Projects

Projects available for MSc Biology of Vision

The final module, occupying half of the duration of the course, consists of an original laboratory-based research or informatics project. 

The current students are involved with some of the projects below.  Click on them for detail.

Please note that these may differ from those available next year.

Role of the MRTFA-SRF pathway in fibroblast to adipocyte transdifferentiation in Thyroid Associated Orbitopathy

Maryse Bailly

Title: Role of the MRTFA-SRF pathway in fibroblast to adipocyte transdifferentiation in Thyroid Associated Orbitopathy

Summary: Increases in orbital connective tissue and fat are responsible for most of the detrimental manifestations of Thyroid Associated Orbitopathy (TAO), as transdifferentiation of orbital fibroblasts into adipocytes leads to the production of fluid-binding glycosaminoglycans, tissue swelling and increased pressure in the orbit. As our previous work on mouse fibroblasts showed that adipogenesis is potentially linked to a down-regulation of the Serum Response Factor (SRF) pathway through decreased expression of its co-activator MRTF-A/MAL, this project aims at testing the relevance of these studies in the context of TAO using primary cell lines established from orbital fibroblasts from patients or normal controls. We will determine the level of expression of MRTF-A/MAL in the different cell lines, their ability to contract collagen gels, as well as the baseline level of activation of MAL/SRF in anchored versus free-floating gels and following serum/drug stimulation. We will then induce in vitro transdifferentiation towards the adipocyte phenotype using well known drug combination, and assess adipocyte commitment using Oil Red O stain to visualise lipid formation, as well as triglyceride production. We expect the patient–derived cell lines to show preferential commitment towards adipogenenesis. We will then test whether altering MRTF-A expression can revert the phenotype of the diseased cells.

Key words: Cell culture, Western blot, gel contraction assays, adipogenesis assays, immunostaining and microscopy, transfection.

No special requirements.

MRTF-A protein (green) is localised in the nucleus where it activates Serum Response Factor in primary human fibrotic orbital fibroblasts. In red, actin filaments showing the fibroblasts’ cytoskeleton.

Cell-cell adhesion in endothelial homeostasis and angiogenic behaviour

Maria Balda

Title: Cell-cell adhesion in endothelial homeostasis and angiogenic behaviour

Summary: Tight junction-associated signalling pathways regulate epithelial behaviour but little is known about the importance of these mechanisms for the regulation of endothelial proliferation, migration, and angiogenesis. Thus, understanding how tight junction-associated transcription factor ZONAB-regulates gene expression of signalling pathways involved in endothelial function and angiogenesis will be important as a basis to develop new endothelial cell-targeting therapies for various diseases. To manipulate ZONAB expression or activity using lentiviral vectors for overexpression and depletion by RNAi in endothelial cells and in such manipulations to test how ZONAB affect endothelial functions and the angiogenic potential. We will determine the role of ZONAB in endothelial cell migration, vascular permeability, proliferation and angiogenesis.

Techniques:

-Primary culture of human microvascular endothelial cells

-Analysis of protein expression and localization by immunoblot and immunofluorescence

-Manipulation of protein expression by RNA interference and viral vectors

-Functional assays such as cell migration, angiogenesis on Matrigel, transepithelial electrical resistance and cell proliferation

Knowledge required: Basic Cell and Molecular Biology as well as Biochemistry and Statistics 

Molecular chaperone function and protein misfolding in retinal degeneration.

Mike Cheetham

Title: Molecular chaperone function and protein misfolding in retinal degeneration.

