Abstracts of research projects offered for October 2016
The research interests of all the members of the Department are available at www.phar.cam.ac.uk/research.
Candidates are advised to read these research interest pages in conjunction with this information.
Dr Matthew Harper:
Platelet heterogeneity in cardiovascular disease
Not all cells behave in the same way, even when they are seemingly of the same type. Heterogeneity in signalling may lead to important biological differences in cell behaviour that may be important in understanding and treating disease.
Platelets play a critical role in arterial thrombosis and myocardial infarction, which is a major cause of death in the UK. During thrombosis, platelets adhere to a ruptured atherosclerotic plaque and become activated. Platelets are short-lived, with a circulating life of 7 – 10 days. Platelets in circulation are a mix of young recently-released platelets, and older platelets. Young platelets are also known as ‘reticulated platelets’. It has been suggested that they are more active and less sensitive to some anti-platelet drugs.
In this project we will study these reticulated platelets. Are they really more active than older platelets? What accounts for their increased activity and drug resistance? Do they play a special role in thrombosis? Are reticulated platelets a potential drug target? To answer these questions, we will use a range of approaches including flow cytometry, fluorescence imaging, protein biochemistry and proteomics.
Keywords: cardiovascular disease; heart attack; cell signalling; cell heterogeneity; thrombosis
Rheumatoid arthritis (RA) is a common, chronic autoimmune disorder resulting in joint inflammation with pain and swelling. Hyperactivity of blood-borne inflammatory cells, such as neutrophils and platelets, has been implicated in progression of the disease and exacerbating joint inflammation. RA sufferers also have an increased risk of arterial thrombosis and heart attacks. This crosstalk between joint inflammation and cardiovascular disease presents a major clinical problem and a major therapeutic opportunity.
This project will take place jointly between labs specialising in arthritis (Dr Smith) and thrombosis (Dr Harper) in the Department of Pharmacology. The student will receive expert training in a wide range of techniques, including in vivo and in vitro disease models, electrophysiology, molecular biology, flow cytometry and imaging.
Keywords: sensory neuron; inflammation; arthritis; cardiovascular disease; thrombosis
Dr Robert 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 Laura Itzhaki:
Tandem-repeat proteins: Folding, function, role in disease and therapeutic intervention
The major focus of our research is a class of proteins with very distinctive architecture, known as tandem-repeat proteins (e.g. ankyrin, tetratricopeptide and armadillo repeats). These proteins are frequently deregulated in human diseases such as cancers and respiratory and cardiovascular diseases. The individual modules of repeat proteins stack in a linear fashion to produce highly elongated, superhelical structures, thereby presenting an extended scaffold for molecular recognition. The term ‘scaffold’ implies a rigid architecture; however, as suggested by their Slinky spring-like shapes, it is thought that repeat arrays utilise much more dynamic and elastic modes of action. For example: stretching and contraction motions to regulate the activity of a bound enzyme; reversible nanosprings to operate ion channels; proteins that wrap around their cargoes to transport them in and out of the nucleus. The modular architecture of repeat proteins makes them uniquely amenable to the dissection of their biophysical properties as well as the rational redesign of these properties. We are interested in understanding how the process of folding and unfolding of this distinctive protein class directs their functions in the cell. We are also looking at small molecule and peptide-based approaches to target these proteins for therapeutic benefit; examples include the development of inhibitors of ankyrin-repeat proteins gankyrin for the treatment of liver cancer and inhibitors of tankyrase for the treatment of breast cancer. Lastly, we are exploiting the design-ability of repeat proteins with the goal of creating artificial proteins with applications in medicine and nanotechnology.
Research in our group is at the interface between biology and chemistry; we also have close collaborations with computational groups and synthetic chemistry groups in Cambridge, and therefore students will be able to learn a broad range of techniques and approaches, including protein engineering, biochemistry and biophysical analysis including single-molecule techniques, cell biology and medicinal chemistry.
Keywords: protein engineering, biophysics, cancer, protein folding, tandem-repeat proteins
Dr Graham Ladds:
The role of cellular chaperones in receptor-signalling bias
G protein-coupled receptors (GPCRs) form the largest protein family in the human genome with ~30% of marketed drugs targetting these receptors. The secretin-like, or family B GPCRs, form a group of 15 receptors that are important therapeutic targets for diabetes, cardiovascular disease, bone disorders, inflammatory pathologies and migraine. Given their therapeutic potential, family B GPCRs have attracted considerable interest from the pharmaceutical industry. However, it has proven difficult to develop useful drugs against these receptors. Family B GPCRs typically show coupling to a range of effectors. Although the best-characterised signalling pathway is to Gαs (for cAMP production), coupling has also been reported to Gαo, Gαi, Gαq and Gαz as well as β-arrestins. Many of the receptors bind multiple ligands and signalling pathways activated are often dependent on the ligand; termed signalling bias. Moreover, it has become apparent that some secretin-like receptors also associate with receptor activity-modifying proteins (RAMPs) and these may influence ligand-specificity for the receptor, thereby modulating their pharmacology. In this project we aim to determine the molecular role that RAMPs perform in modulating signalling bias of secretin-like receptors.
