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NEUROSCIENCES AND MENTAL HEALTH

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					                           NEUROSCIENCES AND MENTAL HEALTH


1)      FORMATTING INSTRUCTIONS AND APPLICATION PROCEDURE
2)      FREQUENTLY ASKED QUESTIONS
3)      PROJECT DESCRIPTIONS




1)      Formatting instructions/email application procedure:

        (a) Email applications should have “PhD Studentship Application” in the subject field, and
            should comprise a covering letter, CV, a summary/abstract of any research project
            undertaken (which should be no more than one side of A4), and the names and
            addresses of two referees.
        (b) Please save your application as a single Word document attachment and call it the same
            as your name, eg John Smith.doc
        (c) Please indicate in your covering letter where you saw the advertisement and also where
            else you have looked for studentships.
        (d) Applications should be sent direct to the Research and Development Office
            (chratapps@ich.ucl.ac.uk).


        Overseas applicants should also see FAQ1 below BEFORE submitting an application.


        Please note that if you apply without following the guidelines given above, your application
        may not be considered.


2)      Frequently Asked Questions

Q1.     Can students from outside the UK apply?

A.      Yes, overseas students have previously been accepted into the programme. The Child Health
Research Appeal Trust (CHRAT) will fully fund UK/EU students. Non-UK/EU students receive the normal
stipend, and the UK/EU component of their fees is paid, but they must pay the extra overseas fees
themselves (the current difference for 2005/06 is £11,165 per year). Furthermore, all candidates who are
selected for the Programme must be interviewed, and we unfortunately have no money to pay for overseas
students to come to interview.

Q2.     Can I come to visit the Institute before the interviews?

A.      Yes, there is an Open Day at ICH on Wednesday 23 November 2005 from 2.00pm onwards when
prospective applicants will meet the Postgraduate Tutors, existing PhD students, and have the opportunity to
take up tours of the facilities.

Q3.   I have a lower second degree but I am now doing an MSc. Is this equivalent to an upper
second?

A.     Assuming you passed your MSc, you could be eligible to hold a PhD studentship. Please note,
however, that in the previous year’s applications nobody with such a background was successful.

Q4.     Does my age matter?

A.      No.


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Q5.   I will be away at the time of the interviews (Monday 30 January and Tuesday 31 January 2006).

A.    Contact us: we may be able to interview early.

Q6.   Do I need to have chosen my project from the portfolio, prior to interview?

A.    No. The best candidates will be selected and offered studentships and we will then arrange for you
      to come back to ICH and visit potential supervisors/labs before you make a final decision. However,
      if only one or two projects would be of interest to you because of your specialist background, please
      say so in your covering letter.

Q7.   I have another PhD offer, which needs a decision before you decide on your studentships.

A.    Ask them to wait (they usually will); if not, contact us.




                                                       2
3)      Project Descriptions:

                                                                                          Page
    Behavioural and electrophysiological responses to social and non-social stimuli in   4
     preschool children with autism spectrum disorder and children with language delay
     (Dr T Charman*/Dr T Baldeweg)

    Inactivation of neurotrophin receptors by tyrosine phosphatases in neuroblastoma     5
     cells (Dr A Stoker)

    Discovering proteins in the pain pathway (Dr K Mills/Professor M Koltzenburg*)       6


    Image guided proteomics in the investigation of hippocampal injury associated with   7
     status epilepticus in rats (Dr R Scott*/Dr M Lythgoe/Dr N Greene)

    Prevention of neural tube defects by inositol: determining the molecular             8
     mechanism (Dr N Greene*/Professor A Copp)

    Characterisation and functional analysis of the HESX1-interacting proteins DNMT1     9
     and SRFBP1 (Dr J P Martinez-Barbera*/Dr M Dattani)

    Autistic symptomatology and impaired social cognition in early-onset antisocial      10
     behaviour (Dr K Lawrence*/Professor D Skuse)

    Regulation of neural stem cell recruitment following stroke                          11
     (Dr T Jacques*/Dr M Lythgoe/Professor A Copp)

    Use of enteric nervous system stem cells as a potential treatment for                12
     Hirschsprung’s disease (Dr A Burns/Dr N Thapar*)


* Principal Supervisor




                                                   3
Behavioural and electrophysiological responses to social and non-social stimuli in preschool children
with autism spectrum disorder and children with language delay

Supervisors: Dr Tony Charman and Dr Torsten Baldeweg

Hypothesis: The study will use behavioural and electrophysiological methods to determine the universality and
specificity of social and non-social impairments in preschool children with autism spectrum disorders in
comparison to children with language delay and further will determine whether both social and non-social
impairments are concurrently and longitudinally associated with language outcomes.

Aims and methods: It is well-established that young children with autism spectrum disorder (ASD) show
impairments in their response to a variety of social stimuli. Behavioural studies have found reduced orienting to
name, impairments in joint attention and abnormal processing of faces (1,2). Individual differences in these
abilities are associated with language and social outcomes in preschoolers with ASD (3). Recent studies
suggest that impairments might not be restricted to the social domain only and that a number of non-social
aspects of auditory and visual processes might also be impaired in ASD (4,5).

