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MOAC and Systems Biology Doctoral Training Centres’

Mini projects 2007

DRAFT

UNRATIFIED DOCUMENT

BP=Biophysics/biophysical chemistry; B=biology; T=mathematics/computing; E=experimental

SB need to say what systems is, what techniques to study and whether it is possible to perturb the system.

Comment Decision

MOAC SB

B BP T E T

1. Steven Brown and Ann Dixon SOLID-STATE NMR AS A STRUCTURAL PROBE OF MEMBRANE PROTEINS

Suggest not SB. Is it?

2. Tim Bugg and Jian-Jun Li Chemical Genetics of Plant Apocarotenoid Natural Products

Need to justify why it is a good SB project

3. Tim Bugg and Sandeep Sandhu Analysis of Bacterial Cell Wall Biosynthesis


4. Kerry Burton Bioinformatics characterisation of fungal sequences regulating protein expression and localization

Kerry should talk to Sascha Ott.Need to justify why it is a good SB project

7. Katherine Denby Analysis of transcription factors with a role in plant defence


8. Ann Dixon and Graham Ladds GPCR SIGNALLING: MAPPING KEY PROTEIN-PROTEIN INTERACTIONS IN THE MEMBRANE


9. Lorenzo Frigerio Imaging the intracellular targeting of vacuolar membrane proteins in living cells

Need to justify why it is a good SB project.

10. Maj Hulten Crossover Hotspots and Genetic Interference: Mathematical Modelling of a Wave Phenomenon & Measurement of Crossover positions by immunofluorescence

Experimental and theoretical projects. Projects need to be defined clearly.

11. Steve Jackson and David Wild Modelling transcriptional networks involved in flower induction


12. Graham Ladds DEVELOPMENT OF FLUORESCENT YEAST STRAINS FOR INVESTIGATIONS INTO POPULATION VARIABILITY


13. Andrea Massiah & Brian Thomas Investigation into floral input pathway activity during vegetative phase maturation


14. Richard Napier (plus Dan Mitchell and Hugo van den Berg) Recording Real-time changes in hormone concentration

Need to justify why it is a good SB project. Note: Linked to 23

15. Joanne Oates (Ann Dixon and Matt Clarify experimental part

Hicks) GLYCOPHORIN A MEMBRANE

INSERTION AND FOLDING BY LINEAR

DICHROISM

16. Martyn Rittman & Alison Rodger Measurement of DNA persistence length

17. Alison Rodger Understanding flow in linear dichroism experiments

What are membrane markers? Need more details

18. David Roper The role of PBPs in Peptidoglycan Biosynthesis

Need to justify why it is a good SB project. Bit ambitious.

19. David Scanlan and Claudia Blindauer

Towards an understanding of multiple paralogues for metal-handling genes in a coastal cyanobacterium

Is this two projects? Rewrite as two.

20. Hendrik Schaefer Bioinformatic and transcriptional analysis of a marine methylotroph

Part 1 too risky without genome. Second part wet lab part OK. What will the student actually do.

21. A Shmygol Decoding Ca2+ signals in human myometrium: confocal imaging and numerical simulation of the Ca2+ release events targeting plasma membrane ion channels.

Is it too ambitious? Is it two projects? Need to be rewritten as separate projects. Or just experimental.

22. Corinne Smith Protein-protein interactions 2: quantitative assessment of protein-protein interactions

Linked to 26. Is it really feasible? Are constructs available. What will student actually do? Focus down. Justify SB

23. Hugo Van den Berg Simulating Real-time changes in hormone concentration from Biacore data

Need to justify why it is a good SB project. Linked to 14

24. Hugo van den Berg and David Whitworth Modelling the photoprotective response in Myxococcus xanthus

Linked with 27

25. Dave Whitworth Predicting Biomolecular Interactions: Comparative Prokaryotic Genomics

Justify why systems biology

26. Dave Whitworth & Corinne Smith Protein-protein interactions 1: Protein expression, purification and activity assays

See 22

27. Dave Whitworth Assaying the photoprotective response in Myxococcus xanthus.


28. David Wild Reconstructing gene regulatory networks with nonlinear state space models


29. David Wild A Bayesian approach to

biological information retrieval

30. David Wild Fast Bayesian clustering for microarray data


31. David Wild Integrating transcriptome and metabolome changes in response to pathogen infection


Clarify FT-IR

32. David Wild (WSB) and Philip McTernan (CSRI). Delineating molecular mechanisms contributing to mitochondrial dysfunction in obesity and diabetes

Write as two projects. Theory first


33. Vicky Buchanan-Wollaston Analysis and modelling of microarray time course data


34. Matthew Turner Protein crystallisation at surfaces in the presence of electric fields

Linked with PRU. Justify why systems biology. Ideally PRU first

35. Sascha Ott Finding Transcription Factor Binding Sites in Co-expressed Promoters


36. Sascha Ott Modelling and Predicting Transcription Factor Binding Sites


37. Sascha Ott Nucleosome Positioning

38. Sascha Ott In Silico Detection of Regulatory Modules


39. Laura Baxter Identification of the host targets of pathogenicity effector proteins

40. Julie V. Macpherson Development of diamond and single walled carbon nanotube electrodes for neurosensing applications

What is the student actually be going to do? What protein?

41. Rand/Carre Analysis of dynamic changes in gene expression under the control of the circadian clock

42. Unwin Protein crystallisation at surfaces in the presence of electric fields

Linked with 34.

43. Colin Robinson Translocation of proteins by a novel TATAC-type bacterial protein transporter

Justification of SB

44. Erwin George and Martin Eigel Simulation of spatio-temproal protein distribution with GDF

Needs clarification. Why consumables? Why SB?

44. Markus Kirkilionis Distribution of proteins in cells

Justification of SB needed. Is it doable? What will the student do. Why consumables?

M1. Stefan Bon Nanoparticles What is biological application? Budget it too high.

M2. Steven Brown SOLID-STATE NMR: A PROBE OF OXYGEN-CONTAINING HYDROGEN BONDS

M3. Ewen Buckling Chromosomes Need to clarify where samples are coming from and what the student will actually be doing.

M4. Tom Drewello Bio-templates for the formation of nano-sized silver and gold particles by electrospray

Budget too high

M5. Jonathan Duffy Glass transition of sugars confined in nanopores

What will the stunt actually do? Clairification of relevance to biology.

M6. Matthew Hicks PCR kinetics in real time What will the student actually do in 8 weeks???

M7. Martyn Lochner Synthesis and electrophysiological characterisation of granisetron derivatives

M9. Magnus Richardson Determinants of firing patterns of cerebellar neurones. Part II: theory

Follows M10

M10. Mark Wall Determinants of firing patterns of cerebellar neurones. Part I: experiment

Precedes M9

M11. V. Zammit & Dr Ann Dixon Protein-protein interactions involved in Carnitine palmitoyltransferase 1 oligomerisation

M12. Professor M. Wills Structure Structure/function studies on ALDC, part 1; Chemistry

Linked with M15 and M16

M13. Andrew Millard Development of comparative genomic hybridisation with a micro-array of the cyanophage S-PM2

M14. Andrew Millard Characterisation of genes in the “ORFanage” region in the cyanophage S-PM2

M15. V.Fulop Structure Structure/function studies on ALDC, part 2; biology

Linked with M12 and M16. What will student do? What if crystals won’t grow? Risk evaluation. What if M12 does not work.

M16. P. M. Rodger Structure Structure/function studies on ALDC, part 3; modelling

Linked with M12 and M15, but can stand alone.

M17. Sascha Ott Modelling the Distribution of Crossovers

M18. Corinne Smith Natively unfolded proteins

M19. Magnus Richardson Neuronal interactions with electromagnetic fields

S1. Rebecca Allen Identification of the host targets of pathogenicity effector proteins

Justify why SB

S2. Vicky Buchanan-Wollaston Analysis of stress related signalling pathways in Arabidopsis leaf development

Justify why SB

S3. Vicky Buchanan-Wollaston Functional analysis of genes that regulate plant stress responses in Arabidopsis

Justify why SB

S4. Chandler, Ryabov, Bittner-Eddy Gene expression profiles in the honey bee

Justify why SB

S5. Peter J. Eastmond Defining sugar-signalling pathways in Arabidopsis seedlings using whole genome micro-arrays

Justify why SB

S6. Magnus Richardson Effects of synaptic amplitude distributions on neuronal firing rates

Justify why SB. MAKE SURE THE CORRECT PROJECT GETS INSERTED!!!!!

S7. Magnus Richardson Extracting Reject Wit

neuronal structure from image data hdrawn

S8. Konstantinos Thalassinos Identification of post translational modifications of proteins using mass spectrometry

Justify why SB. Need to say what the student will do. Needs rewriting.

S9. Matthew Turner Understanding animal behaviour

Justify why SB. What will student actually do?

S10. David Grammatopolous and David Rand Insights of the corticotrophin-releasing hormone (CRH)-MAPK signalling pathway using a systems biology approach

Consumables budget???

MOAC / Systems Biology mini-project proposal 1Submit one page only to Monica Lucena ([email protected]) by Wednesday 14th February, 2007, 5pm).

Project title: SOLID-STATE NMR AS A STRUCTURAL PROBE OF MEMBRANE PROTEINS

Project suitable as: (tick all that apply)

MOAC Mini Project Experimental biology Systems Biology Mini Project Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project

Mathematics/computing

Project timing*

MOAC Mini Project Slot 1 (26/03 to 18/05/2007) Systems Biology Mini Project Slot 1 (26/03 to 22/05/2007)

Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)

Supervisor (the person who will be doing the day to day supervision of the mini-project):

Name: Dr. Steven Brown (SB) and Dr. Ann Dixon (AD)

Department: Physics (SB) and Chemistry (AD)___________ Building, Room: Room 439 (SB) and Room C503 (AD)

E-mail address: [email protected], [email protected] Phone number: 024765 74359 (SB)

Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision support to

the supervisor and also agrees to meet the student briefly at least once a week):

Name:

Department: ______________________________________ Building, Room:

E-mail address: ___________________________________ Phone number:

Project Outline.Introduction. A key element of a cell is its lipid membrane; as well as sequestering the contents of the cell, the membrane, and in particular membrane proteins that are embedded in the membrane, regulate how the cell interacts with its environment. For example, the import and export of specific molecules to the cell as well as the action of hormones attaching to membrane-bound protein receptors cause given chemical reactions to occur inside the cell.

While membrane proteins account for 30% of proteins encoded by the human genome, only 92 membrane protein structures have been successfully solved (as compared with the over 30,000 structures for soluble proteins). Indeed, discoveries related to membrane protein structures have led to three Nobel prizes since 1987. This discrepancy in the number of known structures is due to experimental difficulties, as membrane proteins are difficult to produce in sufficient quantities for structural analysis, difficult to crystallise for X-ray studies, and often produce large aggregates which can exceed the size limit for solution-state NMR spectroscopy. Solid-state NMR (SS-NMR) is a powerful probe of molecular structure, offering atomic-scale resolution [1]. A landmark in the application of solid-state NMR to structural biology was the determination in 2002 by Castellani et al. of the structure of the 62-residue soluble protein, the -spectrin Src-homology (SH3) domain [2]. Solid-state NMR is well suited to the study of membrane proteins in their natural phospholipid bilayer environment, and advanced solid-state NMR methodology has also been applied to membrane proteins.

Aims. This project will focus on developing sample preparation and SS-NMR methods for a well-characterised membrane protein, Glycophorin A (GpA). GpA contains a single transmembrane domain (Fig. 1a) that inserts into membrane bilayers as a stable -helix and drives protein dimerisation (Fig. 1b) [3]. The GpA dimer has been extensively studied, and we have a wealth of knowledge regarding the amino acids that stabilise its folding. One sequence motif that is critical for the correct folding of GpA is the Gly-x-x-x-Gly motif (GG4), where Glycine residues located every four amino acids localise to the same face of the helix and pack tightly against the other helix (Fig. 1b) [4]. These Gly residues can be isotopically labelled, allowing us to investigate the stability of the GpA dimer in membranes by SS-NMR.

* Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

Brief Outline of work. Peptides corresponding to the transmembrane domain of GpA, containing 15N labels on Gly79 and Gly83 (Fig 1a), will be purchased and purified using reversed-phase HPLC. Once purified, the peptides will be solubilised in a variety of lipid formulations. The stability and correct folding of GpA dimers will be analysed using one and two-dimensional SS-NMR spectrometry. Peptide purification and solubilisation will be carried out in the Department of Chemistry. Solid-state NMR experiments will be performed in the Department of Physics.

References: [1] S. P. Brown, & L. Emsley. Solid-State NMR, in Handbook of Spectroscopy, Vo-Dinh and Gauglitz (eds), Wiley (2003). [2] F. Castellani, B. van Rossum, A. Diehl, M. Schubert, K. Rehbein, and H. Oschkinat, Nature 420, 98 (2002). [3] Lemmon, M.A., J.M. Flanagan, J.F. Hunt, B.D. Adair, B.-J. Bormann, C.E. Dempsey, and D.M. Engelman, J. Biol. Chem., 267, 7683 (1992). [4] Russ, W.P. and D.M. Engelman, J. Mol. Biol., 296(3), 911 (2000).

Consumables budget†: £ 200.00 for lipids and other consumables.

† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

http://www.wileyeurope.com/WileyCDA/WileyTitle/productCd-3527297820.html

Chemical Genetics of Plant Apocarotenoid Natural Products 2Prof. Tim Bugga and Dr. Jian-Jun Lib, Department of Chemistry, University of Warwick[collaboration with Dr. Andrew Thompson & Dr. Martin Sergeant, Warwick HRI]a email [email protected], Room C513, ext. 73018;b email [email protected], Room C201, ext. 73822

Apocarotenoids are plant natural products formed by oxidative cleavage of carotenoids such as beta-carotene and lycopene. Several of the apocarotenoids have important biological properties, for example abscisic acid is used as a signalling hormone for drought response and growth. Through a BBSRC-funded SCIBS grant, TDHB and AJT are studying the family of carotenoid cleavage dioxygenases (CCDs), which catalyse the cleavage reactions [1], and have developed selective inhibitors for members of this family of enzymes.

The aim of this project is to develop analytical methods for detection of apocaretonoid natural products arising from 7,8-oxidative cleavage, and then to examine whether treatment of the producing plant with CCD inhibitors affects the production of natural products. The project will be in two stages:

1. Development of methods for isolation & analysis of apocarotenoids. The apocarotenoids crocin and crocetin are produced a) in the red stigmas of the saffron crocus (Crocus sativus); b) in the fruit of Gardenia jasminoides [2]. The by-product of oxidative cleavage, safranal, is also found in the volatiles of Crocus sativus [3]. Methods for extraction of the natural products, and analysis by HPLC and GC-MS, will be developed, using dried plant material.

2. System Perturbation. The apocarotenoid biosynthesis pathway will then be perturbed by addition of a 7.8-selective CCD inhibitor to the growing plant, prior to flowering/fruiting. The plant tissue will then be collected, and analysed for the distribution of natural products.

HO

OH

zeaxanthin

HO

CHOOHC

CHO

7

8

COORROOC

crocin R = (Glc)2crocetin R = H

CHO

safranal

7,8-oxidative cleavage

References.1. M.E. Auldridge, D.R. McCarty & H.J. Klee, Curr. Opin. Plant Biol., 2006, 9, 315-321.2. S. Pfister, P. Meyer, A. Steck & H. Pfander, J. Agric. Food Chem., 1996, 44, 2612-2615.3. M. d’Auria, G. Mauriello, & G.L. Rana, Flavour Fragrance J., 2004, 19, 17-23.

Timetabling. Suitable for MAOC mini-project slot 1,2 or 3; Systems Biology slot 1 or 2. During May-June would be best for plant flowering times, but other times are possible.Consumables. £100 for HPLC solvents, consumables.

mailto:[email protected]


Analysis of Bacterial Cell Wall Biosynthesis 3

Biophysical Chemistry Mini-Project: Professor Tim Bugga and Sandeep Sandhub , Department of Chemistry, University of Warwick [Collaboration with Prof. C. Dowson, Dr. D. Roper, Dr. A.J. Lloyd, Department of Biological Sciences; Dr. M. Chappell, Department of Engineering] aRoom C513, email [email protected], tel 02476-573018; broom C201, email [email protected], tel 02476-573822

Bacterial cell wall peptidoglycan biosynthesis is the site of action of many clinically important antibiotics, including the beta-lactam family of penicillins and cephalosporins, and the vancomycin group of glycopeptide antibiotics [1]. TDHB’s group, in collaboration with Prof. C. Dowson and Dr. D. Roper (Biological Sciences), have considerable experience of studying enzymes on this pathway [2], and are able to produce the intermediates and enzymes for all the cytoplasmic steps (first 6 steps), and the first three steps of the lipid-linked cycle. The aim of the Biophysical Chemistry mini-project is to measure the levels of each intermediate and each enzyme in bacterial cells; the data from this mini-project will then be used to set up a quantitative model of the whole pathway.

MurNAcL-AlaD-GluL-LysD-AlaD-Ala

PP


GlcNAc

PP


GlcNAc

Ser-Ala

PP


GlcNAc

Ser-Ala


GlcNAc

Ser-Ala

PP


GlcNAc

Ser-Ala

PP

P

PP

P

MraY

MurG

UDPGlcNAc

MurM/N

Ala-tRNASer-tRNA

trans-glycosylase

UDP-MurNAc-pentapeptide

UMP

peptidoglycancross-linking

CYTOPLASM

CELL SURFACE

UDP-MurNAc-L-Ala-D-Glu-L-Lys

D-Ala-D-AlaATP

D-AlaDdlB

ATPMurF

MurE

L-LysATP

MurD

D-GluATP

MurC

L-AlaATP

UDPMurNAcMurB

NADPHFADH2

MurA

PEPUDPGlcNAc

Outline: (discipline – biophysical chemistry; techniques – UV/vis/fluorescence, analytical chemistry)Cell extract and membranes will be prepared from Escherichia coli at several time points during logarithmic and stationary growth phases. The concentration of each of the cytoplasmic intermediates (present in soluble cell extract) will be determined by anion exchange FPLC, by comparison with authentic standards, through UV/vis absorption at 260 nm ( = 10,000 cm-1). The concentration of each lipid-linked intermediate (isolated by n-butanol extraction of the membrane fraction) will be determined by modification with Sanger’s reagent (2,4-dinitrofluorobenzene), and detection by reverse phase HPLC at 400 nm, eluting in 20-100% methanol. In vitro assays will also be set up, using the E. coli membranes and authentic undecaprenyl phosphate, in order to generate larger quantities of material for HPLC detection. The activities of each of the biosynthetic enzymes (MurA-F, MraY, MurG) will be determined by UV/vis continuous assays (MurA-F) and fluorescence assays (MraY, MurG).These data will be used in subsequent mini-projects, to set up a quantitative model for peptidoglycan biosynthesis.References [1] “Bacterial Peptidoglycan Biosynthesis and its Inhibition”, T.D.H. Bugg, in “Comprehensive Natural Products Chemistry”, (ed. M. Pinto), Vol. 3, pp. 241-294, Elsevier, 1999. [2] P.E. Brandish, M. Burnham, J.T. Lonsdale, R. Southgate, M. Inukai, and T.D.H. Bugg, J. Biol. Chem., 271, 7609 (1996); N.I. Howard and T.D.H. Bugg, Bio-Org. Med. Chem., 11, 3083 (2003); J.J. Li and T.D.H. Bugg, Chem. Commun., 182 (2004); A.J. Lloyd, P.E. Brandish, A.M. Gilbey, and T.D.H. Bugg, J. Bacteriol., 186, 1747 (2004).Timetabling: Suitable for 8-week MOAC student in slot 1 or 2; or Systems Biology student in slot 1.Consumables budget: £100 (for assay reagents & growth media)



MOAC / Systems Biology mini-project proposal 4Submit one page only to Monica Lucena ([email protected]) by Wednesday 14th February, 2007, 5pm).Project title: Bioinformatics characterisation of fungal sequences regulating protein expression and localization

Project suitable as: (tick all that apply)MOAC Mini Project Experimental biology Systems Biology Mini Project Wet Project(8 weeks) Biophysical chemistry (12 weeks) √ Dry Project

√ Mathematics/computing

Project timing‡

MOAC Mini Project √ Slot 1 (26/03 to 18/05/2007) Systems Biology Mini Project Slot 1 (26/03 to 22/05/2007)

√ Slot 2 (21/05 to 13/07/2007) √ Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Kerry Burton

Department: Warwick HRI____________________________ Building, Room:

E-mail address: [email protected]_____________ Phone number: 75137



Name:



Project outline:

References: : [1] Burns, C., Gregory, K.E., Kirby, M., Cheung, M.K., Riquelme, M., Elliott, T.J., CHALLEN, M.P., Bailey, A.M., and Foster, G.D. (2005) Efficient GFP expression in the mushroom Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genetics and Biology 42, 191–199 / [2] Zhang, C., Odon, V., Kim, H.K., CHALLEN, M., Burton, K. Hartley, D. and Elliott, T. (2004) Mushrooms for Molecular Pharming. Mushroom Science 16, 611-617.

Consumables budget§: £ nil

‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Therapeutic proteins are used in medicine to treat diseases, cancers and inherited conditions. Research to reduce manufacturing costs is assessing the applicability of agricultural organisms. The fungus Agaricus bisporus has cost advantages and has been shown to produce the demonstration proteins Green Fluorescent Protein and Cyanovirin-N. A University of Warwick spin-out company, Prospero Therapeutics Ltd, was formed in 2006 to commercialise the project and it is now seeking equity investment funding to develop the production of two therapeutic proteins.Two approaches are being used to elevate protein yields; the exploitation of gene promoter elements and protein leader sequences. Several high-level promoter sequences have been identified from Agaricus bisporus. Evidence suggests these can be tissue specific, developmentally regulated and are capable of enhancing GFP expression. Information on protein leader sequences, which can direct nascent protein into intracellular locations and affect stability and rate of degradation, is more limited in Agaricus bisporus In this project the student will use a comparative genomics approach to identify additional promoters and leader regulatory sequences from filamentous fungi. This will be achieved through bioinformatic analyses of genome sequences from several closely related fungi: Phanerochaete chrysosporium, Postia placenta, Coprinopsis cinereus and Cryptococcus neoformans for leader sequences of proteins of known intracellular location. Agaricus bisporus has a genome of 34 Mb which equates to ca. 10,000 genes; currently only 10% of the ORFs are available as sequences. Using a wide range of molecular biology algorithms the basidiomycete genome sequences will be screened for promoter and leader sequences. Genes encoding known proteins with well-defined intracellular locations will be examined to identify putative leader sequences. Analagous sequences from different fungi will be compared to identify consensus motifs/similarities that define intracellular location and/or promote gene transcription.Timeframes are flexible, and as such the project will suit both 8-week MOAC and 12-week Systems Biology.


Project title: Analysis of transcription factors with a role in plant defence




Project timing**


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Katherine Denby

Department: Warwick HRI and Systems Biology __________ Building, Room: TPB133 (HRI), Coventry House 327

E-mail address: [email protected]_______________ Phone number: 75097/50251



Name:



Project outline:

References: AbuQamar et al. (2006) Expression profiling and mutant analysis reveals complex regulatory networks involved in Arabidopsis response to Botrytis infection. Plant Journal 48:28-44

Consumables budget††: £ 400 (my two dry projects have budgets of £0!!)

** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.†† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

I am interested in elucidating the gene regulatory networks controlling defence against the fungal pathogen Botrytis cinerea in the model plant Arabidopsis thaliana. High-resolution time course expression profiling experiments are underway to provide data for inferring large-scale network models but in this project we will focus on identifying specific local networks. This information will be used in building network models and to guide future experimental work.

