Institute of Molecular BioSciences

Research Projects for700-level programme

2011

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‘Rules of Thumb’ when choosing a project and

supervisor • Make sure that you are interested in the project and that it will

provide you with a variety of practical and intellectual skills

• Ensure that you get on well with your intended supervisor

• Choose a project that is well resourced: adequate bench space,

equipment and funding for consumables

• Check that you will belong to a research group: regular group

meetings and a supportive intellectual environment will facilitate

productivity

• Choose a supervisor who has a keen interest and active involvement in research

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The Institute of Molecular BioSciences

offers research programmes for PGDipSc, BSc(Hons) and MSc students in:

Plant Development

Molecular Evolution and Ecology

Bioinformatics and Genomics

Gene Expression

Protein Structure and Function

Molecular Genetics

Host-Microbe Interactions

Cell Biology

Cancer Research

A selection of the research projects suitable for advanced study [PGDipSc,

BSc(Hons) and MSc] within the Institute of Molecular BioSciences is

presented in abbreviated form below. Additional projects are also available,

and prospective students are encouraged to discuss these with individual

staff members.

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Associate Professor Rosie Bradshaw http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/bradshaw_rosie.cfm Exploring the genome of a pine needle pathogen, Dothistroma septosporum. Dothistroma needle blight causes severe problems in New Zealand’s commercial forests and is also causing epidemics in Canada and Europe that are linked to climate change. The pathogen responsible is a fungus, Dothistroma septosporum, which makes a toxin called dothistromin that causes the bright red coloured bands seen in diseased needles. Currently the only reliable method of disease control involves spraying forests with fungicide and this is not permitted in some countries. As part of an international collaboration with the Dothideomycetes Comparative Genomics Consortium we have been able to have the D. septosporum genome sequenced by the Joint Genome Institute in the USA. The availability of this genome has opened up a huge wealth of possibilities for understanding, and ultimately controlling, the pine needle blight disease. Depending on interest, numerous projects are available focused on, for example:

- Functional analysis of candidate genes involved in communication between the plant and pathogen such as small protein ‘effector’ molecules.

- Survey and functional analysis of other genes likely to be involved in the disease process, such as those encoding cell-wall degrading enzymes.

- Comparative analysis of genome structure, gene synteny (order) and types of genes between the pine pathogen D. septosporum and closely-related fungi, such as the tomato pathogen Cladosporium fulvum, with a view to understanding host specificity.

- Evolution of gene clusters such as the dothistromin toxin gene cluster of D. septosporum. This toxin is closely related to aflatoxin, a potent carcinogen and toxin made by some fungi in the genus Aspergillus and we already know there are similar toxin genes in these species.

Would suit majors in: Genetics, Microbiology, Biochemistry or Plant Biology (Hons, MSc or DipSci).

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Dr Murray Cox http://massey.genomicus.com/ My research addresses fundamental questions in contemporary population genomics. I have particular interest in modeling genome dynamics — firstly, establishing how genetic variation is distributed within and between individual genomes, and secondly, determining how this diversity changes over evolutionary time. My work draws heavily on statistics and computer science with a solid foundation in genetics and biochemistry. I develop computational methods for genome analysis, largely in the fields of coalescent theory, demographic inference and systems biology. I am currently applying these tools to a series of ongoing projects, which includes using neutral genomic variation to reconstruct the demographic history of humans, linking global patterns of diversity to evolutionary dynamics in small subpopulations, and advancing studies of non-model organisms through de novo genome sequencing and automated gene analysis. More broadly, I am interested in the interface between biology, statistics and computer science, especially where large genetic datasets can be used to address questions of outstanding biological importance. Project 1: Reconstructing Pacific prehistory using human genetic data Models to capture the prehistory of human populations in Island Southeast Asia and the Pacific will be developed using mtDNA, Y chromosome and autosomal datasets. This project will primarily focus on a series of anthropologically interesting communities in eastern Indonesia, especially from the islands of Flores, Sumba and Timor. Project 2: Dissecting the genomes of newly sequenced fungal symbiotes [In collaboration with Assoc Prof Rosie Bradshaw and/or Prof Barry Scott] The genomes of two filamentous fungi with importance to the New Zealand economy have recently been sequenced. The first, Dothistroma septosporum, is a fungal pathogen of pine trees [Rosie Bradshaw]; the second, Epichloë festucae, is a fungal mutualist of perennial ryegrass, an important component of New Zealand pastures [Barry Scott]. Transcriptomes – that is, the mRNA profiles – of these two species have either been sequenced or are currently being generated. This project will be developed around the genome sequences and/or transcriptomes of these organisms. The requirements of these projects vary, but students will need a thorough knowledge of relevant disciplines. Projects may be tailored for honors projects or candidates for the M.Sc./Ph.D. I am also open to supervising students on other research topics that fall within my area of expertise.