Summary: Molecular chaperones facilitate and regulate protein conformational change within cells. This encompasses many fundamental cellular processes: including the correct folding of nascent chains; protein transport and translocation; signal transduction and protein quality control. Chaperones are, therefore, important in several forms of human disease, including neurodegeneration. Within the retina, the highly specialized photoreceptor cell presents a fascinating paradigm to investigate the specialization of molecular chaperone function and reveals unique chaperone requirements essential to photoreceptor function. Mutations in several photoreceptor proteins lead to protein misfolding mediated neurodegeneration. The best characterized of these are mutations in the molecular light sensor, rhodopsin, which cause autosomal dominant retinitis pigmentosa. Rhodopsin biogenesis requires chaperones, while rhodopsin misfolding involves molecular chaperones in quality control and the cellular response to protein aggregation. Furthermore, the specialization of components of the chaperone machinery to photoreceptor specific roles has been revealed by the identification of mutations in molecular chaperones that cause inherited retinal dysfunction and degeneration. These chaperones are involved in several important cellular pathways and further illuminate the essential and diverse roles of molecular chaperones. Projects will be based on the role of chaperones in rhodopsin biogenesis and misfolding or investigation of the function of important photoreceptor proteins.

Techniques: A range of contemporary molecular cell biology techniques potentially including; DNA cloning and purification, cell culture, transfection, immunofluorescence, western blotting, sub-cellular fractionation, immunohistochemistry.

The Neuroprotective Effects of Forskolin in Glaucoma

Francesca Cordeiro

Title: The Neuroprotective Effects of Forskolin in Glaucoma

Summary: Glaucoma is the leading cause of irreversible blindness in the world. This chronic and progressive optic neuropathy is characterized by the loss of retinal ganglion cells (RGC) and their axons.  Elevated intraocular pressure is the primary risk factor for glaucoma, responsible for long-term damage to the optic nerve. Patients with glaucoma typically lose their visual field and become blind if untreated. Reduction of intraocular pressure (IOP), the only modifiable risk factor, slows the onset and progression of the disease yet no actual treatment is available to restore optic nerve damage.  Neuroprotection has gained substantial interest in recent years as a therapeutic approach to prevent neuronal degeneration and loss of function in glaucoma.  Coleus forskohlii is a plant used for several years by the Ayurvedic medicine.  The diterpene forskolin is the principal active molecule of that plant and has been shown to process neuroprotective properties.  The primary mechanism of action of forskolin is to directly increase the activity of the enzyme adenylate cyclase and subsequently increase the level of intracellular cyclic adenosine monophosphate (cAMP).  In the eye, forskolin leads to a transitory decrease of levels of IOP and has also been implicated in neuronal survival suggesting its possible application to glaucoma.  Forskolin appears to be neuroprotective via the stimulation of cAMP.  It is known to promote the survival of several neurons including spinal motor neurons, mesencephalic dopaminergic neurons, serotoninergic and central noradrenergical cells.  Forskolin has also been shown to be neuroprotective in the retina, inhibiting RGCs death in excitotoxic models.                     The proposed project will initially look at the neuroprotective efficacy of forskolin in RGC cultures, before attempting to replicate the results in vivo, with a view to publishing.

Key Words: Glaucoma, neuroprotection, retinal ganglion cells, forskolin, cell culture, apoptosis, necrosis, FACS, MTT assay,

Special techniques: none.

Using human embryonic stem cells to create blood vessels

Marcus Fruttiger (1st of 2)

Title: Using human embryonic stem cells to create blood vessels

Summary: The project will be largely based on in vitro cell culture techniques and aims to differentiate human embryonic stem cells into vascular precursor cells. These cells might later be used for therapeutic purposes. In this project the student will test the potential of such cells to form functional, artificial blood vessels in vitro.

Techniques used: Cell culture, FACS, PCR, Immunohistochemistry

No special requirements

The role of immune cells in retinal neovascularization

Marcus Fruttiger (2nd of 2)

Title: The role of immune cells in retinal neovascularization

Summary: Retinal neovascularization occurs in response to ischemia (e.g. in diabetic retinopathy or retinopathy of prematurity). In a mouse model of oxygen induced retinopathy we found that immune cells have the ability to dramatically alter the neovascular response that normally occurs. The project will be aimed at investigating the molecular mechanisms that underlie this phenomenon. Insights gained might have implications for the treatment of human ischemic retinopathies.

