MolFun is a three year PhD training programme funded by the European Commission in the integrated application of techniques required to elucidate molecular functions on a global scale. The funding will provide each PhD student with a stipend (~£12,000 per year, tax free), mobility allowance and full research costs. The PhD will be entirely in English, the universal language of science and engineering. A course in scientific English will be available for those who require it. Candidates will have (or expect to have by summer 2006) a Masters and/or a strong first degree. Application is by CV, with the names and e-mail of two references to Prof. DG Fernig. Applicants should indicate in a covering letter the project(s) they are interested in.
The MolFun team comprises 13 members of Staff (see list at end of document)
in the School of Biological Sciences (http://www.liv.ac.uk/biolsci/) led
by Prof. David Fernig (dgfernig@liv.ac.uk).
B. Stress responses in C. elegans (ARC, PAM, HHR, TK)
This project will identify genes responding to environmental stressors,
in particular through transcript profiling and exploration of homologues
identified in environmental model species. These will be subject
to siRNA inhibition or mutational knockout in order to explore the phenotypic
consequences of gene manipulation. Changes in protein expression
will be explored using immunoblot and proteomic techniques (MALDI-ToF and
ESI-MS/MS).
C. Functional analysis of protein kinase-A catalytic subunit (PK-A C-subunit)isoforms
in the nematode, Caenorhabditis elegans (MJF, PAM, HHR, TK)
The cyclic AMP-dependent protein kinase (PK-A) plays key roles in many
aspects of cellular activity. We have used bioinformatics, functional genomics
and molecular approaches to show that in the nematode, C. elegans, there
is an unprecedented structural diversity of PK-A catalytic-subunit isoforms.
This diversity arises as a result of alternative splicing of transcripts
from the kin-1 gene. The purpose of this project is to explore the functional
contributions of these variants to intracellular signalling. We will use
RNAi to determine the phenotypic consequences of suppression of specific
isoforms. This approach will be complemented by an analysis of the ability
of transfection with recombinant proteins to rescue phenotype in knock-out
organisms. We will then use isoforms, associated with the most interesting
phenotypes, in studies of protein phosphorylation and mass spectrometric
identification of phosphoproteins.
D. Developing stem cell technology for the treatment of liver failure
(PAM, JET, DGF)
At present, the most successful treatment of acute liver failure is
liver transplantation from cadaveric donors. However, because of the shortage
of available donor organs, mortality rates remain high. The purpose
of this project is to improve culture conditions for deriving hepatocytes
from mouse embryonic stem cells. These conditions will then be applied,
and if necessary, modified, to enable hepatocytes to be derived from human
ES cells. The potential of mouse and human visceral endoderm to transdifferentiate
to hepatocytes will also be determined by investigating mRNA and protein
expression profiles of ES cell derived hepatocytes, visceral endoderm,
embryonic and adult hepatocytes. The benefits of using visceral endoderm
as a source of hepatocytes is that these cells differentiate spontaneously
in mouse and human ES cell cultures and are thought to be non-tumourigenic.
Imaging of molecular function, e.g., acute phase response, in single living
cells will be used to investigate the function of ES cell- and visceral
endoderm-derived hepatocytes and see if this compares with freshly isolated
embryonic and adult liver cells
E. The heparin interactome (MCW, DJR, DGF, EY, JET, TK)
The glycosaminoglycan heparan sulfate (HS) regulates the activity of
the majority of growth factors, cytokines, chemokines and morphogens through
specific interactions with these regulatory proteins. In addtion,
numerous pathogens hijack HS for cell adhesion and penetration. State-of-the-art
technology in glycomics (mass spec, saccharide microarrays, conjugation
chemistry), protein chemistry and proteomics will be used to identify all
of the key HS binding proteins in two model systems; mouse embryonic stem
cell and C. elegans and to identify the regions of the proteins that are
responsible for binding to structurally-defined oligosaccharides.
With the use of a battery of bioinformatics tools, each HS binding protein
will be studied in detail with regard to its structure, function and evolution.
A three-dimensional structural picture of the variation in and common themes
between the set of HS binding sites will facilitate future structure-based
functional annotation and drug discovery.
F. Neuropilin (DGF,JET, EY, /MCW, DJR)
Neuropilin, first described as a co-receptor for semaphorin-3A axon
repellent factor, has now been found to regulate cell function and organogenesis
outside the nervous system by interacting with a wide range of growth factors
and morphogens. In particular, it markedly potentiates the activity
of vascular endothelial growth factor and fibroblast growth factor in endothelial
cells. Mutant recombinant neuropilin will be used to define structurally
and biophysically protein binding sites in neuropilin. Wild-type
and mutant recombinant neuropilin will then be used to establish the profile
of phosphoproteins changes elicited in endothelial cells that occurs when
neuropilin potentiates the activity of vascular endothelial growth factor
and fibroblast growth factor.
G. Proteoglycans in the nervous system (TK, JET, EY, DGF, DGS, DJR)
C.elegans will be used as a model organism to study the role of heparan
sulphate proteoglycans (HSPGs) in the development and function of the nervous
system. C.elegans provides an excellent model to study molecular interactions
and gene function in vivo as the phenotype of the animal is the direct
read out of mutations that disturb function of a gene product. Bioinformatics
will be used to identify all the C.elegans homologues of vertebrate HSPGs.
The biological function of the candidate genes will be studied using existing
mutant alleles and new alleles in these genes. Molecular interactions of
HSPGs and HS binding proteins will be established. Cell specific GFP markers
and cell imaging allow analysis of the role of HS and HSPGs in neuronal
functions.
H. Phytosterol dealkylation in C. elegans (HHR, DJR, TK, PAM)
Most invertebrate species cannot synthesise sterols de novo and must
obtain them from the diet. Since phytophagous species obtain 24-alkylated
plant sterols, which cannot support normal growth and development, they
are dealkylated to cholesterol. The four steps in this pathway have been
primarily elucidated in phytophagous insect species, but have also been
demonstrated in C. elegans. The availability of genome-wide sequences and
post-genomic technologies, now make it timely to isolate the encoding genes
from C. elegans. This will then allow identification of orthologous genes
in phytophagous insect pests and plant-parasitic nematodes and provide
novel targets for new strategies of control. The project will involve,
bioinformatics and molecular modelling, microarray, RNAi and analysis of
phenotype, quantitative RT-PCR, heterologous protein expression, GC/LC-mass
spectrometry of sterols, 'model/species hopping'.