Keywords: Cell signalling, GPCRs, signal bias, RAMPs, arrestins, G proteins
Dr Catherine Lindon:
Ubiquitin-mediated signalling in cell division
Cellular proteostasis depends on ubiquitin-mediated targeting of proteins for destruction at the proteasome, and is a critical element in cell cycle control.
PhD projects are available
(1) to investigate the dynamic relationship between destruction and function of the Aurora kinases, important cell cycle regulators and common drivers in cancer
(2) to identify sites of ubiquitin modification in substrates, and the nature of the ubiquitin chains they are modified with, to further understanding of “the ubiquitin code"
(3) to decipher ubiquitin pathways controlling specific substrates (such as Aurora kinases) throughout the cell cycle
These projects will involve time-lapse imaging of living cells to measure substrate proteolysis and localization, and to examine the functional outcomes of ubiquitination, alongside biochemical strategies to ‘capture’ ubiquitination events in different cell cycle phases. Students will receive training in a wide range of cell culture and molecular cell biology techniques, cellular imaging and quantitative image analysis.
Keywords: Cell cycle, mitosis, ubiquitin, cancer
Dr Ewan Smith:
The molecular basis of sensory neurone function in pain
Pain is initially triggered by activation of sensory neurones. Under basal conditions, a subset of sensory neurones is only activated by noxious stimuli: nociceptors. In recent years it has become clear that sex has a significant impact how pain is perceived and how it is processed in the body. Similarly, it has been shown that neonatal injury can have long-lasting impact on sensory neurone function.
Projects will be developed to investigate the impact of sex and neonatal injury on nociceptor function with a specific focus on ion channel expression and properties.
Techniques used include: molecular biology (RT-PCR, mutagenesis etc.), neuronal culture, immunohistochemistry, real-time fluroescence imaging and whole-cell electrophysiology, and behaviour.
Keywords: pain, nociception, acid, neurobiology, ion channels
Prof. Colin Taylor:
Structure and functions of Ca2+ channels within dynamic organelles
How does Ca2+, an element and the simplest of all intracellular messengers, selectively regulate so many cellular activities? We address this question by examining how the behaviour of intracellular Ca2+ channels, notably IP3 receptors, and the organelles in which they reside lead to complex changes in intracellular Ca2+ concentration. We are exploring the structural basis of IP3 receptor gating and the contribution of dynamic intracellular organelles to shaping cytosolic Ca2+ signals. The ER, within where most IP3 receptors reside, forms intimate contacts with most other organelles. Dynamic regulation of these interactions may be as important as receptor-regulated formation of IP3 in determining how cells generate and respond to cytosolic Ca2+ signals. Furthermore, IP3 receptors move within the ER. We do not understand the relationships between these dynamic IP3 receptors and the much smaller number of immobile IP3 receptors that appear to generate most Ca2+ signals. What licences immobile IP3 receptors to respond? We apply super-resolution optical and electrophysiological methods alongside gene-editing with CRISPR/cas9 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 organelles. We apply these methods to both 'work-horse' cells like HEK293 cells, and to primary cells: bronchial and vascular smooth muscle (cAMP/Ca2+ interactions controlling contraction), astrocytes (spatial organization of intracellular ATP provision) and glioma cells (migration). 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.
Keywords: Cell signalling, patch-clamp recording, super-resolution microscopy, gene-editing, ion channel, Ca2+, cyclic AMP, intracellular organelles, smooth muscle, astrocyte, glioma.
Dr Hendrik van Veen:
Mechanisms of multidrug transport in microorganisms and cancer cells
Multidrug transporters mediate the extrusion of a broad range of drugs from the cell, away from intracellular targets on which these drugs act. These membrane transporters modulate the toxicity and pharmacokinetics of drugs in organisms ranging from bacteria to man, and are a cause of drug resistance in pathogenic microorganisms and cancers. Using structural and biochemical tools, we work on interesting questions: how are drugs recognised by multidrug transporters and how is metabolic energy coupled to transport? Is drug transport the primary physiological role of these systems? Can we use novel mechanistic insights to generate inhibitors and new drugs that bypass recognition and thus improve the drug-based treatment of diseases? Please, contact Dr Hendrik van Veen to discuss the projects offered.
Keywords: Antimicrobial and anticancer drug resistance, membrane transporters, drug efflux, drug recognition, mechanisms of transport, multidrug transport.
Rational drug design
As a research lab, we are essentially 'small molecule-centric'. We employ state-of-the art in silico approaches coupled with prospective, complementary wet experiments (calcium imaging, electrophysiology, biochemical and biophysical assays) to know the molecular mechanisms underlying the ligand recognition, ligand modulation, structural variation and evolution of some calcium-permeable ion channels. We are pursuing rational development of novel chemical probes/modulators of these ion channels. Additionally, we are actively engaged in collaborative research for rational development of novel chemical scaffolds/leads against some proteins implicated in microbial infections, cancer, pain and diabetes. The projects are quite interdisciplinary and will allow the student(s) learn a range of computational and chemical biology techniques.
Keywords: structure- and ligand-based drug design, protein structure modelling, molecular dynamics simulations, drug repositioning and calcium signalling.