Techniques used: Tissue dissection, Immunohistochemistry, qPCR      

Requirements: reasonable dexterity for tissue dissection 

Phagosome maturation in retinal pigment epithelial cells

Clare Futter

Title: Phagosome maturation in retinal pigment epithelial cells

Summary: The daily phagocytosis and degradation of shed photoreceptor outer segments by retinal pigment epithelial (RPE) cells is critical for photoreceptor survival. Products of the failure to completely degrade outer segments build up in aging RPE and are particularly marked in age-related macular disease. This project will analyse the processing of phagocytosed rod outer segments in cultured RPE cells and RPE cell lines. We have shown that sequential stages of phagosome maturation can be identified by monitoring degradation of rhodopsin. This project will determine the role of the actin and microtubular cytoskeleton in regulating phagosome processing by determining the effects of cytoskeleton-disrupting drugs on phagosome movement and rhodopsin degradation.

Techniques: Cell culture, Live cell imaging, Confocal microscopy, Electron microscopy.                                         

Molecular genetics of inherited blinding diseases

Alison Hardcastle

Title: Molecular genetics of inherited blinding diseases

Summary: Our lab specialises in identifying genes and mutations leading to blinding diseases such as retinitis pigmentosa, cone dystrophy and congenital cataract.

The project will focus on identification of causative mutations or genes, and will involve learning some key molecular genetic techniques including PCR, sequence analysis and bioinfomatic analysis to search for loci, genes and mutations in patients. Other techniques such as comparative genomic hybridisation, haplotype and linkage analysis may also be employed.

No statistical knowledge is required for this project.

Molecular genetics of inherited corneal dystrophies

Alison HARDCASTLE: (originally Steve Tuft)

Title: Molecular genetics of inherited corneal dystrophies

Summary: Corneal dystrophies are clinically and genetically heterogeneous. For many common and rare forms of these dystrophies the causative genes remain to be identified. In addition, we now know that mutations in certain genes can cause more than one phenotypic outcome in our patients.

The project will focus on identification of causative loci, mutations or genes, and will involve learning some key molecular genetic techniques including PCR, sequence analysis and bioinfomatic analysis. Other techniques such as comparative genomic hybridisation and linkage analysis may also be employed.

No statistical knowledge is required for this project.        

Manipulating the maturing retina

Glen Jeffery

Project title: Manipulating the maturing retina

Summary: With age there are structural changes in the retina and different cells are recruited from the body to help it adapt to stress and the accumulation of cellular debris. There are ways of moderating the accumulation of debris and reducing stress that impact on visual function and may be significant in the way in which we view age related changes in retinal function. The project will examine these using a range of approaches including real time retinal imaging and an analysis of retinal structure. See: PLoS ONE: Research Article, published 01 Oct 2010 10.1371/journal.pone.0013127.

There are a number of projects that will examine different moderates and different read outs of the maturing retina including those that clear debris and enhance function.

Key words: Retinal imaging, Immunohistochemisty 

Near Infra-red light and ocular inflammation

Glen Jeffery

Project title: Near Infra-red light and ocular inflammation

Summary:Key theories of ageing stress that the process is associated with a decline in ATP production and an increase in ROS, however, this has not been proven in vivo. These elements originate from mitochondria. Mitochondria function can be manipulated with near infra red light (NIR), supposedly improving the ratio of ATP to ROS. NIR has been used to ameliorate a range of induced pathologies.

We have shown that NIR can reduce age related inflammation in the eye. This project will explore whether this reduction is due to changes in ROS and ATP and other similar markers or by a different mechanism. Exposure to NIR will take place in the eye and brain and tissue stained for range of markers that reflect ageing and cell efficiency.

Complexes involved in melanosome biogenesis studied in a model system

Levine-Project-1

Tim Levine

Title: Complexes involved in melanosome biogenesis studied in a model system

Summary: Melanosomes are important components of many ocular structures, with a critical role in the RPE, where they protect against photosensitisation and oxidation, as well as reducing light scatter. Formation of melanosomes away from the bulk of the secretory pathway depends on the actions of many protein complexes, one of which is BLOC-1. Although BLOC-1 was first described 10 years ago, its precise function is not known. We have identified homologues of BLOC-1 in yeast, so for the first time the yeast model system can be used to study this question. The project will study the mechanism of action of yeast BLOC-1 using mutant strains and tagged constructs.

