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Recipients:
University College London

Results

Structural and Functional characterisation of chlamydial inclusion proteins. 25 Jun 2012

The project aims to study the structure and function of two chlamydial inclusion proteins. Chlamydia is a major human pathogen, causing sexually transmitted disease and trachoma (a form of blindness). To replicate inside host cells, chlamydia build a specialised intracellular compartment called an inclusion, which is segregated from the host endocytic system but selectively engages key host organelles and secretory traffic. To achieve this, Chlamydiae deliver a family of hydrophobic inclusion proteins (Incs) into the inclusion membrane, but very little is kown about their structure and function. Exploiting our ability to purify these membrane proteins, the project aims to fully understand i0 the bases of membrane tubulation by IncC, ii) the nature of the novel endoplasmic reticulum retention signal within IncB, iii) the host targets if IncC and IncB and their roles during bacterial infection. the arising data will allow new insights into the molecular mechanism of this medicallu important pathogen.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Modulation of the folding energy landscape of a nascent polypeptide by interactions with the ribosome surface. 25 Jun 2012

He we propose identifying and developing a fast-folding protein domain as the new ribosome-nascent chain complex (RNC) system, and using a series of destabilising mutations to determine the effect of ribosome attachement on the foding eneregy landscape. Key goals in this project are the establishment of a suitable RNC system; development of NMR methodology for the study of surface interactions; and ultimately measurement of the effect ofribosomal attachment on the protein folding energy landscape.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Biophysical and structural investigations of DNA repair modulation by viruses. 25 Jun 2012

Viral proteins subvert various cellular processes to optimise the propogation or stable sojourn of virus genomes. Viral stealth and replication strategies can be compromised by uracil-DNA glycosylase (UDG) activity, making UDG a kown target of viruses infecting both prokaryotes and eukaryotes. In viruses of prokaryotes, UDG is silenced by the diverse protein inhibitors: a deeper investigation of structural diversity in this realm would benefit methodology in low homology bioinformatics, our understanding of protein evaluation, and inform the development of novel inhibitors of DNA repair enzymes. In human HIV-1 infections, UDG is targeted to the proteosome by association with the viral accessory protein Vpr. As a strategy for dealing with incoming virus genomes, it is possible that the UDG's ancillary role in somatic hyper-mutation may be reprised in combination with the anti-viral deoxycytidine deaminase, APOBEC3g (A3G). The HIV-1 accessory proteins Vif (targeting A3G), and Vpr, are therefore appealing anti-viral targets, and therapeutic molecules could likely be developed through biophysical and structural knowledge to their respective interactions. We aim to develop systems to identify novel viral predictors of UDG and to produce viral proteins using Vpr [and Vif with relevance to A3G] stably for biophysical and structural analyses via NMR and x-ray crystallography.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

The Role of Exported Malaria Parasite Proteins in Pathogenesis. 25 Jun 2012

Understanding the molecular mechanism of protein export by the malaria parasite and the role of exported proteins in parasite survival.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

The Secret Life of Proteins - Computational and Experimental Studies of Moonlighting Proteins. 25 Jun 2012

The aim of the project is to further our understanding of protein moonlighting (multiple unrelatedfunctions of a single protein). During this project we will explore a number of questions that relate to the molecular mechanisms and properties that allow or enhance the moonlighting potential of a protein. the project will concentrate on computational approaches wit the aim of producing testable predictions, which we then hope to veryify with experimental studies.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Development of diencephalic asymmetries in zebrafish and chick embryos. 25 Jun 2012

While most of the body is symmetrical with respect to the midline, a few functions, including higher order behaviours and cognitive functions in the brain, have evolved to be concentrated on one (left or right) side. Although the molecular mechanisms underlying left/right asymmetry of body organs are now fairly well understood, we still know little about how lateralised brain functions arise during development. Recent research has discovered that the parapineal, an asymmetrically positioned group of neurons in the left diencephalon, is essential for development of asymmetries in the adjacent epithalamus, but little is known about the molecular mechanisms of this regulation. Also, no region equivalent to parapineal has yet been discovered in most vertebrates including birds and mammals, whereas the molecular pathways leading to epithalamic asymmetries are likely to be conserved. This project explores how epithalamic asymmetries develop in two different species - zebrafish and chicken, first by establishing the molecular mechanisms by which the parapineal regulates this process in zebrafish, then by studying

