chemistry and biophysics to determine how Candidalysin intercalates, destabilises and damages epithelial membranes, and will identify key structural regions in Candidalysin that can be targeted for novel antifungal therapies. Two representative publications from supervisors:

Moyes DL, Wilson D, Richardson JP, Tang SX, Wernecke J, Höfs S, Gratacap RL, Mogavero S, Robbins J, Runglall M, Murciano C, Blagojevic M, Thavaraj S, Förster TM, Hebecker B, Kasper L, Vizcay G, Iancu SI, Kichik N, Häder A, Kurzai O, Cota E, Bader O, Wheeler RT, Gutsmann T, Hube B and Naglik JR (2016). Candidalysin: A fungal peptide toxin critical for mucosal infection. Nature, 532, 64-68

Borysik AJ, Hewitt DJ, Robinson CV (2013). Detergent release prolongs the lifetime of native-like membrane protein conformations in the gas-phase. J Am Chem Soc., 135, 6078-6083.

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31.1 MS1 - a cardiac regulator of development and stress Co-Supervisor 1: Mark Pfuhl Research Division or CAG: Cardiovascular & Randall Division E-mail: mark.pfuhl@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/mark.pfuhl.html Co-Supervisor 2: Alka Saxena Research Division or CAG: NIHR Biomedical Research Center and Medical and Molecular Genetics Email: Alka.Saxena@gstt.nhs.uk www.guysandstthomasbrc.nihr.ac.uk/Professionals/Corefacilities/Genomicsfacility/Genomics-core-facility.aspx Project Description: Ms1/STARS is a stress response protein of cardiac muscle. It was discovered as a protein overexpressed in cardiomyocytes early in the hypertension model of rat aortic banding. It was shown to be important for the maintenance of cardiac development in zebrafish and is upregulated in mouse models of cardiac hypertrophy and in failing human hearts. We determined the structure of the only folded part of the protein and discovered that it resembles the DNA binding domain of transcription factors. Even though it was previously only ever reported to be present in a sarcomeric or cytosolic location we were the first to identify it in the nucleus and to show that it can bind to DNA in a sequence specific manner. We now want to find out what exactly Ms1 is doing in the nucleus by ChIP-Seq and RNA-Seq experiments (year 1). We want to understand the upstream signalling that decides about the subcellular localisation which we have shown to be dependent on its NLS - intriguingly it is in the nucleus in neonatal but in the sarcomere in adult rat cardiomyocytes (see below, year 2). Ultimately we want to understand how the role that Ms1 plays in the nucleus is related to the onset of cardiac hypertrophy and ultimately heart failure by studying cardiomyocytes from mouse models of hypertrophy as well as samples from patients with ventricular hypertrophy (year 3). Training in: cell culture, immunofluorescence, confocal microscopy, ChIP-Seq & RNA-Seq, DNA binding assays, molecular biology, protein chemistry.

Two representative publications from supervisors: M. Zaleska, C. Fogl, A. L. Kho, A. Ababou, E. Ehler, M. Pfuhl, “The Cardiac Stress Response Factor Ms1 Can Bind to DNA and Has a Function in the Nucleus”, PLoSONE DOI: 10.1371/journal.pone.014461410, 2015 C. Fogl, L. Puckey, U. Hinssen, M. Zaleska, M. El-Mezgueldi, R. Croasdale, A. Bowman, A. Matsukawa, N. J. Samani, R. Savva, M. Pfuhl, "A structural and functional dissection of the cardiac stress response factor MS1", Proteins 80, 398-409, 2012

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32.1 Identifying novel molecular mechanisms involved in the generation of new insulin-

producing beta cells from adult stem/progenitor cells.

Co-Supervisor 1: Rocio Sancho

Research Division or CAG: GMM

E-mail: rocio.sancho@crick.ac.uk

Co-Supervisor 2: Ivo Lieberam

Research Division or CAG: GMM

Email: mailto:ivo.lieberam@kcl.ac.uk

Website: http://www.kcl.ac.uk/ioppn/depts/devneuro/Research/groups/lieberam.aspx

Project Description: Diabetes is caused by the irreversible loss of insulin-producing beta cells in the pancreas. Recent

research has revealed that the adult pancreas is capable of surprising cellular plasticity, opening new

possibilities to reprogram adult stem/progenitor cells into insulin-producing cells (Puri et al. 2015;

Sancho et al. 2014). Three key transcription factors are sufficient to initiate a beta cell fate program

when artificially introduced into adult stem/progenitor cells: Ngn3, Pdx1 and MafA. However, the

tight regulation of these factors makes the process inefficient, and the beta cells generated are often

not fully functional.

The goal of the proposed PhD project is to understand how the pro-endocrine factors Ngn3, Pdx1 and

MafA are regulated in adult stem cells, to enable us to optimise the regulating pathways to improve

the efficiency of beta cell generation and achieve fully functional beta cells.

During the rotation project the PhD student will set up pilot screens for novel regulators and interactors of Ngn3/Pdx1/MafA. 0+4 students will also familiarise with all the techniques required for screen validation by characterising a protein already identified as a regulator of Ngn3. The goal of year 1/2 is to perform Crispr/Cas9 screening for Ngn3/Pdx1/MafA-regulators using flow cytometry, and identify Ngn3/Pdx1/MafA-interacting proteins using immunoprecipitation and mass spectrometry. In year 2/3 biochemical and functional validation of the top screen hits will be performed. In the final year functional (insulin release, calcium measuring, in vivo mouse kidney transplantation) and translational assays (human primary cells) will be performed to assess the physiological function of the newly generated beta cells. Two representative publications from supervisors: 1: Sancho R, Gruber R, Gu G, Behrens A. Loss of Fbw7 reprograms adult pancreatic ductal cells into α, δ, and β cells. Cell Stem Cell. 2014 Aug 7;15(2):139-53. doi: 10.1016/j.stem.2014.06.019. PubMed PMID: 25105579; PubMed Central PMCID: PMC4136739. 2: Sancho R, Blake SM, Tendeng C, Clurman BE, Lewis J, Behrens A. Fbw7 repression by hes5 creates a feedback loop that modulates Notch-mediated intestinal and neural stem cell fate decisions. PLoS Biol. 2013;11(6):e1001586. doi: 10.1371/journal.pbio.1001586. PubMed PMID: 23776410; PubMed Central PMCID: PMC3679002

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33.1 Therapeutic use of regulatory T cells in liver disease: Immunoregulation and tissue regeneration Co-Supervisor 1: Dr Marc Martinez-Llordella Research Division/Department or CAG: Liver Sciences Department, Division of Transplantation and Mucosal Biology E-mail: marc.martinez-llordella@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/timb/research/liver.aspx Co-Supervisor 2: Prof Alberto Sanchez-Fueyo Research Division/Department or CAG: Liver Sciences Department, Division of Transplantation and Mucosal Biology – King’s College Hospital, Institute of Liver Studies Email: sanchez_fueyo@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/timb/research/liver.aspx Project Description: Hepatic inflammation of any aetiology is characterized by lymphocyte infiltration. If the inflammation is not controlled it could lead to fibrosis, resulting in cirrhosis or liver failure. The balance of effector and regulatory T cells (Tregs) generally determines the outcome of hepatitis. Tregs are a heterogeneous population with specific properties depending on the homing tissue, including immunoregulation and tissue repair. However, the factors that modulate Treg homeostasis and function in the liver remain unclear. Our previous studies have demonstrated that IL-2 administration preferentially expand Tregs in the liver and increases their suppressive functions. Therefore, we believe that IL-2 therapy can modulate Treg immunoregulation during hepatic inflammation and enhance tissue regeneration. The objectives of this study are: i) to characterise the phenotype and features of intrahepatic Tregs in humans and mice (Year 1); ii) to determine the Treg homeostasis and cell-to-cell interactions during chronic liver inflammation (Years 1-2); iii) to assess the role of Tregs in the tissue regeneration after hepatic injury (Year 2); and iv) to evaluate the benefits of combining IL-2 therapy with hepatocyte transplantation to improve regeneration of end-stage liver damage (Years 2-3). In order to achieve our aims, we will employ animal models of acute hepatitis and liver fibrosis (wild-type and Treg-depleted mice). We will perform isolation and cell culture methodologies from human and mouse (MLR, Treg suppression assays…), cell biology assays (flow cytometry, ELISA…) and molecular analysis (qPCR, microarray, DNA sequencing…). Two representative publications from supervisors: Bailey-Bucktrout SL, Martinez-Llordella M, Zhou X, Anthony B, Rosenthal W, Luche H, Fehling HJ, Bluestone JA. Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response. Immunity. 2013 Nov 14;39(5):949-62. Bohne F, Martínez-Llordella M, Lozano JJ, Miquel R, Benítez C, Londoño MC, Manzia TM, Angelico R, Swinkels DW, Tjalsma H, López M, Abraldes JG, Bonaccorsi-Riani E, Jaeckel E, Taubert R, Pirenne J, Rimola A, Tisone G, Sánchez-Fueyo A. Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation. J Clin Invest. 2012 Jan;122(1):368-82.