Techniques: Molecular biology and molecular genetics; live cell confocal microscopy. No special requirements.

Neutral lipid handling in the retinal pigment epithelium

Levine-Project-2

Tim Levine

Title: Neutral lipid handling in the retinal pigment epithelium

Summary: The RPE handles huge amounts of phagocytosed lipid in its daily salvaging of components from turned-over rod outer segments. Handling of this lipid is key for continued health of the RPE, as lipid deposits between RPE and Bruch’s membrane are hallmarks that lead to AMD, the commonest cause of sight loss in Western societies. This project will study the role of FITM2 in the handling of a lipid load as it is processed by RPE cells. Plasmid borne FITM2 and siRNA will be used to alter levels of FITM2 in RPE cell lines, to study the effect on neutral lipid handling and related cellular pathways such as ER stress, which is a final common pathway underlying RPE dysfunction.

Techniques: Cell culture with standard biochemical/molecular techniques; live cell confocal microscopy.

Regulation of corneal epithelial cell-cell adhesion, migration and inflammation

Karl Matter

Title: Regulation of corneal epithelial cell-cell adhesion, migration and inflammation

Summary: Corneal epithelial cell-cell adhesion and migration are crucial processes for corneal biogenesis, maintenance and repair. Inflammation can cause disruption of adhesion and corneal disease. This project will focus on the functional and molecular analysis of a new regulator of RhoGTPase signalling that regulates all three processes: cell-cell adhesion, migration and inflammation. The purpose of this project is to determine how this RhoGTPase regulator contributes to inflammatory signalling and to design approaches to selectively inhibit its role in inflammatory signalling.

Techniques: Culture of human corneal epithelial cells, Transfection of cDNAs and siRNAs, Analysis of inflammatory signalling, Analysis of protein expression, Immunofluorescence microscopy.

Knowledge required: Basic cell and molecular biology

Manipulation of gene expression in the RPE

Steve Moss

Title: Manipulation of gene expression in the RPE

Summary: The project will involve examination of RPE cells in which specific genes have been deleted. The deleted genes will have roles in cell-cell contact, signal transduction and the complement pathway. Experiments will include confocal and electron microscopy of primary and cultured RPE cells, with morphometric analysis of RPE structure and organelles, and functional analysis with regard to cell signaling.

Key words: Cell culture, microscopy, western blotting, image analysis

No special requirements

Retinal and cortical structure and function in patients with genetic mutations leading to abnormal photoreceptor function

Nardini-Project-1

Marko Nardini

Title: Retinal and cortical structure and function in patients with genetic mutations leading to abnormal photoreceptor function

Summary: A set of rare genetic mutations lead to conditions in which the function of photoreceptors is drastically impaired. For example, several different genetic mutations lead to congenital achromatopsia, a condition in which cone photoreceptors do not function. Patients must rely on rods, which only support vision in low light, and are absent in the fovea. Because the retinal input is abnormal from birth, cortical organisation of visual areas is also altered (http://tinyurl.com/baseler02). Patients with genetic mutations of this kind are a promising target for trials of gene replacement therapy in the near future. Researchers at UCL have just shown restoration of vision in a mouse model of congenital achromatopsia (http://tinyurl.com/carvalho11). We are pursuing several research questions with these patient groups, with the aims to better understand these conditions, to better understand the plasticity of brain organisation and its dependence on retinal input, and to obtain baseline data useful for potential future trials of novel therapies. Some of these topics could form the basis for an MSc project: a. What is the time course of functional organisation of visual cortex of patients with abnormal retinal function in childhood (and in normal development)? b. How does structure and function of retinal rod and cone photoreceptors change over the course of childhood in inherited retinal diseases with a genetic basis (and how do these compare with structure and function in healthy children)? c. What are the behavioural consequences of developing an atypical visual system, i.e. one in which visual cortex represents only the retinal rod input: is patients’ cortex optimised for making use of the retinal input that they have (and can they therefore out-perform controls on some specific tasks)?