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Dr Paul Dijkwel http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/dijkwel_paul.cfm Hunting for the elixir of life for plants Ageing is a universal but poorly understood fact of life. In plants, ageing occurs throughout development and ends with a process called senescence. Leaf senescence can be found in all plant species. The spectacular leaf changes in the autumn demonstrate the importance of leaf senescence for trees. Annual plants undergo leaf senescence mainly during their reproductive stage. Leaf senescence involves the programmed death of leaf tissue and is characterised by change of leaf colour and massive transport of nutrients to other parts of the plant. The reclaimed nutrients are subsequently used for growth in the next year (perennials) or for seed production (annuals). In crop plants, such as lettuce and cauliflower, leaf senescence is also initiated after detachment from the root system. Presumably, the reclaimed nutrients are transported to the growing part of the plant to support growth for as long as possible. Clearly, senescence is of great economic importance and understanding this process allows us ultimately to control senescence and, therefore, improve crop characteristics. We are interested in both fundamental and applied aspects of senescence. From an applied point of view we aim to increase shelf life of fresh produce. In addition we are interested in the regulation of leaf senescence. Fundamental understanding of the process will aid the development of methods to increase shelf life and decrease crop losses. Both the model plant Arabidopsis thaliana and commercially important crops are being used in the research.

To study leaf senescence in Arabidopsis, regulatory genes have been identified that cause an altered leaf senescence phenotype when mutated. The student will help with the analysis of the genes and proteins encoded by the genes, in order to determine how the regulatory genes function. In general students will have the opportunity to work with several different techniques. Research projects are suitable for Honours or Masters students interested in molecular biology, plant physiology, bio-informatics or metabolomics.

Projects can be adjusted to suit students needs but is likely to contain several of the above-mentioned elements.

Artist: Pinot

Wild type Methuselah mutant

Age-defying Arabidopsis mutant: Wild type plants senesce normally. The Methuselah mutant has a mutation in a senescence regulatory gene and remains fully green. Will this mutant lead the way to the elusive elixir?

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Dr Helen Fitzsimons The molecular mechanism of long-term memory storage My research interest is in memory, specifically the molecular basis of long-term memory storage in the brain. Histone modifications, such as acetylation or methylation, have a dramatic effect on chromosome structure and gene expression and these modifications determine how patterns of gene expression are maintained after establishment in early development, so called “developmental memory”. I am studying whether neuronal memory is regulated in a similar manner to developmental memory by investigating the role of histone modifying enzymes in the establishment and maintenance of long-term memories in Drosophila. A project on this topic may involve techniques such as general molecular biology, immunohistochemistry, in situ hybridisation and Drosophila genetic analyses including crosses, overexpression, RNAi knockdown and generation of transgenic flies. Would suit BSc (Hons) or PGDipSci students.