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34.1 Understanding regulation of cellular energy metabolism Co-Supervisor 1: Snezhana Oliferenko Research Division or CAG: Randall Division of Cell and Molecular Biophysics E-mail: snezhana.oliferenko@kcl.ac.uk OR snezhka.oliferenko@crick.ac.uk Website: https://www.crick.ac.uk/research/a-z-researchers/researchers-k-o/snezhana-oliferenko/ Co-Supervisor 2: Simon Ameer-Beg Research Division/Department or CAG: Randall Division of Cell and Molecular Biophysics Email: simon.ameer-beg@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/cell/ameer-beg/index.aspx Project Description: Cellular energy metabolism occurs through oxidative phosphorylation in mitochondria and glycolysis in the cytoplasm. In humans, differentiated cells tend to rely on oxidative phosphorylation but dividing cells or abnormally proliferating cancers often adopt aerobic glycolysis that favours increased macromolecular synthesis, a phenomenon known as Warburg effect. How is energy production regulated? How do cells choose a particular energy generation route, and how do they switch between alternative strategies? How do these metabolic choices affect the rates of cellular growth? We want to answer these fundamental, yet surprisingly poorly understood questions by combining precise spatiotemporal imaging of cellular energy metabolic pathways with the awesome power of genetic engineering. We will visualize energy metabolites such as glucose, ATP, glutamate and others in real time using Förster Resonance Energy Transfer (FRET)-based sensors and probe how fluctuations in nutrient availability, cellular differentiation, chronological aging and genetic perturbations of metabolic regulation affect their intracellular flux. To speed up our research by straightforward genome editing, we will initially use two yeast species that exhibit divergent energy production pathways. We will eventually translate our research to human cells with a view of understanding how altered energy metabolism contributes to disease. Year 1. Development of genetically encoded FRET-based biosensors to report metabolic activity (SO and SAB). Year 2. Understanding spatiotemporal regulation of metabolic sensors at a single-cell level in response to environmental and genetic perturbations (SO and SAB). Years 3-4. Studying the mechanisms underlying regulation of energy metabolism and preparing experimental results for publication (SO and SAB). Two representative publications from supervisors: Makarova, M., Gu, Y., Chen, J-S., Beckley, J., Gould, K. and S. Oliferenko. 2016. Temporal regulation of Lipin activity diverged to account for differences in mitotic programs. Current Biology. 26: 237-243.*Highlighted in Prasad, R. and Y. Barral. 2016. Posttranslational Regulation: A Way to Evolve. Current Biology. 26: R102-124, and Editor’s Choice in Science: 351:828-829 A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging. Poland, S. P., Krstajić, N., Monypenny, J., Coelho, S., Tyndall, D., Walker, R. J., Devauges, V., Richardson, J., Dutton, N., Barber, P., Day-Uei Li, D., Suhling, K., Ng, T., Henderson, R. K. & Ameer-Beg, S. M. 2015. Biomedical optics express. 6: 277-296

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35.1 Mechanism of action of a cancer-selective protein toxin Co-Supervisor 1: Prof Mahvash Tavassoli Research Division or CAG: Mucosal Biology/Cancer E-mail:Mahvash.tavassoli@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/mahvash.tavassoli.html Co-Supervisor 2: Dr Manuel Muller Research Division or CAG: Natural and Mathematical Sciences – Department of Chemistry Email: manuel.muller@kcl.ac.uk Website: http://www.kcl.ac.uk/nms/depts/chemistry/people/core/MullerManuel.aspx Apoptin, a small viral protein, can selectively induce cell death in cancer cells while sparing healthy cell lines. Previous research suggests that selective phosphorylation of apoptin in cancer cells contributes to this fascinating tumour selective cytotoxic property. A number of protein kinases have been implicated in apoptin phosphorylation including protein kinase C and Akt. However, little is known about the functional consequences of apoptin phosphorylation. This projects aims to apply synthetic protein chemistry to address the mechanism of cancer-selective toxicity of apoptin. The student will develop a protein semi-synthesis strategy to access site-specifically phosphorylated apoptin derivatives (Mueller lab). Using state-of-the-art protein transduction methodologies, semi-synthetic apoptin derivatives will be introduced into cancerous and normal cell lines, allowing the student to directly test the effect of phosphorylation on pro-apoptotic activity in a range of primary and metastatic cell lines as well as normal cells from various tissue type (Tavassoli lab). The incorporation of photo-crosslinkers into apoptin variants will enable the identification of phosphorylation-specific down-stream targets, potentially allowing the student to establish a model for the mechanism of action of cancer-specific toxic. The information obtained can also help future design of post-translationally modified proteins with therapeutic potential. Collectively, this interdisciplinary project will provide in-depth training in cancer biology and protein chemistry, and provides training in a range of cellular, molecular, imaging, biochemical and bioinformatics methods. This study is expected to yield crucial mechanistic insight into the function of apoptin with important implications for its potential use as selective cancer therapy therapeutics. Two representative publications from supervisors: Apoptin interacts with and regulates the activity of protein kinase C beta in cancer cells. Bullenkamp J, Gäken J, Festy F, Chong EZ, Ng T, Tavassoli M. Apoptosis. 2015 Jun;20(6):831-42. doi: 10.1007/s10495-015-1120-6. Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication. Kucharski TJ, Ng TF, Sharon DM, Navid-Azarbaijani P, Tavassoli M, Teodoro JG. J Virol. 2016 Sep 29;90(20):9433-45. doi: 10.1128/JVI.00936-16. Print 2016 Oct 15.