Initial work has identified several Arabidopsis transcription factors (TFs) whose expression is significantly upregulated during B. cinerea infection. For 4 of these, we have transgenic lines overexpressing the TF with His and myc tags fused to the protein. These lines enable us to study the effect of increased levels of the TF and the tags allow for easy analysis of protein levels. In this project, the susceptibility of these lines to B. cinerea will be tested compared to wildtype plants. This will involve determination of lesion development, as well as measurement of fungal growth within the plants. The degree of overexpression of TFs will be assessed through immunoblotting with anti-His or anti-myc antibodies and homozygous lines identified with PCR. Microarray experiments will be carried out to determine gene expression profiles, and hence potential target genes, for one or more of the TF overexpressing lines.


Project title: GPCR SIGNALLING: MAPPING KEY PROTEIN-PROTEIN INTERACTIONS IN THE MEMBRANE




Project timing‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr. Ann Dixon (AD) and Dr. Graham Ladds (GL)

Department: Chemistry (AD) and Warwick Medical School (GL) Building, Room: Room C503 (AD), Room M119 (GL)

E-mail address: [email protected] Phone number: 024761 50037



Name: Prof. Alison Rodger

Department: Chemistry______________________________ Building, Room:

E-mail address: [email protected]________________ Phone number: 024765 74696

Project outline:Introduction. G-protein coupled receptors (GPCRs) are the largest family of membrane bound receptors, with over 2000 members in vertebrates.1 Because they control the biological activity of messenger molecules such as hormones, growth factors, and neurotransmitters, GPCRs control many physiological processes linked to disease. For this reason, they are the targets of a large number of drugs. All GPCRs contain a central domain composed of seven transmembrane helices (Fig.1). Apart from this shared feature, the GPCR family contains a very diverse range of sequences and functions. GPCR signalling is further complicated by the fact that these proteins can form oligomeric complexes with other GPCRs (Fig.1),2,3 but the mechanism of this association is unknown. Therefore, to understand GPCR signalling we must not only study their structure and interactions with ligands, but also their interactions with one another.

Aims of Project. The mechanism of GPCR interaction will be investigated for two yeast GPCRs known to interact and form oligomers. For both proteins, amino acid mutations in transmembrane domain I (TM1) have been shown to prevent oligomerisation, either by destabilising intermolecular TM1-TM1 interactions or intramolecular interactions between TM1 and neighbouring TM domains (e.g TM2 and TM7). We will study the self-association of truncated versions of each protein (Fig.2) in an attempt to map the sites of protein-protein interaction.

Assay. Protein self-association will be examined using the TOXCAT assay. TOXCAT measures the relative strength of non-covalent associations between TM helices in a natural membrane bilayer.4 The concept behind the assay is shown in Fig.2. In TOXCAT, a protein containing one, three, five, or all seven GPCR TM domains will be expressed in the inner membrane of E. coli. Association of the TM domain results in activation of a reporter gene (CAT) whose activity is related to the strength of association.

‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

Brief Outline of work. In the first stage, the ability of individual GPCR TM domains to self-associate will be assessed using the TOXCAT assay. At this stage, synthetic primers encoding the entire TM domain will be used to create the constructs (no PCR required). This approach is used routinely in AMD's laboratory and is rapid and simple. In the second stage, truncations containing groups of three and five TM domains will be investigated for their ability to self-associate using the TOXCAT assay. Polymerase chain reaction (PCR) and modern cloning techniques will be required to amplify these longer regions, but it is anticipated that no more

than four of these constructs will be required (TM1-3, TM1-5, TM3-5, and TM3-7).

I. TOXCAT assay used to analyse individual GPCR TM domains: Long DNA primers encoding individual TM domains will be purchased and inserted into TOXCAT plasmid DNA, and the assay performed.

II. TOXCAT analysis of multiple TM regions: Truncations containing groups of three and five TM domains will be prepared using PCR, inserted into TOXCAT plasmid DNA, and the assay performed.

This project will provide students with a basic knowledge of modern cloning techniques as well as methods for handling and analysing membrane proteins.

References: [1] Bockaert, J., Pin, J.P., EMBO, 1999 (18) 1723-1729. [2] Overton, M.C., Chinault, S.L., Blumer, K.J., J. Biol. Chem., 2003 (278) 49369-49377. [3] Thevenin, D., Lazarova, T., Roberts, M.F., Robinson, C.R., Protein Sci., 2005 (14) 2177-2186. [4] Russ, W.P., Engelman, D.M., Proc. Natl. Acad. Sci. U S A, 1999 (96) 863-868.

Consumables budget§§: £150-200 for media, enzymes and consumables needed for cloning and assay.

MOAC / Systems Biology mini-project proposal 9Submit one page only to Monica Lucena ([email protected]) by Friday 9th February, 2007, 5pm).

§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Project title: Imaging the intracellular targeting of vacuolar membrane proteins in living cells


MOAC Mini Project √ Experimental biology Systems Biology Mini Project √ Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project


Project timing***


Slot 2 (21/05 to 13/07/2007) √ Slot 2 (18/06 to 07/09/2007)

Slot 3 (16/07 to 07/09/2007)


Name: Lorenzo Frigerio

Department: Biological Sciences _____________________Building, Room: B122

E-mail address: [email protected]________________Phone number: 02476 523181

Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision

support to the supervisor and also agrees to meet the student briefly at least once a week):

Name:



Project outline:

References:

(1) Brandizzi, F., Fricker, M., Hawes, C. (2002) Nat Rev Mol Cell Biol 3: 520-30. (2) Jauh, G.-Y., Phillips, T.E., Rogers, J.C. (1999) Plant Cell 11: 1867-1882. (3) Patterson, G.H., Lippincott-Schwartz, J. (2002) Science 297: 1873-7. (4) Runions, J., Brach, T., Kuhner, S., Hawes, C. (2006) J Exp Bot 57:43-50

Consumables budget†††: £ 200

Systems Biology Doctoral Training Centre 12-week Miniprojects 10

*** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. Therefore, the final deadline for submission of reports is 18 September 2007.††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Vacuoles are the hallmark of plant cells and the intracellular endpoint of the plant secretory pathway. Very little is known about the process that targets membrane proteins to the tonoplast (vacuolar membrane). We have generated a panel of fluorescent protein[1] fusions to the major tonoplast aquaporins, the tonoplas intrinsic proteins (TIPs)[2]. We intend to study the proces of TIP targeting in living leaf and seed cells. Besides the TIP-GFP reporters, which are useful to mark the steady-state localisation of the proteins, we now have fusions between different TIPs and a photoactivatable form of GFP (PA-GFP)[3]. PA-GFP can be activated with a short burst of 405 nm light whereupon it matures into ‘normal’ GFP. The project makes use of TIP-PA-GFP fusions to study the trafficking of TIPs to the tonoplast by optical pulse-chase. The fusions will be co-expressed with a set of standard xFP reporters to mark the main organelles of the plant secretory pathway. The main aim is to establish a reliable protocol for the activation of TIP-PA-GFP in the endoplasmic reticulum (ER), the port of entry of the secretory pathway. This will require: 1) the localisation and 3D imaging of the ER network both in leaf and developing seed cells. 2) The identification of a small region of interest within the ER network, which will be repeatedly activated. As protein diffusion at the ER membrane is extremely rapid, this will lead to the photoactivation of the whole ER surface within minutes[4]. 3) Loss of fluorescence from the ER and the appearance of TIP-activated GFP at different organelles of the secretory pathway will be monitored over time throughout the cell volume. A successful photoactivation experiment will provide invaluable information on a number of parameters: the route followed by the protein to reach the PSV; the timing of the transport event; the half-life of the protein. Such parameters have so far proven very difficult to determine using standard biochemical methods or immunocytochemistry.The project is multidisciplinary as it combines basic molecular biology, plant genetics, advanced light imaging and image processing.

Type of project: Theoretical and/or practical

Supervisor’s name: Maj Hultén

Crossover Hotspots and Genetic Interference:Mathematical Modelling of a Wave Phenomenon

&Measurement of Crossover positions by immunofluorescence

BACKGROUND TO THE PROJECT The formation of crossovers (also called chiasmata and leading to recombination) between maternal

and paternal chromosomes is a fundamental biological process, obligatory for the reductional cell division, halving the chromosome number in gametes (sperm and eggs in mammals). It is well known that crossovers are not randomly distributed along the length of chromosomes, but accumulate near their ends (crossover hotspots) and at long intervals (crossover/chiasma/genetic interference). This pattern is evolutionary conserved from yeast to humans.

Crossover maps, showing crossover positions can be assessed by family linkage analysis, tracing DNA alleles between generations, but also directly visualised and analysed by microscopy (Review in Hulten 2005; see also popular version http://www.biologia.uniba.it/eca/NEWSLETTER/NS-18/_NS-18.html).

The first hypothesis for the underlying mechanism on the non-random crossover positioning was produced in 1916 by Muller, but this is still an outstanding biological enigma - and in spite of decades of work there is yet no appropriate hypothesis explaining the mechanism underlying the occurrence of crossover hotspots and interference (Browning 2003; review in Hillers 2004).

We have a large crossover database (illustrations included) and material for microscopy images available for analysis by the students.

AIMS OF THIS PROJECT The aim of the theoretical project is to investigate different mathematical approaches to illustrate the

crossover patterns as documented in the computerised database. We suggest that this pattern (which varies slightly between individual sperm- and egg-forming cells) might be explained by the way the chromosome pairs are suspended within the cell nucleus, in combination with vibration of the nuclear membrane, at the time when crossovers are formed. It is hoped that the student will be able to establish a statistically significant mathematical model that can be published in a renowned peer-reviewed journal.

The practical project will involve preparation of microscopy slides for immune-fluorescence microscopy and measurements of crossover positions along the length of individual chromosomes together with statistical analysis.

METHODS TO BE USED In the theoretical project the student will apply different established mathematical models to test the

goodness of fit to the computerised data, under the assumption that the crossover patterns may be recognised as the result of a vibration wave phenomenon. This could be caused by vibration of the nuclear membrane to which the chromosomes are attached during the time period when crossovers are formed.

We are still collecting more raw data, and in the practical project the student would be involved in preparation of microscopy slides as well as recording crossover positions and measuring their positions immuno-fluorescence images.

REFERENCES Browning S (2000) Genetics 155:1955-1960 Hillers KJ (2004) Current Biology 14, 24, R1036 Hultén et al (2005) In Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics (online edition),

Jorde, L.. B., Little, P. F. R.., Dunn, M. J. and Subramaniam, S. (Eds) John Wiley & Sons Ltd: Chichester. DOI: 10.1002/047001153X.g102206, and references therein

TYPE OF PROJECT This project is available as both a miniproject 1 and 2.

Systems Biology Doctoral Training Centre 12-week Miniprojects 11TYPE OF PROJECT : THEORETICALSUPERVISOR’S NAME: Steve Jackson & David WildTITLE OF PROJECT : Modelling transcriptional networks involved in flower

induction BACKGROUND TO THE PROJECT

The power of microarrays are that they provide genome-wide transcription profiles which enable broader, more global questions to be asked about whole systems rather than the usual tight focus on one or two genes. The amount of information arising from just a simple experiment, however, can be vast and is often not fully analysed/utilised. Good bioinformatics and modelling approaches are therefore essential to make use of all the data and to interpret the results.

Timecourse analysis of gene expression in Arabidopsis leaves has been done before on a limited scale, however we are aiming to build the first complete transcription profile of the events going on in a particular leaf throughout its development. This will enable the identification of transcriptional networks controlling specific processes that occur at different stages of development of the leaf (eg. leaf expansion, floral induction, senescence). High quality microarray data has been obtained and some preliminary analysis has been done which shows the expected transcriptional profiles of specific key genes. We are now in a position to explore the huge amount of information contained in this unique set of data.AIMS OF THIS PROJECT

We have data from plants grown in different photoperiodic conditions, in florally inductive long days (LD), non-inductive short days (SD), and from plants that were transferred from SD to LD to induce flowering at a specific time. The aims of this miniproject will be to interrogate these datasets and identify genes that are up-regulated in this leaf upon flower induction. Clusters of genes involved in specific pathways will be identified and modelling will be used to identify control networks and nodal control genes.METHODS TO BE USED

Genes whose expression is significantly modulated over time will be determined by the method of Tai and Speed (2004). Time series clustering of gene expression profiles will be performed using the Bayesian approach of Heard et al. (2005), which has the advantage over other clustering methods in that there is no pre assigned number of clusters. Gene ontology annotation of the clusters will be used to guide the selection of subsets of genes for network inference. We will use the state space modelling approach of Rangel et al. (2004) and Beal et al. (2005) to infer candidate gene regulatory networks involved in flowering and identify candidate nodal (control) genes.TIMING OF PROJECT

This project is available for June.

REFERENCESBeal, MJ, Falciani, F, Ghahramani, Z, Rangel, C, Wild, DL. (2005). Bioinformatics 21:349-356.Heard, NA, Holmes, CC, Stephens, DA. 2006. JASA 101:18-29.

Heard NA, Holmes CC, Stephens DA, Hand DJ, Dimopoulos G. (2005). Proc Natl Acad Sci U S A. 2005 Nov 22;102(47):16939-44

Rangel, C., Angus, J., Ghahramani, Z., Lioumi, M., Sotheran, E., A., Gaiba, A.,.Wild, D.L. and Falciani, F. Bioinformatics, 20(9):1361-1372 (2004).

Tai, YC and TP Speed. Annals of Statistics Vol. 34 (5).

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16287981&query_hl=1&itool=pubmed_docsum

0 M Ligand 10-6M LigandFigure 2

Supervisor1: DR. GRAHAM LADDS, Room M119, Warwick Medical School, 024765 28361 [email protected]

Discipline: Experimental Biology / Biochemistry_________________________________________________________________________________________________________________________________________________

DEVLOPEMENT OF FLUORESCENT YEAST STRAINS FOR INVESTIGATIONS INTO POPULATION VARIABILITY

_________________________________________________________________________________________________________________________________________________

Biological systems are composed of physical constituents that constrain their performance. However, some aspects of system performance, including cell-to-cell variation, are often regulated by active mechanisms. The study of variation in the behaviour of genetically identical cells hopes to address these issues. Recently it has become apparent that the use of fluorescent protein reporters can aid the study of cell-to-cell variation in gene expression. For example, variation in gene expression among genetically identical bacteria has been studied by measuring the correlation in expression of two different fluorescent reporter genes under the control of the same promoter [1]. Similarly Colman-Lerner and co-workers have reported an investigation into cell-to-cell variation within the pheromone-response pathway of the budding yeast Saccharomyces cerevisiae [2]. This response-pathway is built upon the use of a G protein-coupled receptor (GPCR) which upon stimulation by a mating pheromone activates a G protein complex and induces a cell cycle arrest by activation of a MAP kinase cascade. However, while the S. cerevisiae pheromone-signalling cascade is a useful system to investigate it differs wildly from mammalian GPCR signalling networks. For example, within S. cerevisiae it is the G subunits which perpetuate the response following GPCR activation, whereas in higher eukaryotes it is the G subunit which fulfils this function [3]. The fission yeast, Schizosaccharomyces pombe has a pheromone-cascade closely resembling that of mammalian cells and is shown in Figure 1. In Sz. pombe it is the G subunits which perpetuate the response bring about a cells entry into meiosis [3].

● Aims of the project. To data there has not been a study into cell-to-cell variation within the Sz. pombe pheromone-signalling cascade. Building upon the ideas expressed by Colman-Lerner et al., we have developed a Sz. pombe strain which incorporates a pheromone-inducible Green Fluorescent Protein (GFP) reporter. To date we have only performed a preliminary analysis of this reporter which indicates that it is induced by pheromone (Figure 2). Our aim is generate Sz. pombe strains which are ideal for use in cell-to-cell variation experiments. Such strain will enable us to augment our existing computational model of the pheromone-response pathway by introducing stochastic differential equations to account for population variability [4].

● Brief description of the work proposed. In the first stage we will perform time-course experiments using confocal microscopy and flow cytometry to determine the strength and duration of the response using the GFP reporter strain. These studies will be accompanied by the use of immuno-blotting techniques of cell lysates to enable a detailed biochemical analysis of GFP reporter production. During the analysis and characterisation of the GFP reporter we propose to begin the production of our second fluorescent marker which will involve the use of molecular biology techniques to generate a construct suitable for integration into a host yeast strain. This project will enable the student to become familiar with all aspects of yeast genetics, molecular biology and utilise state-of-the-art fluorescent techniques to explore cell-to-cell viability. This project will complement the ongoing work by a second year MOAC PhD student currently working within my laboratory and is suitable for both MOAC and SB students.

● References: [1] Elowitz et al., 2002 Science 297, 1183-1186; [2] Colman-Lerner et al., (2005) Nature 437, 699-706; [3] Ladds et al., 2004 Trends Biotechnol 23: 367-373. [4] Smith et al., - In preparation.Timetabling: This project can be run in any of the project slots.

Figure 1

Consumables budget: £300.00-£350.00 for media, running costs of fluorescent microscope, antibodies, enzymes and consumables needed for cloning and assays.

MOAC / Systems Biology mini-project proposal (cover sheet for above)Submit one page only to Monica Lucena ([email protected]) by Wednesday 14th February, 2007, 5pm).

Project title: DEVLOPEMENT OF FLUORESCENT YEAST STRAINS FOR INVESTIGATIONS INTO

POPULATION VARIABILITY




Project timing‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr Graham Ladds

Department: Warwick Medical School___________________Building, Room: M119 Biological Sciences_________

E-mail address: [email protected]___________ Phone number: 02476 528361_______________



Name: Professor John Davey_________________________

Department: Biological Sciences_______________________ Building, Room: M119 Biological Sciences_____

E-mail address: [email protected]________________ Phone number: 02476 524204_______________

Project outline:

‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

See attached one page outline


References:

Consumables budget§§§: £ 300

Systems Biology Doctoral Training Centre 12-week Miniprojects 13TYPE OF PROJECT: LABORATORYSUPERVISOR’S NAME AND DEPARTMENT: Dr Andrea Massiah & Prof Brian Thomas,

Warwick HRI TITLE OF PROJECTInvestigation into floral input pathway activity during vegetative phase maturation BACKGROUND TO THE PROJECTDuring the early period of vegetative growth following germination, plants are often incapable of responding to an environmental stimulus, such as photoperiod, and initiating flowering. The mechanisms that result in reproductive incompetence during this period, the juvenile phase, are not understood. The question is raised as to whether lack of reproductive competence in the juvenile phase is due to inactivity of floral induction pathways that are established in the adult phase of vegetative growth. In comparison to wildtype Arabidopsis thaliana plants, the recessive hasty (hst-1) mutant displays a loss-of-function phenotype of a shortened juvenile phase, as assessed using juvenile and adult leaf morphological characteristics. The mutant shows accelerated flowering when grown in long day photoperiods and constant light (1, 2) and the reduction in flowering time is attributed to the alteration in juvenile phase length. This mutant provides an opportunity to investigate the association of floral input pathways with the juvenile phase.AIMS OF THIS PROJECT The aim of the project is to obtain expression profiles of key genes involved in the promotion of flowering (GIGANTEA, CONSTANS, FLOWERING LOCUS T, FD, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1), repression of flowering (GIBBERELLIC ACID INSENSITIVE) and floral meristem identity (LEAFY, APETALA 1) during the development of the hasty mutant and wildtype plants through to flowering. In parallel the plants will be assayed for competence to respond to daylength as a floral inductive stimulus and for leaf morphological characteristics. This comparative analysis will test the relationship between the expression of floral input pathways and the duration of the juvenile phase, as well as testing whether juvenility as determined by reproductive competence can be distinguished from juvenility in leaf morphology.METHODS TO BE USED

Transfer experiments between short day and long day photoperiods with flowering time assessments to determine juvenile phase length (weeks 1 – 8)

mRNA extraction and cDNA synthesis (weeks 3 – 4) Primer design (week 1) and quantitative real-time RT-PCR (weeks 5 – 10) Phenotypic analysis of trichome distribution on the abaxial surface of leaves

(week 3) REFERENCE(S)

1. Tefler, A. and Poethig, R.S. (1998) HASTY: a gene that regulates the timing of shoot maturation in Arabidopsis thaliana. Development 125: 1889-1898

2. Bollman, K.M., Aukerman, M. J., Park, M., Hunter, C., Berardini, T. Z. and Poethig, R. S. (2003) HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130: 1493-1504

§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

TIMING OF PROJECTProject available as miniproject 2


Project title: Recording Real-time changes in hormone concentration ______________________________


MOAC Mini Project X Experimental biology Systems Biology Mini Project X Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project


Project timing****


Slot 2 (21/05 to 13/07/2007) XSlot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: Richard Napier (plus Dan Mitchell –Warwick Medical School - and Hugo van den Berg, Maths)

Department: Warwick HRI (Medicine and Maths) _________ Building, Room: Office TP141, (but worek to be done in virus lab

in Biological Sciences)

E-mail address: [email protected]___________ Phone number: 75094_____________________

Project outline:

**** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

Background: Biologists need sensors that allow them to measure concentrations - and changes in concentration - of small signalling molecules (such as hormones) in real time, in living cells. We have developed a dynamic biosensor based on surface plasmon resonance (SPR, Biacore) recordings. The sensor molecule is an antibody making the technology applicable for diverse biological, industrial or environmental monitoring tasks. In order to deal with constantly changing sample concentrations – real-time real-life analysis - we need to develop ways to analyse and model the data outputs (mini-project 1). This project will utilise these routines to record, evaluate and interpret hormonal responses to exemplify and validate the analytical methodology as well as collect data on rapid hormonal fluxes for salient plant and animal signalling systems. Aims and Methods: The project will use data from the Biacore and explore how hormone fluxes can be evaluated in real time in living organisms (plants) and tissues/serum (animals). The experiments will be designed to utilise the analysis subroutine developed in the related mini-project. This is based on the displacement of a specific antibody from the chip surface by free analyte (hormone) sampled using a microdialysis probe. The kinetic displacement data will be converted to real-time concentration data by the subroutine and the system explored to establish suitable flow rates, chip loadings etc. Once optimised, the student can chose either plant or animal model for collecting novel data on hormone movement. Weeks 1 and 2: Familiarisation with the Biacore system (Biological Sciences, Virus lab). Antibodies, antigens, chips and solutions are available. Familiarisation with the Biacore Bia-Evaluation software. Week 3 – 4 Microdialysis will be taught and established, particularly the system requirements for interfacing the probes with the Biacore. Weeks 5- 12. Testing for hormonal concentration changes. Discussion with the student will identify a favoured biological test system from a shortlist for which antibody and antigen (analyte) are available. Eg, for students preferring to sample from plants auxin and ABA, for those opting for a mammalian system the release of the pro-inflammatory signalling peptide C3a (Dr Dan Mitchell (Warwick Medical School) will supervise). The chip will be loaded on all four channels. Channel 1 is the control, 2 -4 are analytical and will be loaded at different densities in order to explore the dynamic range of the system. Baseline data will be recorded and the system stimulated to initiate a response which will be recorded on all channels in real time. Sample flow rates and other parameters will be tested for their effects on signal and sensitivity. Outputs will be compared to prior knowledge (literature or local) and concentration records validated by standard ex-vivo techniques (ELISA, HPLC).Ideally, but not essentially, linked to mini-project ‘Simulating Real-time changes in hormone concentration from Biacore data‘. It also has potential for development as a PhD.