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

The role of c-Jun in controlling the repair-supportive phenotype of Schwann cells in injured nerves. 25 Jun 2012

Work in the Jessen and Mirsky laboratory, using a mouse in which the transcription factor c-Jun has been inactivated in Schwann cells only (c-Jun-cKO mouse), shows that the Schwann cell response to nerve injury depends on activation of the transcription factor c-Jun in Schwann cells, and that this protein specifies the phenotype of the Bunger repair cell, a cell essential for nerve regeneration. Consequently, nerve repair is severely compromised in c-JuncKO mice. This project will address the following issues: 1) Establish an in vitro model of the regeneration deficit in c-Jun-cKO mice, using adult DRG neurons and Scwann cells from injured nerves. 2) Use this model to analyse Schwann cell factors that control axon growth and neuronal survival. 3) Test whether axonalregeneration and neuronal survival can be improved by enhancing Schwann cell c-Jun expression. 4) Determine whether the diminishing ability of distal nerve tosupport repair with time after injury ( the deterioration of the distal stump ) is due to instability of the Bungner repairsupportive phenotype, and its gradual attenuation. 5) Test whether the repair-supportive Schwann cell phenotype

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Morphogenesis underlying choroid fissure fusion. 25 Jun 2012

Coloboma is a defect in the morphogenesis of the eye that results from failureof choroid fissure closure. It is among the most common congenital defects in humans and can significantly impact vision. However, very little is known about the developmental mechanisms regulating choroid fissure fusion. Therefore, I aim to resolve the cellular and molecular mechanisms underlying choroid fissure closure by high-resolution 4D confocal imaging of zebrafish retinal cells during fusion. In particular, I will investigate how cell cycle progression regulates the epithelial remodelling that accompanies fusion.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

PDGF as a cell autonomous regulator of Epithelial-to-Mesenchymal-Transition (EMT) in neural crest cells. 25 Jun 2012

A defining characteristic of neural crest (NC) cells is the epithelial-to?mesenchymal transition (EMT) they undergo to segregate from the neural tube to start migration. EMT is a cellular process converting non-motile epithelial cells to motile mesenchymal cells, showing strikingly common characteristics in metastatic cancer cells and NC cells. Preliminary data suggests that PDGF signalling is required cell-autonomously for NC cell migration in Xenopus laevis embryos whereas PDGF loss-of-function is sufficient to inhibit EMT in in vitro cultures. The proposed project aims to investigate cellular and Molecular mechanisms governed by PDGF during EMT of NC and to extrapolate the gained knowledge onto cancer cell metastasis. We will perform high-resolution time-lapse video analysis of NC EMT comparing gain-of-function and loss-of-function of PDGF in vivo and in vitro. Further study will aim to identify the pathways and downstream targets triggered by PDGF signalling. Finally the gained knowledge will be used to study EMT in cancer cell lines and a transparent zebrafish model allowing the live-imaging

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Blastema formation and skeletogenesis during arm regeneration of the brittle star Amphiura filiformis: cellular and molecular characterization. 25 Jun 2012

The aim of this research project is to understand the initial stages of brittle star arm regeneration in terms of stem cell involvement, cell specification and the earliest activation of the skeletogenic gene regulatory network. The brittle star is a marine organism with a unique capability for regenerating whole arms post-amputation or after injury. To determine whether the regenerative blastema, a mass of proliferative cells giving rise to the entire structure, is composed of stem cells or dedifferentiating cells, molecular tools will be employed for their characterization. Stem cell markers and lineage tracing techniques will be used to identify the nature of the cells, their origins and migratory behaviour. The regenerating arm of the brittle star is contains several skeletal structures and the second aim of this project is to understand the cohort of signalling pathways involved in the early specification of the cell lineages which will develop into this adult tissue. This will be achieved by using molecular techniques and a candidate gene approach for studying the genes that have already been well-characterised in the closely-related sea urchin, , for which a complete gene regulatory network for the embryonic development of skeletogenic cells has been published.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Probing the inhibitory neurosteroid binding site on the GABAA receptor 25 Jun 2012

