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Abstracts of research projects available for October 2015

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

 

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 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, fluorescent pH-imaging 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, real-time pH imaging and whole-cell electrophysiology.

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

 

Professor CW Taylor: Structure and functions of dynamic intracellular Ca2+ channels

How does Ca2+, the simplest of all intracellular messengers, selectively regulate so many cellular activities? Projects in my laboratory address this question by 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 are exploring both the structural basis of IP3 receptor gating and the contribution of dynamic intracellular organelles to shaping cytosolic Ca2+ signals. It is now clear that the ER, within which most IP3 receptors reside, forms intimate contacts with most other intracellular organelles. Dynamic regulation of these interactions may be as important as the receptor-regulated formation of IP3 in determining how cells generate and respond to cytosolic Ca2+ signals. We apply super-resolution optical and electrophysiological methods in combination with gene-editing of native signalling proteins to explore, often at the single-molecule level, the structural basis of IP3 receptor activation and the dynamics of Ca2+-handling organelle. 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, super-resolution microscopy, ion channel, Ca2+, cyclic AMP.