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36.1 Mitochondrial microRNA (MitomiRs) and their relationship to mitochondrial bioenergetics Co-Supervisor 1: Shirin Elizabeth Khorsandi Research Division or CAG: Liver, Renal, Urology, Transplant, Gastro/Gastro Intestinal Surgery CAG E-mail: shirin.khorsandi@kcl.ac.uk http://www.kingshealthpartners.org/clinical-excellence/40-liver-renal-urology-transplant-gastrogastro-intestinal-surgery Co-Supervisor 2: Celine Filippi Research Division or CAG: Child Health CAG Email: celine.filippi@kcl.ac.uk Project Description: How a cell generates energy is fundamental in determining cell phenotype and survival. This aspect is of importance both in cancer and liver transplantation. The cancer cell needs to generate energy in a manner that will support its survival, irrespective of the prevailing oxygen and nutrient availability, to drive cancer cell division, invasion and the fatality of metastasis. While in liver transplantation, the behaviour and resilience of liver mitochondria on reperfusion, determines graft and ultimately, recipient survival. The aim of this work is to look at how mitochondrial microRNA (MitomiRs) define cancer cell behaviour and whether MitomiR species, can be used to assess the transplantability of the human liver. Y1 Establish experience in lab techniques (mitochondrial isolation, MitomiR extraction, mitochondrial bioenergetic measurements, EM architecture). Y2 Analyze tissue samples and interpret data from clinical models of cancer and transplantation Y3/Y4 In vitro validate clinical MitomiR observations. Y4 Computationally explore putative MitomiR clinical applications and write up. Techniques to be utilised will range from basic molecular biology techniques, to functional mitochondrial measurements, microRNA arrays, NGS, transient transfection of microRNA mimics, design and construction of Lentiviral/Adenoviral vectors for RNAi with siRNA or shRNA, and primary human hepatocyte isolation and culture. The Institute of Liver Studies is a unique environment where clinicians and scientists coexist facilitating translational projects. The clinical material available for analysis will extend from the human neonate to adult liver in a variety of cancers and the transplant liver. There will also be the opportunity to visit the microRNA labs at John Hopkins, USA. Two representative publications from supervisors: The microRNA Expression Profile in Donation after Cardiac Death (DCD) Livers and Its Ability to Identify Primary Non Function. Khorsandi SE, Quaglia A, Salehi S, Jassem W, Vilca-Melendez H, Prachalias A, Srinivasan P, Heaton N. PLoS One. 2015 May 15;10(5):e0127073. doi: 10.1371/journal.pone.0127073. Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure. Jitraruch S, Dhawan A, Hughes RD, Filippi C, Soong D, Philippeos C, Lehec SC, Heaton ND, Longhi MS, Mitry RR. PLoS One. 2014 Dec 1;9(12):e113609. doi: 10.1371/journal.pone.0113609. eCollection 2014

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37.1 Using chemical modification of anticancer drug scaffolds to study efflux mediated chemoresistance in cancer stem cells Co-Supervisor 1: Dr Khondaker Miraz Rahman Research Division or CAG: Institute of Pharmaceutical Science E-mail: k.miraz.rahman@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/ips/about/people/Rahman/index.aspx Co-Supervisor 2: Professor Ben Forbes Research Division or CAG: Institute of Pharmaceutical Science Email: ben.forbes@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/ips/about/people/Forbes/index.aspx Project Description: Cancer stem cells (CSCs) are key tumor-initiating cells that may play an integral role in disease recurrence following chemotherapy. To improve clinical outcomes for cancer patients, treatments must have the ability to kill the entirety of cancer cells, including CSCs. One of the key mechanisms of chemoresistance in CSCs is the overexpression of ABC transporters such as P-glycoprotein (P-gp). Although a relationship between P-gp expression and drug resistance is widely accepted in cancer cells, the situation is less well defined for CSCs. We have recently identified a number of putative P-gp inhibitors with the help of advanced computational modelling. Some of these molecules are regulatory agency approved molecules with known safety profiles. The PhD project aims to use these molecules as chemical tools to understand the role of efflux modulation in reversing chemoresistance in CSCs. We aim to identify specific pharmacophores that can be chemically linked to existing chemotherapeutic agents and alter their propensity to be substrates for the P-gp efflux pumps. The student will explore the phenomenon of efflux transporter associated resistance, and the findings from the study could help the researchers to understand the role of P-gp in conferring multi-drug resistance in CSCs and develop targeted therapies to overcome efflux mediated chemoresistance. Specific deliverables and work plan of the project includes - Year1: In silico validation of the identified lead structures and medicinal-chemical modification to develop suitable chemical tools. Year 2: Use the identified and synthesised compounds as chemical tools to study the role of P-gp modulation on chemoresistance in CSCs using biochemical and cellular assays. Year 3 & 4: Develop pharmacophore-drug hybrids and evaluate their ability to overcome efflux-mediated resistance in CSCs. Two representative publications from supervisors: Jamshidi, Shirin, J. Mark Sutton, and Khondaker M. Rahman. "An overview of bacterial efflux pumps and computational approaches to study efflux pump inhibitors." Future Medicinal Chemistry 8.2 (2016): 195-210. Madlova M, Bosquillon C, Asker D, Dolezal P, Forbes B. In vitro respiratory drug delivery models possess nominal functional P-glycoprotein activity. Journal of Pharmacy and Pharmacology 61 (2009): 293-301

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38.1 Role of sphingosine-1-phosphate (S1P) signalling in cochlear homeostasis and progressive hearing loss Co-Supervisor 1: Professor Karen P Steel Research Division or CAG: Wolfson Centre for Age-Related Diseases E-mail: Karen.steel@kcl.ac.uk http://www.kcl.ac.uk/ioppn/depts/wolfson/research/Deafness/Index.aspx Co-Supervisor 2: Professor Andrea Streit Research Division or CAG: Department of Craniofacial Development and Stem Cell Biology Email: andrea.streit@kcl.ac.uk http://www.kcl.ac.uk/dentistry/research/divisions/craniofac/ResearchGroups/StreitLab/StreitLab.aspx Project Description: Progressive hearing loss is very common with a major impact on quality of life. One form of pathology involves loss of cochlear homeostasis. The fluid bathing sensory hair cells is maintained at 100mV (endocochlear potential, EP) by the stria vascularis, and this is critically dependent on its blood supply. EP provides a voltage gradient across hair cell transduction channels, facilitating synaptic activation. S1P signalling is required for cochlear homeostasis. In mouse, mutations in genes involved in S1P signalling (S1pr2 and Spns2) cause rapid loss of EP and worsening hearing. Spns2 transports S1P out of cells that generate it and S1pr2 is the major S1P receptor required for normal hearing. S1PR2 and SPNS2 are involved in human hearing. The project asks if strial dysfunction in mice with abnormal S1P signalling is mediated by abnormal capillary function, and involves: • Analysis of the capillaries of the stria vascularis in S1P-mutant and control mouse cochleas using gene expression, confocal and 3D ultrastructural methods to identify pathological changes (Y1); • Isolation of fresh strial explants and exposure in vitro to a series of pharmacological agents known to affect S1P signalling to investigate their effects on capillary constriction/dilation (Y2); • Using intravenous tagged markers to determine the permeability of mutant and control strial capillaries combined with exposure to vasoactive agents to ask if normal capillary permeability can be restored to mutants (Y3). • Testing effects of suitable pharmacological agents on hearing in mutants (Y4). The findings will have obvious translational potential in cases of deafness due to strial dysfunction. Two representative publications from supervisors: Ingham NJ, Carlisle F, Pearson S, Lewis MA, Buniello A, Chen J, Isaacson RL, Pass J, White JK, Dawson SJ and Steel KP. (2016) S1PR2 variants associated with auditory function in humans and endocochlear potential decline in mouse. Scientific Reports, 6:28964. Steventon B, Mayor R, Streit A. (2016) Directional cell movements downstream of Gbx2 and Otx2 control the assembly of sensory placodes. Biol Open. bio.020966.

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39.1 Characterising the epigenome in mice after exposure to environmental carcinogens Co-Supervisor 1: Dr. Volker M. Arlt Research Division or CAG: Analytical and Environmental Sciences Division/ MRC-PHE Centre for Environment & Health/ NIHR Health Protection Research Unit in Health Impact of Environmental Hazards E-mail: volker.arlt@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/volker.arlt.html Co-Supervisor 2: Prof. David H. Phillips Research Division or CAG: Analytical and Environmental Sciences Division/ MRC-PHE Centre for Environment & Health/ NIHR Health Protection Research Unit in Health Impact of Environmental Hazards Email: david.phillips@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/david.phillips.html Project Description:

Modern life involves unavoidable exposure to environmental carcinogens. These can damage cellular DNA, which can lead to mutations, and mutations in critical genes are characteristic features of tumours. Despite linear relationships between dose, DNA damage in target tissues and tumour incidence, identifying which tissues are targets for carcinogenicity by an agent is problematic. This suggests the importance of events subsequent to DNA damage and mutation in determining organotropism, which may include epigenetic alterations (e.g. DNA methylation). The methylation status in DNA can regulate the expression of genes, i.e. if they are switched on or off within the body and this regulation is impacted by the exposure to environmental carcinogens. For example, it has been suggested that epigenetic changes induced by combustion-derived polycyclic aromatic hydrocarbons (PAHs) may contribute to carcinogenesis, although a direct cause and effect relationship has yet to be established.

