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Abstracts of research projects available for October 2014

The research interests of all the members of the Department are available on the world wide web here

Candidates are advised to read these research interest pages in conjunction with this information

Dr Laura Itzhaki: Tandem-repeat proteins: Folding, function, role in disease and therapeutic intervention

A major focus of research in our group is a class of proteins with very distinctive architectures, known as tandem-repeat proteins. Tandem-repeat proteins, such as ankyrin repeats, tetratricopeptide repeats, armadillo repeats and HEAT repeats, are frequently deregulated in human diseases including cancer and respiratory and cardiovascular diseases. They are made up of small structural motifs repeated in tandem to produce elongated and superhelical structures. They function as scaffolds for protein-protein interactions by providing extended surfaces for molecular recognition, but their Slinky spring-like shapes suggest a rather dynamic, elastic mode of action. The modular architecture of these proteins make them uniquely amenable to the dissection of their biophysical properties and to the rational redesign of these properties. We are interested in understanding how the process of folding and unfolding of this distinctive class of proteins directs their functions in the cell.  We are also looking at small molecule and peptide-based approaches to target these proteins and their binding partners for therapeutic benefit. 

We use a range of approaches, including protein engineering, biochemistry and biophysical analysis including single-molecule techniques, cell biology and medicinal chemistry. For all projects, we collaborate closely with computational groups to gain atomic-level insights into protein dynamics and function.

Key words: protein engineering, biophysics, cancer, protein folding, tandem-repeat proteins

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Dr R Henderson:Nanoengineering cellular environments

We are looking principally at two systems at present.  In the first we are making scaffolds that bear sequences of DNA that bear recognition sites for DNA-binding proteins (for example proteins involved in gene transcription and DNA replication and repair). We are using these to quantify the dynamics and kinetics of protein interaction and target location on the DNA.  How do proteins rapidly and accurately find their relatively short target sequences on an enormously long DNA molecule?  This topic has exercised interest for many years, but the data so far available remains inconclusive.  The real-time imaging afforded by fast-scan AFM provides a powerful new technique to study the process directly. The second set of experiments also addresses a question that has proved difficult to resolve using other methods, namely the spatial relationship between the differing elements of the adenylyl cyclase (AC)/protein kinase A (PKA)/phosphodiesterase (PDE) signalling system. The successful operation of this system requires correct positional arrangement of the different components to allow, first, cAMP to be generated, then to allow it to interact with PKA, and finally, PDE must be in appropriate proximity to allow regulated degradation of cAMP. The exact geometrical relation between these components still is unknown. We are generating origami scaffolds, and attach each of the elements of the AC/PKA/PDE system in differing orientations and examine the role of orientation on activation by looking at changes in the structure of the PKA, which alters during the signalling process.

Primary field: Cell biology

Keywords: Cell signalling, DNA, DNA-binding proteins, imaging, atomic force microscopy, scanning probe microscopy

 

 

Dr. WT Khaled: Cellular and molecular characterization of breast cancer heterogeneity

Breast cancer is the most common type of cancer in the UK with an estimated 4000 new cases every month. However, the heterogeneous nature of the disease means that an effective “one size fits all” treatment remains elusive. Thus understanding the cellular and molecular basis of breast cancer heterogeneity is essential for the development of more effective treatments. Our research focuses on deconstructing the molecular basis of breast cancer heterogeneity and the identification of mammary epithelial cell fate regulators that drive tumour heterogeneity with the aim of developing targeted therapeutics for breast cancer.

Microarray studies have shown that there are multiple subtypes of breast cancer with varying degrees of prognosis. It is known that the luminal subtype has the best prognostic outcome while the basal like breast cancer (BLBC) subtype has the worst outcome. There is evidence that such tumour heterogeneity could be attributed to the tumour’s cell of origin. For example, luminal breast cancer is thought to arise from differentiated luminal cells while BLBC is thought to arise from undifferentiated progenitor and stem like cells. Such model would necessitate the expression and function of cell type specific factors which determine the cells’ fate. We have recently identified the transcription regulator BCL11A as a BLBC oncogene and a mammary stem cell regulator (in press). By using various mouse models we have shown that deletion of BCL11A significantly reduced the number of mammary stem cells and incidences of BLBC.

Part of my lab is aiming to develop anti-BCL11A therapeutic approaches for the treatment of BLBC. The proposed PhD project will be part of this effort and will focus on unravelling the molecular functions of BCL11A in BLBC and exploiting such functions for therapeutic development.

Primary field: Cancer biology, Cancer therapeutics

Keywords: Molecular biology, proteomics, genetic screens, Chip-seq.

 

Dr Jenny Pell: Regulation of adult stem cell fate by signalling and epigenetic mechanisms