Techniques: a. neuroimaging: functional magnetic resonance imaging (fMRI), EEG, near infra-red spectroscopy (NIRS); b. retinal structure and function: optical coherence microperimetry, tomography (3D scans of the retina), c. behavioural (psychophysical) testing.

Special requirements: A student could contribute to this work in one of two ways. 1. A student with knowledge of programming could help to develop algorithms to analyse neuroimaging data or to correlate retinal structural and functional data. 2. A student confident in interacting with young children and their parents could help to collect psychophysical or imaging data. In either case, knowledge of psychophysical methods, neuroimaging or retinal physiology would be an advantage.

Integration of information for 3D shape perception in the developing visual brain

Nardini-Project-2

Marko Nardini (2nd of 2)

Title: Integration of information for 3D shape perception in the developing visual brain

Summary: Visual processing involves a hierarchy of functionally specialized cortical areas which can be localised and studied using functional magnetic resonance imaging (fMRI). In human adults, 3D shape perception depends on integrating stereoscopic information with “monocular” perspective information. While processing of 3D shape based on these separate information sources is associated with “early” retinotopic visual areas, integrated percepts of shape based on both stereo and perspective are associated with higher visual areas LOC and hMT+/V5 (http://tinyurl.com/welch2005). In my recent studies, children below 12 years did not integrate stereoscopic and perspective information to judge 3D shape (http://tinyurl.com/pnas10). One hypothesis is that in younger children these high-level visual areas do not yet carry out integration of depth cues. An MSc project could be based around: a. investigating 6-12 year old children’s abilities to make 3D shape discriminations (e.g. compare the slant of two surfaces viewed stereoscopically using ‘3D glasses’), b. relating these perceptual abilities to activation of visual areas while children view 3D stimuli in the fMRI scanner, c. Collecting EEG measures of cortical activity in response to single or combined depth cues with younger children.

Techniques: psychophysics, fMRI, EEG, stereo vision

Special requirements: knowledge or previous experience of psychophysical or fMRI methods would be ideal, otherwise a strong technical aptitude and a willingness to learn these methods is needed. You should also be confident in your ability to test young children (aged ~10 years), and to motivate them to complete repetitive psychophysical tasks.

Functional analysis of Xenopus TGFBI in corneal development

Shin-ichi Ohnuma (1st of 3)

Title: Functional analysis of Xenopus TGFBI in corneal development

The mechanism of Cyclin Dependent Kinase inhibitor in Xenopus retinogenesis

Shin-ichi Ohnuma (2nd of 3)

Title: The mechanism of Cyclin Dependent Kinase inhibitor in Xenopus retinogenesis

Summary: Cell cycle coordination with neurogenesis is essential for formation of functional eye. Cyclin Dependent Kinase inhibitor has very important roles in the co-ordination. This project aims to examine the molecular mechanism using Xenopus embryonic eye as a model. Especially, the student will examine functional interaction of Cyclin Dependent Kinase inhibitor with novel binding partners, which have been isolated in our lab.

Key words: Xenopus, neural development, retina

Re-establishment of Vision in blind cavefish

Shin-ichi Ohnuma

Title: Re-Establishment of Vision in blind cavefish

Summary: Cavefish lost eye during very long living in dark cave. We aim to re-construct functional eyes using transplantation techniques. To this end, functional eye precursor tissues and some related tissues are isolated from normal fish embryos and transplant it into cavefish embryos. Then, the structure and function of transplanted eye are investigated by a combination of histological analysis and functional examination. Also, this project will investigate the roles of some factors such as eye formation genes in functional eye formation/transplantation.