Aims: to investigate: • whether epigenetic changes occur in mice in response to exposure to environmental carcinogens (e.g. PAHs) (YEAR 1+2). • if changes to the epigenetic (methylation) marks on DNA can act as biomarkers of carcinogen exposure (YEAR 1+2). • if such epigenetic effects distinguish target and non-target organs for tumour formation (YEAR 3+4).

Genome-wide analysis of various tissues from mice exposed to different environmental carcinogens will identify changes in DNA methylation status in all genes by reduced representation bisulphite sequencing. Results will be validated by pyrosequencing measuring methylation at specific genes. Bioinformatics analysis will identify preferential targets of hypo- or hypermethylation and concomitant gene dysregulation related to carcinogenesis. Two representative publications from supervisors: Krais AM, Speksnijder EN, Melis JP, Indra R, Moserova M, Godschalk RW, van Schooten FJ, Seidel A, Kopka K, Schmeiser HH, Stiborova M, Phillips DH, Luijten M & Arlt VM (2016) The impact of p53 on DNA damage and metabolic activation of the environmental carcinogen benzo[a]pyrene: effects in Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice. In: Archives of Toxicology, 90, 839-851. Alexandrov LB, Ju YS, Haase K, Van Loo P, Martincorena I, Nik-Zainal S, Totoki Y, Fujimoto A, Nakagawa H, Shibata T, Campbell PJ, Vineis P, Phillips DH, Stratton MR (2016) Mutational signatures associated with tobacco smoking in human cancer. In: Science, in press.

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40.1 Understanding immune responses in intestinal homeostasis and disease Co-Supervisor 1: Patricia Barral Research Division or CAG: Division of Immunology, Infection and Inflammatory Diseases E-mail: patricia.barral@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/immunobiology/research/Patricia-Barral/index1.aspx Co-Supervisor 2: Jo Spencer Research Division or CAG: Division of Immunology, Infection and Inflammatory Diseases Email: jo.spencer@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/immunobiology/research/JoSpencer/index.aspx Project Description: Humans carry 10 times more bacteria in the intestine than total cells constituting the body. These commensal bacteria influence health and disease through complex interactions with the immune system. Alterations in the crosstalk between commensals and immune cells can lead to the development of inflammatory and autoimmune diseases including asthma, multiple sclerosis and inflammatory bowel disease (IBD). This project seeks to understand the mechanisms that control the crosstalk between commensals and immune cells and how those modulate health and disease. We will focus in a population of intestinal T cells (called NKT cells) that recognise lipids from commensal bacteria. NKT cells collaborate to control commensals in the healthy intestine, but they also accumulate in the intestine of IBD patients, which suggests that they are important for the onset and/or progression of the disease. In this project we will investigate the mechanisms that control the function of intestinal NKT cells in health and IBD. We will isolate NKT cells from the intestine of mice and humans and perform functional assays to define their features, distribution and functions (year 1). Using mouse models and samples from IBD patients we will study how NKT cell features are altered during inflammation (year 2). Finally, we will explore the cellular and molecular mechanisms that lead to NKT cell activation in IBD and how these are regulated by commensals (years 3 and 4). These experiments will provide insight into the mechanisms of inflammatory diseases and guide the search for new therapies. Training: cell isolation and culture, flow-cytometry, imaging techniques, qPCR, mouse genetics, models of disease. Two representative publications from supervisors: Saez de Guinoa J, Jimeno R, Farhadi N, Jervis PJ, Cox LR, Besra GS & Barral P. CD1d-mediated activation of group 3 Innate Lymphoid Cells drives IL-22 production. EMBO Reports (2016) in press Vossenkämper A, Blair PA, Safinia N, Fraser LD, Das L, Sanders TJ, Stagg AJ, Sanderson JD, Taylor K, Chang F, Choong LM, D'Cruz DP, Macdonald TT, Lombardi G, Spencer J. A role for gut-associated lymphoid tissue in shaping the human B cell repertoire. J Exp Med. (2013) 210:1665-74

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41.1 Broadly neutralizing antibody responses against HIV Co-Supervisor 1: Dr Katie Doores Research Division or CAG: Immunology, Infection and Inflammatory Diseases E-mail: katie.doores@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/infectious/research/doores/index.aspx Co-Supervisor 2: Dr Julie Fox Research Division or CAG: GRIDA, Guy’s and St Thomas’ NHS Foundation Trust Email: julie.fox@kcl.ac.uk; Julie.fox@gstt.nhs.uk http://www.kcl.ac.uk/medicine/research/divisions/diiid/departments/infectious/research/juliefox.aspx Project Description: Approximately 10-30% of HIV infected individuals generate antibodies that are capable of neutralizing a broad range of HIV isolates and these antibodies have been shown to protect against SHIV challenge in Macaque models. Isolation and characterisation of these antibodies has revealed regions of the HIV envelope glycoprotein, gp120/gp41, that are susceptible to antibody binding and re-eliciting these antibodies may be a key step for a successful HIV vaccine. Gp120 is heavily glycosylated with host-derived N-linked glycans and it was previously thought that these glycans shield conserved protein regions from the immune system. However, we have recently shown that a number of the HIV broadly neutralizing antiodies (bnAbs) bind directly to these glycans highlighting them as potential targets for HIV vaccine design. Using unique longitudinal samples from acutely HIV infected patients in the SPARTAC study (N Engl J Med 2013;368:207-17) we will investigate the development of glycan-binding bnAbs in vivo using in vitro neutralization assays, antigen-specific B cell sorting and single genome amplification. We will determine how the evolving glycan shield impacts and directs bnAb development. Ultimately these studies will be used to design immunogens and immunization strategies aimed at re-eliciting these bnAbs through vaccination. Two representative publications from supervisors: L. M. Walker,* M. Huber,* K. J. Doores,* E. Falkowska, R. Pejchal, J.-P. Julien, S.-K. Wang, A. Ramos, P. Y. Chan-Hui, M. Moyle, J. L. Mitcham, P. W. Hammond, O. A. Olsen, P. Phung, S. Fling, C.-H. Wong, S. Phogat, T. Wrin, M. D. Simek, Protocol G Principal Investigators, W. C. Koff, I. A. Wilson, D. R. Burton, P. Poignard, Broad neutralization coverage of HIV by multiple highly potent antibodies, Nature, 2011, 477, 466-470.

S. A. Krumm, H. Mohammed, K. M. Le, M. Crispin, T. Wrin, P. Poignard, D. R. Burton, K. J. Doores, Mechanisms of escape from the PGT128 family of anti-HIV broadly neutralizing antibodies, Retrovirology, 2016, 13(1), 8.