Consumables budget††††: £ 300 (Biacore chips and coupling reagents)


Project title: GLYCOPHORIN A MEMBRANE INSERTION AND FOLDING BY LINEAR DICHROISM




Project timing‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr. Joanne Oates (Dr. Ann Dixon) and Dr. Matt Hicks (Prof. Alison Rodger)

Department: Chemistry______________________________ Building, Room: Room C503 (AD)

E-mail address: [email protected] Phone number: 024761 50037



Name:___________________________________________

Department:_______________________________________ Building, Room: __________________________

E-mail address:____________________________________ Phone number:___________________________

Project outline.Background. Correct insertion, folding, and interactions of proteins in biological membranes are critical steps in a wide variety of biological processes. Surprisingly, very little detail is available addressing the mechanisms or kinetics of these essential processes. Thus far, the intensive study of -helical membrane proteins such as Glycophorin A (GpA, Fig. 1a) and Bacteriorhodopsin has supported a widely-accepted model for protein folding and interactions in membranes, namely the two-stage model of membrane protein folding (Fig. 1b) [1]. In this model, folding is described as occurring in two thermodynamically distinct stages: in stage 1, unfolded protein partitions into the membrane and forms stable -helices across the bilayer; in stage 2, individual -helices associate laterally in the membrane to form the functional state of the protein. Clearly, this model is incredibly simplistic and does not allow for probable folding intermediates, such as the example shown in Figure 1b (denoted 1a), where a partially folded structure first forms on the surface of the bilayer before insertion.

This overall lack of detailed information on membrane protein folding is due to the challenges one faces when attempting to acquire meaningful biophysical data for these very hydrophobic systems. Membrane proteins are notoriously difficult to work with in solution, and are typically studied in detergents and solvent mixtures with the hopes that these mimic the membrane bilayer environment. Such a hope is not always well-founded and,

†††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

ideally, we would like to investigate their properties in membrane bilayers. However, at present very few techniques can accommodate measurements in bilayers. At Warwick, we have targeted development of a technique that shows huge promise for working with bilayer systems: linear dichroism (LD) [2]. In LD we flow orient liposomes, and from the LD measurement one can monitor insertion and protein conformation simultaneously - this is one of the major advantages of LD over other (e.g. fluorescence-based) methods.

To date, technical developments have been made using model peptide and protein systems such as the antibacterial peptide gramicidin [3] and the light-harvesting membrane protein bacteriorhodopsin [4]. There is great potential to develop LD methods in order to obtain information on the kinetics of protein insertion, the stability of a particular protein fold, the orientation of proteins and co-factors in membranes and possibly protein-protein interactions in the membrane.

Aims of Project. We will analyse the well-characterised membrane protein Glycophorin A (GpA) in liposomes, using LD in conjunction with complementary biophysical and biochemical techniques. GpA contains a single transmembrane domain (Fig. 1a) that is known to insert into bilayers as a stable -helix and consequently drive dimerisation of the full-length protein (Fig. 1b) [5]. GpA has been extensively studied and a great deal is known about the amino acids that interact to stabilise the GpA dimer.

Much of the work on GpA thus far has been carried out in detergent micelles. Micelles are commonly used to solubilise membrane proteins, but they lack a bilayer structure and contain a high degree of curvature. This high curvature creates a lateral pressure that can affect the stability of protein folds and the degree to which membrane proteins interact. Liposomes (which we will use in this study), on the other hand, contain a bilayer structure and have a much lower degree of curvature, providing an environment that more accurately represents a natural membrane. We will look at the influence of lipid type on the formation and stability of GpA, a factor implicated in a number of other experimental systems [6]. Investigations of GpA will enable a detailed assessment of the advantages of liposomes as a valuable tool in the future study of less well-characterised transmembrane proteins of biological interest. Additionally the potential of LD to unravel the more complex issues of membrane protein folding will be addressed.

Brief Outline of Work. A peptide corresponding to the transmembrane domain of GpA will be purified using reversed-phase HPLC. Once purified, the peptide will be inserted into liposomes of varying lipid composition. Lipid membranes occur naturally in animals, bacteria and plants; the composition of these membranes varies with cellular location, cell type and between species. This is important, for example in antibacterial peptides - because bacterial membranes have more negatively charged lipids than human membranes. In this study we will use different proportions of phophatidyl choline which has an overall neutral charge, and phosphatidyl serine which has an overall negative charge. In addition to this we will explore the effect of cholesterol, which makes membranes more rigid by filling the gaps between the hydrocarbon chains. Time allowing it may also be possible to explore the effects of the degree of saturation of the fatty acyl chains of the lipids by using different proportions of dimyristoyl phosphatidyl choline (saturated) and palmitoyloleoyl phosphatidyl choline (one acyl chain unsaturated). Liposomes can be prepared in different ways, we will prepare them using a method comprising of freeze-thaw cycles and extrusion through filters with a pore size of 100 or 200 nm to give unilamellar liposomes of a uniform size. The secondary structure of the peptide will be measured using circular dichroism, and the oligomeric state will be assessed using chemical cross linking followed by SDS-PAGE analysis. Finally, LD measurements will be used to evaluate the insertion and conformation of the GpA TM peptide.

References. [1] Popot, J.L. and D.M. Engelman, Biochemistry, 29, 4031 (1990).[2] Dafforn, T. R., and Rodger, A. (2004) Current Opinion In Structural Biology 14, 541-546.[3] Rodger, A., Rajendra, J., Marrington, R., Ardhammar, M., Norden, B., Hirst, J. D., Gilbert, A. T. B., Dafforn, T. R., Halsall, D. J., Woolhead, C. A., Robinson, C., Pinheiro, T. J. T., Kazlauskaite, J., Seymour, M., Perez, N., and Hannon, M. J. (2002) Physical Chemistry Chemical Physics 4, 4051-4057.[4] Rajendra, J., Damianoglou, A., Hicks, M., Booth, P., Rodger, P. M., and Rodger, A. (2006) Chemical Physics 326, 210-220.[5] Lemmon, M.A., J.M. Flanagan, J.F. Hunt, B.D. Adair, B.-J. Bormann, C.E. Dempsey, and D.M. Engelman, J. Biol. Chem., 267, 7683 (1992). [6] Hong, H. and L.K. Tamm, PNAS, 101, 4065, (2004) Consumables budget§§§§. £150-200 for lipids and consumables needed for peptide purification.

§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects


Project title: Measurement of DNA persistence length____________________________________________




Project timing*****


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Martyn Rittman______________________________

Department: MOAC_________________________________ Building, Room: Chemistry dept, B608________

E-mail address: [email protected]_______________ Phone number: 23293_____________________



Name: Alison Rodger_______________________________

Department: MOAC/chemistry________________________ Building, Room: Chemistry dept, B607________

E-mail address: [email protected]________________ Phone number: __________________________

Project outline:

***** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

DNA persistence length is a widely accepted concept that measures flexibility of DNA. A recent review (in preparation), however, has highlighted difficulties in experimental measurement and conflicts within the definition or even the underlying concepts of persistence length.

The project proposed would entail carrying out measurements of DNA persistence length using stopped flow linear dichroism spectroscopy (SFFLD). To do this the DNA is passed through a narrow tube at a high rate to orient it, then stopped and allowed to relax back into a randomly ordered state. LD measures orientation by recording the difference between absorption of parallel and perpendicularly polarised light that has passed through a sample. For the case of DNA we expect to see relaxation curves for which a half-life can be measured and fitted to several known models.

Once the principle has been established it will be possible to extend the concept. The dependence of persistence length on any one of a number of factors can be investigated, for example DNA base sequence, temperature, salt concentration or DNA length.

Experience will be gained in handling biomolecule samples, biophysical instrumentation, modelling data, and literature searching. It will require sufficient mathematical expertise to understand the origins of polymer modelling as relates to the definitions of persistence length.

It is not linked with any other project.

References: See Martyn Rittman or Alison Rodger for draft copy of the review in preparation.

Consumables budget†††††: £ _______


Project title: Understanding flow in linear dichroism experiments___________________________________




Project timing‡‡‡‡‡


By negotiation Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

By negotiation Slot 3 (16/07 to 14/09/2007)


Name: Alison Rodger______________________________

Department: Chemistry______________________________ Building, Room: B607_____________________

E-mail address: [email protected]________________ Phone number: 74696_____________________

Project outline:

References: CH921 lectures

Rodger and Nordén, Circular dichroism and linear dichroism, OUP 1997

Consumables budget§§§§§: £ 150____††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Flow linear dichroism (LD) is a useful technique for determining relative orientations of sub-units of long molecular systems of molecules attached to something else (such as liposomes) which can be flow oriented. However, to use the data quantitatively we need to understand how the molecules are oriented. We have recently been looking for molecular markers to determine the degree of orientation. An alternative approach is to use models of flow and collect data at different flow rates. For membrane proteins, long DNAs and fibrous proteins linear dichroism is becoming a key structural technique, in part because others such as X-ray crystallography and NMR do not provide the level of information we require to understand the structure and function of biomolecular systems.

This project will involve designing experiments to provide LD data using DNA, liposomes and fibrous proteins as a function of flow rate and viscosity and relating those data back to flow models available in the literature. Comparison with results achieved via the molecular marker approach will be undertaken.

Experience will be gained in handling biomolecule samples, biophysical instrumentation, modelling data, and literature searching.

It is not linked with any other project.


Project title: The role of PBPs in Peptidoglycan Biosynthesis


MOAC Mini Project X Experimental biology Systems Biology Mini Project X Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project


Project timing******


X Slot 2 (21/05 to 13/07/2007) X Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr David Roper______________________________

Department: Biological Sciences_______________________ Building, Room: B126_____________________

E-mail address: [email protected]_____________ Phone number: 024 7652 8369______________



Name:___________________________________________

Department: ______________________________________ Building, Room: __________________________

E-mail address: ___________________________________ Phone number: __________________________

Project outline:

References:

Consumables budget††††††: £ 250____

****** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.†††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The role of PBPs in Peptidoglycan Biosynthesis(see project description)

The role of PBPs in Peptidoglycan Biosynthesis

Biochemical Mini-Project: Dr David I RoperA, & Professor C.G.DowsonB Department of Biological Sciences, University of Warwick

[Collaboration with Professor T.D.H Bugg, Department of Chemistry, & Dr M. Chappel, Department of Engineering]

A Room B126, email [email protected] tel 024 7652 8369B Email [email protected] tel 024 7652 3534

Bacterial resistance to antibiotics is a serious medical issue that complicates chemotherapy of infectious disease treatments and adds significantly to healthcare costs[1,2]. Although problems or resistance are common to many groups of bacteria they are particularly acute for noscomial infections caused by Gram positive bacteria[1,2]. Although several areas of bacterial metabolism can be exploited for antimicrobial intervention, peptidoglycan synthesis is an invaluable and validated target area in this respect. The collaborating groups of Roper, Bugg and Dowson and considerable experience, expertease and track record in analysis of this process3-7. At present we have a good understanding of the cytoplasmic processes that lead to the formation of the peptidoglycan precursor UDP-MurNac-Pentapeptide. Less is known about the subsequent membrane bound and cell surface processes that are required to produce the mature form of peptidoglycan which is extensively crosslinked by a group of enzymes known and the PBPs (Penicillin Binding Proteins)8.

We are addressing this area with an interdisciplinary approaching involving scientists from the physical, engineering and mathematical sciences.

The PBPs encompasses a large number of enzymes spread throughout the bacterial species, they are as the name implies, the target of the beta-lactam antibiotics including penicillin and therefore constitute a validated and well known drug target. There collective action is to catalyse transglycoslyase and transpeptidase reactions that link individual peptidoglycan monomers together to produce a three dimensional network. In most organism the function of a subset of PBPs has been shown to be essential, highlighting their utility as drug targets. These are mostly bifunctional proteins, catalyzing the polymerization of both sugar and peptide moieties of the peptidoglyan substrates using separate and independent enzymatic domains. Thus far only the extracellular portions of these proteins have been studied at the molecular level and these studies and been largely restricted to the transpeptidase domain of bifunctional PBPs: very little is known about the transglycoslyases. These PBPs contain and anchoring trans-membrane tail which probably has a functional role as well. We have developed a high expression system for membrane proteins which allows rapid cloning and expression in a form that can be purified by highly selective affinity chromatography which has thus far not been applied to the study of PBPs9.

The project will require1. Cloning a selection of absolutely required PBPs from E.coli, S.pneumoniae and S.aureus into a high

expression vector system for membrane proteins developed in our laboratory.2. Expression and purification studies of these proteins into detergent micelles for subsequent study.3. Functional characterization of PBP catalysis using prepared peptidoglycan intermediates.

Relationship to other projectsThis project is related to “Systems Analysis of Bacterial Cell Wall Biosynthesis” (Prof T.Bugg, Chemistry)



Students are strongly encouraged to complete both projects.

Timetabling: Suitable for 8-week MOAC student in slot 1 or 2; or Systems Biology student in slot 1.Consumables budget: £500 (for molecular biology reagents & growth and purification media)References:

1. Levy, B.L. & Marshall, B. 2004 Nature Med. 10, S122;2. Walsh C.T. 2000 Nature 406, 65; 3. Bugg TDH (1999) Comp Nat Prod Chem 3, 241;4. Schouten, J.A., et al (2006) Molecular Biosystems 2 484-491.5. Bugg, T.D.H. et al (2006) Infectious Disorders - Drug Target. 6,(2), 85-1066. Kishida,H. et al, (2006) Biochemistry, 45, 783-792 7. Besong, G.E.,et al .(2005) Angew. Chem. Int. Ed. Engl. 44(39):6403-6.8. Di Guilmi A.M. et al (2002) Current Pharmaceutical Biotechnology, 2002, 3, 63-759. Henderson, P.J.F. et al (2005). Biochem Soc Trans 33: 867-872.

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32

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Project title: Towards an understanding of multiple paralogues for metal-handling genes in a coastal cyanobacterium




Project timing‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr. David Scanlan and Dr. Claudia Blindauer_______

Department: Biological Sciences and Chemistry__________ Building, Room: __________________________

E-mail address: [email protected], [email protected]

Phone number: 28363 (D. Scanlan) or 28264 (C. Blindauer)_



Name:___________________________________________



Project outline:

Towards an understanding of multiple paralogues for metal-handling genes in a coastal cyanobacterium

Every living organism requires certain metal ions for survival, and a number of mechanisms for uptake, storage, and excretion have evolved to ensure an adequate supply with these nutrients. An intriguing family amongst the plethora of metal-handling proteins are the metallothioneins. These are small (usually 5-10 kDa), cysteine-rich proteins with the capability to bind high amounts of the essential Zn(II) and/or Cu(I), as well as the toxic Cd(II).

Recent research has revealed that a significant number of both freshwater and marine cyanobacterial strains contain genes for metallothioneins [1]. The structure of the prototype of these proteins, SmtA from Synechococcus PCC 7942, has been determined [2], and it has been shown that bacterial MTs differ significantly in structure, metal binding thermodynamics, and dynamics from eukaryotic MTs. To a large extent, these differential properties are owed to the presence of a zinc finger fold (shown in cyan and red in the figure) and the participation of two histidine residues in metal ion binding.

Multiple sequence alignment of all available bacterial MTs reveal that variations occur in the region around the two histidine residues, and it is predicted that these variation will have a substantial impact on the metal binding properties of the expressed protein.

In most cases, cyanobacterial genomes appear to contain only one gene for metallothionein, but the coastal strain Synechococcus CC9311 has no less than four different genes (see below for predicted protein sequences):

‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.



9 14 32 36 40 49 52Syn. PCC 7942 MTSTTLVKCACEPCLCNVDPSKAIDRNGLYYCSEACADGHTGGSKGCGHT---GCNCHG Syn. CC9311 MTVTVVKCACSSCTCEVSSSSAISRNGHSYCSDACASGH-RNNEPC-HDAAGACGCNCSyn. CC9311 MATSNQVCACDPCSCAVSVESAVQKDGKVYCSQPCADGH-SGSDEC-C---KSCDCSyn. CC9311 MNEVLLLCDCSLCKRSVEESRSIRIGGQHFCSESCAKGH-PNMEPC-DGERDGCNCG...Syn. CC9311 MTTNLVRCDCPPCTCSIEEATAAMYGNKLFCSEACATAH-INQEPSNSAEHTECSC

This was recognised recently by Paulsen [3], together with similar observations for other metal-handling genes. It has been postulated that this species in its coastal habitat might either have a greater need for metals, or a greater need to respond to excess metal levels, or that the cells experience episodic metal concentrations. In this context, it is interesting to note that the four paralogues are very likely to have different metal clusters, and hence metal content: One has ligands similar to Synechococcus PCC7942 SmtA, another lacks His49, but has a Cys in position 48, and the two others are expected to bind less than four zinc ions, with one of them lacking both His49 and Cys16 (site C), and another missing Cys47, which bridges sites B and C.

We hypothesise that different MTs, with different metal binding properties, are expressed in response to changes in the environment. It is therefore highly desirable to explore the relationship between the biophysical properties of the four homologues and their expression pattern under different conditions.

In a first step, it is planned to - produce expression constructs for each of the four genes- express the respective proteins in E. coli- purify the expression products by FPLC.

Genomic DNA, isolated from cultured Synechococcus CC9311, will be available in Dr. Scanlan's lab, and the genes of interest will be amplified by PCR. Subsequently, they will be cloned into PET29a, following an established procedure [1]. Methods for expression and purification are also established in-house.

The expressed proteins will be subjected to an initial characterisation by- elemental analysis (atomic absorption spectroscopy and inductively-coupled plasma mass spectrometry) for identifying

which metals are bound- mass spectrometry, which will allow the determination of the number of bound metal ions- if time and protein yields permit, 1H NMR experiments will be carried out. These experiments will

a) establish how well the proteins are folded, and b) give qualitative information on pertinent differences between the homologues.

We consider this pilot study as an ideal precursor for a subsequent interdisciplinary Ph.D. project, which would include culturing Synechococcus CC9311 in defined media with differing trace element composition, establishing differential expression of metal-handling genes, and ultimately linking environmental factors, expression patterns, and biophysical properties, including metal thermodynamics and dynamics and structure of expressed proteins.

References:

1. Blindauer CA, Razi, MT, Campopiano, DJ, Sadler, PJ, (2007) J Biol Inorg Chem, available online: http://www.springerlink.com/content/h46631w453431m63/?p=3296037ac21f4eac8ecbf7f45d57d59e&pi=0

2. Blindauer CA, Harrison MD, Parkinson JA, Robinson AK, Cavet JS, Robinson NJ, Sadler PJ (2001) Proc Natl Acad Sci USA 98, 9593-9598

3. Palenik B, Ren Q, Dupont CL, Myers GS, Heidelberg JF, Badger JH, Madupu R, Nelson W , Brinkac LM, Dodson RJ, Durkin AS, Daugherty SC, Sullivan SA, Khouri H, Mohamoud Y, Halpin R, Paulsen IT (2006) Proc Natl Acad Sci USA 103:13555-13559

Consumables budget§§§§§§: £ 300

Important notes:The work proposed above would either be suitable for a 12-week "wet" Systems Biology project, carried out in both Biological Sciences and Chemistry Departments, or for two MOAC projects, with the Molecular Biology parts carried out in Biological Sciences in Dr. Scanlan's group, and the Biophysical part in Chemistry in Dr. Blindauer's lab. The required work-load can be adapted according to available time and progress, for example by restricting the studies to less than four proteins. The Biophysical Chemistry part depends on the successful outcome of the Molecular Biology part. However, should the production of DNA constructs fail completely, Dr. Blindauer has expression constructs for several other bacterial MTs available, and the biophysical part of the project could then be carried out with these.

§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

http://www.springerlink.com/content/h46631w453431m63/?p=3296037ac21f4eac8ecbf7f45d57d59e&pi=0


Project title: Bioinformatic and transcriptional analysis of a marine methylotroph_______________________




Project timing*******


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Hendrik Schaefer_____________________

Department: Biological Sciences___________________ Building, Room: BioSci, B-177______________

E-mail address: [email protected]____________ Phone number: 02476-574208_____________



Name: J Colin Murrell_______________________

Department: Biological Sciences___________________ Building, Room: BioSci_____________________

E-mail address: [email protected]_____________ Phone number: 02476-523553______________

Project outline:

References:

Consumables budget†††††††: £ 200___

******* Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.††††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Methyl bromide and dimethylsulfide are two significant atmospheric trace gases that play important roles in ozone depletion and climate regulation, respectively. Recently, marine methylotrophic bacteria were isolated that degrade these compounds.

Methylophaga sp. DMS010 is an obligate methylotroph able to degrade the climate-cooling gas dimethyl sulfide (Schäfer, Appl Environ Microbiol., in press) Strain DMS010 also grows on a range of other C1-compounds (methanol, methylated amines). The genome of strain DMS010 is currently being sequenced and it is expected that a draft genome sequence will be available early in 2007 (February/March). This project will combine bioinformatics analyses and experimental work. The genome sequence of Methylophaga sp. DMS010 will be searched for genes encoding enzymes of primary one-carbon compound degradation (such as methanol dehydrogenase etc.), formaldehyde dissimilation and assimilation pathways and those of sulfur compound degradation. Subsequently, the expression of some key genes of C1 and sulfur metabolism during growth on methanol and dimethylsulfide (the biomass is already available, but the student could perform culture work if interested) will be investigated using reverse transcriptase (RT-) PCR using gene specific primers. This will aid in identification of the central metabolic pathways of this important marine methylotrophic bacterium.

Alternatively (e.g. should the genome sequence not yet be available), a methyl bromide degrading isolate can be studied. Roseovarius sp. 217 (Schäfer et al., 2005, Env Microbiol 7, 839-853) appears to degrade methyl bromide by an as-yet-uncharacterised pathway. A draft genome sequence for this organism is available and has facilitated the identification by mass spectrometry of polypeptides that were induced during growth on methyl bromide. On the basis of SDS-PAGE and mass spectrometry analyses a potential degradation pathway is suggested. Due to the toxicity of methyl bromide the biomass yield on this substrate is small, making it impossible to perform the relevant enzyme assays with methyl bromide grown cultures. Therefore, a transcriptional approach will be used here. The transcriptional regulation of candidate genes will be investigated by reverse transcriptase PCR using gene specific primers to be designed based on the genome sequence.

Supervisor: Dr. A. Shmygol. 21Associate Professor of Reproductive MedicineRoom 00108,Clinical Sciences Research Institute, Warwick Medical School.Tel.: (024)76968702

Discipline: Experimental Biology /Mathematical modelling.Co-supervisor: TBAProject Title:

Decoding Ca2+ signals in human myometrium: confocal imaging and numerical simulation of the Ca2+ release events targeting plasma membrane ion channels.

Project outline: Calcium is a ubiquitous intracellular signal, controlling a vast variety of cellular processes including muscle contraction, neurotransmitter release, gene expression, etc [1]. Many hormones and neuromediators elicit their regulatory action on target cells by releasing Ca2+ from the intracellular stores, i.e. endo-/sarcoplasmic reticulum (SR). Such release is mediated by the “second messenger” molecules, namely inositol 1,4,5-trisphosphate (InsP3), which is produced inside the cell in response to agonist binding to the surface membrane receptors (usually belonging to the GPCR family) [2]. InsP3 interacts with the InsP3-sensitive Ca2+ channels clustered on the membrane of the SR. A wave-like increase in the intracellular Ca2+ propagating throughout the entire cell is called “global Ca2+ signal” as opposed to localized, non-propagating Ca2+ signalling events called “Ca2+ sparks” and “Ca2+ puffs”. The global Ca2+ signals trigger contraction of smooth muscle cells and may be involved in the activation of gene expression. The localized Ca2+ release events are believed to target the clusters of Ca2+-activated ion channels on the surface membrane of the cell. Depending on the type of ion channels targeted, this may increase or decrease cellular excitability therefore facilitating or hampering the generation of global Ca2+ signals. The localized Ca2+ signalling events can only be visualised and studied in detail by using a high-resolution confocal microscopy. Progress in confocal microscopy of living cells suggests that Ca2+

sparks and puffs are composed of even smaller events called “Ca2+quarks” and “Ca2+ blips” [3]. The recruitment of the elementary Ca2+ release sites upon increase in the InsP3 level is stochastic and involves small-scale binding-diffusion processes. As a result, the amplitude of the local Ca2+ signalling events has a graded size, ranging from elementary blips, which involve the opening of only one release channel to moderately larger puffs, which result from the concerted opening of a few channels within the same cluster [4]. The resultant Ca2+ signal therefore depends on many factors including the size of the clusters and their distribution, strength of the stimulatory input and the filling state of the store [5]. Since it is often difficult to directly observe the elementary release events and virtually impossible to measure the binding of Ca2+ to cytoplasmic buffers and Ca2+ up-take by neighbouring organelles (e.g. mitochondria) on such a small scale, the study of localized Ca2+ signalling requires in-silico experimentation based on mathematical modelling. Numerical simulation of the multiple molecular complexes involved in Ca2+

signalling will help to discern the limiting steps in the whole process and will provide experimentally testable predictions. To date, most of the research on Ca2+ signalling has been conducted on immortalized cell lines [2-5]. Although these are convenient model systems for studying the phenomenology of Ca2+

signalling, it is often difficult to extrapolate the conclusions made on cell lines to cells in human body. For this reason, the researchers in medical sciences are shifting their attention to biopsies taken from patients undergoing surgical procedures in order to understand physiology and especially pathophysiology of Ca2+ signalling.