The present study will combine both behavioural and electrophysiological methods to measure social and non-
social processing abilities in preschool children with autism spectrum disorders. Further, it will test whether these
measures of social and non-social processing are associated with severity of language delay and severity of
autism. Finally, the study will test whether these associations are specific to ASD by also measuring the same
abilities in preschool children with language delay but not autism.

The student will be involved in the development of both the behavioural and electrophysiological paradigms that
will require adaptation for use with preschool children with ASD and language delay. The behavioural measures
will include aspects of joint attention behaviour, social orienting, preference for motherese and face processing.
The neurophysiological measures will include event-related potentials (ERP) during face processing tasks (in
collaboration with Dr. Michelle de Haan, DCNU) (6,7) and low-level auditory responses measured by ERP using
a mismatch negativity design to subtle changes in tone frequency and duration (8). The study will provide
important information on the neural maturation of the brains of children with ASD and children with language
impairment and help determine whether this underlies the delay in language onset that is seen in both groups of
children.

References:
1 Charman, T. (2003) Why is joint attention a pivotal skill in autism? Philosophical Transactions of the Royal
   Society of London Series B-Biological Sciences, 358, 315-324.
2 Dawson, G. et al. (2004) Early social attention impairments in autism: Social orienting, joint attention, and
   attention to distress. Developmental Psychology, 40, 271-283.
3 Charman, T. et al. (2003) Predicting language outcome in infants with autism and pervasive developmental
   disorder. International Journal of Language & Communication Disorders, 38, 265-285.
4 Kuhl, P. K. et al. (2005) Links between social and linguistic processing of speech in preschool children with
   autism: behavioral and electrophysiological measures. Developmental Science, 8, F1-F12.
5 Bertone, A. et al. (2005) Enhanced and diminished visuo-spatial information processing in autism depends
   on stimulus complexity. Brain, 128, 2430-2441.
6 de Haan, M. et al. (2003) Development of face-sensitive event-related potentials during infancy: a review.
   International Journal of Psychophysiology, 51, 45-58.
7 McPartland, J. et al. (2004) Event-related brain potentials reveal anomalies in temporal processing of faces
   in autism spectrum disorder. Journal of Child Psychology and Psychiatry, 45, 1235-1245.
8 Baldeweg, T. et al. (1999) Impaired auditory frequency discrimination in dyslexia detected with mismatch
   evoked potentials. Annals of Neurology, 45, 495-503.




                                                           4
Inactivation of neurotrophin receptors by tyrosine phosphatases in neuroblastoma cells

Supervisor: Dr Andrew Stoker

Hypothesis: Trk neurotrophin receptors are tyrosine kinases that regulate neuronal survival and influence the
development of neuroblastoma and sensory neuropathies. The hypothesis is that Trk receptors require negative
control through tyrosine dephosphorylation and this research project aims to define those tyrosine phosphatases
that selectively inactivate Trk receptors.

Aims and Methods: Neurotrophin receptors of the Trk family (TrkA-C) are receptor tyrosine kinases (RTKs) with
roles in neuronal differentiation and survival (1). Trk members are also now considered potential therapeutic targets
for neuronal tumours, including the childhood cancer neuroblastoma (2-4). Trk dysfunction is also implicated in
other clinical areas such as sensory neuropathies (5, 6). Given this wide range of developmental and clinical
involvement, it is paramount that we understand how Trk signalling is molecularly controlled. Trk enzymes are
activated by neurotrophins, inducing autophosphorylation on tyrosine residues and transmission of downstream
signals. The attenuation of kinase signalling is also critical and this occurs through receptor dephosphorylation by
protein tyrosine phosphatases (PTPs) (7). PTPs play many roles in neural development (7-9) and some have been
implicated as tumour suppressors. However, their molecular functions and regulation remain poorly understood. In
particular, the range and specificity of receptor-like PTPs (RPTPs) towards the Trk family of proteins is not known.
This project therefore aims to define the RPTPs expressed in neuronal tumour cell lines and to understand which
RPTP members selectively target the Trk receptors. These data will provide new insight into the importance of
negative control of Trk RTKs and whether this could be harnessed in the future to control the behaviour of neuronal
tumours.

(A)       We will first define which RPTP genes are expressed in a range of human neuroblastoma cell lines. RT-
          PCR will be used, with PCR primers specific for all members of the RPTP gene family.
(B)        RPTPs showing significant expression in the cells will be targeted by selectively increasing or reducing
          their expression in cells. Over expression will use expression vector transfection, whereas reduced
          expression will be achieved using state-of-the-art RNA interference technology (RNAi). The consequences
          for Trk biochemical signalling, cell differentiation and proliferation will be analysed.
(C)       To assess whether candidate RPTPs directly bind to and act directly upon Trk receptors, we would also
          carry out co-immunoprecipitation assays between each RPTP and their putative Trk target protein.