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42.1 The CXCL8-producing T cell: function in health and carcinogenesis Co-Supervisor 1: Dr Deena Gibbons Research Division or CAG: DIIID E-mail: deena.gibbons@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/immunobiology/research/gibbons/index.aspx Co-Supervisor 2: Dr Susan John Research Division or CAG: DIIID Email: susan.john@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/diiid/departments/immunobiology/research/SusanJohn/index.aspx Project Description: The human neonatal immune system is not just an immature version of that of the adult but is qualitatively and quantitatively different. Our research focuses on understanding immune cell development and function in the human neonate and how this impacts on disease. We have identified a novel T cell effector function in neonates that contrasts with T cell biology in adults. Furthermore, this discovery dispels the long held belief that the newborn immune system is anti-inflammatory. This effector function, the ability to produce CXCL8 (aka interleukin-8, IL8), is imprinted in the thymus during T cell development and represents an intermediate function en route to classic adaptive immune cell functions such as IFN production. This raises a number of important questions that form the basis of this PhD project: 1. What is the function of CXCL8 during T cell development in the thymus and does this play a role in the transformation of progenitors to T cell acute lymphocytic leukaemia (T-ALL) or in its maintenance? 2. What is the fate of these cells as the human immune system develops post-natally and do these cells determine how infants respond to infection? 3. What are the signals and signalling pathways that allow conversion of CXCL8-producing T cells to IFN -producing cells and other T cell lineages? Methods will involve multiple cellular and molecular techniques as well as work both in vitro and in vivo mouse models. Two representative publications from supervisors: Deena Gibbons, Paul Fleming, Alex Virasami, Marie-Laure Michel, Neil Sebire, Kate Costeloe, Robert Carr, Nigel Klein, Adrian Hayday. Interleukin-8 (CXCL8) Production is the signatory T cell effector function of human newborn infants. Nature Medicine 20, 1206-10 (2014) Rani A, Afzali B, Kelly A, Tewolde-Berhan L, Hackett M, Kanhere A.S, Pedroza-Pacheco I, Bowen H, Jurcevic S, Jenner R.G., Cousins, D., Ragheb J.A., Lavender Pand John S. IL-2 regulates expression of c-maf in human CD4 T cells. 2011. J. Immunol. 187(7): 3721-9.

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43.1 Elucidating the crosstalk between lymphocytes and intestinal epithelial cells using human mini-guts Co-Supervisor 1: Graham Lord Research Division or CAG: Division of Transplantation Immunology and Mucosal Biology E-mail: graham.lord@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/timb/about/people/profiles/grahamlord.aspx Co-Supervisor 2: Joana F Neves Research Division or CAG: Division of Transplantation Immunology and Mucosal Biology Email: joana.pereira_das_neves@kcl.ac.uk Project Description: Studies, by us and others, have found that maintaining intestinal homeostasis depends on the interactions between the gut epithelium, the intestinal microbiota and the gut-associated immune system. Disrupting this delicate balance usually results in intestinal inflammation, which is associated with several diseases such as Inflammatory Bowel Disease (IBD) and cancer. We recently developed a novel in vitro system of lymphocyte culture in intestinal organoids (“mini-guts”) that mimics the intestinal environment. This pioneer system allows us to identify and dissect the mechanisms that govern the crosstalk between lymphocytes and epithelial cells at the intestinal barrier. Certain populations of a recently discovered group of lymphocytes, called Innate Lymphoid Cells (ILC) are increased in IBD patients. Thus, we are particularly interested in studying the interaction of ILC with intestinal epithelial cells. Aim-1) Ascertain the differences between intestinal organoids established from inflamed and non-inflamed tissue of IBD patients and their effect on the function and differentiation of ILC (Years 1-2). Aim-2) Understand the effect of ILC on the biology of intestinal epithelial cells (Years 2-3). Aim-3) Identify the molecular pathways that regulate the crosstalk between ILC and intestinal epithelial cells (Years 3-4). This study can lead to the identification of novel targets to modulate ILC and intestinal epithelial cells in order to promote intestinal homeostasis. During this project the student will acquire a wide range techniques such as flow cytometry, imaging, molecular biology (including CRISPR), transcriptomic, bioinformatics and lymphocyte biology and mucosal immunology techniques (mouse/human). The supervisors and their established collaborations have vast expertise these areas. Two representative publications from supervisors: Powell N, Lo JW, Biancheri P, Vossenkämper A, Pantazi E, Walker, AW, Stolarczyk E, Ammoscato F, Goldberg R, Scott P, Canavan JB, Perucha E, Garrido-Mesa N, Irving PM, Sanderson JD, Hayee B, Howard JK, Parkhill J, MacDonald TT, Lord GM. Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation. Gastroenterology. 2015 149(2):456-67. Olszak T*, Neves JF*, Dowds CM*, Baker K, Glickman J, Davidson NO, Lin CS, Jobin C, Brand S, Sotlar K, Wada K, Katayama K, Nakajima A, Mizuguchi H, Kawasaki K, Nagata K, Müller W, Snapper SB, Schreiber S, Kaser A, Zeissig S*, Blumberg RS*. Protective mucosal immunity mediated by epithelial CD1d and IL-10. Nature, 2014; 509 (7501): 497-502. *These authors contributed equally to this work

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44.1 Elucidating the molecular mechanisms of peanut allergies Co-Supervisor 1: James McDonnell Research Division or CAG: Randall Division E-mail: james.mcdonnell@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/james.mcdonnell.html Co-Supervisor 2: Alexandra Santos Research Division or CAG: Allergy, Asthma and Lung Biology Email: alexandra.santos@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/alexandra.santos.html Project Description: The incidence of allergic disorders is increasing worldwide and now affects more than a quarter of the population in industrialised countries and there is an urgent need for better treatments. Immunoglobulin E (IgE) is the central player in allergic reactions, recognising allergens both as a B cell receptor (BCR) and bound to high-affinity receptor FcεRI on effector cells and antigen presenting cells (APCs). Allergic reactions are rapidly generated when allergens bind and crosslink IgE-FcεRI complexes on mast cells and basophils, resulting in cell degranulation and release of inflammatory mediators. This project seeks to understand the mechanisms of allergic reactions against peanut allergens, one of the most common and most dangerous types of food allergies. Recent work by Dr. Santos has demonstrated that not all anti-peanut IgE antibodies found in patients are pathogenic. Using a set of patient derived anti-peanut IgE antibodies, this project will test the hypothesis that a number of factors - including allergen epitope, epitope number (valency), spatial arrangement and IgE affinity - contribute to triggering of allergic responses and thus determine IgE pathogenicity. The project will offer training in methods of antibody discovery/expression/characterisation, cellular immunology, bioinformatics, molecular interaction analysis, and molecular structure determination. Early priorities in the project will be to produce a representative set of patient-derived pathogenic and non-pathogenic IgE antibodies, using in-house protocols for highly efficient single B cell cloning and antibody production, which will then be used to test hypotheses on the molecular mechanisms that determine the allergy triggering properties of IgE antibodies. Two representative publications from supervisors: Santos et al. (2014) Basophil activation test discriminates between allergy and tolerance in peanut sensitized children. J. Allergy Clin. Immunol. 134:645-652. Drinkwater et al. (2014) Human immunoglobulin E flexes between acutely bent and extended conformations. Nature Struct. Mol. Biol. 21:397-404