Uterine myocytes are among the best endowed with sarcoplasmic reticulum (see [6] for recent review), yet many details of the Ca2+ signalling in these cells remain unknown.

This project will address the problem of how the InsP3 mediated Ca2+ signals are decoded into changes in membrane excitability in human uterine myocytes.HypothesisThe Ca2+ signal decoding in human myometrial cells is achieved via differential Ca2+ sensitivity of potassium and chloride channels and via spatio-temporal properties of localized Ca2+ signals.Aims

1. To build an investigative model of localized Ca2+ release events coupled to clusters of Ca2+

activated ion channels based on spatial distribution of the InsP3 and ryanodine receptors and Ca-

activated chloride and potassium channels in human myometrial cells and spatio-temporal properties of localized Ca2+ signalling events.

2. Using numerical simulation experiments on the model, to investigate the role of the Ca2+ release channels clustering, micro-scale Ca2+ diffusion and buffering in the preferential activation of Ca2+-dependent chloride channels.

MethodsData sources: The student will utilize experimental data from a number of different sources. The ion channel expression and distribution data, as well as parameters of myometrial Ca2+ signals are currently investigated by the Warwick Parturition Group. The student will have an opportunity to take part in this “wet-lab” research. Our own data will be supplemented by current data in the literature on human myometrium, rat myometrium, and where no other data is available approximations from cardiac/other smooth muscles will be made.Modelling: The student will construct biophysically detailed models of the localized SR Ca2+ release events and activation of Ca2+ dependent ion channels based on spatial distribution of ion channels within the myometrial cell. The model will incorporate the time and the space dimensions and will allow to investigate the role of ion channels clustering and to estimate diffusional distances between the source of Ca2+ release and the target cluster of Ca2+-dependent chloride and/or potassium channels. The model will be developed from experimental data, as much as possible using data obtained from our previous and on-going studies but also using data from the literature (concerning the Ca2+ buffering kinetics and buffer densities within the microdomains). The models will help to determine conditions under which the localized Ca2+ release event can preferentially target one or another type of Ca2+-activated ion channels.References:

1. E. Carafoli (2002). Calcium signalling: A tale for all seasons. PNAS, 99, 3, pp. 1115-1122.2. M. J. Berridge (1993) Inositol trisphosphate and calcium signalling. Nature, 361, pp.315-325.3. M. D. Bootman & M.J. Berridge (1995). The elemental principles of calcium signalling. Cell, 83, pp 675-678.4. S. Swillens, G. Dupont, L. Combettes & P. Campeil (1999). From calcium blips to calcium puffs: theoretical analysis

of the requirements for interchannel communications. PNAS, 96, pp. 13759-13755. 5. M. D. Bootman, M.J. Berridge & P. Lipp (1997). Cooking with calcium: the recipes for composing global signals

from elementary events. Cell, 91, pp367-373.6. A. Shmygol & S. Wray (2004). Functional architecture of the SR calcium store in uterine smooth muscle. Cell

Calcium, 35, pp.501-508.

Project title: Protein-protein interactions 2: quantitative assessment of protein-protein interactions. 22Project suitable as: (tick all that apply)

MOAC Mini Project Experimental biology Systems Biology Mini Project √ Wet Project(8 weeks) √ Biophysical chemistry (12 weeks) Dry Project


Project timing


√ Slot 2 (21/05 to 13/07/2007) √ Slot 2 (25/06 to 14/09/2007)

√ Slot 3 (16/07 to 14/09/2007)


Name: Corinne Smith_______________________________

Department: Biological Sciences_______________________ Building, Room: __________________________

E-mail address: [email protected]____________ Phone number: 22461_____________________

Project outline:

References:

Consumables budget: £ 200 - Ni columns, PAGE consumables etc.

This project is one of a pair of linked projects (Biology - Dave Whitworth / Biophysical chemistry - Corinne Smith), although either project could be undertaken in isolation if necessary.

In the first of the linked projects, expression vectors will be engineered for production of seven regulatory proteins (three PhoR homologs and four PhoB homologs). In this project the resulting proteins will be tested for their ability to participate in biomolecular interactions. In homologous systems, PhoBs can homodimerise, PhoRs can homodimerise and PhoRs can interact with PhoBs. In addition to these protein-protein interactions, PhoBs can bind to DNA. Candidate target DNA sequences can be easily generated in vitro by PCR of the promoter regions upstream of Pho regulon genes.

PhoB homodimerisation, PhoR-PhoB interaction and PhoB-DNA interaction are usually dependent on the phosphorylation state of the involved proteins. Phosphorylation of PhoR and PhoB can be altered at will through the addition of specific phosphate-donating molecules. Typically, PhoBs can be phosphorylated by adding acetylphosphate (AcP), while PhoRs can be phosphorylated in vitro by the addition of ATP.

The project aims to characterise the affinity and/or kinetics of interactions for all pairwise combinations of PhoR/PhoB and PhoB/DNA, and then test whether the association parameters are affected by phosphorylation state. In this way it will be possible to characterise the connectivity of the Pho regulon interaction map, and provide values for many of the key parameters affecting dynamical behaviour.

Initially, one homologue each of PhoR and PhoB will be purified and characterised by estimating their state of association using light scattering and by assessing the secondary structure content using circular dichroism. The parameters for monitoring binding interactions between PhoR/PhoB and PhoB/ DNA using isothermal titrating calorimetry (ITC) will then be determined. Obtaining binding constants for the interaction between PhoB-PhoR and PhoB-DNA together with characterisation of the PhoB association state will complete the first stage of the project. If time permits, the project will be expanded to investigate all seven PhoB and PhoR homologues by setting up an affinity column assay using, for example, 6His-PhoR bound to a nickel column, and then washing over a mixture of all 4 PhoBs. The proteins which bind will then be identified using SDS page and subsequent mass spectrometry analysis of each band on the gel. The results of the affinity assay will provide a basis for further ITC studies to determine the binding constants for all possible pairwise interactions of the PhoB/PhoR homologues.


Project title: Simulating Real-time changes in hormone concentration from Biacore data


MOAC Mini Project Experimental biology Systems Biology Mini Project Wet Project(8 weeks) Biophysical chemistry (12 weeks) X Dry Project

X Mathematics/computing

Project timing‡‡‡‡‡‡‡

MOAC Mini Project X Slot 1 (26/03 to 18/05/2007) Systems Biology Mini Project X Slot 1 (26/03 to 22/05/2007)

X Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Hugo van den Berg (and Richard Napier)

Department: Maths (Warwick HRI)____________________ Building, Room: Maths Institute, (TP 141)

E-mail address: [email protected] (Richard.napier @...Phone number: 23698 (75094)_________



Name:___________________________________________



Project outline:

Consumables budget§§§§§§§: £ 150 (Biacore chip and reagents)

‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Background: Biologists need sensors that allow them to measure concentrations - and changes in concentration - of small signalling molecules (such as hormones) in real time, in living cells. We have developed a dynamic biosensor based on surface plasmon resonance (SPR, Biacore) recordings. The sensor molecule is a recombinant antibody for which we know the kinetic constants and we have detailed calibration data. However, at present the Biacore analysis software is designed for batch mode measurements, treating each sample as a discrete record. In order to deal with constantly changing sample concentrations – real-time real-life analysis - we need to develop a protocol to analyse and model the data outputs.AIMS and METHODS: The project will explore how data from the Biacore can be evaluated in real time to allow recordings of continually changing hormone concentrations in living organisms. The kinetic displacement data can be readily linearised, but account needs to be made of baseline (control channel) subtraction, small time delays between channels and the changing signal to noise ratios over the decay curve of antibody displacement. You will find the time period over which a statistically-robust rate can be determined and develop routines for iterative, incremental calculation of concentration from these conditions. These forward calculations will be compared to routines of, for example, reverse fit which will model the dissociation over iterative increments of the data and test fit, deriving the concentrations of analyte and presenting the data in real time. The routine found to work best will be prepared as a software subprogramme to work with the Biacore. The system will be tested using artificial analyte gradients. Weeks 1 and 2: Familiarisation with the Biacore system (Biology Dept, Flu lab). We have antibodies and biotinylated antigens, chips and solutions available for this. The chip will be loaded on two channels (channel 1 as control, 2 as analytical), a set of calibration sensorgrams taken. Weeks 3 - 12. Using the sensorgrams and the known kinetic constants for the antibody-antigen pair, the mathematical handling of the Biacore output will be explored and challenged. A dynamic model of binding kinetics in the Biacore chamber will be constructed to allow the reconstruction of the input signal (concentration in the analyte line) from the observed SPR signal.


Modelling the photoprotective response in Myxococcus xanthus 24Project type: THEORETICAL

Supervisor: Hugo van den Berg (Systems Biology) [email protected] +23698

Co-supervisor: David Whitworth (Biology)

BACKGROUND TO THE PROJECT Cells of the microorganism Myxococcus xanthus soon perish when they are illuminated by bright light: the energy in the light is transferred to oxygen by a component in the respiratory chain, and this very reactive (singlet) oxygen plays havoc with the cellular machinery. To avert these lethal effects, M. xanthus expresses the crt-genes which encode enzymes that catalyse the biosynthesis of carotenoids, lipid-like molecules which act as photoprotective pigments by absorbing the energy from the singlet oxygens and dissipate it as heat.

The expression of the crt-genes is regulated by a sensor in the membrane. In particular, the protein carQ, which is normally bound to carR, another membrane component, is liberated in the presence of singlet oxygen (which leads to the destruction or inactivation of carR). The free carQ is a transcription factor for one of the crt-genes (crtI). The remaining crt-genes lie together on the crtEBDC operon, which is constitutively inhibited by carA, a protein that controls the expression of crtEBDC through negative feedback. The protein carA lies on the crtEBDC operon but has, in addition, a leaky constitutive expression.

Besides stimulating crtI expression, carQ also serves an autotranscription factor, stimulating its own expression in a positive feedback loop. The sequestering protein carR is found on the same operon. A third protein on this operon, carS, disinhibits the expression of the crtEBDC operon: carS down-modulates the tonic feedback inhibition exerted by carA on the crtEBDC promoter.

Three promoters are thus involved in the photoprotection response: carQRS, crtI, and crtEBCD. A time course of the activity of each of these promoters following a photo-irradiation challenge can be measured in a system where one of these promoters has been artificially coupled to the gene encoding LacZ, which is used as a reporter of promoter activity. The central problem is to reconstruct the regulatory dynamics of the photoprotection response from these LacZ time courses. A mathematical model might help to determine which experimental perturbations give the most information about this dynamics; mathematical modelling also helps to interpret the experimental findings.

AIMS The objectives of this project are to develop a mathematical model of the photoprotective response in M. xanthus and explore its role in suggesting & interpreting experiments. The project is offered in conjunction with an experimental project in which the student will have the opportunity to carry out some of the protocols suggested by his or her analysis of the mathematical model.

RESEARCH METHODS Analysis of non-linear dynamics; numerical solution of initial value problems; system identification tools.

TIMING OF PROJECT Both periods are negotiable, but see note below.

IMPORTANT NOTE While this project can be pursued in its own right, it is a companion to an experimental project offered by Dr David Whitworth. A student choosing to do both a theoretical and an experimental project on this subject is advised to take the theoretical project first, in order to benefit optimally from the opportunities offered by the laboratory environment.


Project title: Predicting Biomolecular Interactions: Comparative Prokaryotic Genomics 25Project suitable as: (tick all that apply)

MOAC Mini Project Experimental biology Systems Biology Mini Project Wet Project(8 weeks) Biophysical chemistry (12 weeks) √ Dry Project

√ Mathematics/computing

Project timing


√ Slot 2 (21/05 to 13/07/2007) √ Slot 2 (25/06 to 14/09/2007)

√ Slot 3 (16/07 to 14/09/2007)

Supervisor: Name: Dave Whitworth_____________________________Department: Biological Sciences______________________ Building, Room: B008____________________

E-mail address: [email protected]___________ Phone number: 74738____________________

Supervisor’s advisor:Name: David Hodgson / Peter Cock___________________

Department: Biological Sciences / MOAC_______________ Building, Room: _________________________

E-mail address: [email protected]______________ Phone number: 50059____________________

Project outline:

References:

Consumables budget: < £ 50 - Stationary, software and media…

One of the biggest challenges of computational biology is to be able to correlate genome sequence with function. While it is currently possible to predict functional classes for most gene products, the role of particular proteins within the cell is often obscure - particularly with respect to the specificity of reactions catalysed, or position within the interaction network.

We have been developing computational approaches to predict interactions between conserved proteins, based entirely on their sequence. Using a database of paired proteins thought to interact, it is possible to identify residues involved in conferring specificity to a biomolecular interaction using a variety of approaches (information theory, evolutionary tracing, co-variation analysis, etc.). When this information has been acquired, interactions can then be predicted using a generalised linear model (GLM) and validated. The ability to make predictions is vitally important in those (common) cases where multiple candidates for interaction partners exist. Suitable interactions for study include the -factor/anti--factor, PhoB/pstS, -factor/DNA, and HisKA/HisKA interactions.

The project will involve developing a database of interacting protein pairs (or DNA-protein pairs). Initial searches will use RPS-BLAST to screen all completed bacterial genomes for proteins containing particular Pfam domains, while DNA regions of interest will be isolated based on proximity to conserved genes. While the results of these searches will be interesting in their own right, hits will then be filtered by a variety of user-proscribed rules - based on biological understanding of the systems of interest. For instance, most -factor genes are found adjacent to those of their partner anti--factors, while PhoBs are known to bind DNA upstream of the pstS gene. Multiple sequence alignments will then be derived, enabling the degree and nature of conservation at particular sequence positions to be determined. Specificity-conferring residues will be identified, and mapped onto solved structures where available. A GLM will be then be developed to model the interacting biomolecules, enabling predictions of partnerships. Predictions will be made for key systems, and results compared to available literature.

The project will involve a lot of coding, and so would be good for someone with programming know-how, but it would also be appropriate for someone wanting to develop their programming skills.

Project title: Protein-protein interactions 1: Protein expression, purification and activity assays. 26Project suitable as: (tick all that apply)



Project timing


√ Slot 2 (21/05 to 13/07/2007) √ Slot 2 (25/06 to 14/09/2007)

√ Slot 3 (16/07 to 14/09/2007)


Name: Dave Whitworth_____________________________



Supervisor’s advisor:Name: Corinne Smith_______________________________



Project outline:

References:

Consumables budget: £ 200 - expression vectors, cloning enzymes and reagents, Ni spin columns...

This project is one of a pair of linked projects (Biology - Dave Whitworth / Biophysical chemistry - Corinne Smith), although either project could be undertaken in isolation if necessary.

Protein-protein interactions are of fundamental importance for biomolecular network formation and resulting dynamic behaviour. This project aims to quantify the interactions between seven two-component system proteins known to act within the Pho regulon of Myxococcus xanthus.

In most bacteria the uptake of phosphate is mediated by a low-affinity high-capacity transporter (Pit). However, upon phosphate limitation, expression of a high affinity transporter (Pst) is induced. Pst induction is regulated by a two-component signal transduction system (PhoBR). PhoR is a sensor kinase which indirectly senses phosphate availability. When phosphate levels drop PhoR interacts with and phosphorylates the response regulator PhoB. PhoB-P then activates transcription of the pstSCAB operon (encoding Pst).

In the complex social predator Myxococcus xanthus the Pho regulon includes at least three PhoR homologs and four PhoB homologs, in addition to Pst and Pit transporters. Three phoB genes are adjacent to phoR genes, implying functional association. Two-hybrid interaction assays suggest that as well as the three PhoR-PhoB interactions predicted from the genome, other signalling interactions occur between paired-gene systems, resulting in a complicated network architecture. Towards a complete description of the Pho system, we wish to quantify the interaction affinity for all pairwise combinations of PhoR and PhoB homologs.

During this project a series of protein expression vectors will be constructed using standard molecular biology techniques (PCR, DNA sequencing, restriction enzyme digestions, DNA ligation, bacterial transformation, agarose gel electrophoresis, DNA extractions etc.). The expression vectors will be engineered to express proteins as fusions to a 6His tag, for one-step column purification. We currently have clones which express interacting domains (transmitter and receiver domains) of the PhoR and PhoB homologues as GST fusions, and the cloned regions of these constructs will be sub-cloned into an appropriate 6His vector. Whole length protein fusions will also be expressed by PCR-cloning of full length proteins (for those proteins without transmembrane helices). Once expression vectors have been constructed protein expression will be induced in E. coli. Localisation and solubility of expressed protein will be determined by cell fractionation. Expression conditions will be varied to maximise the amount of soluble protein produced, and fusion proteins will be purified using nickel columns. Some of the purified fusion proteins will then have their 6His tags removed with appropriate protease digestions. In the paired project expressed proteins will then be tested for biomolecular interactions using a variety of assay techniques.

Project title: Assaying the photoprotective response in Myxococcus xanthus . 27Project suitable as: (tick all that apply)



Project timing


√ Slot 2 (21/05 to 13/07/2007) √ Slot 2 (25/06 to 14/09/2007)

√ Slot 3 (16/07 to 14/09/2007)

Supervisor: Name: Dave Whitworth_____________________________

Department: Biological Sciences______________________ Building, Room: B008_____________________

E-mail address: [email protected]_____________ Phone number: 74738____________________

Supervisor’s advisor:Name: David Hodgson______________________________

Department: Biological Sciences______________________ Building, Room: _________________________

E-mail address: [email protected]____________ Phone number: 23559____________________

Project outline:

The Car regulon of Myxococcus xanthus controls a light-protective, light-induced behaviour. Light causes cellular production of the reactive oxygen species singlet oxygen. In response, enzymes (crt) are expressed which generate a class of compounds called carotenoids. Carotenoids quench singlet oxygen, protecting cells from further illumination.

Expression of the crt genes is controlled by four regulatory (car) genes. One regulator is encoded in an operon with the crt genes (carA), and the gene product represses the crtEBDC promoter. The other three regulators are found together in the carQRS operon. CarQ is a sigma factor, CarR is an anti-CarQ factor while CarS is an anti-CarA factor. CarQ stimulates transcription of carQRS and crtI, while CarR is light (singlet oxygen) – sensitive. This system is inactive in the dark with crtEBDC being repressed by CarA, however in the light, repression of crtEBDC expression is relieved and crtI expression is stimulated, resulting in production of photoprotective carotenoids.

The promoters within the Car regulon can be assayed using a genetic construct called a promoter probe. Promoter probes are integrated into the chromosome and position a promoterless lacZ gene under the control of the promoter of choice. Promoter probes are available for all three Car regulon promoters, in a variety of genetic backgrounds. Typically, promoter activity assays have been performed by exposing a dark-adapted culture to continuous bright light at T=0. Samples are periodically removed for protein and LacZ assays.

The project will entail generating time-courses of LacZ expression from promoter probe-containing strains of M. xanthus. Irradiation schemes will be varied, as will nutrient availability and genetic background. Carotenoids can be also be extracted from samples, providing additional insights into the dynamics of the Car regulon.

IMPORTANT NOTE: While this project can be pursued in its own right, it is a companion to a theoretical project offered by Dr Hugo van den Berg. A student choosing to do both theoretical and experimental projects on this subject is advised to take the theoretical project first, to benefit optimally from the opportunities offered by the laboratory environment.

References: Browning, Whitworth and Hodgson 2003 Mol Microbiol 48: 237-251Whitworth and Hodgson 2001 Mol Microbiol 42: 809-819

carQ carR carS

CarR

CarQ

1O2 PPIX

LIGHT

CarS

crtEBDC/carA operon

CarA

Carotenoids

(Photosensitiserprotoporphyrin IX)

(TOXIC)

carQ carR carS

CarR

CarQ

1O21O2 PPIX

LIGHT

CarS

crtEBDC/carA operoncrtEBDC/carA operon

CarA

Carotenoids


(TOXIC)

crtI

carQ carR carS

CarR

CarQ

1O2 PPIX

LIGHT

CarS

crtEBDC/carA operon

CarA

Carotenoids


(TOXIC)

carQ carR carS

CarR

CarQ

1O21O2 PPIX

LIGHT

CarS

crtEBDC/carA operoncrtEBDC/carA operon

CarA

Carotenoids


(TOXIC)

crtI

Consumables budget: ~ £150 – Growth media, assay components, enzyme substrates...


Project title: Reconstructing gene regulatory networks with nonlinear state space models




Project timing********


Slot 2 (21/05 to 13/07/2007) X Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: David Wild

Department Systems Biology Building,Room Coventry House , 332.

E-mail address [email protected]________________Phone number: 50242

Project outline:

References: Beal, MJ, Falciani, F, Ghahramani, Z, Rangel, C, Wild, DL. (2005). Bioinformatics 21:349-356. Rangel, C., Angus, J., Ghahramani, Z., Lioumi, M., Sotheran, E., A., Gaiba, A.,.Wild, D.L. and Falciani, F. Bioinformatics, 20(9):1361-1372 (2004).

******** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

A major challenge in systems biology is the ability to model complex regulatory interactions. In previous work we have described the use of Linear-Gaussian state-space models (SSMs), also known as Linear Dynamical Systems (LDS) or Kalman filter models to ‘reverse-engineer’ regulatory networks from high-throughput data sources, such as microarray gene expression profiling (Rangel et al., 2004; Beal et al. 2005). SSM models are a subclass of dynamic Bayesian networks used for modeling time series data and have been used extensively in many areas of control engineering and signal processing. Although the linear Gaussian state-space model has provided a natural starting point for our modelling approach, we wish to explore nonlinearities in the dynamics and output processes of the model, focusing on incorporating nonlinearities that reflect the underlying biological mechanisms. Another valid criticism of the simple linear-Gaussian dynamical system model is that its parameters are stationary— the dynamics and output mechanisms are assumed to be the same at any point in time along the gene expression time course. We therefore propose to investigate time varying models which may more faithfully reflect these aspects of true biological processes. This project will focus on simulation studies based on synthetic mRNA data generated from a model which contains definite non-linearities in the dynamics of the hidden factors (arising from the oligomerization of transcription factors). The Matlab Rebel toolkit of functions and scripts (http://choosh.ece.ogi.edu/rebel/), designed to facilitate sequential Bayesian inference (estimation) in general state space models will be used, so some facility with Matlab programming is essential.

http://choosh.ece.ogi.edu/rebel/


Project title: A Bayesian approach to biological information retrieval




Project timing††††††††


Slot 2 (21/05 to 13/07/2007) X Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: David Wild



CollaboratorName: Zoubin Ghahramani

Department:: Engineering, University of Cambridge

E-mail address:[email protected] Phone number: 01223-764-093

Project outline:

References: J. Cheng and P. Baldi, (2006) “A machine learning information retrieval approach toprotein fold recognition”, Bioinformatics, 22, 1456–1463Z. Ghahramani and K.A. Heller, (2006) "Bayesian Sets", In Advances in Neural Information Processing Systems (NIPS 2005).