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Dr Tracy Hale http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/hale_tracy.cfm Exploring Chromatin Structure and Function Project 1: How do mitotic chromosomes condense? In order to fit into the nucleus, genomic DNA is packaged through the binding of both histone and architectural proteins into the highly ordered structure of chromatin. Faithful transmission of the genome during cell division requires dramatic changes in chromatin architecture. Of importance is the compaction of chromosomes during mitosis, as failure to properly condense causes instability leading to birth defects and cancer. A major structural component of chromatin is the linker histone H1, which accounts for 10% of the total protein content of mitotic chromosomes. While recently demonstrated to be essential for condensing mitotic chromosomes, the role of histone H1 in this process has long been contentious. Histone H1 becomes highly phosphorylated during mitosis and it is thought this phosphorylation is responsible for maintaining the window of chromosome condensation, however the events that regulate the timing of this phosphorylation are unknown. We have identified a phosphorylated residue on the N-terminal tail of histone H1 that is only present during mitosis. We demonstrate that Aurora B, the kinase that orchestrates vital events in mitosis including chromosome condensation, is responsible for this phosphorylation. Taking advantage of an extensive range of histone H1 and Aurora B mutant proteins, immunofluorescent confocal imaging of cells will be used to explore Aurora B phosphorylation of histone H1 in vivo. This will allow us to address if this phosphorylation mark has a regulatory role in H1 mitotic function and is therefore involved in controlling the timing of chromosome compaction.

Project 2: Is loss of HP1 required for tumour progression? Members of the Heterochromatin Protein 1 family (a, b, g), regulate heterochromatin formation and therefore play an important role in gene expression, genomic stability and DNA repair. Work with pathologists from the Baylor College of Medicine has shown a reduction of HP1 during the progression of a variety of tumours, with metastatic tumours often exhibiting a complete loss of HP1 expression. Using molecular and cellular techniques this project will explore the pathways that regulate HP1 expression and the contribution each family member makes in suppressing tumour growth.

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Professor Michael McManus http://www.massey.ac.nz/massey/learning/departments/institute-molecular biosciences/staff/en/mcmanus_michael.cfm Project I: Characterisation of white clover PAP26-like gene in response to P supply Plants show well defined responses to phosphate (P) deprivation. These include secretion of acid phosphate enzymes (APase) to mobilise soil P from bound forms. In studies with Arabidopsis, a P-supply regulated gene has been identified (AtPAP26) and we have recently identified a PAP26-like gene from the pasture legume white clover (Trifolium repens L.). The identification of a PAP26-like gene in white clover will, for the first time, allow for more definitive studies on the enzyme with regard to P-supply. The following is how the project may develop:

1. Expression of the gene in bacteria – (and for MSc studies) production of antibodies to monitor protein induction.

2. Kinetic characterisation of the recombinant enzyme – this will provide some evidence of function – ie. non-specific or specific enzyme.

3. Examination of the glycosylation status of the enzyme. Project 2: Characterisation of novel sulfur (S)-assimilation enzymes in unicellular organisms (marine alga; cyanobacteria) Lower organisms, including marine alga, contain some novel polyproteins that ‘bridge’ the evolution of the S-assimilation pathway between bacteria and higher plants. In higher plants, the enzymes in the S assimilation pathway exist as single proteins, whereas in some lower organisms two enzyme domains occur on the same protein. The question, therefore, is whether both domains of the polyprotein are active as enzymes and how they are regulated. The project may develop as follows:

1. Expression of polyproteins in yeast (including plasmid handling and transformation).

2. Enzyme assays of recombinant proteins including partial purification. 3. Western analysis 4. (For MSc), production of transgenic tobacco for in planta expression studies.

Both projects can be adapted to become suitable for either BSc(Hons) or MSc students.