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45.1 Targeting PSK kinases for breast cancer therapy Co-Supervisor 1: Dr Jonathan Morris Research Division or CAG: Cancer Studies E-mail: jonathan.morris@kcl.ac.uk Website: http://www.kcl.ac.uk/lsm/research/divisions/cancer/research/groups/cspcc.aspx Co-Supervisor 2: Dr John Maher Research Division or CAG: Cancer Studies Email: john.maher@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/john.maher.html Project Description: Drugs that target and inhibit microtubule dynamics (eg. taxanes) provide one of the most effective classes of therapeutics for the front-line treatment of metastatic breast cancer, but these compounds also produce debilitating side effects and patients relapse and become resistant within 3-8 months. Microtubules are therefore proven targets for chemotherapy and their disruption works well in the clinic, but additional and better strategies are now needed to target these structures in dividing breast cancer cells to provide more effective and longer lasting treatment. In seeking candidates for intervention we have identified an unusual family of proteins called prostate-derived sterile 20-like kinases (PSKs), which bind microtubules and regulate their stability and organisation. PSKs are activated catalytically in dividing breast cancer cells and this activity is required for their proliferation. The initial project will use novel small molecule inhibitors for PSKs to assess the requirements for these proteins during mitosis and for cell proliferation The PhD objectives will be to: • Identify biological functions for PSKs and their mechanisms of action in breast cancer cells • Inhibit PSK activity and downstream substrates in breast cancer cell models in order to alter cell proliferation • Characterise novel chemical inhibitors for PSKs and establish their biological effects on malignant cells • Use breast cancer tissue arrays to identify patient subtypes suitable for kinase inhibition and therapy The results will establish whether PSKs offer suitable targets for breast cancer therapy. Skills: • Expertise in cellular and molecular biology techniques (Morris) • Knowledge of cancer cell biology and treatment (Maher) Two representative publications from supervisors: Wojtala RL, Tavares IA, Morton PE, Valderrama F, Thomas NS, Morris JD. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) are activated in mitosis and contribute to mitotic cell rounding and spindle positioning. 2011. J. Biol. Chem. 286. 30161-70. Tavares IA, Touma D, Lynham S, Troakes C, Schober M, Causevic M, Garg R, Noble W, Killick R, Bodi I, Hanger DP, Morris JD. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) phosphorylate tau

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protein and are activated in tangle-bearing neurons in Alzheimer Disease. 2013. J. Biol. Chem. 288. 15418-29. 46.1 How do mutations in MYO18B lead to muscle diseases? Co-Supervisor 1: Dr. Julien Ochala (basic scientist) Research Division or CAG: Centre of Human and Aerospace Physiological Sciences E-mail: julien.ochala@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/julien.ochala.html Co-Supervisor 2: Dr. Heinz Jungbluth (clinician/clinician scientist) Research Division or CAG: Randall Division of Cell and Molecular Biophysics Email: Heinz.jungbluth@gstt.nhs.uk Website: https://kclpure.kcl.ac.uk/portal/heinz.1.jungbluth.html Project Description: Myopathies are life-threatening diseases characterized by devastating muscle weakness. The understanding of this group of disorders has advanced in recent years through the identification of the causative gene mutations such as MYO18B. The aim of the present project is to underpin the hitherto pathophysiological mechanisms by which MYO18B mutations lead to muscle weakness. MYO18B encodes a member of the myosin superfamily, Myo18b. As this protein is involved in muscle trafficking, the overall hypothesis of the present project is that MYO18B mutations alter the organisation and function of myosin molecules in muscle, disrupting the force production mechansims. To prove this, the present project will be divided into three sub-objectives: Sub-objective 1 (year 1, with Dr. Jungbluth): Determining how MYO18B mutations affect myosin arrangement using muscle samples from human patients and advanced microscopy. Sub-objective 2 (years 2, with Dr. Ochala): Characterising how MYO18B mutations modify myosin motor function and force production using muscle tissue from human patients and motility assays. Sub-objective 3 (years 3 and 4, with Dr. Ochala and Jungbluth): Identifying the time course of above changes using muscles from mice with a specific Myo18b deficiency. This will allow a clear genotype-phenotype correlation, potentially helping the design of efficient therapeutic interventions. At the end of this project, the student should be able to: - Master biophysical techniques and advanced microscopy, - Apply his newly developed skills to normal and diseased cells/molecules, - Generate and analyse large sets of data, - Synthetize the findings by writing scientific papers Two representative publications from supervisors:

Cullup T, Kho AL, Dionisi-Vici C, Brandmeier B, Smith F, Urry Z, Simpson MA, Yau S, Bertini E, McClelland V, Al-Owain M, Koelker S, Koerner C, Hoffmann GF, Wijburg FA, ten Hoedt AE, Rogers RC, Manchester D, Miyata R, Hayashi M, Said E, Soler D, Kroisel PM, Windpassinger C, Filloux FM, Al-Kaabi S, Hertecant J, Del Campo M, Buk S, Bodi I, Goebel HH, Sewry CA, Abbs S, Mohammed S, Josifova D, Gautel M, Jungbluth H. (2013) Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy. Nat Genet. 45: 83-87.

Ochala J, Gokhin DS, Penisson-Besnier I, Quijano-Roy S, Monnier N, Lunardi J, Romero NB, Fowler VM. (2012) Congenital myopathy-causing tropomyosin mutations induce thin filament dysfunction via distinct physiological mechanisms. Hum Mol Genet. 21: 4473-4485.

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47.1 Developmental basis of skin diversity Co-Supervisor 1: Tanya Shaw Research Division or CAG: Immunology, Infection & Inflammatory Disease E-mail: Tanya.shaw@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/diiid/centres/cmcbi/research/shaw/DrTanyaShaw.aspx Co-Supervisor 2: Anthony Graham Research Division or CAG: Centre for Developmental Neurobiology Email: Anthony.graham@kcl.ac.uk Website: http://www.kcl.ac.uk/ioppn/depts/devneuro/Research/groups/graham.aspx Project Description: The skin exhibits anatomical diversity, with different regions fulfilling different functions; the skin over our heads is covered in hair, the skin of our hands and feet is quite different that of our abdomen. Regional differences in the skin emerge during development and these are imparted by the dermis, which has a complex developmental history. Specifically, the dermis of the back is derived from somites, derivatives of the paraxial mesoderm, the dermis of the limbs is derived from lateral plate mesoderm and the dermis of the face from neural crest cells. We hypothesize that the positionally distinct dermal features (including repair and regeneration potential, and susceptibility to site-specific skin diseases) reflect the developmental origin of the tissue. Having discovered significantly different gene expression profiles of dermis from various anatomical sites in homeostasis, the objectives of this project are to: 1. Determine if dermis of different embryonic origins has different properties during wound healing. 2. Investigate whether the anatomical distribution of site-specific skin diseases reflects the distinct histories of the tissue. This project will use a combination of human samples and in vivo models; the student will be trained in many cellular and molecular biology techniques, including primary tissue culture, gene expression profiling (e.g. RNA-seq), western blotting, immunohistochemistry, and microscopy. Two representative publications from supervisors: EJ Fitzgerald O'Connor, II Badshah, LY Addae, P Kundasamy, S Thanabalasingam, D Abioye, TJ Shaw. Histone deacetylase 2 is upregulated in normal and keloid scars. J Invest Dermatol 2012, 132, 1293-6. Shone, V. and Graham, A. Endodermal/ectodermal interfaces during pharyngeal segmentation in vertebrates. Journal of Anatomy 2014, 225, 479-491.

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48.1 Molecular mechanisms underlying the contractile dysfunction in ageing-related muscle weakness Co-Supervisor 1: Dr. Yin Biao Sun (basic scientist) Research Division or CAG: Randall Division of Cell and Molecular Biophysics E-mail: yin-biao.sun@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/yin-biao.sun.html Co-Supervisor 2: Prof. Steve Harridge (basic scientist) Research Division or CAG: Centre of Human and Aerospace Physiological Sciences E-mail: s.harridge@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/stephen.harridge.html Project Description: Ageing-related muscle weakness represents a major, and growing, healthcare burden, which leads to declines in physical function, independency and quality of life. The molecular mechanisms underlying such phenomena remain unclear but potentially include a dysfunction of the molecular motor, myosin, that can only partly explain the decline in the force-generating capacity. In the present project, we aim to precisely characterise such myosin dysfunction. Understanding the mechanisms underpinning such contractile dysfunction is essential in the development of effective ‘preventative’ or ‘therapeutic’ interventions. We are going to apply two of the state-of-the-art techniques, the Fluorescence for In Situ Structure (FISS) and the Small-angle X-ray scattering, to study both structural and functional effect of aging on myosin in human skeletal muscle cells. We will use muscle biopsy specimens that we have already obtained from healthy young adults, and three groups of older individuals aged over 75 years who represent the health and activity span. Performing FISS and X-ray experiment requires a unique combination of expertise, ranging from molecular biology, protein biochemistry, muscle physiology to biophysics of data interpretation. Depends on the student’s background and previous experience, he/she will have opportunities to be trained in all these skills. - Years 1 and 2 (with Dr. Sun): trainings in basic skills required for the experiment and performing FISS to complete the control experiments using myosin probes. - Years 3 and 4 (with Dr. Ochala and Dr. Sun): specifically characterise the alterations in myosin function in aging muscle cells using both X-ray and FFSS techniques. Two representative publications from supervisors: Kampourakis T, Sun YB, Irving M. (2013) Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments. PNAS. 113: E3039-3047. Li M, Ogilvie H, Ochala J, Artemenko K, Iwamoto H, Yagi N, Bergquist J, Larsson L. (2015) Aberrant post-translational modifications compromise human myosin motor function in old age. Aging Cell. 14: 228-235.