Consumables budget‡‡‡‡‡‡‡‡: travel between Warwick and Cambridge for collaborative meetings with Prof. Ghahramani

†††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Humans readily learn new concepts after observing a few examples and show extremely good generalization to new instances. In contrast, search tools on the internet exhibit little or no learning and generalization. Recent work by Ghahramani and Heller (2006) shows that information retrieval can be firmly grounded in a Bayesian statistical model of human learning and generalization. Given a set of items, the system finds other items that belong to the same concept. For example, given Monday, Wednesday, it should return the days of the week; given three Jim Carrey movies, it should return other Jim Carrey movies; given a couple of proteins with some function, it should return other proteins with a similar biological function. Implementations of the algorithm exist for binary data (such as images) and discrete categorical data. The aim of this project is to extend this approach to data types of relevance to bioinformatics problems. Depending on the interests of the student, the project could develop in one of two directions. The first would be a more empirical data analysis project which would involve extending the method to the problem of protein fold recognition (recognizing proteins that have similar tertiary structures based on a database of known protein structures). This problem was recently cast in the framework of information retrieval by Chen and Baldi (2006), who have made a suitable data set available. The second direction would be more analytical and involve extending the theory to the problem of sequence similarity searching, based on a first or second order Markov chain model of biological sequences (di- or tri- peptides or nucleotides). This would involve extending the existing implementation for sparse binary data, which relies on the Bernoulli-Beta conjugate pair of distributions to the Multinomial-Dirichlet conjugate pair, with the goal of producing a prototype software implementation.


Project title: Fast Bayesian clustering for microarray data




Project timing§§§§§§§§


Slot 2 (21/05 to 13/07/2007) X Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: David Wild



CollaboratorName: Zoubin Ghahramani

Department:: Engineering, University of Cambridge

E-mail address:[email protected] Phone number: 01223-764-093

Project outline:

References: de la Cruz, B.J., Rasmussen, C.E., Ghahramani, Z., Wild, D.L. A Bayesian Approach to Modeling Uncertainty in Gene Expression Clusters Submitted. K.A. Heller and Z. Ghahramani (2005) "Bayesian Hierarchical Clustering", In Proceedings of the Twenty-second International Conference on Machine Learning (ICML 2005). K.A. Heller, Z. Ghahramani, D. Wild (2006), “Efficient Bayesian Hierarchical Clustering for Gene Expression Data”, Valencia Bayesian Statistics Meeting 8, Benidorm, Spain.

Consumables budget*********: travel between Warwick and Cambridge for collaborative meetings with Prof. Ghahramani

§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.********* Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The use of clustering methods has rapidly become one of the standard computational approaches of understanding microarray gene expression data. In clustering, the patterns of expression of different genes across time, treatments, and tissues are grouped into distinct clusters (perhaps organized hierarchically) in which genes in the same cluster are assumed to be potentially functionally related or to be influenced by a common upstream factor. Such cluster structure can be used to aid the elucidation of regulatory networks. However, commonly used clustering methods either require the number of clusters to be predefined, or rely on the setting of some heuristic score threshold to distinguish members of a particular cluster from non-members, making the determination of the number of clusters arbitrary and subjective. In previous work (de la Cruz et al., 2007), we have described a novel approach to the problem of automatically clustering gene expression profiles, based on the theory of infinite mixtures. However, this work, like most Bayesian approaches, is based on sampling using Markov Chain Monte Carlo (MCMC) methods. While MCMC has useful theoretical guarantees, its applicability to vast post-genomic datasets is limited by its speed. In this project, we propose to apply a fast novel algorithm for agglomerative hierarchical clustering (Heller and Ghahramani, 2005), which is based on evaluating the marginal likelihoods of a probabilistic model, and may be interpreted as a bottom-up approximate inference method for a Dirichlet process (i.e. countably infinite) mixture model. The project will involve adapting the method to the problem of clustering gene expression profiles from a variety of experimental data sets.


Project title: Integrating transcriptome and metabolome changes in response to pathogen infection




Project timing†††††††††


Slot 2 (21/05 to 13/07/2007) X Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: David Wild__________________________________

Department: Systems Biology ________________________ Building, Room: Coventry House ____________

E-mail address: [email protected]_________________ Phone number: __________________________

Second supervisor:Name: Katherine Denby_____________________________

Department: Warwick HRI and Systems Biology __________ Building, Room: TPB133, Coventry House 326__


Project outline:

References Beal, MJ, Falciani, F, Ghahramani, Z, Rangel, C, Wild, DL. (2005). Bioinformatics 21:349-356.

Heard, NA, Holmes, CC, Stephens, DA. 2006. JASA 101:18-29. Rangel, C., Angus, J., Ghahramani, Z., Lioumi, M.,

Sotheran, E., A., Gaiba, A.,.Wild, D.L. and Falciani, F. Bioinformatics, 20(9):1361-1372 (2004). Tai, YC and TP

Speed. Annals of Statistics Vol. 34 (5).

Consumables budget‡‡‡‡‡‡‡‡‡: £ 0____

††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The aim of this project is to integrate gene expression data and metabolite profiles of Arabidopsis leaves infected with Botrytis cinerea (a fungal pathogen) to identify novel associations between gene expression and metabolite levels. Such associations may be based on function (eg. a gene encodes an enzyme involved in synthesis of a particular metabolite) or regulation (eg. a gene encodes a transcription factor regulating expression of a biosynthetic pathway producing a particular metabolite). The data for this project will be a time series of 24 samples taken every 2 hours following B. cinerea infection. One leaf from each plant is harvested for expression profiling and three other similar sized leaves will be used for measuring metabolites. Metabolite profiles will be generated using FT-IR (which gives fingerprints without identifying particular compounds) and ESI-MS. In this miniproject genes whose expression is significantly modulated over time and between biological conditions (control and pathogen infected) will be determined by the method of Tai and Speed (2004). Time series clustering of gene and metabolites profiles will be performed using the Bayesian approach of Heard et al. (2005), which has the advantage over other clustering methods in that there is no pre-assigned number of clusters. Gene ontology annotation of the clusters will be used to guide the selection of subsets of genes and metabolites for network inference. We will use the state space modelling approach of Rangel et al. (2004) and Beal et al. (2005) to integrate gene expression and metabolomic data and to infer candidate joint gene and metabolite regulatory networks.

Systems Biology Doctoral Training Centre 12-week Miniprojects 32TYPE OF PROJECT (LABORATORY OR THEORETICAL): BothSUPERVISOR’S NAME: David Wild (WSB) and Philip McTernan (CSRI). TITLE OF PROJECT: Delineating molecular mechanisms contributing to mitochondrial dysfunction in obesity and diabetes.BACKGROUND TO THE PROJECT

Obesity is by far the single most important risk factor for type 2 diabetes (T2DM). Whilst there is genetic deposition for this it is clear that environmental factors play a significant role in its aetiology. However, the mechanisms for this link between obesity and its metabolic dysfunction remain unclear. Studies suggest that mitochondrial dysregulation may have an important impact on diabetic risk. The integrity of mitochondria (the power house of the cell) in (fat) adipose tissue in obesity and T2DM has recently been investigated in animal models but to date no data is available in humans. The primary defect(s) causing bioenergetic dysfunction may reside in nonbioenergetic pathways such as signalling between mitochondria and nucleus or in overall mitochondrial biogenesis or degradation pathways impacted by inflammation and our current studies in adipose tissue taken form obese patients suggest that the expression of genes related to energy metabolism in the mitochondria appear to decrease with a similar pattern observed in adipose tissue Abd Sc AT from T2DM subjects. As such these data suggest that as well as adipose tissue being important in regulating other organ systems these studies suggest a role for mitochondria in human adipose tissue which appear dysregulated in obesity and diabetes. In clinical practice, pathological phenotypes are often labelled with ordinal scales rather than binary, e.g. the Gleason grading system for tumor cell differentiation or the BMI scale for obesity. However, in the literature of microarray analysis, these ordinal labels have been rarely treated in a principled way. In preliminary work (Chu et al. (2005)) we have developed a gene selection algorithm based on Gaussian processes to discover consistent gene expression patterns associated with ordinal clinical phenotypes. The technique of automatic relevance determination is applied to represent the significance level of the genes in a Bayesian inference framework. The usefulness of this algorithm for ordinal labels has been demonstrated by the gene expression signature associated with the Gleason score for prostate cancer data. These results also demonstrated how multi-gene markers that may be initially developed with a diagnostic or prognostic application in mind are also useful as an investigative tool to reveal associations between specific molecular and cellular events and features of tumor physiology. This algorithm can also be applied to microarray data with binary labels with results comparable to other methods in the literature.

AIMS OF THIS PROJECTMicroarray gene expression data for adipose tissue obtained from lean, obese and T2DM subjects, as well as data from matched paired fat depots from BMI and gender matched subjects already exists. The goal of this project is to identify and validate functionally relevant gene regulatory networks important to the pathobiology of obesity and type 2 diabetes. To accomplish this goal, our project has the following specific aims: Identify gene expression signatures that reliably differentiate these data sets and identify and validate the regulatory networks to which

the genes comprising these signatures belong. Validate the expression of the genes identified in the microarray studies, using a variety of lab based molecular biology techniques

HypothesisWe hypothesize that mitochondrial genes related to energy homeostasis are differentially expressed in obesity and T2DM and that thealteredexpressionofthesegenesreflectsfunctionallyimportantgeneregulatory networkscriticaltothepathobiologyofobesity and T2DM.MethodsSubjects:. All samples have appropriate approval from the local ethical committee guidelines, with individual consent given. Anthropometric data has been collected including age, height, weight, waist, systolic and diastolic blood pressure measurements.Data: Microarray data will be collected from a lean non-diabetic cohort (BMI: 22.2±(SD)2.1 Kg/m2 age: 39.8±(SD)8.7 yrs), an obese non-diabetic cohort (BMI: 30.1± 2.81Kg/m2 age: 47.3±11.5 yrs) and a T2DM cohort (BMI: 59.9±7.5 Kg/m2 age: 37.15± 7.8 yrs) of human adipose tissue (AT). Paired fat samples will also be collected from BMI and gender matched subjects.Bioinformatics Analysis (Miniproject 1): Data will be analysed using the algorithms described above in 2 separate studies: paired samples for Sc versus Om AT (binary labels) and Abd Sc AT for lean, obese and T2DM samples (ordinal labels) to obtain gene expression signatures which reliably differentiate the data sets. The statistical machine learning models will be trained and rigorously cross validated and the gene expression signatures validated using independent data sets. Gene Ontology analysis, coupled with the use of pathway databases, will be applied to identify the functional role and putative pathways of the genes identified.Validation (Miniproject2): Real time PCR will be undertaken to follow up the genes which have been identified as altered by microarray data, along with Western blot techniques which will examine the expression of mitochondrial proteins important in energy metabolism. Taken together these will be used to validate the expression of the genes identified in the microarray studies. This work will involve undertaking lab based molecular biology techniques within a team of researchers working in this field as part of the Diabetes & Metabolism team based at the CSRI Warwick Medical School

REFERENCES1. Kusminski CM, Harte AL, Fisher ff. M, Creely SJ, da Silva NF, Baker AR, Kumar S, McTernan PG (2006) The in vitro effects of resistin on the innate immune signaling pathway in isolated human subcutaneous adipocytes. JCEM, Oct 24; [Epub ahead of print].

2. Chu W., Ghahramani, Z., Falciani, F. and Wild, D.L. Biomarker Discovery in Microarray Gene Expression Data with Gaussian Processes. Bioinformatics , 21: 3385-3393 (2005).

TIMING OF PROJECTBoth


Project title: Analysis and modelling of microarray time course data




Project timing§§§§§§§§§


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)

Supervisors (the person who will be doing the day to day supervision of the mini-project):

Name: Vicky Buchanan-Wollaston_____________________

Department: Warwick HRI and Systems Biology__________ Building, Room: TPB128 at HRI; at WSB____

E-mail address: [email protected]_________ Phone number: 75136/50249________________

Name: Katherine Denby_____________________________

Department: Warwick HRI and Systems Biology__________ Building, Room: TPB133 (HRI), 325 at WSB____

E-mail address: [email protected]_______________ Phone number: 75097 (HRI), 50251 (WSB)____

Name: Andrew Mead________________________________

Department: Warwick HRI and Systems Biology__________ Building, Room: DLE189 (HRI)______________

E-mail address: [email protected]____________ Phone number: 750202 (HRI)_______________

Project outline:

References: Buchanan-Wollaston V, et al.(2005) Comparative transcriptome analysis reveals significant differences in gene expression and signalling

pathways between developmental and dark/starvation induced senescence in Arabidopsis. Plant J 42: 567-585.Lim et al. (2007) Leaf senescence Ann Rev Plant Biol 58:115–136

Consumables budget**********: £_______

§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.********** Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Leaf senescence is a key step in plant development during which nutrients from the leaf are mobilized for use in further growth or for seed development. The process of leaf senescence is controlled by internal or external factors and can be very variable, depending on environmental conditions or the phase of development (Buchanan-Wollaston et al, 2005; Lim et al, 2007).

At Warwick HRI we have recently carried out detailed time course experiments where the same leaf (leaf 7) has been harvested at set time points during development and the plant material subjected to microarray analysis. In one experiment the plants were grown in long days – in this case leaf 7 is one of the largest leaves and senescence is instrumental in mobilization of nutrients to developing seeds. In the other experiment plants were grown in short days – these plants flowered much later with many more leaves with leaf 7 shaded by bigger leaves and senescing well before any seed was developing. Thus senescence is being induced by different signals in the two experiments and a comparison of the gene expression patterns will be very informative as to the signaling pathways involved.

The student will use the extensive data sets for the 2 experiments (176 2-colour slides for long day and 152 2-colour slides for the short day) and will use analytical tools to identify and compare differentially expressed gene sets. They will use and compare a number of analytical software tools for this analysis including GeneSpring, Timecourse, and Splinecluster, and follow the gene clusters with functional analysis using programs such as MapMan and AraCyc. In this way, genes and pathways that are common or specific to each type of senescence will be identified.

Four individual leaves were harvested at each time point and array data is available for 4 technical replicates for each leaf. As the leaf ages there are many other variables that affect the progression of senescence and the biological replicates become more diverse, possibly reflecting a difference in the biological age of the leave, despite being harvested at the same physical times. The student will investigate statistical modeling approaches that will allow the estimation of the true biological age of leaves whilst predicting a smooth gene expression response over biological time. This will involve the use of multivariate statistical methods (possibly including principal component analysis, cluster analysis, discriminant analysis) together with iterative regression model fitting.


Project title: Protein crystallisation at surfaces in the presence of electric fields________________________




Project timing


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Matthew Turner______________________________

Department: Physics / Systems Biology_________________ Building, Room: Physical Sciences, PS142____

E-mail address: [email protected]______________ Phone number: x22257____________________



Name:___________________________________________



Project outline:

References: 1. R. J. Hunter, Foundations of Colloid Science (Oxford University Press, USA; 2nd edition, 2001)2. D.H. Everett, Basic Principles of Colloid Science, (The Royal Society of Chemistry, London, 1988).

Consumables budget: £ 50

Aggregation at surfaces is ubiquitous in nature. Solutions of proteins usually crystallise by a first order process, also known as "nucleation and growth". Such aggregates include the protein fibrils or clumps characteristic of Alzheimer's, Huntingdon's or Sickle cell disease. In such cases supersaturated solutions can persist for long periods. At these high concentrations the equilibrium state would involve coexistence between molecules in both a solid and a suspended phase but the system doesn't have an easy (fast) way to reach this equilibrium state. The solid phase is often observed to first form at surfaces, e.g. of the containing vessel or of pre-existing aggregates, as these provide the appropriate nucleation substrate. A simple model for the formation of a solid phase at a surface would involve several stages. The first of these would involve the nucleation of the first critical (stable) patch of molecules in the solid phase at the surface. This, in turn, depends on the microscopic free energy barriers that must be overcome and hence also on the concentration of proteins. The energy barriers can be traced to the sub-optimal packing associated with a small (sub-critical) aggregate. The concentration dependence can be related to the rate of many body collisions, these being necessary to form the critical aggregate. The second stage would be the growth of these patches by diffusive-limited accretion to the surface. The effect of surface fields on the proteins can be dealt with by using a suitable Fokker-Planck equation.

Coordinating with experimental work in the lab of Prof Unwin we plan to construct a simple model for the surface-induced aggregation of small protein molecules. This is to be extended to treat the effect of electric fields on the alignment and distribution of these molecules. For example, in the simplest case, dipolar molecules must pay an activation energy to align into the configurations required to form the crystal.

Finally, the successful student will consider the implications for the size and shape of such surface aggregates as well as possible extensions to the crystallisation of membrane proteins near electrodes.


Project title: Finding Transcription Factor Binding Sites in Co-expressed Promoters




Project timing††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Sascha Ott__________________________________

Department: Warwick Systems Biology Centre____________ Building, Room: Coventry House, Room 327___

E-mail address: [email protected]___________________ Phone number: 024-761-50258______________



Name: Katherine Denby_____________________________

Department: Warwick HRI and Systems Biology__________ Building, Room: TPB133 (HRI), Coventry House 326 (WSB)

E-mail address: [email protected]_______________ Phone number: 75097/50251________________


Department: Warwick HRI and Systems Biology__________ Building, Room: TPB (HRI), Coventry House (WSB)

E-mail address: [email protected]_________ Phone number: 02476 575136_______________

Project outline:

Consumables budget‡‡‡‡‡‡‡‡‡‡: £ 0____

†††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

We are interested in elucidating the gene regulatory networks controlling plant senescence and defence in the model plant Arabidopsis thaliana. Large-scale network models are being inferred from high-resolution time course expression data but in this project we will focus on identifying specific local networks. This information will be used to retrain our network models and to guide future experimental work.

We are investigating plant gene expression in response to senescence (aging of the leaf) and infection by a fungal pathogen, Botrytis cinerea. Initial work has identified several Arabidopsis transcription factors (TFs) whose expression is significantly upregulated during these processes. Transgenic lines overexpressing the individual TFs have been generated and gene expression profiles obtained. The aim of this miniproject is to use this expression data to identify target genes and potential binding sites of these TFs.

The first goal will be to identify sets of genes that are differentially regulated in the overexpressing lines compared to wildtype and hence, potential TF targets. These groups of genes may be further refined by co-expression in large sets of publicly available expression data, co-expression in senescence and B. cinerea infection expression data and/or analysis of function (eg. in same biochemical pathway). Once sets of co-expressed genes have been identified, we will evaluate the promoter regions for overrepresentation of (consensus) sequences known to bind particular TFs. As these promoters might harbour yet undescribed binding sites as well, we will also use motif finding methods such as MEME or NMica to find overrepresented sequences in these tightly co-regulated groups of genes.

This project may be linked with the experimental project “Identification of Transcription Factors with a Role in Plant Defence”.


Project title: Modelling and Predicting Transcription Factor Binding Sites




Project timing§§§§§§§§§§


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Sascha Ott__________________________________



Co-supervisor Name: John Reid___________________________________

Department: Crystallography Dept., Birkbeck, University of London Building, Room: Malet Street, London WC1E 7HX

E-mail address: [email protected]______________ Phone number: 020-7631 6814______________



Name: David Wild__________________________________


E-mail address: [email protected]_________________ Phone number: 024-761-50242______________

Project outline:

Consumables budget***********: £ 70 (two return tickets to London)

§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.*********** Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The event of transcription factors (TFs) binding to certain recognition sequences in the promoters of regulated genes is at the core of the transcriptional regulation of genes. For hundreds of TFs we have descriptions of the sequences they recognise in the form of consensus sequences and position weight matrices. Furthermore, information about which pairs of TFs (composite pairs) have a preference to bind to neighbouring sites at what distance is being accumulated in the TransCompel database. A frequent task in Systems Biology is to predict which TF might bind to a given piece of DNA sequence based on existing knowledge. In this project we seek to enhance the predictive power and usefulness of existing software by a) increasing the number of weight matrices used, b) evaluating the effective number of weight matrices taking into account highly similar database entries, c) integrating data on composite elements into the predictions, and/or d) evaluate the impact of different models of background DNA on the prediction accuracy.

The student will build on software that was developed by John Reid and has proved useful and effective in guiding experimentalists to new hypotheses which they test in experiments. Feeding further information (from the Jaspar and Matbase databases) into this tool, expanding the mathematical model by incorporating composite elements, and determining the best way of modelling background DNA will lift this analysis software to a new level. Furthermore, evaluating the overlaps between similar weight matrices will facilitate the integration of sequence data with other data sets (such as microarray data, ChIP-on-chip data) in future work. Having a good command of TF binding site prediction will facilitate PhD projects in the field of gene regulatory networks.(David Wild will be away from mid-May to June, so some advisor meetings might have to be rescheduled accordingly.)


Project title: Nucleosome Positioning




Project timing†††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Sascha Ott__________________________________





Name: David Wild__________________________________



Project outline:Nucleosomes play a central role in the organisation of DNA within the nucleus of eukaryotic cells. About 146 bases of DNA can wrap around a nucleosome circling it about 1.75 times (see structure “1AOI” in PDB). Nucleosomes influence the accessibility of DNA and, therefore, its function. Recently significant progress was made in the in silico detection of preferred nucleosome positions. The aim of this project is to use available software tools for the prediction of nucleosome positions in order to address a number of questions of DNA organisation: What are – on average – the preferred positions of nucleosomes within promoters? How about (mammalian) bidirectional promoters or the recently discovered 3’-UTR-promoters? Does expression strength relate to this question?

Do different functional classes of promoters show distinct patterns of preferred nucleosome positioning? Do enhancer regions tend to be unoccupied by nucleosomes? Are there patterns of nucleosome positioning that are evolutionarily conserved between species? Do exon/intron-boundaries affect nucleosome positioning? How about matrix attachment regions? Are there dependencies between transcription factor binding sites and nucleosome positions?The student will tackle some of the above questions and possibly other aspects of nucleosome occupancy as the project develops. The software to be used includes Segal et al.’s nucleosome predictor as well as a predictor by Morozov et al. that is based on molecular modelling.(David Wild will be away from mid-May to June, so some advisor meetings might have to be rescheduled accordingly.)

References:

“A genomic code for nucleosome positioning”, E. Segal et al., Nature 442:750-2.

Consumables budget‡‡‡‡‡‡‡‡‡‡‡: £ 0___

††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects


Project title: In Silico Detection of Regulatory Modules




Project timing§§§§§§§§§§§


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Sascha Ott__________________________________





Name: David Wild__________________________________



Project outline:

References: “Evaluation of regulatory potential and conservation scores for detecting cis-regulatory modules in aligned mammalian genome sequences”, D.C. King et al., Genome Research, 2005.