The laboratory has many years of experience working with RPTPs, molecular cell biology and cell culture models of
neuronal differentiation and axonogenesis (9, 10). We also have recent experience with RNAi. The project will



References:

1.    E. J. Huang, L. F. Reichardt, Annu Rev Biochem 72, 609 (2003).
2.    A. Eggert et al., Oncogene 19, 2043 (Apr 13, 2000).
3.    R. Ho et al., Cancer Res 62, 6462 (Nov 15, 2002).
4.    E. Lavenius, C. Gestblom, I. Johansson, E. Nanberg, S. Pahlman, Cell Growth Differ 6, 727 (Jun, 1995).
5.    Y. Yajima et al., J Neurochem 93, 584 (May, 2005).
6.    P. Anand, Prog Brain Res 146, 477 (2004).
7.    A. W. Stoker, J Endocrinol 185, 19 (Apr, 2005).
8.    S. E. Ensslen-Craig, S. M. Brady-Kalnay, Dev Biol 275, 12 (Nov 1, 2004).
9.    K. G. Johnson, D. Van Vactor, Physiol Rev 83, 1 (Jan, 2003).
10.   F. Rashid-Doubell, I McKinnell, A. R. Aricescu, G. Sajnani, A. W. Stoker, J. Neurosci. 22, 5024 (2002).




                                                           5
Discovering proteins in the pain pathway

Supervisors: Dr Kevin Mills and Professor Martin Koltzenburg

Hypothesis: Proteins are differentially expressed in cells and we want to discover those that are specifically
expressed in pain-signaling neurons with the view to harness this for the development of analgesic drugs.

Aims and methods: Despite significant advances in our understanding of the neurobiology of nociception, chronic
pain continues to be a leading health problem with an estimated prevalence of 19% of the general population. Pain
is signalled by a dedicated chain of specific neurons that start in the periphery with so called nociceptors, neurons
that have the remarkable ability to detect tissue damaging stimuli. Peripheral nociceptive sensory neurons are
housed in the dorsal root ganglia (DRG) and are intermingled with other sensory neurons and this anatomical
situation has impeded their detailed biochemical analysis. Recent molecular discoveries have, however, greatly
improved our understanding about the identity and function of nociceptors and this work has shown that these cells
express a unique set of genes, perhaps best exemplified by TRPV1, the receptor for capsaicin, the hot ingredient in
chilli. We can therefore use capsaicin as a tool to selectively identify nociceptors and compare them with cells that
have non-nociceptive functions which is the prerequisite to identify molecules that are selectively expressed in
peripheral nociceptors.

Capsaicin-sensitive nociceptors will be studied in two ways. First, we will use systemic treatment of capsaicin in
neonatal animals to selectively destroy nociceptors. DRGs from both treated and non-treated animals will
subsequently analysed using 2D-PAGels followed by MALDI-mass spectrometry, an analytical technology that is
well established in the laboratory. In a second approach we will use calcium imaging techniques to identify
capsaicin sensitive neurons in culture. Using laser capture microdissection, identified nociceptors will be harvested.
These cells will then be analysed using SELDI mass spectrometry an exciting new technology that allows analysis
and quantification of proteins in minute quantities of starting material. The project will involve a number of
transferable skills and techniques that are of general applicability to many areas of modern neurobiological
research. This includes tissue dissection, cell culture, vital staining, calcium imaging, and a range of analytical
proteomic technologies including MALDI-TOF and SELDI-TOF spectometry. The student will obtain hands-on
experience in all of these techniques and will be taught the theoretical background. The combination of laser
microdissection and SELDI-TOF spectrometry is a novel emerging technology that has the promise to obtain new
insights into the protein expression pattern in specific neuronal pathways on a level of detail previously not feasible.

References:

1) Ball HJ, Hunt NH (2004) Needle in a haystack: microdissecting the proteome of a tissue. Amino Acids 27:1-7.
2) Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M, Basbaum AI,
   Julius D (2000) Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science
   288:306-313.
3) Hanz S, Perlson E, Willis D, Zheng JQ, Massarwa R, Huerta JJ, Koltzenburg M, Kohler M, van Minnen J, Twiss
   JL, Fainzilber M (2003) Axoplasmic importins enable retrograde injury signaling in lesioned nerve. Neuron
   40:1095-1104.
4) Julius D, Basbaum AI (2001) Molecular mechanisms of nociception. Nature 413:203-210.
5) Mantyh PW, Clohisy DR, Koltzenburg M, Hunt SP (2002) Molecular mechanisms of cancer pain. Nat Rev
   Cancer 2:201-209.
6) Mills K, Mills PB, Clayton PT, Johnson AW, Whitehouse DB, Winchester BG (2001a) Identification of alpha(1)-
   antitrypsin variants in plasma with the use of proteomic technology. Clin Chem 47:2012-2022.
7) Mills PB, Mills K, Johnson AW, Clayton PT, Winchester BG (2001b) Analysis by matrix assisted laser
   desorption/ionisation-time of flight mass spectrometry of the post-translational modifications of alpha 1-
   antitrypsin isoforms separated by two-dimensional polyacrylamide gel electrophoresis. Proteomics 1:778-786.
8) Payne Smith M, Beacham DW, Ensor L, Koltzenburg M (2004) Cold-sensitive, menthol-insensitive neurons in
   the murine symathetic nervous sytem. NeuroReport 15:1399-1403.