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49.1 Linking Genotype to Phenotype in Psoriatic Arthritis: determining the role of disease-associated genes in Tc17 cell function and development Co-Supervisor 1: Prof Leonie Taams Research Division or CAG: DIIID / GRIID E-mail: leonie.taams@kcl.ac.uk www.kcl.ac.uk/lsm/research/divisions/diiid/centres/cmcbi/research/taams/index.aspx Co-Supervisor 2: Dr Francesca Capon Research Division or CAG: GMM/GRIID Email: francesca.capon@kcl.ac.uk Website: http://tinyurl.com/CaponLab Project Description: Psoriatic arthritis (PsA) is an inflammatory disease of the joints, with frequent skin involvement. The disease is typically associated with HLA class I, suggesting a role for CD8+ T cells. We previously demonstrated that the inflamed joints of patients with PsA contain higher frequencies of CD8+ T cells expressing the pro-inflammatory cytokine IL-17. These IL-17+ CD8+ T cells (Tc17 cells) correlate with clinical parameters of disease activity. Tc17 cells are also found in the skin of patients with psoriasis. In addition to particular HLA class I associations, a number of other genetic variants have been associated with PsA and psoriasis. Interestingly, some of these genetic variants relate to the IL-17 pathway (e.g. IL23R, TRAF3IP2). Together, this leads us to hypothesize that (i) the presence of Tc17 cells in the inflamed joint is driven by particular HLA class I associations, and (ii) IL-17 production by CD8+ T cells can be influenced by particular non-HLA genetic variants. Overarching objectives: Year 1: determine HLA genotypes and other genetic variants in existing samples from patients with PsA, and link this information to Tc17 frequency. Year 2/3: assess gene/protein expression of PsA-associated genetic variants that are associated with the IL-17 pathway in relevant immune cells. Year 3/4: determine the functional effects of these genetic variants on the expression of IL-17 by CD8+ (and CD4+) T cells, and Tc17 development and function. Skills training: mononuclear cell isolation, cell culture, intracellular cytokine staining, multi-colour flow cytometry, ELISA, Western blotting, DNA/RNA extraction, qPCR, genotyping, overexpression/knockdown, bioinformatics. Two representative publications from supervisors: Menon B, Gullick NJ, Walter GJ, Rajasekhar M, Garrood T, Evans HG, Taams LS*, Kirkham BW* (*joint senior authors). Interleukin-17+CD8+ T Cells Are Enriched in the Joints of Patients With Psoriatic Arthritis and Correlate With Disease Activity and Joint Damage Progression. Arthritis Rheumatol. 66 (5): 1272–1281 (2014) Mahil SK, Twelves S, Farkas K, Setta-Kaffetzi N, Burden AD, Gach JE, Irvine AD, Képíró L, Mockenhaupt M, Oon HH, Pinner J, Ranki A, Seyger MM, Soler-Palacin P, Storan ER, Tan ES, Valeyrie-Allanore L, Young HS, Trembath RC, Choon SE, Szell M, Bata-Csorgo Z, Smith CH, Di Meglio P, Barker JN, Capon F. AP1S3 mutations cause skin autoinflammation by disrupting keratinocyte autophagy and up-regulating IL-36 production. J Invest Dermatol Epub ahead of print 4th July 2016; doi: 10.1016/j.jid.2016.06.618

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50.1 TRPV1 and TRPA1 ligands as P-gp substrates: a wide-reaching investigation from basic molecule through to animal models of disease Co-Supervisor 1: Sarah A. Thomas BSc, PhD (Reader in Physiology) Research Division or CAG: Institute of Pharmaceutical Science E-mail: sarah.thomas@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/ips/about/people/Thomas/index.aspx Co-Supervisor 2: Julie Keeble BSc, PhD (Lecturer in Pharmacology and Named training and competency officer) Research Division or CAG: Institute of Pharmaceutical Science Email: julie.keeble@kcl.ac.uk http://www.kcl.ac.uk/lsm/research/divisions/ips/research/pharmathera/Staff/Keeble.aspx Project Description:

Transient Receptor Potential Vanilloid 1 (TRPV1) receptors are intrinsically involved in pain and inflammation (Keeble et al., 2005). TRPV1 ligands appear to act on the intracellular side of the channel, in contrast to ligands of other ligand-gated ion channels. This suggests that ligands need to pass through the cell membrane in order to access their binding site and for metabolism/excretion from cells, perhaps via passive diffusion due to their lipid solubility. However, it is also possible that TRPV1 ligands are transported by an efficient biological system. Indeed, a recent article in Nature Neuroscience identified a FAAH-1 variant as a driver for anandamide transport into neuronal cells (Fu et al., 2012). Preliminary experiments in our laboratory show for the first time that anandamide, an endogenous TRPV1 agonist, is a P-glycoprotein (P-gp) efflux transporter substrate (Brown et al., 2015).

The aims of this multi-disciplinary PhD project will therefore be to (1) characterise the transport of various TRPV1 and TRPA1 agonists via P-gp and other ABC transporters through the use of cell culture and assay studies, (2) carry out a study of co-expression of TRPV1 with the various transporters in different tissues, e.g. blood-brain barrier, neurons and inflammatory cells, using immunohistochemical, PCR and Western blot techniques and (3) perform an in vivo investigation into the physiological/pathophysiological implications of TRPV1/TRPA1 ligand transport by P-gp/other transporters, e.g. effects of transporter inhibitors in murine models of pain, inflammation and thermoregulation.

Gannt chart 0-6 months

6-12 months

12-18 months

18-24 months

24-30 months

30-36 months

36-42 months

42-48 months

Cell culture and assay studies

x x x

Transporter and TRPV1/TRPA1 co-expression

x x x

In vivo studies x x x

Writing up x

SAT will supervise the in vitro component and JEK will supervise the in vivo component. Brown et al. (2015) E-journal of the British Pharmacological Society 279P. Fu et al. (2012). Nat Neurosci, 15(1):64-69. Keeble et al. (2005). Arthritis Rheumatism, 52(10):3248-56. Thomas (2012) Brain Res1436:111 Thomas (2011) J.Pharm.Exp.Ther336:506.

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Two representative publications from supervisors: Watson, CP, Pazarentzos, E., Fidanboylu, M., Padilla, B., Brown, R and Thomas S.A. The transporter and permeability interactions of asymmetric ADMA and L-arginine with the human blood-brain barrier in vitro. Brain Research 1648 (2016) 232-242. Alawi, KM, Aubdool, AA, Liang ,L, Wilde, E, Vepa, A, Psefteli, MP, Brain, SD and Keeble JE (2015). The sympathetic nervous system is controlled by transient receptor potential vanilloid 1 in the regulation of body temperature. FASEB J. 29(10):4285-98.