Key words: cavefish, neural development, eye 

Defining the optimal ontogenetic stage of donor cell for efficient rod photoreceptor transplantation

Rachael Pearson

Title: Defining the optimal ontogenetic stage of donor cell for efficient rod photoreceptor transplantation

Summary: Retinal degenerations leading to the loss of photoreceptors are a major cause of untreatable blindness in the developed world. Restoration of vision by replacement of the lost photoreceptors by cell transplantation is an exciting potential therapeutic strategy. We have previously demonstrated proof of principle for rod and cone photoreceptor transplantation, leading to restoration of (rod-mediated) vision. Importantly, we found that the developmental stage of the donor cell is critical; they must be post-mitotic photoreceptor precursor cells. Despite these very encouraging results, a significant proportion of the cells transplanted still fail to integrate within the recipient retina. For clinical application, it will be necessary to transplant only those cells at precisely the right stage in development for effective transplantation. While we have defined one critical check point for the donor cells - that they must be post-mitotic - it is still unclear firstly if the beginning of this transplantation-competent window in development can be better defined and more importantly what (if anything) defines the end of this window. In this project, we will look for markers that define the beginning and end of the developmental transplantation-competent window. This includes assessing the precise developmental expression of 4 markers of rod photoreceptor maturation, nrl (rod-specific transcription factor), rhodopsin, peripherin and rod-alpha-transducin. These are expressed across the period in development known to generate transplantation-competent rods and thought to occur in the temporal order nrl>rhodopsin>peripherin>transducin. We will then compare the integration efficiency of post-mitotic precursors selected on the basis of expression of each of these markers. This will be done by administering in utero viral vectors driving the expression of RFP under the control of the promoters for each of the 4 genes and using FACS-sorting to isolate the relevant populations of donor cells for transplantation. The student will design and make one vector (AAV2/9.Gnat1.RFP). The rest are already available. Techniques to be covered include cloning, transfection, virus production, cell dissociation, transplantation, cryosectioning, fluorescent and confocal microscopy, RT-PCR and qRT-PCR, Western blot, immunohistochemistry. 

Group II metabotropic glutamate receptor (mGluR) function in the retina.

Tom Salt

Title: Group II metabotropic glutamate receptor (mGluR) function in the retina.

Summary: The project will aim to utilise an in vitro retinal preparation to determine how Group II mGluRs might modulate or contribute to function in the mouse retina. Extracellular electrophysiological recordings will be made from the retina in response to photic stimulation. Pharmacological agents that are active in the mGluR domains will be used as tools to elucidate function.

Key Words: retina, in vitro electrophysiology, glutamate receptors,

Special Requirements: This project will involve delicate dissection and handling of delicate equipment. Students must be willing and capable to learn these techniques. Students should have a grasp of GCSE-level physics and chemistry.

The sluggishness of colour perception

Andrew Stockman

Title: The sluggishness of colour perception.

Summary: The primary goal of this project will be to understand more about how chromatic signals are processed and in particular why chromatic signals are processed much more slowly than achromatic ones. Despite the many recent advances in physiology, anatomy, fMRI, and genetics, colour processing in humans-at the systems level at least-remains relatively little understood. We will take advantage of a novel chromatic phenomenon in which the average colour appearance of rapidly-on and slowly-off sawtooth flicker |\|\|\|\|\ can be markedly different from that of slowly-on and rapidly-off sawtooth flicker /|/|/|/|/|. The conventional view of the determinants of chromatic temporal sensitivity, which is inconsistent with this phenomenon, is that chromatic mechanisms are slow because they are subject to greater temporal integration or averaging. We propose to investigate a new model of chromatic processing that can account not only for the differences in the appearances of these waveforms, but also for the well-known sluggishness of chromatic perception. The model that will be tested is based on the simple hypothesis that colour mechanisms are limited by the rate at which they can signal changes in colour; i.e., they are limited by a "slew-rate."

The main observer in the experiment will be the MSc student, who will also run other observers.

Key words: vision research, colour vision, visual psychophysics, flicker sensitivities

The human short-wavelength sensitive cone inputs to luminance and colour perception.

Andrew Stockman with Caterina Ripamonti

Title: The human short-wavelength sensitive cone inputs to luminance and colour perception.