Consumables budget************: £ 0_____

§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.************ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

A current challenge in Bioinformatics is the detection (and annotation) of (cis-)regulatory modules (ReMos or CRMs) in DNA sequences around target genes. These regions are characterised by conservation across those species that conserved the function of a ReMo in the transcriptional regulation of a gene to some extent. This conservation can, in a vast number of cases, be detected as sequence conservation, the conservation of certain binding sites across species, and/or the enrichment of transcription factor binding site sequences in a small stretch of DNA.Numerous methods for this task have been proposed. While any method should be capable of detecting ReMos that show a high or even extreme degree of sequence conservation, the task becomes challenging for ReMos that give only a weak (but still significant) signal of sequence conservation. Meeting this challenge is of particular interest as ReMos of low sequence conservation could form the majority of regulatory regions, especially if distant species such as mouse and chicken are compared. The target of this project is to evaluate the potential of existing methods to detect weakly conserved ReMos.The project will progress as follows. First the student will compile a set of experimentally verified ReMos in mouse using existing databases and web-pages. Then various methods will be used to detect the orthologous loci of these ReMos in various species ranging from mammals to very distant species such as fish. As the correct loci in those species will be unknown a priori, we will apply each method to randomly generated DNA as well as background DNA to evaluate the likelihood to achieve a given score by chance. We will only accept predictions as correct if their associated score is sufficiently unlikely to be found by chance. We will compare the results of several methods of ReMo-detection and evaluate the robustness of some methods under varying parameter settings. The emphasis will be on sequence conservation-based methods, but binding site-based approaches may also be considered.Time permitting the student could implement and evaluate their own ideas to detect ReMos in silico. As the detection of ReMos is of fundamental importance there are various possibilities to deepen the work started in this project in future research.Skills in computer programming are necessary to pursue this project. If you are interested in this project but unsure whether your programming skills are sufficient, please discuss it with me (Sascha) anytime.(David Wild will be away from mid-May to June, so some advisor meetings might have to be rescheduled accordingly.)


Project title: Identification of the host targets of pathogenicity effector proteins_________________________




Project timing††††††††††††


X Slot 2 (21/05 to 13/07/2007) X Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: Dr. Laura Baxter_____________________________

Department: WarwickHRI____________________________ Building, Room: __________________________

E-mail address: [email protected]____________ Phone number: __________________________



Name:Prof. Jim Beynon_____________________________

Department: Warwick HRI/ Warwick Systems Biology______ Building, Room: __________________________

E-mail address: [email protected]______________ Phone number: 02476575141_______________

Project outline:

References: Allen, R.L., Bittner-Eddy, P.D., Grenville-Briggs, L.J., Meitz, J.C., Rehmany, A.P., Rose, L.E. and Beynon, J.L. (2004) Host-parasite co-evolutionary conflict between Arabidopsis and downy mildew. Science 306, 1957-1960Birch, P.R.J., Rehmany, A.P., Pritchard, L., Kamoun, S. and Beynon, J.L. (2006) Trafficking Arms: Oomycete Effectors Enter Host Plant Cells. Trends in Microbiology 14, 8-11.Consumables budget‡‡‡‡‡‡‡‡‡‡‡‡: £ 0

†††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Plants and animals have innate immune systems that prevent the majority of organisms from invading. To overcome this innate immunity some organisms have developed a suite of effector proteins that suppress the host resistance mechanisms. We know these organisms as pathogens. Our group studies the interaction between the downy mildew parasite Hyaloperonospora parasitica and the model plant Arabidopsis. We have cloned two effector proteins by traditional means and have been analyzing their role in the invasion process. To complement this we are part of a team that have sequenced the genome of the pathogen and are in the process of annotation. As part of these studies we are working with the Sanger Centre to sequence 30,000 ESTs (expressed sequence tags). These are cDNAs that represent expressed gene sequences. These will be used for genomic annotation purposes to define the open reading frames on the assembled sequence contigs.

In this project you will join the annotation team in the analysis of the EST dataset. You will take a set of ESTs and carry out the following analyses: determine whether they are full length, asses relationship with genome sequence, ascertain intron structure, define the nature of the proteins coded by the ESTs, carry out clustering of the functions of the encoded proteins, define functional domains within the proteins and define intron/exon splice junction sites. Finally you will add the annotated ESTs to the genome sequence and carry out manual annotation of this new genome.

This project will involve the use of a range of bioinformatic software to analyse a unique data set and contribute to our analysis and understanding of the genome of this plant pathogen.


Project title:

Development of diamond and single walled carbon nanotube electrodes for neurosensing applications


MOAC Mini Project Experimental biology Systems Biology Mini Project X Wet Project(8 weeks) X Biophysical chemistry (12 weeks) Dry Project


Project timing§§§§§§§§§§§§

MOAC Mini Project Slot 1 (26/03 to 18/05/2007) Systems Biology Mini Project X Slot 1 (26/03 to 22/05/2007)

X Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Julie V. Macpherson__________________________

Department: Chemistry______________________________ Building, Room: A106_____________________

E-mail address: [email protected]____________ Phone number: 02476 573886_______________

Project outline:

References: [1]. T.M. Day, N. R. Wilson and J. V. Macpherson, J. Am. Chem. Soc. 2004, 126, 16724; [2]. A.L.

Colley, U. D’Haenens Johansson, M. E. Newton, P. R. Unwin, C. G. Williams, N. R. Wilson and J. V.

Macpherson, Anal. Chem. 2006, 78, 2539; [3]. N. Dale, S. Hatz, F. Tian and E. Llaudet, Trends in Biotech.

2005, 23, 420.

Consumables budget*************: £ _____

§§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.************* Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Chemical signaling underlies every function of the nervous system, from the imperceptible, such as the control of the heart, to higher cognitive functions including, for example, emotions, learning and memory. Neurotransmitters and neuromodulators mediate communication between neurons and between neurons and non-neural cells such as glia and muscle. Many neurotransmitters are electroactive i.e. can be detected by an electrode, and recording the current response at an electrode is one of the main ways for detecting their activity. By making the electrode as small as possible it is also possible to spatially map the response of the neurotransmitter, in imaging applications. Traditionally carbon fibres or graphitic electrode materials are used for sensing due to their biocompatibility, however, they can suffer from limited detection sensitivity due to the high background signals which can mask the signal of interest and interference signals arising from other species in the media. Very recently we have been developing a new breed of carbon based materials, namely single walled carbon nanotubes (in a network arrangement)1 and conducting diamond as electrode materials.2 To date these electrodes have not been tested for neurotransmitter detection. However, given their high sensitivity due to their low background currents and resistance to fouling, we expect these materials to be extremely promising in the field of neurology. During this project we will explore the capability of these materials for neurotransmitter detection, initially will we focus on the catecholamines, hormones released by the adrenal glands in situations of stress such as psychological stress or low blood sugar levels, including dopamine, epinephrine (adrenalin) and norephinephrine. We will assess detection levels, long term stability, and response in the presence of interfering media, similar to that found in the human body. If time allows we will look at the development of small electrodes made from these materials and the possibilities for imaging in-vivo in conjunction with Prof. Nick Dale (neuroscientist, Biological Sciences, Warwick).3

http://en.wikipedia.org/wiki/Low_blood_sugar

http://en.wikipedia.org/wiki/Fight-or-flight_response

http://en.wikipedia.org/wiki/Adrenal_glands

http://en.wikipedia.org/wiki/Hormones

Parts A and B 41

ANALYSIS OF DYNAMIC CHANGES IN GENE EXPRESSION UNDER THE CONTROL OF THE CIRCADIAN CLOCK

Joint experimental-theoretical miniprojects. Experimental supervisor: Isabelle Carré. Theoretical supervisor: David RandThis project aims to apply novel statistical tools to the analysis of dynamic changes in gene expression. These tools will be to extract information about the transcription factor activities mediating 24 hour oscillations in LHY gene expression under the control of the circadian clock.Circadian clocks are biological oscillators that function with a period of approximately 24 hours. These oscillators are normally entrained (synchronised) to the day-night cycle caused by the rotation of the earth, yet their rhythms persist in the laboratory in the absence of environmental time cues. In plants, the circadian clock controls many aspects of physiology and metabolism and is thought to allow preparation of the organism in anticipation of predictable changes in light and temperature conditions. Some of the genes that compose the circadian clock have been identified in Arabidopsis thaliana, a commonly used model system for plant molecular genetics. The LHY gene functions together with two other components known as CCA1 and TOC1 as part of a negative transcriptional-translational feedback loop which is thought to mediate oscillatory behaviour. Transcription of the LHY gene exhibits circadian rhythmicity, peaking at dawn, as well as light-inducibility. In order to understand how TOC1 and other clock-associated genes modulate transcription of LHY, we have constructed transgenic plants carrying luciferase (luc) reporter constructs under the control of the LHY promoter. Luciferase is an enzyme from firefly which emits light when supplied with its substrates, luciferin, oxygen and ATP. Thus, it is possible to monitor activity of the LHY promoter in these plants in real time, by monitoring the number of photons emitted using specialised cameras. In order to identify regions within the promoter, that contribute to the rhythmic pattern of LHY transcription, a number of altered LHY:luc fusion constructs have been created, which carry deletions or point mutations within specific sequences. Two sequence motifs have been identified, that are essential for rhythmic transcription of LHY, as well as a region that is important for accurate waveform of the oscillation. We wish to map the effects of different upstream regulators of LHY onto different regions of the promoter. The “wild-type” LHY:luc transcript has been introduced into a number of mutant backgrounds. The first aim of this project will be to acquire new luciferase data from these mutant plants for analysis using the mathematical methods so as to compare expression patterns in mutant backgrounds to the mutated LHY:luc reporter constructs analysed above. The second aim of this project will be to apply mathematical tools to extrapolate information about transcription factor binding from the patterns of rhythmic transcription of the wild-type or mutated promoter fragments. It is expected that the student will use the MCMC approach developed by Alex Morton, Baerbel Finkenstadt and David Rand. There is a significant suite of software available and therefore it is expected that a student with the appropriate skills will be able to progress quickly.

(a) Luminescence counts of CAB::luc expression for two wt Arabidopsis seedlings in 16L:8D cycles imaged approximately every 35 minutes. (b) (top) Mean data, processed and detrended. (middle) Transcription rate of the CAB gene together with 95% confidence limits. (c) Gradient of the transcription rate with 95% confidence limits.

MOAC / Systems Biology mini-project proposal M1Submit one page only to Monica Lucena ([email protected]) by Wednesday 14th February, 2007, 5pm).

Project title: Nanoparticles


MOAC Mini Project Experimental biology Systems Biology Mini Project Wet Project(8 weeks) X Biophysical chemistry (12 weeks) Dry Project


Project timing†††††††††††††

MOAC Mini Project X Slot 1 (26/03 to 18/05/2007) Systems Biology Mini Project Slot 1 (26/03 to 22/05/2007)

X Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name:Dr.ir. Stefan Bon

Department: Chemistry______________________________ Building, Room: A422

E-mail address: [email protected]__________________ Phone number: 74009



Name:



Project outline:

References:

Consumables budget‡‡‡‡‡‡‡‡‡‡‡‡‡: £ 1500

††††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The aim of this miniproject is the design nanoparticles that have a Janus (two face) type of morphology. One side of the particle will by hydrophobic in nature, the other side of the particle hydrophilic. Such particles will be able to undergo assembly, like conventional surfactants. We are interested to see how these particles behave on a water-oil interface, or whether these particles can aggregate into vesicular type of supracolloidal structures.

The particles will be prepared via (mini)emulsion polymerization of styrene/divinyl benzene in stage one, which will create a network. Entropic driven phase separation of the second monomer vinylbenzyl chloride can lead to a peanut-shaped particle, one lob styrene rich, the other vinylbenzyl chloride rich. The latter can be quaternized with tertiary amines to yield a hydrophilic structure.

You will learn how to perform miniemulsion polymerization. How to characterize particles size distributions with dynamic light scattering, FEGSEM, (cryo)-TEM and AFM.

Self-assembly of particles will be studied with confocal microscopy, wet-AFM and (cryo)-TEM.

More info on our work on supracolloidal polymer chemistry: Supracolloidal structures through liquid-liquid interface driven assembly and polymerization Patrick J. Colver, Tao Chen and Stefan A. F. Bon*, Macromol.Symp. 2006, 245-246, 34-41. (online feb. 7. 2007). Organic/inorganic Hybrid Hollow Spheres Prepared from TiO2-stabilised Pickering Emulsion Polymerization Tao Chen, Patrick J. Colver and Stefan A. F. Bon* accepted by Advanced Materials, 2007. ASAP soon available. Colloidosomes as Micronsized Polymerization Vessels to create Supracolloidal Interpenetrating Polymer Network Reinforced Capsules Stefan A.F. Bon,* Séverine Cauvin and Patrick J. Colver, Soft Matter, 2007, 3, 194-199.


Project title: SOLID-STATE NMR: A PROBE OF OXYGEN-CONTAINING HYDROGEN BONDS




Project timing§§§§§§§§§§§§§


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr. Steven Brown)

Department: Physics _______________________________ Building, Room: Room 439

E-mail address: [email protected] Phone number: 74359

Project Outline.

Oxygen-containing hydrogen bonds, e.g., OH…N or OH…O play key roles in determining the shape and hence function of biomolecules, e.g., proteins, nucleic acids, saccharides. Solid-state Nuclear Magnetic Resonance (NMR) is a powerful site-specific probe of molecular-level structure that is being increasingly applied to provide answers to questions in structural biology, e.g., applications to amyloid fibrils associated with prion disease,1 Alzheimer's disease,2 and Parkinson's disease3 as well as the toxin-induced conformational changes in a potassium ion channel.4

This project will develop 17O-1H solid-state NMR methods for looking at oxygen-containing hydrogen bonds. Oxygen is a challenge for NMR, since the only NMR-active isotope of oxygen, 17O, is, first, spin I = 5/2 and hence the spectra are broadened by the strong quadrupolar interaction and, second, the low natural abundance (0.037 %) necessitates the preparation of 17O-labelled samples. Nevertheless, the combination of high magnetic field (14T and above) with methodological developments is making biological structural applications of 17O solid-state NMR possible. This project involves adapting a 23Na-1H high-resolution solid-state NMR experiment5 for application to 17O-labelled biomolecules. Solid-state NMR experiments will be complemented by computer simulations of the NMR experiment using standard packages that are based on the quantum-mechanical description of the NMR experiment.

References:1 C. Ritter, M.-L. Maddelein, A. B. Siemer, T. Lührs, M. Ernst, B. H. Meier, S. J. Saupe, and R. Riek, Nature 435,

844 (2005).2 A. T. Petkova, R. D. Leapman, Z. Guo, W.-M. Yau, M. P. Mattson, and R. Tycko, Science 307, 262 (2005).3 H. Heise, W. Hoyer, S. Becker, O. C. Andronesi, D. Riedel, and M. Baldus, Proc. Natl. Acad. Sci. U.S.A. 102,

15871 (2005).4 A. Lange, K. Giller, S. Hornig, M. F. Martin-Eauclaire, O. Pongs, S. Becker, and M. Baldus, Nature 440, 959

(2006).5 A. Lupulescu, S. P. Brown, and H. W. Spiess, J. Magn. Reson. 154, 101 (2002).

Consumables budget**************: £ 150 for solid-state NMR cryogen costs

§§§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.************** Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects


Project title: Chromosomes




Project timing††††††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Ewen Buckling

Department: Biological Sciences_______________________ Building, Room: Biological Sciences M014

E-mail address: [email protected]_____________ Phone number: 024 76522444



Name: Maj Hulten

Department: Biological Sciences_______________________ Building, Room: Biological Sciences B121

E-mail address: [email protected]______________ Phone number: 02476 528 9076

Project outline:

Consumables budget‡‡‡‡‡‡‡‡‡‡‡‡‡‡: £ 0

††††††††††††††

‡‡‡‡‡‡‡‡‡‡‡‡‡‡

Crossovers between maternal and paternal chromosomes are laid down at meiotic cell divisions during the formation of sperm and eggs. This ensures the appropriate segregation so that sperm and eggs will contain the normal chromosome number.

The distribution of crossovers along the length of chromosomes can be identified by linkage analysis, tracing DNA markers/alleles between generations, but also directly by microscopy analysis. This allows an overview of crossover positions in individual cells that cannot be obtained in any other way. A large data set on crossover positions is already available in humans, but there is a lack of the corresponding data in mice.

The first step of the project will be to produce preparations containing the relevant cells from mice. Cells will then be examined by immuno-fluorescence microscopy, highlighting the positions of crossovers using an antibody for a protein that is involved in the last step in their formation. Suitable images of cells will be stored on the computer and computer-assisted measurements then performed of crossover positions along the length of individual chromosomes.

This new dataset in mice would be particularly valuable for computer modelling of the non-random crossover positions, which is conserved during evolution. All mouse chromosomes have their movement centre (centromere) near one end, which simplifies this challenge. In spite of decades of work there is no adequate mathematical model for the non-random crossover distribution.

This project can be linked to the theoretical project on crossovers.


Project title: Bio-templates for the formation of nano-sized silver and gold particles by electrospray


MOAC Mini Project Experimental biology Systems Biology Mini Project Wet Project(8 weeks) * Biophysical chemistry (12 weeks) Dry Project


Project timing§§§§§§§§§§§§§§


* Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Tom Drewello

Department: Chemistry______________________________ Building, Room: Chemistry, C516

E-mail address: [email protected]______________ Phone number: 24934



Name:



Project outline:

References:

Consumables budget***************: £ 300

§§§§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.*************** Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Bio-templates for the formation of nano-sized silver and gold particles by electrospray.

The project is concerned with the use of amino acids, peptides and DNA fragments as templates for the formation of nano-sized silver and gold clusters applying electrospray ionisation mass spectrometry. Recent research in our group has shown that when a silver salt solution is electrosprayed together with certain biological molecules, the latter can function as a reducing agent whereby silver cations are reduced and aggregation occurs, so that silver clusters of nano-size dimensions are formed. Both the use of a biological template and the formation of nano-sized silver clusters are topics of enhanced research interest at present and this project combines both areas. Your task will be to learn to work with an electrospray mass spectrometer (ion trap), to investigate different biological molecules evaluating their use as templates for the silver cluster production and to extent your work on to the formation of gold clusters applying what you have learnt form your studies on silver. The project is ideal for an 8 week period and an experienced PhD student will support your efforts.


Project title: Glass transition of sugars confined in nanopores




Project timing†††††††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: Dr Jonathan Duffy

Department: Physics______________________________Building, Room: Room P457

E-mail address: [email protected]_______________ Phone number: 22410



Name:



Project outline:

References:

Consumables budget‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡: £ 250


††††††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The goal of this project is to study the properties of sugar solutions confined in nanoporous hosts. The ability of sugar solutions to form glassy states rather than crystalline solids is important in biology, medicine and the food industry. The phase behaviour of material adsorbed into nanometre-sized pores poses an interesting fundamental problem in physics and chemistry as well as being of immense technological importance. For example, it is well known that the freezing point of a confined liquid is often depressed to a lower temperature than for the bulk liquid. Hysteresis between the freezing and melting temperatures is normally associated with this confinement. Furthermore, confinement can have a significant effect on glass-forming liquids, such as changing the temperature of the glass transition, and modifying the nature of the confined material. Such differences can be attributed to “finite size” effects: in systems of such small effective size, it is no longer possible to consider them in the same manner as a macroscopic system. By using porous glass, we can create a “large” sample (several mm3) containing very many material-filled pores. Whilst porous glass samples are already available, it is hoped that samples with different pore sizes could be grown as part of the project, in order to study size effects systematically. We will also investigate size effects by use of mono-, di-, and tri-saccharide samples.

Project title: PCR kinetics in real time




Project timing§§§§§§§§§§§§§§§


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Matthew Hicks

Department: MOAC_________________________________ Building, Room: Chemistry dept, B608

E-mail address: [email protected]___________ Phone number: 23293



Name: Alison Rodger

Department: MOAC/chemistry________________________ Building, Room: Chemistry dept, B607

E-mail address: [email protected]________________ Phone number: 23234

Project outline:

References: Real-time PCR tutorial: http://pathmicro.med.sc.edu/pcr/realtime-home.htm

Rodger and Nordén, Circular dichroism and linear dichroism, OUP 1997Consumables budget****************: £ 150


Project title: Synthesis and electrophysiological characterisation of granisetron derivatives§§§§§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.**************** Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Development of a novel method for detecting PCR products in real timeThe polymerase chain reaction (PCR) is used to amplify DNA. This has many uses including diagnosis of disease, forensic applications in crime detection and manipulation of DNA for molecular biology applications. Standard PCR detects the product upon completion of the reaction. However, real time PCR (RT-PCR) measures the amount of amplified DNA in the sample during the reaction. This is useful because the rate of amplification is exponential and therefore dependent upon the amount of starting material. Here we present a novel method for RT-PCR detected by flow linear dichroism (fLD). PCR is most commonly carried out using a thermocycler that changes temperature rapidly. However, we have taken an alternative approach and used transport of the sample between regions of a capillary that are held at different temperatures. This has the advantage of a fast change between the different temperatures required during the PCR cycle. After the elongation phase of the PCR, the sample is pumped through a quartz flow cell. This aligns (by shear flow) any amplimer in the reaction, because it is relatively long (i.e. has a high aspect ratio). The deoxynucleotide triphosphates and the primers are not aligned. For an aligned sample there is a differential absorbance of light polarized parallel and perpendicular to the sample orientation axis. This phenomenon is called linear dichroism (LD). Since the amplimer is the only molecule that will align in the PCR the increase in the LD signal is proportional to the amount of amplimer present. This method does not require any dyes or markers to be added to the sample and the measurement is non-destructive. This means that the sample can be recovered and used for any other downstream applications, for example DNA sequencing or other molecular biology applications.

Experience will be gained in handling biomolecule samples, biophysical instrumentation and literature searching.

http://pathmicro.med.sc.edu/pcr/realtime-home.htm




Project timing††††††††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr Martin Lochner

Department: Chemistry______________________________ Building, Room: Engineering A421___________

E-mail address: [email protected]_______________ Phone number: x50170____________________



Name: Prof Alison Rodger____________________________

Department: Chemistry/MOAC________________________ Building, Room: Chemistry B607/MOAC_______

E-mail address: [email protected]________________ Phone number: x23234/x74696______________

Project outline:

References: (1) P. Schumann et al., Bioorg. Med. Chem. Lett. 2001, 11, 1153 (2) G. Luo et al., J. Org. Chem. 2006, 71, 5392 (3) P. Fludzinksi et al., J. Med. Chem. 1987, 30, 1535 (4) A. J. Thompson et al., Br. J. Pharmacol., 2007, in press.

Consumables budget‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡: £ 150


Project title: Measurement of DNA persistence length____________________________________________


†††††††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Introduction – Granisetron (1) is a high-affinity inhibitor of the ligand-gated serotonin (5-hydroxytryptamine) type-3 receptor (5-HT3R). The 5-HT3R is a transmembrane protein that is involved in signal transmission in the central and peripheral nervous system. The binding site for granisetron is not well characterised and there is an ongoing debate in the literature about the exact binding location and orientation of this inhibitor. In order to shed light into this we are interested in synthesising photoaffinity probes based on the granisetron structure (e.g. 2) and with this to be able to map the binding site.

Project objectives – The photoactive group is rather bulky (see example 2) and we would like to establish which positions of the granisetron skeleton will tolerate chemical modifications without dramatic loss of binding affinity. We are currently underway to synthesise granisetron derivatives with a methoxy group at C5 and C6 (9 synthetic steps from commercially available starting material).1-3 The objective of the mini project is to synthesise granisetron derivatives 3a and/or 3b with a methoxy group at C4 and C7 and to test these compounds on the 5-HT3R (in collaboration with Dr A. J. Thompson, Biochemistry Dept., Cambridge).4 This small structure-activity relationship exercise on the aromatic ring of granisetron will be very valuable in guiding futures syntheses of photoaffinity probes such as 2.

Training provided – The proposed mini projects includes training in modern synthetic organic chemistry. We have experience with this chemistry and it is straightforward. Once the compounds are obtained they can be tested on the biological target which will provide an insight to electrophysiology to the student.



Project timing§§§§§§§§§§§§§§§§


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Martyn Rittman______________________________

Department: MOAC_________________________________ Building, Room: Chemistry dept, B608________




Name: Alison Rodger_______________________________

Department: MOAC/chemistry________________________ Building, Room: Chemistry dept, B607________

E-mail address: [email protected]________________ Phone number: __________________________

Project outline:

DNA persistence length is a widely accepted concept that measures flexibility of DNA. A recent review (in preparation), however, has highlighted difficulties in experimental measurement and conflicts between definitions of persistence length.