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51.1 Role of Genetic Regulation of Gene Expression in Type 2 Diabetes and Obesity. Co-Supervisor 1: Kerrin Small

Research Division or CAG: Genetics and Molecular Medicine

E-mail: Kerrin.small@kcl.ac.uk

http://www.kcl.ac.uk/lsm/research/divisions/gmm/departments/twin/research/small/index.aspx

Co-Supervisor 2: Dr Alan Hodgkinson

Research Division or CAG: Genetics and Molecular Medicine

Email: alan.hodgkinson@kcl.ac.uk

http://www.kcl.ac.uk/lsm/research/divisions/gmm/departments/mmg/researchgroups/Hodgkinson

Group.aspx

Project Description:

Genome-wide association studies (GWAS) have identified thousands of regions of the genome that are associated with disease, including Type 2 Diabetes and other obesity-related traits. However, GWAS studies only identify a region of the genome, not the casual gene or underlying molecular mechanism. An important tool for interpreting GWAS loci are eQTL studies, which identify genetic variants that regulate gene expression. We have previously shown that eQTLs in adipose (fat) tissue mediate a subset of Type 2 Diabetes loci, including a master trans-regulator at the KLF14 locus. This project will seek to identify novel regulatory variants and use them to further interpret disease associations, with a particular focus on Type 2 Diabetes and obesity-related traits. In particular this project will focus on two under-explored classes of regulatory variants, rare variants and trans-eQTLs. The student will utilize a unique multi-tissue RNAseq data set from deeply-phenotyped twins from the TwinsUK cohort, and integrate this newly generated matched whole genome sequence data. The student will be taught how to analyze high-throughput sequencing data to answer important biological questions. More broadly the student will undergo training in genomic analysis, bioinformatics (including programming) and scientific writing.

Objectives:

Year 1: Identify regulatory variants (eQTLs, rare variants and splicing) utilizing RNAseq data from

multiple tissues and matched whole genome sequence.

Year 2: Identify trans-eQTLs in adipose tissue in a large multi-center dataset. Year 3: Integrate identified regulatory variants with Type 2 Diabetes and obesity-related traits to elucidate underlying regulatory mechanisms mediating disease risk and response.

Two representative publications from supervisors:

Glastonbury C, Vinuela A, Buil A, Halldorsson, G, Thorleifsson, G, Helgason. H, Thorsteinsdottir U,

Stefansson K, Dermitzakis ET, Spector TD, Small KS Adiposity-dependent regulatory effects on

multi-tissue transcriptomes. Am J Hum Genet. 2016 Sept 1;99(5):567-79. doi:

10.1016/j.ajhg.2016.07.001

Hodgkinson, A., Idaghdour, Y., Gbeha, E., Grenier, J.C., Hip-Ki, E., Bruat, V., Goulet, J.P., de Malliard, T. and Awadalla, P. 2014. High-Resolution Genomic Analysis of Human Mitochondrial RNA Sequence Variation. Science 344: 413-415.

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52.1 Identifying novel therapeutics to target pancreatic cancer metastasis Co-Supervisor 1: Dr. Claire Wells (basic scientist) Research Division or CAG: Cancer Studies E-mail: mailto:claire.wells@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/claire.wells.html or http://wellslaboratory.blogspot.co.uk/

Co-Supervisor 2: Dr. Debashis Sarker (clinical)

Research Division or CAG: Cancer Studies

Email: mailto:debashis.sarker@kcl.ac.uk

Website: https://kclpure.kcl.ac.uk/portal/debashis.sarker.html

Project Description:

Pancreatic cancer (PC) survival is devastatingly low with few patients surviving more than 5 years post diagnosis due to the prevalence of metastatic disease. Currently there are no treatment options that target metastasis and thus novel therapeutic approaches are urgently required. We have recently completed a screen of existing drugs to identify novel therapeutic compounds that can block PC invasion. We now need to validate these hits and elucidate the molecular mechanism that underpins suppression of invasion. One of our top screen hits was a p-21 activated kinase inhibitor. The PAK family are associated with cancer cell invasion and PAK is amplified in PC. Interestingly we have recently published that PAK activity is linked to cancer cell invasion via promotion of a cellular process called invadopodia. However, there is much we still don’t understand about PAK in invadopodia and how this process is regulated. This project aims to bring together our screen discoveries with our existing expertise in PAK biology to develop novel therapeutics to target pancreatic cancer invasion. Project Outline Rotation: Identify PC cell lines that form invadopodia and correlate with PAK activity Year 1: Follow up top 5 hits (including PAK) – expand screen to include invadopodia studies. Identify most promising lead to take into year 2 Year 2: dose responses , invadopodia life cycle analysis, organotypic validation Year 3: Define the biochemical signalling nexus that underpins suppression of invasion. Test findings in clinical material. Training: high resolution live/fixed cell imaging, cell biology, biochemistry, translational medicine techniques. Two representative publications from supervisors:

Nicole S. Nicholas, Aikaterini Pipili, Simon M. Ameer-Beg, Jenny L. C. Geh, Ciaran Healy, Alistair D.

MacKenzie Ross, Maddy Parsons, Frank O. Nestle, Katie E. Lacy and Claire M. Wells (2016) PAK4

suppresses PDZ-RhoGEF activity to drive invadopodia maturation in melanoma cells Oncotarget

DOI: 10.18632/oncotarget.12282

Anna Dart, Gary Box , William Court , Madeline Gale, John Brown, Sarah Pinder, Sue Eccles and Claire M Wells (2015) PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion. J Cell Biol. 211:863

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53.1 The mechanism through which complex patterns of genetic variation within the large

inversion on chromosome 8 contribute human disease.

Co-Supervisor 1: Dr Anders Eriksson Research Division or CAG: Department of Medical and Molecular Genetics

E-mail: anders.eriksson@kcl.ac.uk Website: https://kclpure.kcl.ac.uk/portal/anders.eriksson.html

Co-Supervisor 2: Professor Michael Simpson

Research Division or CAG: Department of Medical and Molecular Genetics

Email: michael.simpson@kcl.ac.uk

Website: https://kclpure.kcl.ac.uk/portal/michael.simpson.html

Project Description:

On the short arm of chromosome 8 in the human genome resides a large inversion that arose in the

lineage leading to anatomically modern humans, probably around 400-500 thousand years ago,

leading to a largely parallel evolution of the inverted and non-inverted versions due to lack of

homologous recombination in this region. The inversion and other genetic variants within this

region have been associated with a range of human diseases including several inflammatory

diseases such as severe acne, psoriasis, lupus as well as neurological conditions including

depression.

The beta defensin gene cluster, located within the inverted region, encode critical components of

the innate immune system is. This cluster of genes is also is duplicated, commonly leading to a wide

array of copies of beta defensin genes and variable orientation of these genes within the

population. This project aims to understand the interaction of the orientation of the common

inversion and the number of copies of the beta defensin cluster contribute to the level of expression

of beta defensins and risk of disease.

In order to achieve the aims of this project the student will develop a range of skills relating to

- Bioinformatics analysis of genome and transcriptome next-generation sequencing datasets.

- Genetic annotation and functional analysis of genetic variants.

- Population genetic statistical analysis.

Project Outline

Year 1: Integration of population scale genotype data with whole genome sequence data

Year 2: Evaluation of an association between changes in the regulatory network of beta defensins

and expression levels of these genes in multiple tissues.

Years 3 and 4: Integration of the association of molecular consequences of genetic variation in this

region with the disease specific datasets

Two representative publications from supervisors:

Navarini et al. Genome-wide association study identifies three novel susceptibility loci for severe

Acne vulgaris. Nature Communications. 2014. 5:4020

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Clemente et al. A Selective Sweep on a Deleterious Mutation in CPT1A in Arctic Populations. Am J

Hum Genet. 2014 95:584