Neurosteroids are naturally occurring potent modulators of type A GABA receptors in the brain. Although there are many neurosteroid metabolites, these can be characterised into two distinct classes - those that potentiate GABAA receptor function and those that inhibit. Previously we deduced where potentiating neurosteroids bind on the GABAA receptor, but the inhibitory neurosteroids remain unaffected if this site is disrupted. This concurs with the belief that such inhibitory neurosteroids bind to another discrete site onthe GABAA receptor. Its discovery will allow the function of the inhibitory neurosteroids to be explored in the brain. We will select a GABA receptor thatlacks sensitivity to inhibitory neurosteroids and use this structure in combination with GABAA receptor subunits to make chimeric and eventually pointmutated receptors to identify the inhibitory neurosteroid binding site. Once the site is found, we will disrupt its function and then observe the consequences for inhibitory synaptic and tonic inhibition. Overall, this studywill use structural, electrophysiological, imaging, pharmacological and molecular approaches with GABA receptors. This study will bring much need clarity to the role and importance of inhibitory neurosteroids in the brain.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Single neuron connectomics and functional mapping in visual cortex 25 Jun 2012

Understanding how cortical connectivity organizes cortical computations is a long-standing challenge in neuroscience. I propose to investigate the relationbetween circuit connectivity and circuit function in the primary visual cortex(V1). To this end, I will deploy a novel monosynaptic retrograde tracing strategy based on rabies virus to identify and functionally characterize the network of neurons that connect to a given postsynaptic V1 neuron. This network constitutes the neuron's "presynaptic connectome": it determines the neuron's functional responses and coding strategy. To measure and manipulate neuronal activity with single cell resolution, I will combine this approach with two-photon imaging of genetically encoded calcium indicators, with optogenetics, and with single-cell patch clamp recordings. My first aim is to establish how cortical connectivity governs sensory representation by population of neurons. To what extent do synaptic connections in V1 determine which neurons will fire together in response to sensory input? Recurrent connectivity in V1 microcircuits may constrain population responses to stereotyped patterns of activation (sensory representations) coding for specific features in the visual scene. My second aim is to elucidate how receptive field properties of V1 neurons originate from the interplay between neuronal microcircuits, and within them, from synaptic interactions among different cell classes.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Development, plasticity, energy supply and pathology of myelinated axons 25 Jun 2012

The white matter of the CNS allows rapid transmission of information at low energetic cost. Despite its importance for brain function, the development, plasticity, energetics and pathology of the white matter are poorly understood. We will investigate, in vitro in brain slices and in vivo in zebrafish: (1) how electrical activity in oligodendrocyte precursors regulates myelination; (2) mechanisms underlying the tuning of conduction speed of myelinated axons; (3) mechanisms localising mitochondria in oligodendrocyte lineage cells; (4) how microglia survey the white matter in health and disease.

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Mechanisms of grid cell processing: interactions at the network level 25 Jun 2012

The primary goal of this project is to establish the role of cholinergic signalling in the modulation of grid cell firing patterns. In particular, to study the relationship between Acetylcholine concentration in the Entorhinal Cortex and grid scale. Acetylcholine is a putative signal of novelty and uncertainty, and has been shown to affect the theta-band frequency of grid cell oscillations in-vitro. In parallel, grid scale is known to increase in novel situations. As such, we aim to demonstrate a role for Acetylcholine and its effects on theta-firing as a mechanism underlying changes in grid scale during situations of novelty and uncertainty. Examination of the biological basis of the rodent cognitive map is perhaps themost promising available model system in which we can bridge the gap from behaviour and cognition to neurons and circuits. Understanding how spatial firing in the entorhinal cortex and hippocampus is generated and modified in novel environments would be an important extension of our knowledge of spatialcognition, and more generally to our understanding of the process of memory formation

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Glial regulation of neural signalling in health and disease with an emphasis on understanding its possible role in psychotic-like states 25 Jun 2012

Astrocytes respond to neurotransmitters released from neurons with a rise of [Ca2+]i. It is becoming increasingly clear that these [Ca2+]i rises can in turn evoke neurotransmitter release from astrocytes, which modulates neuronal function, and that microglial cells can regulate this signalling from astrocytes. We will use patch-clamping, calcium imaging and calcium uncaging in brain slices, to examine the following aspects of these newly discovered signalling pathways, which have been little studied and which may be relevant to understanding both the normal function of the CNS and the changes of function which lead to psychotic-like states. (1) How do the amine transmitters serotonin, dopamine and noradrenaline modulate astrocyte [Ca2+]i and thus synaptic currents in nearby neurons? (2) How do the main excitatory and inhibitory transmitters glutamate and GABA modulate astrocyte [Ca2+]i and thus synaptic currents in nearby neurons? (3) How do endocannabinoids modulate astrocyte [Ca2+]i and thus synaptic currents in nearby neurons? (4) How do agents known to induce psychotic-like states, such as amphetamine, LSD, ketamine and exogenous cannabinoids, modulate astrocyte [Ca2+]i and thus synaptic currents in nearby neurons? (5) What is the role of ATP and adenosine in the above phenomena? (6) How do microglia regulate this signalling?