                                                           6
Image guided proteomics in the investigation of hippocampal injury associated with status epilepticus in
rats

Supervisors: Dr Rod Scott, Dr Mark Lythgoe and Dr Nick Greene

Hypothesis: Acute hippocampal injury associated with status epilepticus is associated with increases in the
abundance of kinases, proteases, lipases and caspases and the abundance is related to the degree of hippocampal
abnormality identified on magnetic resonance imaging. Subsequently, the abundance of growth factors (e.g. brain
derived neurotrophic factor), proteins that regulate axonal growth and regeneration (e.g. alpha-tubulin) and proteins
associated with signal transduction (e.g. GTP binding protein, calmodulin3) correlate with changes in hippocampal
tissue status during development of MTS.

Aims and Methods: Mesial temporal sclerosis (MTS) is the most common lesion identified in patients that undergo
surgery as treatment for epilepsy. There is a long standing hypothesis that status epilepticus can cause acute
                                                                                                  1
hippocampal injury that matures into MTS associated with difficult to treat temporal lobe epilepsy . An understanding
of the mechanisms of brain injury and subsequent epileptogenesis could lead to novel therapies that prevent the
development of epilepsy. Therefore, magnetic resonance (MR) and proteomic approaches will be used to
investigate the pathophysiological and molecular mechanisms underlying the sequence of events from acute
hippocampal injury associated with status epilepticus, to the development of MTS, in a rat model of status
           2
epilepticus . The specific aims of the project are:

1.    to characterise the temporal evolution of structural changes in the hippocampus from acute injury, through a
      ‘silent’ period to the development of spontaneous recurrent seizures;

2.    to determine whether these MR changes are associated with alterations in the abundance and/or post-
      translational modification of specific proteins and;

3.    to assess which primary specific functional changes in the proteome are directly involved in the pathogenic
      processes leading to irreversible neuronal damage and epileptogenesis, and which changes are secondary
      consequences of these primary damage-causing events.

The rat pilocarpine model of status epilepticus, which is established in our laboratory, will be used. MR
investigations will be carried out during the acute, seizure-free and spontaneous recurrent seizure phases at 1, 3, 7,
14, 21 days. A control group will undergo all of the same investigations as the experimental animals. Five animals
                                                                                                3
will be investigated at each timepoint and the hippocampi will be used for proteomic analyses . Hippocampal tissue
samples will be collected from affected and control animals and the protein profile analysed by two dimensional
polyacrylamide gel electrophoresis (2-DE), an established technique in our laboratory. Preliminary experiments
confirm that proteomics approach will be useful as known epilepsy markers are up-regulated on 2D gels. Gels will
be analysed using PDQUEST software and differentially represented protein spots will be excised from gels,
subjected to tryptic digestion, and identified by mass spectrometry. Identification will be based on sequence
information generated by tandem mass spectrometry coupled to reversed-phase liquid chromatography (LC-
MS/MS). Relationships between MR and proteomic abnormalities will be investigated using standard statistical
techniques.

References:
1.   Cavanagh JB, Meyer A. Aetiological aspects of Ammon's horn sclerosis associated with temporal lobe
     epilepsy. British Medical Journal 2, 1403-1407. 1956.
2.   Cavalheiro EA. The pilocarpine model of epilepsy. Ital J Neurol Sci 1995; 16(1-2):33-37.
3.   Greene ND, Leung KY, Wait R, Begum S, Dunn MJ, Copp AJ. Differential protein expression at the stage of
     neural tube closure in the mouse embryo. J Biol Chem 2002; 277(44):41645-41651.




                                                          7
Prevention of neural tube defects by inositol: determining the molecular mechanism
Supervisors: Dr Nick Greene and Professor Andrew Copp