The health of adults depends in large part on the function of adult stem cells to maintain
tissue homeostasis. These fulfill two functions: i, differentiation along a specific lineage to
repair and regenerate tissue and ii, renewal of the stem cell population. The balance of these
is essential; if self-renewal is favoured, then tumours can result but insufficient self-renewal
will result in tissue degeneration, as in ageing.
Stem cell fate is dictated by extrinsic signals from the microenvironment (soluble or
physical), or intrinsic (from within the cell). Both will result in modifications of epigenetic
‘marks’ and ultimately control chromatin structure, gene expression and cell phenotype. In
our lab we investigate signalling pathways that regulate epigenetic fate restriction (see
Woodhouse et al 2012 J Cell Sci 126:565. Ezh2 maintains a key phase of muscle satellite cell
expansion but does not regulate terminal differentiation; Brien et al 2013 Stem Cells In press.
p38α MAPK regulates adult muscle stem cell fate by restricting progenitor proliferation
during postnatal growth and repair). We have developed elegant experimental systems that
combine the strengths of genetically modified animals and the isolation of primary cells with
sophisticated cell signalling/imaging techniques, next generation sequencing and regeneration
models in order to recapitulate in vivo physiology as faithfully as possible.
Our particular interests at present focus on PRC2 (polycomb repressive complex 2; catalyses
histone H3K27me3)-mediated gene silencing; the importance of p16Ink4a in PRC2 action;
signalling to regulate PRC2 and p16 by the p38 family of MAPKs (possible tumour
suppressors), importance of histone H3 phosphorylation.
This project will expose students with state-of-the-art molecular, cellular and in vivo animal
genetic techniques, including next generation sequencing approaches such as ChIP-seq and
RNA-seq. By the end of the project, the student will be equipped to make informed decisions
of future PhD choice. The ethos in our lab is to provide each member with a unique yet
complementary project so that all can work together in a constructive environment.
Dr Pell is an Affiliated Member of the Wellcome-MRC Cambridge Stem Cell Institute which
provides an excellent and thriving science environment, complementing that in
Pharmacology. Informal enquiries/visits are most welcome.
Key words: stem cell, cell signalling, epigenetics, myoblast, ChIP-seq, RNA-seq

 

Dr HW van Veen: Multidrug transporters: from microorganisms to man

We live in an era when drug resistance has spread at an alarming rate and when dire predictions concerning the lack of effective antimicrobial and anticancer drugs occur with increasing frequency. One very powerful mechanism of drug resistance in microorganisms and human cells is based on the transport of the drugs out of the cell by integral membrane proteins with an extraordinarily broad drug specificity. In this project, we ask a few simple questions about these multidrug transporters: how do they work; how can these systems be inhibited; what is their origin.

We will study the binding and transport of cytotoxic drugs by the clinically important ATP-binding cassette transporter PatAB in Streptococcus pneumoniae, which is an important cause of community-acquired respiratory tract infections. We will extend our investigations to the ion-coupled MATE transporter NorM from Vibrio cholerae. Homologues of NorM are essential for the detoxification of metabolic products in plants and for the excretion of toxins by the liver and kidney in mammals. Our studies of NorM can therefore provide intricate details regarding the properties of MATE transporters in many kingdoms in life. Both NorM and PatAB will be studied while expressed in a non-pathogenic host. As reliable structure models of both transporters are available, we can examine detailed structure-function relationships using structural biology as a basis. We will apply a variety of techniques in the areas of biochemistry, electrophysiology, molecular biology, and structural biology.

Key words: Antibiotic resistance, membrane transporters, drug efflux, drug recognition, mechanisms of transport, multidrug transport.

 

Dr E Smith: The impact of tissue acidosis on neuronal excitability

Homeostatic control of pH is vital to normal bodily function. We are interested in examining the mechanisms by which neurones detect pH changes with a focus on two main areas, pain and control of breathing:

1) A subset of sensory neurones detects purely noxious stimuli, so-called nociceptors, but how they are activated by acid is not fully understood. Painful inflammatory conditions, such as rheumatoid arthritis, are associated with tissue acidosis, which is likely a key component in driving pain. We use a combination of molecular biology, immunohistochemistry and whole-cell electrophysiology techniques to examine: how nociceptors respond to acid, the nature of the ion channels involved and the impact of inflammation upon nociceptor excitability.

2) Changes in CO2 concentration modulate breathing, possibly due to CO2-evoked changes in pH. The naked mole-rat has adapted to living in a high CO2/low O2 environment and we will employ a comparative physiology/molecular biology approach to learn how naked mole-rats deal with living in such extreme conditions. By understanding how organisms are adapted to living in extreme conditions it is possible to learn more about how organisms living in “normal” conditions function.

Techniques used include: molecular biology (RT-PCR, mutagenesis etc.), cell culture, transfection, immunohistochemistry and whole-cell electrophysiology.

Key words: pain, nociception, acid, neurobiology, ion channels, breathing

 

Professor CW Taylor: Structure and function of intracellular Ca2+ channel

How does Ca2+, the simplest of all intracellular messengers, selectively regulate so many cellular activities? Projects in my laboratory address this question be attempting to define how the behaviour of intracellular Ca2+ channels, notably IP3 receptors, and the organelles within which they reside lead to complex changes in intracellular Ca2+ concentration. We apply the most advanced optical and electrophysiological methods to explore, often at the single-molecule level, the structural basis of IP3 receptor activation and their interactions with other Ca2+-handling proteins. Our work, including that of PhD students, is supported by extensive national and international collaborations with chemists, structural biologists and mathematicians, and with partners in industry.

 Key words: Cell signalling, patch-clamp recording, fluorescent microscopy, ion channel, Ca2+, cyclic AMP.

 

Dr T Rahman: Development of novel pharmacological tools for studying intracellular calcium channel

Calcium is a universal and versatile intracellular messenger, regulating almost every aspect of cellular life. To generate calcium signals, cells derive calcium both from the extracellular space as well as some internal stores, using various calcium permeable ion channels. Of the multiple ways of intracellular Ca2+ mobilisation, the one involving IP3 receptors (IP3R) has possibly the least useful membrane-permeant pharmacology. Activating IP3Rs in intact cells has been made possible due to availability of membrane-permeant IP3 esters that produce IP3 with or without flash photolysis. However, the choice of inhibiting these receptors in intact cells is more limited. The few inhibitors of IP3R that are available, there are concerns over issues like non-specificity, impermeability through cell membrane and unknown mechanism of action. The major aim of my proposed project is to rationally design/explore and develop novel, cell-permeant chemical tools that will allow selective and significant inhibition of IP3R with known mechanism of action. The work will involve primarily in silico approaches (structural modelling and screening) followed by quantitative pharmacological assays.

Key words: Calcium signalling, molecular modelling, virtual screening, ion channel.