Summary: Human vision depends initially on the transduction of light by three light-sensitive photoreceptors: the long- (L), middle- (M) and short-wavelength sensitive cone types. The L- and M-cones contribute both to our luminance (or achromatic) perception and to our colour (or chromatic) perception. By contrast, S-cones contribute mainly to chromatic perception. Yet, a small S-cone contribution to luminance can be found if the eye is adapted to chromatic light (to, for example, yellow light).

In this project, we will use special “isolation” techniques to ensure that what we detect depends only on S-cone signals. We will then investigate and contrast the properties of our visual performance when the conditions are such that the S-cone signals feed into the luminance pathway with when they are such that the signals feed into chromatic pathways. These results will be compared to similar measurements made in people with only S-cones (blue-cone monochromats) vision. How might their visual systems differ from normals?

The main observer in the experiment will be the MSc student, who will also run other observers.

Key words: vision research, visual psychophysics, S-cones, flicker sensitivities.

The role of Alex proteins in epithelial differentiation

Patric Turowski

Title: The role of Alex proteins in epithelial differentiation

Summary: Retinal pigment epithelial (RPE) cells are highly differentiated. They differ considerably from other epithelia in our body. Importantly for this study, RPE rarely undergo EMT and do not readily produce carcinomas. We have identified gene products that are up-regulated during matrix-induced RPE differentiation. Significantly, some of these gene products are also lost in carcinomas, suggesting that they might play a reciprocal role in epithelial differentiation and EMT/carcinogenesis. ARMCX genes (also called Alex), a narrow cluster of six armadillo repeat-encoding genes on the X chromosome, fall into this category. The aim of this project is to prepare molecular tools to Alex proteins and study their expression and localization in RPE cells and prostate epithelial cells of varying degrees of transformation. This should give some clear indication of their function and seed future in-depth analyses. So far there are no specific antibodies recognizing Alex and only five publications that specifically study this protein family. Therefore any additional information concerning this family of proteins will be a significant step forward.

Techniques to be used: RPE cell culture and transfection, Prostate epithelial cell culture and transfection, Molecular biology: cDNA subcloning and preparation of eukaryotic expression plasmids, Antibody characterization by western blot and indirect immunocytochemistry (incl. confocal microscopy), semi-quantitative and quantitative PCR

Special requirements: none

VEGF receptor signalling in brain and retinal microvascular endothelial cells

Patric Turowski

Title: VEGF receptor signalling in brain and retinal microvascular endothelial cells

Summary: Besides being the central angiogenic factor in health and disease, vascular endothelial growth factor A (VEGF-A) can trigger vascular permeability and is linked to interstitial fluid accumulation in tumours and tissue oedema in retinal neovascular disease. Accordingly, targeting VEGF-A has become a major focus in the treatment of human cancers and ocular neovascular disease. However, systemic anti-VEGF-A therapies are associated with endothelial dysfunction leading to bleeding, inflammation, hypertension, proteinuria and even lethality, since VEGF-A also acts as a trophic factor and regulates normal vascular tone. These observations call for a better understanding of the signalling processes that distinguish between the pathological and homeostatic functions of VEGF-A.

Previously, we have show that the permeability-enhancing action of VEGF-A is restricted to the abluminal side of the microvascular endothelium in the brain and retina. Permeability is induced through basal VEGF receptor 2 and is accompanied by rapid p38 and ERK activation and a significant reduction in junctional claudin-5. In sharp contrast, luminal VEGF-A stimulation does not perturb microvascular barriers, but led to VEGF receptor 1-mediated AKT activation and enhanced endothelial cell survival.

This project will analyse the signalling downstream of VEGF receptors 1 and 2 and determine its importance to endothelial hyperpermeability and cytoprotection.

Techniques: Isolation of cerebral and retinal endothelial cells, Endothelial cell culture, Permeability assays (by measuring dextran flux), Apoptosis assays, Protein analysis by western blot and indirect immunocytochemistry (incl. confocal microscopy)

Special requirements: none