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Dr Gill Norris http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/norris_gill.cfm Research interest: Protein structure and function Project 1: Only 4CC1’s is looking like these…(with Dr Mark Patchett) Glycocins are a novel and diverse class of ribosomally synthesised glycopeptide antibiotics secreted by bacteria to inhibit the growth of competing microbes. Two Bacillus thuringiensis strains (BGSC 4CC1 and BGSC 4Y1), two thermophilic acidophiles (Bacillus tusciae and Alicyclobacillus acidocaldarius), the human comensal Lactobacillus ultunensis, and Streptococcus suis, are each predicted to secrete a glycocin. You will develop activity (bio)assays for at least one of these secreted glycopeptides, determine the growth conditions for optimal production, then purify and characterise the mature modified peptide(s) using enzymatic dissection with HPLC, circular dichroism spectroscopy, mass spectrometry, and, if time allows, use 1H NMR spectroscopy to identify the sugar and sugar linkage to the peptide. Project 2: A Morphing PNGase: Is it real or just a sham? (with Dr Mark Patchett) PNGases are enzymes that cleave the intact sugar chains from proteins. The structures of both wildtype and recombinant wildtype and mutant PNGase F from Flavobacterium meningosepticum have been solved in our laboratory. We have made several mutants designed to elucidate both the catalytic mechanism of, and the binding of substrate by, the enzyme. We have also produced a soluble recombinant orthologue from Deinococcus radiodurans (Dr PNG) that unexpectedly has no activity. We want to know, is Dr PNGase a real PNGase, or has it morphed into a carbohydrate binding protein? There are differences in sequence of the residues making up the outer sphere of the active site, which may or may not be responsible for this loss of activity. Your task, should you accept it, will be to turn PNGase F into Dr PNGase and vice versa by making two amino acid substitutions in each enzyme. You will produce and purify recombinant protein, and monitor the effects of the mutations using enzymatic assays. We have an efficient system for producing recombinant protein in place, as well as a sensitive and reliable assay for measuring activity. Crystallisation trials will be undertaken on the purified mutant protein. Crystals that diffract will be analysed using x-ray diffraction techniques and the structure solved using molecular replacement techniques. Both projects are suitable for M.Sc./B.Sc. (Hons) students interested in protein structure/function, molecular recognition and molecular microbiology, with skills in protein purification, biochemistry and molecular biology.

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1 - pln No enz Ctr l ~45 uL UV_VIS _22 - 1m g/ml FL 30uL GcnA NaOAc pH 4 .5 o/n Run 2 UV_VIS _23 - N-Frag Ctrl 2mb/ml 50 uL UV_VIS _24 - N-Frag pln GcnA Deglyco pH 4.5 Run 7 a fte r 36h 500 uL inj'd UV_VIS _2mAU

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Dr Jeong Park http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/park_research.cfm Project 1: Anti-Cancer Effects of Oleic Acid We are interested in molecular mechanism of oleic acid-driven anti-cancer effects. You will test whether oleic acid serves as a regulatory signal to activate two key proteins, glioma amplified sequence 41 (GAS41) and protein phosphatase 2C (PP2C), both of which have been implicated in cancer through different mechanisms and targets. GAS41 is a nuclear protein that plays a role in various cancers, probably through interactions with other proteins including p53. PP2C has been shown to be stimulated up to 15 fold by oleic acid and to induce cell death in various cell types. Based on our preliminary studies which suggest the GAS41/PP2C protein complex removes phosphate from p53, we hypothesize that a novel GAS41/PP2C complex is the underlying molecular mechanism for oleic acid-driven anti-cancer effects (Figure). Your research will test the hypothesis that oleic acid and GAS41 are positive regulators of PP2C, resulting in p53 stabilization and cell death. You will use in vitro and in vivo systems to functionally characterize the interaction between GAS41 and PP2C in parallel with the activation of p53 protein. Ultimately, you will test an oleic acid-GAS41 mix in a tissue culture model system to see whether stimulation of PP2C activity can alter the activation of p53 and more importantly the sensitivity of cells to radiation and chemotherapeutic agents.