The project proposed would entail carrying out measurements of DNA persistence length using stopped flow linear dichroism spectroscopy (LD). To do this the DNA is passed through a narrow tube at a high rate to orient it, then stopped and allowed to relax back into a randomly ordered state. LD measures orientation by recording the difference between absorption of parallel and perpendicularly polarised light that has passed through a sample. For the case of stopped flow DNA, we expect to see relaxation curves for which a half-life can be measured and fitted to several known models (as shown in the Figure).

The dependence of persistence length on any one of a number of factors can be investigated, for example DNA base sequence, temperature, salt concentration or DNA length.

References: Linear dichroism of biomolecules, which way is up?, Curr Opin Struct Biol. 2004 Oct;14(5):541-6;

DNA persistence length revisited, Biopolymers. 2001-2002;61(4):261-75.

Consumables budget*****************: £ 150_


Project title: Determinants of firing patterns of cerebellar neurones. Part II: theory


MOAC Mini Project Experimental biology Systems Biology Mini Project Wet Project

§§§§§§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.***************** Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.2 0.4 0.6 0.8 1

1ml.sec-17ml.sec-1

LD26

0

Time / seconds

(8 weeks) Biophysical chemistry (12 weeks) Dry Project Mathematics/computing

Project timing†††††††††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr Magnus Richardson ________________________

Department: Warwick Systems Biology Centre____________ Building, Room: __________________________

E-mail address: [email protected]_______ Phone number: 024 761 50250______________



Name: Dr Mark Wall________________________________


E-mail address: [email protected]_______________ Phone number: 024 765 73772______________

Project outline:

References:

Consumables budget‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡: £ 0 (theoretical project)


Project title: Determinants of firing patterns of cerebellar neurones. Part I: experiment



††††††††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Neurones may be divided into two classes, depending on the effect their synapses have on target cells. Excitatory cells increase the voltage of post-synaptic neurones, whereas inhibitory cells decrease post-synaptic voltages and are therefore thought to reduce the firing rate of target cells. However, there is increasing evidence that the role of inhibition is more subtle and that the principal effect of inhibition may be the patterning of the output of excitatory cells.

The cerebellum is a densely populated and connected part of the nervous system - the principal excitatory neurones, the Purkinje cells, have a highly branched structure and receive upwards of 100,000 synapses. In the laboratory of Dr Mark Wall, experiments have been performed in cerebellar slices that examined the change in the firing patterns of Purkinje cells as the inhibitory balance is altered pharmacologically. A surprising result seen in these preliminary experiments was that inhibition rarely just reduced the firing rate of Purkinje cells, but acts rather to modulate their firing patterns, which passed between tonic firing and bursting states. The standard spike-sorting analysis software packages do not provide a means for experimentalists to analyse the higher-order statistics of such spike trains. In these linked experiment-theory mini-projects the student will measure firing patterns of cerebellar cells experimentally and then model their own and existing data. Theoretical methods could include construction of solvable and biophysically-detailed cell models and coupled microcircuits. The higher-order statistics of the spike trains will be analysed to infer the modulatory effect of inhibition on Purkinje cell firing patterns.


Project timing§§§§§§§§§§§§§§§§§


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr Mark Wall________________________________


E-mail address: [email protected]_______________ Phone number: 024 765 73772______________



Name: ___________________________________________



Project outline:

References:

Consumables budget******************: £ 450 (or as near to that as the requirements for the third slot allows)


Project title: Mapping protein-protein interactions involved in Carnitine palmitoyltransferase 1 oligomerisation



§§§§§§§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.****************** Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Neurones may be divided into two classes, depending on the effect their synapses have on target cells. Excitatory cells increase the voltage of post-synaptic neurones, whereas inhibitory cells decrease post-synaptic voltages and are therefore thought to reduce the firing rate of target cells. However, there is increasing evidence that the role of inhibition is more subtle and that the principal effect of inhibition may be the patterning of the output of excitatory cells.

The cerebellum is a densely populated and connected part of the nervous system - the principal excitatory neurones, the Purkinje cells, have a highly branched structure and receive upwards of 100,000 synapses. In the laboratory of Dr Mark Wall, experiments have been performed in cerebellar slices that examined the change in the firing patterns of Purkinje cells as the inhibitory balance is altered pharmacologically. A surprising result seen in these preliminary experiments was that inhibition rarely just reduced the firing rate of Purkinje cells, but acts rather to modulate their firing patterns, which passed between tonic firing and bursting states. The standard spike-sorting analysis software packages do not provide a means for experimentalists to analyse the higher-order statistics of such spike trains. In these linked experiment-theory mini-projects the student will measure firing patterns of cerebellar cells experimentally and then model their own and existing data. The experimental methods used will depend on the student's interests, but may include preparation of cerebellar slices, extracellular measurements of spike trains and the potential for performing some intracellular patch-clamp recordings.


Project timing††††††††††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Prof. Victor Zammit (VZ) and Dr. Ann Dixon (AD)____

Department: Warwick Medical School (VZ) and Chemistry (AD) Building, Room: Chemistry C503 (AD)________

E-mail address: [email protected], [email protected] Phone number: 024 7696 8583 (VZ)__



Name: ___________________________________________



Project outline:Introduction. Carnitine palmitoyltransferase 1 (CPT 1) is an enzyme whose activity is important for key functions in the cell, such as fuel selection for tissues, and is involved in appetite control and development of diabetes. CPT 1 occurs in two isoforms, both of which are integral membrane proteins ([1]; see Fig. 1a) and have very high sequence similarity (~65%). Despite this high similarity, these two proteins are found in different tissues (CPT 1A in the liver and kidneys, CPT 1B in muscle) and have markedly different sensitivity to their inhibitor malonyl-CoA, a molecule that is very important in the mounting of a response to diabetes by the liver [2].

In contrast to the very high sensitivity of CPT 1B to malonyl-CoA, CPT 1A has a very low and variable sensitivity to the inhibitor, which suggests that the molecule is quite flexible and may undergo large conformational changes as part of its biological function [3]. CPT 1A has also recently been found to exist as trimers and possibly higher order oligomers (Fig. 1b), however the molecular basis for this oligomerization is unknown. Also unknown is whether CPT 1B is oligomeric, and therefore whether the inherent differences in malonyl-CoA sensitivity could be linked to differences in oligomeric state.

Aims of Project. The first aim of this project is to determine which domains of CPT 1A (e.g. N-terminal, transmembrane (TM), loop (L), C-terminal: see Fig. 1a) drive the oligomerisation of this protein. Inspection of the sequences of the TM domains of CPT 1A and CPT 1B highlights the presence of several GxxxG motifs, motifs that are known to drive the oligomerisation of TM -helices [4]. Therefore, the TM domains may play a role in oligomerisation of these proteins. The N- and C-terminal domains may also play a role, as may the loop region connecting the two TM domains, and all of these domains will be examined independently for their effect on oligomerisation of wild-type CPT1A. The second aim of this project is to determine whether or not wild-type CPT 1B also forms oligomers.

Brief Outline of Work. Oligomerisation of wild-type CPT 1A and various mutants will be assessed using native gel electrophoresis followed by Western blots to detect monomeric and oligomeric forms of CPT 1A and B, using specific antibodies. This project will make use of constructs which have already been prepared in the laboratory of Prof. Victor Zammit, therefore no cloning will be required. To address whether TM1 or TM2 determines oligomeric state, constructs have been prepared where each TM domain in CPT 1A is independently substituted with the corresponding TM from CPT 1B. A similar construct has been prepared to analyse the role of the loop region. Deletion mutants of the N- and C-terminal domains of CPT 1A have also been prepared and will be tested for their ability to self-associate. Finally, a construct containing wild-type CPT 1B will also be tested.

†††††††††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

The DNA of each construct will be expressed in yeast, where it is transcribed and the corresponding protein is translated and inserted in the mitochondrial outer membrane. As yeast do not express endogenous CPT, the properties of the expressed protein can be studied without background interference. Yeast mitochondria will be prepared and their proteins separated under ‘native’ conditions that preserve the inter-molecular interactions that are involved in the formation of oligomers.

This project will provide students with a basic knowledge of modern protein analysis techniques as well as several methods for handling and detecting membrane proteins, and elucidating their structure-function relationships.

References:[1] Fraser, F., Corstorphine, C. G., and Zammit, V. A. (1997) Biochem J 323, 711-718[2] Zammit. V A (1994) Diabetes 2, 132 – 155[3] Zammit, V.A., rice, N.T., Fraser, F., and Jackson, V. N. (2004) Biochem Soc Trans 29, 287 – 290[4] Russ, W.P. and D.M. Engelman, J. Mol. Biol., 296(3), 911 (2000).

Consumables budget‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡: £ 150-200 for antibodies and consumables needed for electrophoresis.

‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects


Project title: Structure Structure/function studies on ALDC, part 1; Chemistry.




Project timing§§§§§§§§§§§§§§§§§§

MOAC Mini Project X Slot 1 (26/03 to 18/05/2007) Systems Biology Mini Project Slot 1 (26/03 to 22/05/2007)

Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Professor M. WillsDepartment: Chemistry___________________________Building, Room: Chemistry room C504

E-mail address: [email protected]_________________Phone number: (024 765)23260

Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision support to the supervisor and also agrees to meet the student briefly at least once a week): n/a

Project outline:

References1). Dolin, M.I. and Gunsalus, I.C. (1951). J. Bact. 62, 199-214. 2). Løken, J.P. and Størmer, F.C. (1970). Eur. J. Biochem. 14, 133-137. 3). Crout, D.H.G. and Rathbone, D.L. (1988). J. Chem. Soc., Perkin Trans. 1, 98-99. 4). Crout, D.H.G., Rathbone, D.L. and Lee, E.R. (1990). J. Chem. Soc., Perkin Trans. 1, 1367-1369. 5) Najmudin, S., Andersen, J. T., Patkar, S. A., Borchert, T. V., Crout, D. H. G. and Fülöp, V. (2003). Acta. Cryst. D59, 1073-1075.

Consumables budget: £ 200 for reagents.

§§§§§§§§§§§§§§§§§§ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

Context: This mini-project proposal is the first of three directed towards the study of the mechanism of action of the enzyme ADLC. The objective of this mini-project will be to prepare two substrate and transition state analogues for co-crystallisation with the isolated enzyme. The second mini-project will be the X-ray crystal structure analysis of ALDC bound to the analogues (Professor V. Fulop). The third mini-project will involve a molecular modelling study of the enzyme, (Professor P. M. Rodger).

Acetolactate decarboxylase (ALDC) catalyses the decarboxylation of (S)--acetolactate [(S)-2-hydroxy-2-methyl-3-oxobutanoate] 1 and (S)--acetohydroxybutyrate [(S)-2-ethyl-2-hydroxy-3-oxobutanoate] 2 (Scheme 1),1,2 the biosynthetic precursors of valine 3 and isoleucine, 4 (Scheme 1). The products of decarboxylation of 1 and 2 are (R)-acetoin 5 and (R)-3-hydroxypentan-2-one 6 respectively. The enzyme catalyses also the decarboxylation of the corresponding (R)-enantiomers. Decarboxylation of both enantiomers of -acetolactate leads to a single (R)-enantiomer of 5, by the mechanism shown in Scheme 2, by first decarboxylating the (S)-enantiomer 1 with overall inversion of configuration at the -centre and then by catalysing a tertiary ketol rearrangement of the (R)-enantiomer 7 with migration of the carboxylate group to give 8.3-4

The full length (260 amino acid residues) of ALDC from Bacillus brevis has been cloned, expressed and purified by Novozymes A/S (Denmark) to homogeneity and provided to us for crystallization trials.5 A major question concerns the location of the active site. It will be necessary to synthesise substrate or product analogues that can be co-crystallised for X-ray structural studies. Specific synthetic targets to be made in this project are shown in Figure 1. Analogues 9 should bind but cannot undergo decarboxylation. The use of product analogue diols of type 10 will also be explored.



Project title: Development of comparative genomic hybridisation with a micro-array of the cyanophage S-PM2_____




Project timing*******************


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Andrew Millard_______________________________

Department: Biological Sciences______________________ Building, Room: c126______________________

E-mail address: [email protected] ______________ Phone number: 22572_____________________



Name:Nick Mann___________________________________


E-mail address: [email protected] ______________ Phone number: 44 (0)24 7652 3526__________

Project outline: Development of comparative genomic hybridisation with a micro-array of the cyanophage S-PM2.

References: Wommack, K. E. and R. R. Colwell (2000). "Virioplankton: viruses in aquatic ecosystems." Microbiol Mol Biol Rev 64(1): 69-114.

Mann, N. H., M. R. J. Clokie, A. Millard, A. Cook, W. H. Wilson, P. J. Wheatley, A. Letarov and H. M. Krisch (2005). "The Genome of S-PM2, a "Photosynthetic" T4-Type Bacteriophage That Infects Marine Synechococcus Strains." Journal Of Bacteriology 187: 3188-3200.

Consumables budget†††††††††††††††††††: £ £300

*******************

††††††††††††††††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The virus S-PM2 infects marine cyanobacterium Synechococcus. This cyanobacterium and the related Prochlorococcus dominate the oligotrophic regions of the world’s oceans, where their co-occurring viruses (cyanophages) are equally abundant (Wommack & Colwell, 2000) . Cyanophages are known to play an important role in the microbial loop as they divert the flow of nutrients, they influence the population structure of their hosts. A total of 6 cyanophages have had their genomes sequenced, however, only 2 isolated on Synechococcus have had their genomes sequenced, one of these is S-PM2 [1]. A third cyanophage S-WHM1 has had its genome partially sequenced (approximately 80 %, this lab). Analysis of this partial genome reveals many genes in common with S-PM2, yet there are a number of genes that are novel to each organism. A fundamental question is what are core “cyanophage genes”. Analysis of the genome sequence of cyanophages by Sullivan et al 2005 has identified some core genes which were common to three cyanophage genomes. A far larger number of genomes is needed to draw any firm conclusions.

The time and cost to sequence the genome of a large number of cyanophages is prohibitive. An alternative technique is to use comparative genomic hybridisation (CGH) to identify genes common to all cyanophages. A custom array microarray has already been successfully designed and used to monitor gene expression of S-PM2. This array can also be used for CGH studies.

The aim of the project is to develop the technique of CGH using S-PM2 and S-WHM1. As the probes were designed against the S-PM2 sequence, genomic DNA from this phage will be used as a control and S-WHM1 DNA will be used as the test to determine whether the array can be used in CGH. As the partial sequence of S-WHM1 is known, it will be possible to determine if probes designed against S-PM2 can also detect homologous genes in S-WHM1. The project would involve purification of cyanophage isolates S-PM2 and S-WHM1 using CsCl gradients followed by the extraction of DNA. Phage DNA will be labeled with the fluorescent dyes Cy3 and Cy5. Hybridisation of Cy labeled genomic DNA to the array will allow quantification of the binding of probes to the target genomic DNA.

This will allow the identification of genes in S-WHM1 that are also present in S-PM2. The procedure will be optimised to determine the quantity of DNA that is needed for hybridisation. Data analysis will then determine the signal threshold values needed to distinguish the actual presence of that gene from the background noise. There will then be the opportunity to test the CGH on other phages for which no genomic information is available.



1. Mann, N.H., et al., The Genome of S-PM2, a "Photosynthetic" T4-Type Bacteriophage That Infects Marine Synechococcus Strains. Journal Of Bacteriology, 2005. 187: p. 3188-3200.


Project title: Characterisation of genes in the “ORFanage” region in the cyanophage S-PM2_________________________




Project timing‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Andrew Millard_______________________________

Department: Biological Sciences______________________ Building, Room: c126______________________

E-mail address: [email protected] ______________ Phone number: 22572_____________________



Name:Nick Mann___________________________________


E-mail address: [email protected] ______________ Phone number: +44 (0)24 7652 3526_________

Project outline: Characterisation of genes in the “ORFanage” region in the cyanophage S-PM2

References: N.M Mann, MRJ Clokie, A Millard, A Cook, WH Wilson, PJ Wheatley, A Letarov, HM Krisch (2005). The

Genome of S-PM2, a "Photosynthetic" T4-Type Bacteriophage That Infects Marine Synechococcus Strains . J.

Bacteriol. 187, 3188-3200. Consumables budget§§§§§§§§§§§§§§§§§§§: £

£300


Project title: Structure Structure/function studies on ALDC, part 2; biology.

‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§§§§§§§§§§§§§§§

Cyanophages are viruses that are capable of infecting cyanobacteria. Cyanobacteria of the genera Synechococcus and Prochlorococcus are dominant in the world’s ocean and are responsible for greater than 60% primary production in some regions. Cyanophages are known to influence carbon cycling, host population dynamics and host evolution. A number of cyanophages have now been isolated and characterised to increase the understanding of cyanophage/cyanobacterial interactions. The cyanophage S-PM2 is the most well characterised cyanophage, the genome has been completely sequenced and oligonucleotide array has also been produced to study phage gene expression during infection of its host Synechococcus WH7803. The results of genome annotation revealed a number of small contiguous ORFs that are database orphans [Mann et al, 2005]. The results of microarray analysis to monitor gene expression during infection revealed that the genes in the “ORFanage” region are expressed during the infection cycle [Millard et al unpublished results]. The cloning of groups 5 gene genes into an expression vector, under the control of an arabinose inducible promoter has revealed that the expression of some these are toxic when expressed in E.coli. [Mahon, unpublished data]. The aim of this work is to further investigate the gene within the orfanage region and determine exactly which gene produces proteins that are toxic to E.coli and to determine if they are also toxic when expressed in Synechococcus. This will involve the use of the following techniques: PCR, cloning, SDS-PAGE




MOAC Mini Project X Experimental biology Systems Biology Mini Project Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project


Project timing********************


X Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name:Professor V.Fulop

Department: Biol Sci________________________________Building, Room: Biol SCi , Room B128

E-mail address: [email protected]__Phone number: (024 765)72628Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision support to

the supervisor and also agrees to meet the student briefly at least once a week): n/a

Project outline:

References: : Najmudin, S., Andersen, J.T., Patkar, S.A., Borchert, T.V., Crout, D.H.G. & Fülöp, V. (2003). Purification, crystallisation and preliminary X-ray crystallographic studies on acetolactate decarboxylase. Acta Cryst. D59, 1073-1075

Consumables budget: £ 200 for reagents.


Project title: Structure Structure/function studies on ALDC, part 3; modelling.


******************** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

Acetolactate decarboxylase (ALDC) has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. The enzyme decarboxylates the normal substrate (S)--acetolactate. It then catalyses tertiary ketol rearrangement of the (R)-enantiomer with the migration of the carboxylate group. Because this degenerate rearrangement proceeds via a transition state with a syn arrangement of the oxygen functions, the product is (S)--acetolactate which is then decarboxylated in the normal way. The enzyme also catalyses the decarboxylation of (S)--acetohydroxybutyrate. (S)--Acetolactate and (S)--acetohydroxybutyrate, the products of decarboxylation of -ketocarboxylates are the biosynthetic precursors of the amino acids valine and isoleucine .

Details of the overexpression, purification and crystallization of -acetolactate decarboxylase are given in Najmudin et al. (2003). We recently solved the crystal structure of the enzyme from a SAD experiment to give a partial model to 2.3Å. The structure was completed using higher resolution data (2.0Å) in 3 different crystal forms and the resolution at the moment is being extended to 1.1Å. -Acetolactate decarboxylase is a 2 domain α/β protein with no significant structural homology to any other protein. The N-terminus domain comprises a 7 β-strand mixed β-sheet, which is extended into second molecule of the dimer related by 2-fold symmetry to give a 14 β-strand β-sheet. The C-terminus domain is a β-cylinder comprising 5 anti-parallel β-strands and a long α-helix. It provides the three highly conserved histidines, which coordinate the metal ion (Zn). The coordination of the metal is completed by a conserved glutamate from the C-terminus and two water molecules. The likely catalytic site is completed by the highly conserved arginine, threonine and a further glutamate in the vicinity of the metal.

The project shall require the preparation two substrate and transition state analogues for co-crystallization experiments. The synthetic chemistry, first part of the project, will be supervised by Professor M. Wills in the Department of Chemistry. The second mini-project (this one) will be aimed at the X-ray crystal structure analysis of analogues bound to the enzyme. The third mini-project will involve a molecular modelling study of the enzyme, in order to help elucidate its mechanism and will be supervised by Professor P. M. Rodger, Department of Chemistry. The three mini-projects thus provide a coherent series of studies with training in synthetic chemistry, enzyme crystallography and molecular modelling. It is anticipated that the student will go on to use this blend of skills in further independent research work. The project has potential to lead to a full PhD programme by systematic mutagenesis/kinetics/crystallography complemented by modeling substrate entry and turnover at the catalytic site using computational techniques.




Project timing††††††††††††††††††††


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name: Professor P. M. Rodger

Department: Chemistry______________________________Building, Room: Chemistry room B110.

E-mail address: : [email protected]_______Phone number: (024 765)2 3239


the supervisor and also agrees to meet the student briefly at least once a week): n/a

Project outline:

References Najmudin, S., Andersen, J.T., Patkar, S.A., Borchert, T.V., Crout, D.H.G. & Fülöp, V. (2003). Purification, crystallisation and preliminary X-ray crystallographic studies on acetolactate decarboxylase. Acta Cryst. D59, 1073-1075

Consumables budget: £50 for data archiving media.


Project title: Modelling the Distribution of Crossovers



†††††††††††††††††††† Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

ContextThis mini-project is the last of a three-component project directed towards the study of the mechanism of action of the enzyme Acetolactate decarboxylase (ADLC). The first two mini-projects involved the preparation of two substrates and then gathered experimental data on the subsequent kinetics of ADLC and the ADLC-substrate crystal structure. In this, the third, phase, molecular modelling methods will be used to characterise the nature of the substrate-ligand interaction, and hence will seek to elucidate the mechanism by which ADLC catalyses the decarboxylation of acetolactate, and its unusual chiral specificity.BackgroundThe background to the project has been described in the descriptions of the first two mini projects (M. Wills, and V. Fülöp) Briefly, ALDC has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. The crystal structure of ALDC has recently been solved at Warwick, which opens up the possibility of obtaining an understanding at the atomic level of the way in which ALDC recognises potential substrates, and predisposes decarboxylation to occur in such an enantiomerically specific manner.

Aims of the Project: molecular characterisation of substrate-enzyme interactionVarious molecular modelling methods will be used to characterise the interaction of the two substrates prepared in the first mini-project with ADLC. Molecular dynamics (MD) simulations will be performed on the uncomplexed ADLC, using the published crystal structure as a starting point. Simulations on the ADLC/substrate complex will be performed in two ways. Firstly, MD simulations will be performed using the crystal structures obtained in the second mini-project. Secondly, Monte Carlo (MC) simulations will be performed to identify possible binding sites for the substrates within the unbound ADLC, and then these used to initiate further MD simulations. In all cases, the simulations will be analysed to characterise the energetics of binding, to identify any specific recognition motifs in the enzyme/substrate interaction, and to determine the extent of enzyme relaxation induced by the substrate.



Project timing‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Sascha Ott__________________________________





Name: David Wild__________________________________



Project outline:

References:

Consumables budget§§§§§§§§§§§§§§§§§§§§: £ 0


Project title: ____________________________________________________________________________



‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§§§§§§§§§§§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The formation of crossovers between maternal and paternal chromosomes is a fundamental biological process. Crossovers are known to occur more frequently near the ends of chromosomes than in central regions and the position of the centromere is thought to influence the positions of crossovers as well. Crossovers can be observed directly by immunostaining, or indirectly by tracing alleles through generations. As the direct observations show co-occurring crossovers along a chromosome they could harbour more information than the data obtained by indirect methods. The aim of this project is to evaluate competing models of crossover distributions using an unpublished dataset of direct observations in Maj Hulten’s lab.