Hypothesis: Therapeutic doses of the vitamin inositol can act through a mechanism that involves specific protein
phosphorylation pathways, to reduce the risk of severe birth defects of the central nervous system.
Aims and Methods: Formation of the neural tube is a crucial process during embryonic development, this structure
being the precursor of the brain and spinal cord. Consequently, failure of neural tube formation results in severe
birth defects, termed neural tube defects (NTD). NTD are among the most common of birth defects in humans, with
risk determined by genetic and environmental factors. Currently, the only preventive therapy for NTD is the use of
maternal folic acid supplements (1). However, it is clear that many NTD (perhaps 30-50%) are not sensitive to folic
acid and additional approaches are needed to prevent these defects (2).
Using a genetic model of folic acid-resistant NTD in the mouse, we found that the vitamin, inositol, was protective
against NTD (3). These studies have recently led us to initiate a clinical trial of inositol in humans. Our experimental
studies have focussed on identifying the molecular causes of folic acid-resistant NTD and defining the mechanism
by which inositol prevents NTD. In summary, we know that therapeutic doses of inositol stimulate inositol
phospholipid metabolism, resulting in activation of specific isoforms of protein kinase C that are required for
prevention of NTD (4). Protein kinase C acts to phosphorylate target proteins and the aim of the studentship will be
to identify key target proteins and to establish their function in neural tube formation.
The project will initially focus on two candidate proteins that we have already found may be abnormally
phosphorylated in mouse embryos that are at high risk of NTD due to a genetic defect. The student will test whether
these proteins become phosphorylated following inositol treatment and may therefore be involved in the protective
mechanism. In addition, a proteomics screen of phosphoproteins will be performed using 2D protein gels and
fluorescent stains that label phosphorylated proteins (5). The proteins that are phosphorylated following inositol
treatment will then be identified by mass spectrometry, in the recently upgraded ICH Proteomics Facility. The
function of these candidate proteins in neural tube closure will then be tested in by examining the mRNA expression
and protein localisation and by functional studies using a combination of molecular and embryological approaches
such as chemical inhibition, RNA interference and genetic manipulation.
Dr Greene and Prof Copp’s research group comprises several post-doctoral research workers, PhD students and
research assistants based within the laboratories of the Neural Development Unit. In the course of the project the
student will become familiar with techniques in developmental biology, use of genetic mouse models (6) and
application of state-of-the-art proteomics approaches (including 2D gel electrophoresis and mass spectrometry).
References:
1. Copp AJ, Greene NDE, Murdoch JN. The genetic basis of mammalian neurulation. Nat. Rev. Genet. 4: 784-
   793, 2003.
2. Greene NDE, Copp AJ. Inositol prevents folate-resistant neural tube defects in the mouse . Nature Med 1997;
   3: 60-6.
3. Cogram P, Tesh S, Tesh J et al. D-chiro-inositol is more effective than myo-inositol in preventing folate-
   resistant mouse neural tube defects. Hum Reprod 2002; 17: 2451-8.
4. Cogram P, Hynes A, Dunlevy LPE, Greene NDE, Copp AJ. Specific isoforms of protein kinase C are essential
   for prevention of folate-resistant neural tube defects by inositol. Hum Mol Genet 2004; 13: 7-14.
5. Greene NDE, Leung KY, Wait R, Begum S, Dunn MJ, Copp AJ. Differential protein expression at the stage of
   neural tube closure in the mouse embryo. J Biol Chem 2002; 277: 41645-51.
6. Greene NDE, Copp AJ. Mouse models of neural tube defects: Investigating preventive mechanisms. Am. J.
   Med. Genet. Part C 2005; 135C: 31-41.




                                                           8
Characterisation and functional analysis of the HESX1-interacting proteins DNMT1 and SRFBP1
Supervisors: Dr Juan Pedro Martinez-Barbera and Dr Mehul Dattani

Hypothesis: Which proteins do interact with Hesx1?

Aims and Methods: The overall aim of this project is to gain insight into the molecular functions of Hesx1 in
forebrain and pituitary formation in mammals. Hesx1/HESX1 (mouse/human) is a transcriptional repressor, which is
expressed in the rostral region of the vertebrate embryo during gastrulation and neurulation. Our previous studies
have shown that Hesx1/HESX1 is essential for normal development of the forebrain and pituitary gland in both
mouse and humans. However, little is known about the functions of Hesx1/HESX1 at a molecular level, i.e., about
its regulators, target genes and interacting proteins.

To gain further knowledge on the molecular basis of forebrain and pituitary development in mouse and gain insights
into the mechanisms underlying congenital hypopituitarism and Septo-Optic dysplasia (SOD) in humans, we have
recently carried out a yeast two-hybrid screen and identified five Hesx1-interacting proteins. Two of the interactors
are nuclear proteins that can repress transcription. The PhD student will focus on these two interactors and he/she
will carry out the following experiments:

1. An analysis of the intracellular localisation of Hesx1 and its intercators.
2. A functional analysis, in vitro and in vivo, of the Hesx1-interactor partnerships.
3. An analysis of the molecular effects of HESX1 mutations associated with disease in humans on the binding to
   the HESX1 partners.

The student will use a combination of molecular, cellular and embryological techniques to investigate this project.
Molecular and cellular techniques, include PCR amplification, cloning, sequencing, yeast-two hybrid, in vitro
translation, luciferase assays, GST-pull down experiments, and co-immunoprecipitation, among others. As an in
vivo system, the student will use the mouse embryo. The analysis of compound embryos deficient for Hesx1 and its
interactors will reveal the biological significance of the interactions. The student will also learn about confocal
microscopy, embryonic stem (ES) cell targeting and mouse genetics.

The results obtained from the proposed research will have a relevant impact on basic and medical research. Firstly,
it will lead to a better understanding, at the molecular level, of how Hesx1 performs its functions. Secondly, it will
reveal how particular HESX1 mutations affect the interactions with its partners. These experiments will lead to a
better understanding of the pathogenesis of these human disorders by establishing a correlation between HESX1
mutation, disruption of protein-protein interactions, and phenotype. Finally, it might be the basis for a mutational
analysis the interactors in patients with congenital hypopituitarism or SOD. Therefore, the proposed research is
important not only for extending our knowledge of the molecular mechanisms underlying forebrain and pituitary
development in mammals, but also the obtained basic information will be utilised to unravel the complexity of
human conditions affecting the forebrain and associated structures.