Would suit students enrolling for Msc (Hons) students

Project 2: In vitro epigenetic studies in cell free system The compaction of eukaryotic DNA within chromatin structures allows intricate multilevel regulatory response to diverse environmental signals through various chromatin modifications. The epigenetic regulatory mechanisms of gene expression include post-translational modifications of histone tails and specific incorporation of histone variants such as H2A.Z into the genome. We are interested in the molecular mechanism of how a site-specific localization of H2A.Z and its histone tail modifications can modulate gene expression (Figure). To this end, purified recombinant proteins and artificially assembled chromatin templates will be used to analyze site-specific enrichment of H2A.Z and chromatin modification kinetics at the promoter region. The project includes the production of p400 ATPase from baculoviral expression system in insect cells and in vitro chromatin assembly using purified protein components. Would suit students enrolling for BSc (Hons) students

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Dr Mark Patchett http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/patchett_mark.cfm Bacterial War – what is it good for? (with Dr Gill Norris)

Human activities have provided bacteria with recent opportunities and incentives for biochemical innovation, but they’ve been fighting among themselves for billions of years. The bacterial weapons of choice are bacteriocins, a vast array of ribosomally synthesised secreted peptides that typically exhibit a narrow phylogenetic range of toxicity and have potential therapeutic and food preservation applications. We don’t fully understand the molecular ecology of bacteriocins, and have only scratched the surface of their structural and functional diversity (Schmidt EW (2010) The hidden diversity of ribosomal peptide natural products. BMC Biol 8:83).

Students and staff in IMBS/IFS recently characterised the first example of a glycopeptide bacteriocin (glycocin A, GccA) and identified putative glycocin gene clusters in many bacteria. In glycocin biosynthesis peptide scaffolds are morphed (Nolan EM, Walsh CT (2009) How Nature Morphs Peptide Scaffolds into Antibiotics. ChemBioChem 10:34-53) into antibacterial species by enzyme-catalysed glycosylation of serine, threonine and/or cysteine side chains.

Projects that address questions about glycocin molecular biology include:

1. Determining abundance and phylogenetic distribution of glycocin gene clusters.

2. Investigating the molecular biology and ecology of glycocin diversification processes, including the isolation and characterisation of phosphoglycocin A.

3. Testing the hypothesis that a phosphotransferase system is the GccA receptor by gene disruption. [read Kjos M, Nes IF, Diep DB (2009) Class II one-peptide bacteriocins target a phylogenetically defined subgroup of mannose phosphotransferase systems on sensitive cells. Microbiol 155:2949-2961.]

4. Determining the phylogenetic range of toxicity of GccA and derived peptides.

5. Testing the hypothesis that GccI is sufficient to confer immunity to GccA by regulated heterologous expression of this protein in susceptible bacteria. An extension would be the purification and structural characterisation of GccI.

6. Developing activity (bio)assays for secreted glycopeptides and identifying growth conditions that optimise glycopeptide production.

7. Developing homologous and/or heterologous glycocin expression systems with the potential to establish a new field of research - glycocin engineering.

Bioinformatics methods include comparative analysis of whole genome datasets. Laboratory techniques include isolation of pure bacterial cultures, DNA cloning, gDNA and peptide purification and characterisation, chemical cross-linking, cell fractionation, western blotting, lectin affinity and proteomics (mass spectrometry).

For MSc/BSc(Hons) students interested in molecular recognition, molecular micro-biology, protein structure/function and evolvability, who are keen to develop skills in protein biochemistry, molecular biology, bacterial genetics and bioinformatics.

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Dr Jasna Rakonjac http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/rakonjac_jasna.cfm Project 1: Mini-virus nanoparticles Mini-M13 virions, the smallest virions ever reported, have been constructed recently in the lab. The aim of this project is to turn them into diagnostic probes or drug delivery nanoparticles. To this end, detector or targeting proteins will be displayed at the tip of fluorescently labelled or drug-conjugated viruses. These functionalized mini-viruses will then be tested in home diagnostic devices and as targeted anti-cancer drug delivery particles.