Amount: £162,047
Funder: The Wellcome Trust
Recipient: University College London

Mechanisms and plasticity of dendritic temporal sequence processing. 13 Feb 2012

The mechanism by which neurons detect, organize and process numerous chemical and electrical signals is a fundamental problem in modern neuroscience. For example, the ability to discriminate spatiotemporal sequences of synaptic input has been suggested to be important for generating efficient coding schemes, and input sequence detection in general is a computation essential for animal behaviour. Previous work showed that dendrites could be responsive to the "direction" of temporally spaced inputs due to their cable filtering properties, suggesting that single neurons could act as basic sequence detectors. This "passive" direction selectivity was confirmed experimentally by Branco and colleagues, who also discovered that "active" NMDA receptor conductance recruitment dramatically enhances the direction sensitivity of somatic responses. In addition, Branco et al. 3 found that dendrites could discriminate not only directional sequences, bu1 also a wide range of spatiotemporal patterns. However, the role and impact of dendritic sequence processing is unknown for computation in different cortical areas and under different conditions. I plan to concentrate on two broad questions addressing how dendritic mechanisms can optimally discriminate sequences and potentially store selected input patterns. First, I will concentrate on the types of spatiotemporal patterns that can be distinguished from each other. Second, I will investigate whether specific input sequences can be stored, by probing plasticity processes triggered by different input patterns. Optimal conditions for dendritic pattern recognition. What is the optimal spatial distribution of inputs? What is the "optimal dendrite"? Interaction of patterns on the dendrite. The interaction of backpropagating action potential and patterned inputs. Influence of background noise. Sources of calcium ? pharmacology. Tthe effects of neuromodulation. Plasticity triggered.by patterned input. Plasticity induction readouts. Plasticity induction protocols - repetition of the same input pattern and pattern and action-potential combination Effect of neuromodulation in regulating of pattern-induced plasticity.

Amount: £14,356
Funder: The Wellcome Trust
Recipient: University College London

Olig2 and the regulation of neural stem cell fate. 27 Jan 2012

During development of the central nervous system (CNS), neuroepithelial stem cells (NSCs), residing in the ventricular zones (VZ) of the embryonic brain and spinal cord, divide and differentiate to generate all the neurons and glial cells (astrocytes and oligodendrocytes) of the mature CNS. Typically, neurons form before glia. In the ventral spinal cord, for example, embryonic NSCs of the pMN progenitor domain generate several subtypes of motor neurons (MNs) before switching abruptly (at E12.5 in mouse) to production of oligodendrocyte precursors (OLPs). The OLPs then migrate away from the VZ into all parts of the spinal cord before associating with axons, differentiating into post-mitotic oligodendrocytes (OLs) and synthesizing myelin. We aim to address how NSCs switch at a predetermined time from neuron to glial production ? specifically, the mechanism of the MN-OLP fate switch in the ventral spinal cord. Broadly, the proposed project aims to characterize the role and regulation of the basic helix-loophelix transcription factor Olig2 in the MN-OLP fate-switch. Recent work from the Richardson lab showed that a specific serine residue (S147) in Olig2 is phosphorylated during MN specification and de-phosphorylated at the switch to OLP production. What triggers de-phosphorylation of Olig2 at the time of the MN-OLP switch? The sequence surrounding S147 conforms to a protein kinase-A target site, but the identity of the putative phosphatase responsible for dephosphorylation has not been established. What are the targets and co-factors of Olig2 in its different phosphorylated states that coordinate the temporally-defined switch in NSC fate? And what are the functions of the other predicted Olig2 phosphorylation states? This project will involve three distinct lines of investigation. 1: Characterizing the expression of candidate phosphatases/phosphatase inhibitors in the ventral spinal cord at the time of the MNOLP fate-switch. 2: Identifying target genes of Olig2 in its phosphorylated and de-phosphorylated states. 3: Characterizing the developmental function of another Olig2 phosphorylation site, S263, a potential target of p38 mitogen-activated protein kinase (MAPK).

Amount: £31,882
Funder: The Wellcome Trust
Recipient: University College London