References:
1. Dattani, M.H., Martinez Barbera, J.P., Thomas, P.Q., Brickman, J.M., Gupta, R., Martensson, I.-L., Toresson,
   H., Fox, M., Wales, J.K.H., Hindmarsh, P.C., Krauss, S., Beddington, R.S.P. and Robinson, I.C.A.F. (1998).
   Mutations in the homeobox gene Hesx1/HESX1 associated with septo-optic dysplasia in human and mouse.
   Nat. Genet. 19, 125-133.
2. Martinez-Barbera, J.P., Rodriguez ,T.A. and Beddington, R.S. (2000). The homeobox gene Hesx1 is required in
   the anterior neural ectoderm for normal forebrain formation. Dev. Biol. 223, 422-430.
3. Martinez Barbera, J.P and Beddington, R.S.P. (2001). Getting your head around Hex and Hesx1: forebrain
   formation in mouse. Int. J. Dev. Biol. 45, 327-336.
4. Dasen, J.S. Martinez Barbera, J.P., Hernan, T.S., O’Connel, S., Olson, L., Ju, B., Tollkuhn, J., Buek, S.H.,
   Rose, D.W. and Rosenfeld, M.G. (2001). Temporal switching of a paired-like homeodomain repressor/TLE
   corepressor complex for a related activator mediates pituitary organogenesis. Genes and Dev. 15, 3193-3207.
5. Dattani, MT. (2004). Novel Insights into the Aetiology and Pathogenesis of Hypopituitarism. Hormone Research
   62, 1-13.




                                                          9
Autistic symptomatology and impaired social cognition in early-onset antisocial behaviour

Supervisors: Dr Kate Lawrence, Professor David Skuse and Dr Jane Gilmour

Hypothesis: A significant proportion of children with early onset antisocial behaviour will have an undiagnosed
autistic spectrum disorder (ASD) and social cognition impairments that are similar in extent and quality to those
of children with ASD.

Aims and Methods: A minority of adults with antisocial behaviour are described as being ‘life-course persistent’
individuals, manifesting antisocial behaviour since early-childhood (Moffitt & Caspi, 2001). It is increasingly
thought that there may be a neurocognitive component critical in these cases that is not present in individuals
whose anti-social behaviour occurs only in adolescence. We propose that 'autistic' features of cognitive
processing are causally linked to the emergence and persistence of antisocial behaviour, because children with
these neurocognitive deficits are vulnerable to the effects of particular environmental risks - within the family and
wider social environment. Apart from a scattering of single case reports (Silva, Ferrari, & Leong, 2003; Scragg &
Shah, 1994) no research has thus far explored the links between ASD and antisocial or criminal behaviour.

Over one third of children excluded from Primary schools in a deprived London borough have been found to
have deficits in pragmatic skills that were as severe, and similar in profile, to children with ASD (Gilmour, Hill,
Place, & Skuse, 2004). We have also conducted preliminary neurocognitive investigations of incarcerated
adolescents with severe antisocial behaviour, within a Young Offenders Institute. These have shown that a
substantial proportion of such young offenders have significant and severe deficits in social cognition, of an
autistic-type.

The proposed investigation plans to look further at these samples. Extent of autistic symptomatology will be
assessed using clinical assessments including the Autism Diagnostic Observation Schedule (ADOS) and the
Developmental, Dimensional and Diagnostic Interview (3Di). Furthermore, we plan to conduct neurocognitive
tests to assess social cognition skills in this population. The student will be responsible for putting together a
battery of neuropsychological tests but these will include the Schedule for the Assessment of Social Intelligence
(SASI). This computerized schedule assesses the perception and interpretation of social information from faces
and other non-verbal stimuli (Skuse, Lawrence, & Tang, 2005).

Professor Skuse’s group is well set up to assess autistic symptomatology and social cognition in antisocial
individuals. Dr Lawrence has extensive experience investigating social cognition in both clinical and typically
developing populations (Lawrence, Kuntsi, Coleman, Campbell, & Skuse, 2003; Wade, Lawrence, Mandy, &
Skuse, 2005). Dr Lawrence and Professor Skuse have developed a battery of tests to assess social cognition
and Professor Skuse has developed a diagnostic parental autism interview (Skuse et al., 2004). We have
existing links with schools in Hackney and an institute for young offenders. Professor Skuse and Dr Gilmour both
have clinical experience with autistic children and have conducted previous research with early onset anti-social
individuals. In addition there are a number of research assistants and PhD students in the department
researching social cognition in different clinical and typically developing groups.

References:
1) Gilmour, J., Hill, B., Place, M., & Skuse, D. H. (2004). Social communication deficits in conduct disorder: a
   clinical and community survey. Journal of Child Psychology and Psychiatry, 45, 967-978.
2) Lawrence, K., Kuntsi, J., Coleman, M., Campbell, R., & Skuse, D. (2003). Face and Emotion Recognition
   Deficits in Turner Syndrome: A Possible Role for X-Linked Genes in Amygdala Development.
   Neuropsychology, 17, 39-49.
3) Moffitt, T. E. & Caspi, A. (2001). Childhood predictors differentiate life-course persistent and adolescence-
   limited antisocial pathways among males and females. Developmental Psychopathology, 13, 355-375.
4) Scragg, P. & Shah, A. (1994). Prevalence of Asperger's syndrome in a secure hospital. British Journal of
   Psychiatry, 165, 679-682.
5) Silva, J. A., Ferrari, M. M., & Leong, G. B. (2003). Asperger's disorder and the origins of the Unabomber.
   American Journal of Forensic Psychiatry, 24, 5-43.
6) Skuse, D., Lawrence, K., & Tang, J. (2005). Measuring social-cognitive functions in children with
   somatotropic axis dysfunction. In press with Hormone Research
7) Skuse, D., Warrington, R., Bishop, D., Chowdhury, U., Lau, J., Mandy, W. et al. (2004). The developmental,
   dimensional and diagnostic interview (3di): a novel computerized assessment for autism spectrum disorders.
   Journal of the American Academy of Child and Adolescent Psychiatry, 43, 548-558.
8) Wade, A. M., Lawrence, K., Mandy, W., & Skuse, D. (2005). Charting the development of emotion
   recognition from 6 years of age.
   In Press with the Journal of Applied Statistics