Project 2: Sensitising bacteria to antibiotics Pathogenic bacteria fashion surprisingly complex and versatile molecular machines, which lead the assault on their hosts. Though these machines are diverse in their structure and function, each has an obligatory exit port or secretion channel, called SECRETIN. Giants among the channels, SECRETINS have tightly regulated GATES or valves that release toxins and other substrates, whilst maintaining the resistance of bacterial envelope to antibiotics. Despite its importance in bacterial disease, little is known about the secretin gate or its opening mechanism. Using simple “lazy man’s” random mutagenesis approach, this project will identify the gates from Salmonella’s secretin InvG. The role of the gates in the assault of the pathogens on the host cells will further be examined. Identifying the gates of the secretin channels will ultimately lead to new antibiotics and vaccines that will combat food poisoning.

Gate ?

Secretin channel structure and potential position of the gate. Spagnuolo et al., Molecular Microbiology (2010), 76:133–150

M13 virus decorated with CoPt nanoparticles. Mao, et al., Science (2004), 303:213

Nanoparticle

M13 virus

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Professor Barry Scott http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/scott_barry.cfm I am interested in understanding the molecular and cellular basis of plant-microbe symbiosis. My group studies the biology of the mutualistic interaction between Epichloë festucae and perennial ryegrass. We have cloned endophyte genes required for the synthesis of a number of bioprotective molecules and shown these genes are highly expressed in planta but not in axenic culture. We are investigating the signalling mechanisms between endophyte and host responsible for this symbiosis-specific gene expression. Using insertional and targeted mutagenesis we have shown that endophyte production of reactive oxygen species (ROS) controls mutualistic growth of hyphae. We are now dissecting the molecular and cellular basis of this new role for ROS. We have also isolated a range of new symbiotic mutants that are yet to be characterised and carried out a high throughput mRNA sequencing project that identifies a candidate set of 1200 fungal genes required for the symbiosis. There is considerable scope for further bioinformatics on this data set and functional analysis of some of the candidate genes. Project 1: Functional analysis of an Epichloë festucae symbiosis gene Using Agrobacterium T-DNA mutagenesis a battery of Epichloë festucae mutants have been isolated that are defective in their ability to establish a mutualistic symbiosis with perennial ryegrass. The aim of this project will be to clone and characterise the gene tagged in one of these mutants and confirm by complementation that the T-DNA insertion is responsible for the symbiotic phenotype. The symbiotic phenotype of the mutant will be determined using a range of biochemical and microscopic approaches. This project will provide training in plant and fungal molecular biology using techniques such as gene cloning, PCR analysis, transformation, plant inoculations, microscopy and biochemical analysis. Would suit students enrolling for BSc (Hons) or MSc (Hons) students Project 2: What is the function of a nitrogen metabolism/transport gene cluster? We have recently carried out a high throughput mRNA sequencing analysis of a WT and MAP kinase mutant association and found that several gene clusters for secondary metabolites associated with the symbiosis are ‘shut-down’ in the mutant compared to WT. Several clusters of unknown function, including one proposed to be involved in nitrogen transport/metabolism was shut down. This results suggests this cluster of genes has a key role in the symbiosis. The aim of this project will be to make knock-outs of one or two genes in this cluster and test their symbiotic phenotype. This project will provide training in plant and fungal molecular biology using techniques such as gene cloning, PCR analysis, transformation, plant inoculations, microscopy and biochemical analysis. Would suit students enrolling for BSc (Hons) or MSc (Hons)

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Dr Jan Schmid http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/schmid_jan.cfm Students interested in participating in the research areas described on Jan’s web page should see him to discuss possible projects of mutual interest.

