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 David Belin:
(1) Role of the anterior insular cortex in impulsivity and the associated vulnerability to develop compulsive disorders
The existence of specific somatic states associated with different emotions, as proposed by the somatic marker hypothesis, has long remained controversial. In the last decade human studies have revealed that interoceptive mechanisms contribute to subjective feeling, thereby influencing executive functions such as impulse control and decision-making. Dysregulation of interoceptive awareness has been suggested to contribute to several neuropsychiatric disorders including drug addiction, obsessive-compulsive disorder and anxiety. It is therefore of primary importance to better understand the neurobiological mechanisms supporting interoceptive influences over perception of somatic states and executive functions. At the neurobiological level, activation of the anterior insula in humans has been correlated with interoceptive awareness and associated emotional processes alongside impulse control and decision making. However, the neurocircuitry whereby the insula may support interoceptive control over executive functions and associated vulnerability to develop compulsive disorders remains poorly understood. This may stem from the fact that interoceptive awareness and its influence over executive functions has so far been characterized only in humans, thereby limiting causal investigations of neural networks. Rats offer the opportunity to causally identify the neural substrates of complex psychological mechanisms such as impulse control and decision-making. However, if rats show both conditioned responses to interoceptive cues and interoceptive awareness, whether they are capable of using interoceptive awareness to control executive functions remains unknown. Functional interactions between the insula and the nucleus accumbens shell and core, the basolateral amygdala or the anterior cingulate cortex have been suggested to subserve its influence over executive functions. Using a unique combination of pathway specific opto- and chemogenetic manipulations in freely moving rats we will identify the respective contribution of each of these pathways to interoceptive awareness and its control over executive functions and then investigate their respective contribution to impulsivity and associated vulnerability to compulsive disorders.
Key words: experimental psychology, neuropharmacology, impulsivity, addiction, Obsessive Compulsive Disorder, schedule-induced polydipsia, chronic self-administration, insula, circuit mapping, in situ hybridisation, qPCR, western blot, optogenetics, DREADDs
Dr David Belin:
(2) Role of noradrenergic mechanisms in the nucleus accumbens shell in impulse control and compulsivity
Impulsivity represents an endophenotype of vulnerability to develop compulsive disorders such as drug addiction and Obsessive Compulsive Disorder (OCD), which place a heavy burden on modern societies. However, the neural mechanisms whereby impulsivity contributes to an increased vulnerability to develop compulsive behaviours remain unknown, thereby preventing the development of new effective therapeutic strategies. We have recently demonstrated that the selective noradrenaline reuptake inhibitor atomoxetine prevents the transition from impulsivity to compulsivity in highly impulsive rats. The aim of this project is to investigate the contribution of the noradrenergic innervation of the nucleus accumbens shell (AbcS) from the nucleus tractus solitarius (NTS) to impulsivity and the associated increased vulnerability to develop OCD- and addiction-relevant compulsive behaviours in the rat. The key goals of this project are (i) to demonstrate that noradrenergic mechanisms in the AcbS are involved in the well established expression of compulsivity as measured using schedule-induced polydipsia (SIP), an established model of compulsivity, (ii) identify noradrenergic correlates of impulsivity and compulsivity in the AcbS and (iii) demonstrate that specific chemogenetic and optogenetic manipulation of the NTS-AcbS pathway influences impulsivity, compulsive adjunctive behavior under SIP and compulsive relapse to cocaine seeking in rats.
Key words: experimental psychology, neuropharmacology, impulsivity, addiction, Obsessive Compulsive Disorder, schedule-induced polydipsia, chronic self-administration, nucleus tractus solitarious, nucleus accumbens, noradrenaline, in situ hybridisation, qPCR, western blot, optogenetics, DREADDs
Dr Matthew Harper:
Platelet heterogeneity in cardiovascular disease
Platelets are essential to normal haemostasis but also 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. Most activated platelets aggregate together but an important subpopulation instead become pro-coagulant. They interact with the coagulation cascade and generate a burst of thrombin that is responsible for forming a stable, occlusive thrombus.
In this project we will study these pro-coagulant platelets. What intracellular and extracellular signals determine whether a platelet aggregates or becomes pro-coagulant? How do pro-coagulant platelets contribute to thrombosis? Can we prevent platelets becoming pro-coagulant, and would this reduce thrombosis? We will use a range of approaches including flow cytometry, fluorescence imaging, protein biochemistry and proteomics.
Keywords: cardiovascular disease; heart attack; Ca2+; cell signalling; cell heterogeneity; cell death
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.
Key words: 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
We study cell division (mitosis) in human cells, to better understand the contribution of ubiquitin-mediated signaling. PhD projects are currently available to investigate (1) the dynamic relationship between ubiquitin-mediated proteolysis and function of mitotic kinases Aurora A and Aurora B, well-known targets of the anaphase promoting complex (APC/C) ubiquitin ligase at the end of mitosis and common drivers of cancer, and (2) the role of ubiquitination in the dynamic behaviour of nuclear lamins during assembly of the nuclear envelope after mitosis.
These projects will rest on 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 cell division. Students will receive training in a wide range of cell culture and molecular cell biology techniques, cellular imaging and quantitative image analysis.
Key words: Cell division, 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.
Three possible projects are as follows:
1) How do other cell types influence sensory neurone function during disease? Sensory neurones do not exist in empty space, but are embedded in tissues, such as the synovium, skin and muscle – how do these cell types influence sensory neurone function under physiological and pathophysiological conditions?
2) How does neonatal injury influence the development of sensory neurone function? Neonatal injury is known to influence development of the nociceptive system and pain in later life. To what extent does neonatal injury influence the function of primary sensory neurones?
3) How do sensory neurones regulate intracellular pH? Regulation of intracellular pH is critical to maintain normal cellular function and an increase in intracellular proton concentration can generate action potentials. Inflammation is associated with tissue acidosis – how does this influence regulation of intracellular pH in sensory neurones?
Techniques used include: molecular biology (RT-PCR, mutagenesis etc.), cell culture, transfection, immunohistochemistry, real-time pH imaging and whole-cell electrophysiology, and behaviour.
Key words: pain, nociception, acid, neurobiology, ion channels
Prof. Colin 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 organelles. 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, intracellular organelles
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.
Key words: Antimicrobial and anticancer drug resistance, membrane transporters, drug efflux, drug recognition, mechanisms of transport, multidrug transport