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Regulation of neural stem cell recruitment following stroke

Supervisors: Dr Tom Jacques, Dr Mark Lythgoe, Professor Andrew Copp

Hypothesis: This project will test the hypothesis that activation of specific signalling pathways in the stem cell
compartment of the brain can enhance the recruitment of endogenous stem cells following stroke.

Aims and methods: Acute brain injuries such as stroke are a major cause of disability in children and adults.
Traditionally, the brain was thought to have little capacity to repair itself. However, it is now apparent that there is a
population of stem cells within the brain that can proliferate and migrate towards the site of injury (Parent, 2003). In
most cases however, this response is insufficient to produce a complete recovery. Furthermore, very little is known
about the molecular mechanisms that regulate this recruitment or what limits the response of these cells to injury.
Therefore a major challenge is to understand the molecular mechanisms that regulate these endogenous neural
stem cells. An understanding of these processes will lead to both insights into brain injury but also open the
possibility of promoting brain repair by these endogenous stem cells.

The aims of this project are to establish a novel approach to genetically target the stem cell compartment in a model
of stroke and to use this approach to test the hypothesis that the phosphatase, PTEN, regulates recruitment of stem
cells following stroke.

As most, if not all, genes involved in the regulation of postnatal stem cells have critical functions during early
development of the brain, standard approaches to genetically target these cells are limited. To circumvent the
limitations of conventional genetic approaches, we have developed a technique which takes advantage of the
anatomical localisation of the stem cell compartment in combination with the Cre-Lox system of conditional genetics
(Sauer, 1998). We have used this approach to develop a reproducible model of tumour formation from the stem cell
compartment (Jacques et al., 2005).

In this project, we will use a similar approach to genetically target the stem cell compartment in a model of stroke.
The technique for inducing stroke is well established within the RCS Biophysics Unit at ICH (Lythgoe et al.,
2003;West et al., 2004;van der Weerd L. et al., 2005). Once established, this approach will be used to test the
hypothesis that signalling through the phosphatase, PTEN (‘phosphatase and tensin homologue deleted on
chromosome 10’) regulates stem cell recruitment in stroke. PTEN is a regulator of the cellular PI (phosphoinositide)
3-kinase signalling pathway (Leslie and Downes, 2004). Several lines of evidence indicate that the PTEN/PI3
kinase pathway is an important regulator of neural stem cell function and may regulate the response of endogenous
stem cells to stroke (Katakowski et al., 2003;Groszer et al., 2001).

In addition to the substantial clinical importance of stroke, this model provides a well defined injury with which the
methodology can be established. However, once proven in this setting, the methods developed would then be
available to investigate how endogenous stem cells are regulated in a much broader range of brain diseases and by
a much broader range of signalling molecules.

Reference List

(1) Groszer M, Erickson R, Scripture-Adams DD, Lesche R, Trumpp A, Zack JA, Kornblum HI, Liu X, Wu H (2001)
     Negative Regulation of Neural Stem/Progenitor Cell Proliferation by the Pten Tumor Suppressor Gene in Vivo.
     Science 294:2186-2189.
(2) Jacques TS, Ikenberg K, Reynolds S, Naumman H, Brandner S (2005) A novel model of tumour formation by
     neural stem cells. Neuropathology and Applied Neurobiology 31:228.
(3) Katakowski M, Zhang ZG, Chen J, Zhang R, Wang Y, Jiang H, Zhang L, Robin A, Li Y, Chopp M (2003)
     Phosphoinositide 3-kinase promotes adult subventricular neuroblast migration after stroke. J Neurosci Res
     74:494-501.
(4) Leslie NR, Downes CP (2004) PTEN function: how normal cells control it and tumour cells lose it. Biochem J
     382:1-11.
(5) Lythgoe MF, Sibson NR, Harris NG (2003) Neuroimaging of animal models of brain disease. Br Med Bull
     65:235-257.
(6) Parent JM (2003) Injury-induced neurogenesis in the adult mammalian brain. Neuroscientist 9:261-272.
(7) Sauer B (1998) Inducible gene targeting in mice using the Cre/lox system. Methods 14:381-392.
(8) van der Weerd L., Lythgoe MF, Badin RA, Valentim LM, Akbar MT, de Belleroche JS, Latchman DS, Gadian DG
     (2005) Neuroprotective effects of HSP70 overexpression after cerebral ischaemia--an MRI study. Exp Neurol
     195:257-266.
(9) West DA, Valentim LM, Lythgoe MF, Stephanou A, Proctor E, van der WL, Ordidge RJ, Latchman DS, Gadian
     DG (2004) MR image-guided investigation of regional signal transducers and activators of transcription-1
     activation in a rat model of focal cerebral ischemia. Neuroscience 127:333-339.
                                                            11
Use of enteric nervous system stem cells as a potential treatment for Hirschsprung’s disease

Supervisors: Dr Alan Burns and Dr Nikhil Thapar

Hypothesis: Significant numbers of newborn babies suffer developmental disorders of the enteric nervous system
(ENS), the nervous system of the gut. We will test the hypothesis that ENS stem cells isolated and transplanted into
defective gut will replace the missing ENS and restore gut function. The results will lay the basis for future stem cell
replenishment strategies to treat children with ENS disorders and avoid inadequate surgical treatments.