The dataset gives approximate crossover positions, chromosome lengths, and centromere positions of several thousand cases. Most of the data is from human chromosomes, but a smaller part of the data is from mouse. The first step of the project is to formulate mathematical models of crossover-distributions that reflect the different hypotheses that experimentalists have. The second step is to fit each model to the data and evaluate the support the data gives to each hypothesis. Time permitting the results obtained from this unpublished dataset can be compared to results that can be obtained using indirect observations only.

This project can be linked to the experimental project on crossovers.(David Wild will be away from mid-May to June, so some advisor meetings might have to be rescheduled accordingly.)


Project timing*********************


X Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

X Slot 3 (16/07 to 14/09/2007)


Name:Dr Corinne Smith _____________________________


E-mail address: [email protected]___________ Phone number: 02476 522461______________



Name:___________________________________________



Project outline:

References:

Dafforn, T. R. and C. J. Smith (2004). "Natively unfolded domains in endocytosis: hooks, lines and linkers." EMBO Rep 5(11): 1046-52.Dunker, A. K., C. J. Brown, et al. (2002). "Intrinsic disorder and protein function." Biochemistry 41(21): 6573-82.Dunker, A. K., J. D. Lawson, et al. (2001). "Intrinsically disordered protein." J Mol Graph Model 19(1): 26-59.Luo and Baldwin, (1997) ‘Mechanism of helix induction by trifluoroethanol: A framework for extrapolating the helix-forming properties of trifluoroethanol /water mixtures back to water. Biochemistry 36 8413 Scheele, U., J. Alves, et al. (2003). "Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin." J Biol Chem 278(28): 25357-68.Tompa, P. (2002). "Intrinsically unstructured proteins." Trends Biochem Sci 27(10): 527-33.

Consumables budget†††††††††††††††††††††: £ 300

********************* Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.††††††††††††††††††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

It has been accepted dogma that a protein must adopt a compact ordered tertiary structure in order to have function. Recently this view has been challenged and the importance of natively unfolded proteins in biology is becoming recognised (Dunker, Lawson et al. 2001; Dunker, Brown et al. 2002; Tompa 2002). We have highlighted the role of natively unfolded domains in the field of endocytosis where some key endocytic proteins were shown to be deficient in traditionally folded structure and to harbour important binding motifs within their unstructured linker regions (Dafforn and Smith 2004). This project will focus on a detailed biophysical characterisation of 6 peptides which relate to unfolded regions of three important endocytic proteins, auxilin, clathrin and beta-adaptin, together with peptides with well-known extended structures such as those relating to the collagen sequence and poly-proline. Trifluoroethanol is widely used to induce helical structure in peptides and some native unfolded domains have been observed to be resistant to such induction. To our knowledge, this property has never been systematically studied.

The aim of this project is therefore to test whether response to trifluoroethanol is a good measure of the degree of ‘unfoldedness’ of natively unfolded protein domains. Initially, the secondary structure of the selected peptides will be analysed using circular dichroism and the effect of temperature on their conformation monitored. Then trifluoethanol will be titrated into each peptide and the effect on conformation observed. Analysis of these results using the method of Luo and Baldwin,(1997) will then be carried out and compared to results obtained for more conventional folded regions.

Should time permit, the study can be extended to investigate whether the presence of a binding motif within a peptide alters its conformational properties in comparison with peptides from regions of sequence close by which do not contain a binding motif. This will help answer the question of whether the binding motifs found in extended regions in endocytic proteins influence the folding properties of these regions.


Project title: Neuronal interactions with electromagnetic fields




Project timing‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)



Department: Warwick Systems Biology Centre____________ Building, Room: 339_______________________




Name:___________________________________________



Project outline:

References:

Consumables budget§§§§§§§§§§§§§§§§§§§§§: £ 0 (theoretical project)

‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§§§§§§§§§§§§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Neurones are electrically active cells; they interact with externally applied electromagnetic fields, such as in therapeutic deep-brain stimulation, and also generate their own fields, which though individually weak, can be measured through the scalp by EEG when large populations of neurons fire in synchrony.

The standard method for modelling the electrophysiology of neurones - cable theory - treats each element of the cell, the dendrites and axons for example, as small coupled resistors. Though this is a powerful method for modelling the response of isolated neurones, it ignores electromagnetic interactions and so cannot be used to model the effects of external stimulation or how the neurone's own field affects the conductive extra-cellular medium and neighbouring cells.

In this project the student will develop more complete neurone models that capture their electromagnetic interaction with the environment. Simplified models could include spherical and cylindrical geometries, relevant to cell bodies and dendrites. These analytical results will be compared to finite-element numerical simulations, which are also to be constructed by the student. The project would suit someone with a background in mathematics or physics and with an interest in neuroscience.

MOAC / Systems Biology mini-project proposal S1Submit one page only to Monica Lucena ([email protected]) by Wednesday 14th February, 2007, 5pm).

Project title: Identification of the host targets of pathogenicity effector proteins_________________________


MOAC Mini Project Experimental biology Systems Biology Mini Project X Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project


Project timing**********************


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr. Rebecca Allen____________________________

Department: WarwickHRI____________________________ Building, Room: __________________________

E-mail address: [email protected]___________ Phone number: __________________________



Name:Prof. Jim Beynon_____________________________

Department: Warwick HRI/ Warwick Systems Biology______ Building, Room: __________________________

E-mail address: [email protected]______________ Phone number: 02476575141_______________

Project outline:

References: Allen, R.L., Bittner-Eddy, P.D., Grenville-Briggs, L.J., Meitz, J.C., Rehmany, A.P., Rose, L.E. and Beynon, J.L. (2004) Host-parasite co-evolutionary conflict between Arabidopsis and downy mildew. Science 306, 1957-1960Birch, P.R.J., Rehmany, A.P., Pritchard, L., Kamoun, S. and Beynon, J.L. (2006) Trafficking Arms: Oomycete Effectors Enter Host Plant Cells. Trends in Microbiology 14, 8-11.Consumables budget††††††††††††††††††††††: £ 100

********************** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.†††††††††††††††††††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Plants and animals have innate immune systems that prevent the majority of organisms from invading. To overcome this innate immunity some organisms have developed a suite of effector proteins that suppress the host resistance mechanisms. We know these organisms as pathogens. Our group studies the interaction between the downy mildew parasite Hyaloperonospora parasitica and the model plant Arabidopsis. We have cloned two effector proteins by traditional means and have been analyzing their role in the invasion process. To complement this we are part of a team that have sequenced the genome of the pathogen and are in the process of annotation. We have used protein motif searching scripts to identify the effector complement of the genome. From this analysis we have decided to target 60 effector proteins for further analysis.

We now wish to know what their role is in planta. We would predict that they target plant proteins that are involved in immune responses and attempt to suppress their function. Therefore, we are using the yeast-2-hybrid screen to identify the interacting proteins. From these studies we will construct models of the protein interaction networks involved in host immunity.

The project will involve cloning a particular effector into a yeast-2-hybrid bait vector and carrying out a screen for interacting proteins in a prey library. Interacting proteins will be identified by using a range of reporter genes. Positive clones will be purified and sequenced to identify the nature of the potentially interacting protein. Informatic analyses will then be carried out to assess the potential relevance of the interacting proteins to host/pathogen interactions.


Project title: Analysis of stress related signalling pathways in Arabidopsis leaf development______________




Project timing‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)





Supervisor’s advisor (for non-permanent members of staff or those on probation: academic who agrees to provide supervision support to the supervisor and also agrees to meet the student briefly at least once a week):

Name:___________________________________________



Project outline:


pathways between developmental and dark/starvation induced senescence in Arabidopsis. Plant J 42: 567-585.Lim et al. (2007) Leaf senescence Ann Rev Plant Biol 58:115–136 Lorenzo and Solano(2005) Current Opinion in Plant Biology 8:532–540

Consumables budget§§§§§§§§§§§§§§§§§§§§§§: £ 450

‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§§§§§§§§§§§§§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Stress responses in plants are dependent on a complex network of signaling pathways controlled by a variety of plant hormones. The two hormones, jasmonic acid (JA) and salicylic acid (SA) are key in the expression of many stress response genes but their influence is often conflicting, with increased expression of genes involved in SA signalling resulting in reduced expression of JA response genes and vice versa (e.g. Lorenzo 2005).

SA and JA are also important in regulating gene expression during developmental leaf senescence (e.g. Buchanan-Wollaston et al., 2005, Lim et al 2007). At Warwick HRI, we have been investigating the effects of mutants in the SA and JA signaling pathways on the progression of leaf senescence. We have also investigated the effects of spraying the hormones on leaves at different stages of development. Microarray and chlorophyll fluorescence analysis has been applied and there are considerable data sets available that have not been fully analysed.

In this project the student will choose to investigate either SA or JA signaling, and identify mutants and treatments accordingly. Experiments will be designed following analysis of the data sets that are already available. This will include a detailed evaluation of the microarray data that has been generated to decide on the most effective growth experiment and microarray analysis to undertake. The student will grow Arabidopsis plants (wild type and mutants) and treat a proportion of these with hormones at different time points. They will carry out phenotype analysis, chlorophyll assays, chlorophyll fluorescence measurements etc. At the same time, leaf material will be collected for RNA isolation and microarray analysis. The effects of the mutations and/or treatments on phenotype during senescence will be assessed and a small array experiment (up to 16 slides) will be carried out on one treatment/mutant comparison at a single timepoint.

The student will gain experience in plant growth, phenotype analyses including chlorophyll fluorescence measurements, microarray hybridization and will analyse extensive microarray data, both from their own experiments and also from the previous experiments already carried out. Comparison of results with available data on the signaling pathways and senescence will allow hypotheses to be drawn on the timing and effect of the SA or JA signal in senescence.


Project title: Functional analysis of genes that regulate plant stress responses in Arabidopsis____________




Project timing***********************


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)





Project outline:


pathways between developmental and dark/starvation induced senescence in Arabidopsis. Plant J 42: 567-585.Lim et al. (2007) Leaf senescence. Ann Rev Plant Biol 58:115–136 Van der Graaff et al, 2006 Transcription Analysis of Arabidopsis Membrane Transporters and Hormone Pathways during Developmental and Induced Leaf Senescence Plant Physiology, 141: 776–792

Consumables budget†††††††††††††††††††††††: £ 450

*********************** Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.††††††††††††††††††††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Environmental stress has a severe effect on plant growth and plants often respond by entering premature senescence. Signalling pathways that control gene expression during leaf senescence have many overlapping features with pathways that are expressed during plant stress responses (Buchanan-Wollaston et al, 2005; Lim et al., 2007; van der Graaff et al, 2006).

At Warwick HRI we have identified a number of regulatory factors that show enhanced expression during plant senescence and have generated knock out and overexpressing transgenic Arabidopsis lines for many of these. Many of these genes are also increased in expression in response to a variety of different stresses including low nitrogen, drought, high light, osmotic stress etc. The aim of this project is to investigate the effects of a selection of the mutations on the plant stress response.

Plants will be grown (wild type, knock out and overexpression mutants (3-6 different genes)) under a variety of experimental stress conditions. High light and drought treatments will be applied in the glasshouse, low N conditions will be applied on agar plates etc. measurements will be taken to compare the effects of the stress on physiological parameters in the wild type and the mutants. These measurements will include chlorophyll fluorescence using the CF imager, chlorophyll and protein assays and protein gels to measure RUBISCO levels etc. This data will indicate the links between each gene studied and different stress responses.

Microarray analysis (up to 16 slides) will be carried out on one stress and mutant combination (this will be a 4 way comparison; mutant v wild type and stress v unstressed). The mutant and stress to be studied will be selected following the analysis described above. The data will show the effect of the mutation on gene expression in untreated plants, the effect of the stress on gene expression in wild type plants and the differential expression in the mutant compared to the wild type following the stress treatment. Data will be analysed using GeneSpring and differentially expressed genes will be examined for changes in significant pathways using programs such as GOstat, MapMan etc. Publicly available data will be mined to investigate the potential role of the selected gene in plant development and stress response.

MOAC / Systems Biology mini-project proposal S4Submit one page only to Monica Lucena ([email protected]) by Wednesday 14th February, 2007, 5pm).Project title: Gene expression profiles in the honey bee: a model for studying the interaction of innate immunity and socially mediated defences against microbial diseases


MOAC Mini Project Experimental biology Systems Biology Mini Project √ Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project


Project timing‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) √ Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr David Chandler / Dr Eugene Ryabov / Dr Peter Bittner-Eddy

Department: Warwick HRI___________________________Building, Room: WHRI TPB 49

E-mail address: [email protected] ___________Phone number: 02476 575 041



Name:



Project outline:

References:

Consumables budget§§§§§§§§§§§§§§§§§§§§§§§: £ 250

‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§§§§§§§§§§§§§§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

There is a rapidly developing interest in the use of insects as model eukaryote animal hosts for infectious diseases. This is driven by the parallels between the innate immune response of insects and of vertebrates, together with the practical advantages of insects as experimental tools.

We are interested in the European honey bee (genome sequence published at end of 2006) as a potential model system for studying the genomics of disease resistance in highly social organisms, including humans. Honey bees are one of a select number of organisms with a highly evolved and complex social structure. The high density populations in honey bee colonies provide ideal conditions for the spread of microbial diseases, and hence there should be a strong selection pressure on the bee innate immune system. However, the honey bee genome encodes relatively few proteins associated with immune pathways. It has been postulated that this shortfall is compensated by socially-mediated defences. However, the role of most of the innate-immunity components remains to be validated, along with understanding how gene expression varies in different tissues and in response to different pathogens. These need to be addressed as a matter of priority before social defences can be understood.

For this project, we want you to assess the potential of the honey bee as a model system, using gene expression profiling. Bees will be challenged with pathogenic microorganisms, and expression of innate immunity genes will be evaluated using a microarray and quantitative RT-PCR. We want you to investigate how expression profiles vary in different tissues and with different types of pathogen. This exciting project will give you the opportunity to contribute in a meaningful way to a newly developing field.


Project title: Defining sugar-signalling pathways in Arabidopsis seedlings using whole genome micro-arrays.


MOAC Mini Project Experimental biology Systems Biology Mini Project X Wet Project(8 weeks) Biophysical chemistry (12 weeks) Dry Project


Project timing************************

MOAC Mini Project Slot 1 (26/03 to 18/05/2007) Systems Biology Mini Project X Slot 1 (26/03 to 22/05/2007)

Slot 2 (21/05 to 13/07/2007) X Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Peter J. Eastmond____________________________

Department: Warwick HRI____________________________ Building, Room: TPB 134___________________




Name:___________________________________________



Project outline:

References:

Consumables budget††††††††††††††††††††††††: £ 400

************************ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.†††††††††††††††††††††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Background: Plants sense the supply of sugars produced by photosynthesis and adapt their metabolism and development accordingly. Research into “sugar-signaling” in Arabidopsis thaliana suggests that perception probably occurs via multiple mechanisms, but to date the only sensor that has been identified is hexokinase1 (HXK1)/GIN2 (Rolland et al., 2006). This enzyme catalyses the phosphorylation of glucose to glucose-6-phosphate and has recently been shown to perform a dual role as a glucose receptor (Moore et al., 2003; Cho et al., 2006). Whole genome micro-array experiments on Arabidopsis seedlings have shown that the transcript abundance of many genes either increase or decrease (within 30 min) following a change in sugar availability (Blasing et al., 2005; Osuna et al., 2007). On a genome-wide scale it remains to be determined which of these early response genes react to a HXK-mediated signal and which are regulated by alternate sugar-signaling pathways.Aims: The aim of this project is to perform whole genome micro-array experiments on Arabidopsis seedlings under sugar-deprived and excess conditions, and use the gin2-1 mutant (and treatment with the sugar analogue 2-deoxyglucose) to distinguish which sugar-responsive genes (and therefore processes) are regulated by a HXK-mediated signal and which are regulated independently.Methods: Wild type and gin1-2 mutant seedlings will be starved of sugars and then re-supplied with glucose (or 2-deoxyglucose) using conditions similar to those described by Osuna et al., (2007). RNA will be extracted and micro-array experiments will be performed using CATMA arrays (http://www.catma.org/). Data will be analysed using GeneSpring and MapMan software. Responses of selected genes will be confirmed using real-time RT-PCR.References: Rolland et al., (2006) Annu Rev Plant Biol. 57, 675. Moore et al., (2003) Science 300, 332. Cho et al., (2006) Cell 127, 579. Blasing et al., (2005) Plant Cell 17, 3257. Osuna et al., (2007) Plant J. 49, 463.


Project title: Neuronal interactions with electromagnetic fields




Project timing‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)







Name:___________________________________________



Project outline:

References:

Consumables budget§§§§§§§§§§§§§§§§§§§§§§§§: £ 0 (theoretical project)

‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.§§§§§§§§§§§§§§§§§§§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

Neurones are electrically active cells; they interact with externally applied electromagnetic fields, such as in therapeutic deep-brain stimulation, and also generate their own fields, which though individually weak, can be measured through the scalp by EEG when large populations of neurons fire in synchrony.

The standard method for modelling the electrophysiology of neurones - cable theory - treats each element of the cell, the dendrites and axons for example, as small coupled resistors. Though this is a powerful method for modelling the response of isolated neurones, it ignores electromagnetic interactions and so cannot be used to model the effects of external stimulation or how the neurone's own field affects the conductive extra-cellular medium and neighbouring cells.

In this project the student will develop more complete neurone models that capture their electromagnetic interaction with the environment. Simplified models could include spherical and cylindrical geometries, relevant to cell bodies and dendrites. These analytical results will be compared to finite-element numerical simulations, which are also to be constructed by the student. The project would suit someone with a background in mathematics or physics and with an interest in neuroscience.


Project title: Extracting neuronal structure from image data




Project timing*************************


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)







Name:___________________________________________



Project outline:

References:

Consumables budget†††††††††††††††††††††††††: £ 0 (theoretical project)

************************* Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.††††††††††††††††††††††††† Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects

The structure of neurones is closely related to their function. They typically comprise a highly branched dendritic tree attached to the cell body (the input structures) and an axon that branches to make contacts with other neurones (the output structure). Because the electrical response properties of neurones are highly influenced by their shape, accurate geometric reconstructions of neurones are crucial components for the construction of detailed models of neuronal microcircuits and networks.

Experimentalists typically have access to the structure of neurones via the infrared microscope images they use in the process of making intracellular recordings. Though these images are of low resolution, they nevertheless contain much information on the structure of cells and surrounding tissue.

In this project the student will explore whether advanced image analysis techniques can be used to extract more information from these images. One candidate method is super-resolution in which a number of low resolution images are combined to produce a higher-resolution composite image in which finer details of neuronal structure might be seen. The project would be ideal for someone with a background in mathematics, physics or computer science.


Project title: Identification of post translational modifications of proteins using mass spectrometry___________




Project timing‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Dr. Konstantinos Thalassinos__________________

Department: Biological Sciences______________________ Building, Room: C115_____________________

E-mail address: [email protected]____________ Phone number: 024761 50439______________



Name: Prof. Jim Scrivens____________________________

Department: Biological Sciences______________________ Building, Room: C113_____________________

E-mail address: [email protected]_____________ Phone number: 02476 74189_______________

Project outline:

Consumables budget§§§§§§§§§§§§§§§§§§§§§§§§§: £ 500

‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡‡ Students may choose to take up to 2 weeks holiday during the duration of their projects by arrangement with all affected supervisors and the respective Course Director. If students choose to do this then their relevant project(s) will be extended accordingly. The absolute final deadline for reports is 28 September 2007. Supervisors may refuse to mark the last project before 1 October so do not rely on this option.

Introduction - Post translation modifications (PTMs), are chemical modifications of protein products which occur after translation of the mRNA product. PTMs are extremely important and greatly enhance the diversity and functionality of proteins; can determine the cellular localisation of a protein, its interaction with other proteins, its turnover and whether it is active or inactive. Mass spectrometry has become an important method for the characterisation of PTMs (in particular phosphorylation and glycosylation) due to the sensitivity, speed of analysis and the ability to characterise complex mixtures.

Methods - In order to characterise a post translationaly modified protein one needs to determine the nature, the site or sites and the extent of modification. Phosphorylation involves the covalent attachment of a phosphate group in phosphor-mono-ester linkage to the side chain oxygen of serine, threonine or tyrosine. Characterisation using mass spectrometry involves enzymatic digestion of the protein to give a mixture of peptides and phosphopeptides. The phosphopeptides are then separated using chromatographic techniques and the resulting modified peptides characterised using a combination of MALDI mass spectrometry and tandem mass spectrometric approaches. One of the challenges in phosphopeptide analysis is the fact that phosphorylated peptides often exhibit lower intensities than their non-phosphorylated counterparts. Various enrichment protocols have been developed and these will be tested.

Aims - This project is designed to establish methods for the enrichment and characterisation of phosphorylated proteins and implement them, initially on standard material, and then on systems of biological interest.

Techniques to be used - Gel electrophoresis, affinity chromatography, MALDI-MS, ESI-MS/MS and appropriate software to interpret the experimental data.

References: 1) Annan, R. S. and Carr, S. A. (1997). The essential role of mass spectrometry in characterizing protein structure: mapping posttranslational modifications. J Protein Chem 16: 391-402.2) Neubauer, G. and Mann, M. (1999). Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations. Anal Chem 71: 235-42.3) Areces, L. B., Matafora, V. and Bachi, A. (2004). Analysis of protein phosphorylation by mass spectrometry. Eur J Mass Spectrom (Chichester, Eng) 10: 383-92.


Project title: Understanding animal behaviour__________________________________________________§§§§§§§§§§§§§§§§§§§§§§§§§ Please estimate the consumables required for the proposed project. Please note that each student will be allocated a total budget of £450 to cover consumables for all three of their mini-projects




Project timing


Slot 2 (21/05 to 13/07/2007) Slot 2 (25/06 to 14/09/2007)

Slot 3 (16/07 to 14/09/2007)


Name: Matthew Turner______________________________

Department: Physics / Systems Biology_________________ Building, Room: Physical Sciences, PS142____

E-mail address: [email protected]______________ Phone number: x22257____________________



Name:___________________________________________



Project outline:

References: 1. Topaz C.M., Bertozzi A.L. Swarming patterns in a two-dimensional kinematic model for biological groups, SIAM J. App. Math. 65 (1): 152-174, 2004.2. Stephens D.W. and Krebs J.R. 1986. Foraging theory. Princeton, NJ: Princeton University Press.

Consumables budget: £ 50

The behaviour of groups of animals can sometimes be described by appropriate continuum models. Such models describe the variation of average quantities such as density and sometimes orientation, velocity or behavioural state. These are then treated as fields that vary in time and space but, within this approach, no attempt is made to trace the location or identity of individual animals. The models are typically constructed as coupled differential or integro-differential equations. The resulting equations are usually local in space and time since each animal can only sense nearby events and is only affected by its recent experiences. The inter-animal interactions then govern changes in the behaviour of the society in a way that can be analogous to, e.g. phase transitions in physics.

Modern farming practices present a highly controlled environment in which many animals are kept under precisely controlled environmental conditions. These therefore represent ideal experimental systems for the refinement and application of such models. Farmers typically wish to maximise some quantity, such as the rate of increase in weight of their livestock. However, this depends on their collective behaviour, including their motility and resulting feeding patterns. The farmer is also subject to constraints, such as legislation specifying the maximum average density. How should the farmer stock his animals ? Should he sometimes seek to engineer non-uniform density ? Should feed always be provided continuously and should the feedings stations always be distributed homogenously ?

The successful student will undertake the following:1. Literature search and a comparison and criticism of existing approaches to modelling animal behaviour.2. Construction and analysis of a simple model for the behaviour, and development, of farmed chickens.3. A study of possible approaches for comparison of such a model with experimental data, extracted by video using animal recognition software.

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