Aims and Methods: ENS developmental disorders such as Hirschsprung’s disease (HSCR) occur commonly in
newborn babies. The cellular basis of HSCR is incompletely understood, but the absence of nerve ganglion cells
(aganglionosis) in the distal gut it is thought to result from a failure in the migration, proliferation or survival of the
                                                1                                                     2
neural crest cells that give rise to the ENS , or from a defect in the local gut environment . Although surgical
treatments relieve associated life threatening intestinal obstruction they are otherwise inadequate and
                                                                                                 3
unsatisfactory (incontinence, growth retardation and poor postoperative quality of life ). Alternative forms of
treatment such as the use of ENS stem cells are required to improve outcome. Recent animal studies, including
those by the research group have demonstrated that ENS stem cells, that retain the ability to form the ENS when
transplanted to uncolonised, or aganglionic gut, are present within the gastrointestinal tract during development and
                          4-6
into early post-natal life . These cells are contained within neurosphere-like bodies (NLBs), which are generated in
cultures from dissociated gut from embryonic and post-natal mice of up to two weeks of age. When pieces of NLBs
were transplanted into aganglionic early embryonic mouse gut, progenitors were able to colonise the gut and
                                                                                  4
differentiate into appropriate ENS phenotypes, at the appropriate locations . These investigations suggest that
                                                                                       7
transplantation of ENS stem cells could be a viable alternative treatment for HSCR .
This project, however, aims to address a number of important unanswered questions before these exciting
observations can be developed further towards therapeutic trials. In particular it is not known if ENS stem cells can
be isolated from post-natal human gut although preliminary data suggests this will be possible. Furthermore it needs
to be established whether gut, at later developmental stages, or post-natally, when HSCR is diagnosed, provides a
receptive environment for transplanted ENS stem cells. Using a method that has successfully been employed by us
                           4
in post-natal mouse gut , human NLBs will be generated from gut tissue obtained from children undergoing co-
incidental surgery. Ethical approval has already been obtained. The tissue will be isolated, dissociated into cell
suspension, plated into culture and maintained in conditions that favour the generation of ENS stem cell containing
neural crest derived NLBs. Human and mouse NLBs will then be transplanted into recipient human and mouse
aganglionic gut obtained from patients with HSCR, and an established mouse model of HSCR (miRet51)
respectively. We propose to extend transplantation studies on embryonic gut by utilizing mouse and human gut
obtained at later stages of development. Once transplanted, the recipient gut will be placed in organ culture utilizing
a system of culturing transplanted gut on the chorioallantoic membrane (CAM) of developing chick embryos. After
14 days the extent of ENS colonisation will be assessed by immunohistochemical/in-situ hybridisation approaches.
Measurement of gut functionality will be assessed by in vitro measurements of propagative peristalsis (gut transit
time) and/or contractility (isometric contraction) and by the use of monoclonal antibodies with double-label immuno-
fluoresence to detect co-localisation of transplanted neurons (TuJ1) with other components of the enteric
neuromusculature i.e. smooth muscle (-SMA) and interstitial cells of Cajal (c-kit).


References
1. Burns, A. J. & Le Douarin, N. M. The sacral neural crest contributes neurons and glia to the post- umbilical gut:
   spatiotemporal analysis of the development of the enteric nervous system. Development 125, 4335-47. (1998).
2. Taraviras, S. & Pachnis, V. Development of the mammalian enteric nervous system. Curr Opin Genet Dev 9,
   321-7. (1999).
3. Tsuji, H., Spitz, L., Kiely, E. M., Drake, D. P. & Pierro, A. Management and long-term follow-up of infants with
   total colonic aganglionosis. J Pediatr Surg 34, 158-61; discussion 162 (1999).
4. Bondurand, N., Natarajan, D., Thapar, N., Atkins, C. & Pachnis, V. Neuron and glia generating progenitors of
   the mammalian enteric nervous system isolated from foetal and postnatal gut cultures. Development 130, 6387-
   400 (2003).
5. Kruger, G. M. et al. Neural crest stem cells persist in the adult gut but undergo changes in self-renewal,
   neuronal subtype potential, and factor responsiveness. Neuron 35, 657-69 (2002).
6. Natarajan, D., Grigoriou, M., Marcos-Gutierrez, C. V., Atkins, C. & Pachnis, V. Multipotential progenitors of the
   mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture. Development 126,
   157-68. (1999).
7. Burns, A. J., Pasricha, P. J. & Young, H. M. Enteric neural crest-derived cells and neural stem cells: biology and
   therapeutic potential. Neurogastroenterol Motil 16 Suppl 1, 3-7 (2004).



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