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Associate Professor Kathryn Stowell http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/stowell_kathryn.cfm What I do in my spare time: translational medical research where lab-based research carried out in consultation with clinicians can ultimately inform clinical practice. Project 1: What causes malignant hyperthermia? In collaboration with Dr Neil Pollock, Consultant Anaesthetist, Palmerston North Hospital. Malignant hyperthermia is a disorder of skeletal muscle calcium homeostasis that is triggered by inhalational anaesthetics and depolarizing muscle relaxants. Worldwide it is a rare genetic disorder affecting 1 in ~ 50,000 people undergoing anaesthesia. The incidence at Palmerston North hospital is 1 in ~200 with ~50 families affected in New Zealand to date. We are working towards identifying causative mutations and determining their functional effects on calcium homeostasis in skeletal muscle. Techniques include recombinant DNA techniques, site-directed mutagenesis, genotyping, real time PCR for allele discrimination, primary human cell culture and in vivo calcium release assays using fluorescent calcium indicators. Would suit BSc(Hons) or MSc in Biochemistry or Genetics. Project 2: Why is colorectal cancer so difficult to treat? In collaboration with Dr Richard Isaacs, Consultant Medical Oncologist, Palmerston North Hospital. Colorectal cancer is a major cause of cancer-related deaths in New Zealand, with ~120 new cases per annum in the lower North Island alone. Treatment is often compromised by the development of drug resistance. We have used next generation sequencing technology to analyse the transcriptome of colon cancer cell lines before and after treatment with the most commonly used chemotherapy drug to identify differentially expressed genes. We are now working towards understanding the relationships between subsets of differentially expressed genes, the pathways in which they are involved and cell survival and drug resistance. Techniques include mammalian cell culture and response to drug treatment regimen, RT-qPCR for analysis of gene expression, and immunoblotting. The work may be extended to human cancer biopsy samples for an MSc student. Would suit BSc(Hons) or MSc in Biochemistry or Genetics.

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Dr Andrew Sutherland-Smith http://www.massey.ac.nz/massey/learning/departments/institute-molecular-biosciences/staff/en/sutherland-smith_andrew.cfm Project 1: What effects do disease-associated mutations have on filamin protein structure and function? Filamins are large dimeric proteins that cross-link actin filaments within the cytoskeleton. Mutations within filamin cause bone malformation diseases during embryonic development. We are interested in how these inherited filamin mutations cause disease as well as understanding the roles filamin has within cells. We aim to determine the structural and functional effects of filamin mutations compared to the wild type protein. This project would suit a student with interests in protein structure and function. Techniques used include, PCR sub-cloning, recombinant protein expression, protein purification, protein-binding assays, circular dichroism spectroscopy, protein crystallisation and x-ray crystallography. Project 2: Do filamin disorder patient cells respond differently on 3D deformable substrates compared to wild type control cells? Developmental and cellular processes involving the cytoskeleton are modulated in response to external force and the stiffness of the extracellular (EC) substrate. For example the load-dependent growth of bone has been established for over 100 years. Current evidence suggests filamin acts as an essential component of a finely tuned mechanosensor system associated with F-actin, membrane EC receptors and signalling proteins to facilitate force and matrix stiffness sensing mechanisms. We will compare the behaviour (cellular & biochemical) of fibroblast cells from filamin disorder patients (OPD2) vs controls when cultured on deformable 3D gel substrates that more closely mimic tissues. Cell growth on 3D substrates, of varying rigidity, allows analysis of contractility-based mechanosensing mechanisms in contrast to 2D plastic culture plates that are essentially rigid. This project would suit a student with interests in protein biochemistry and cell biology. Techniques used would include mammalian tissue culture, western blotting, immunoprecipitation and microscopic analysis.

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Dr Claudia Voelckel http://www.massey.ac.nz/massey/index.cfm?42AD9910-0EF8-A568-D34A-795564A869F9 Our research group studies the ecology and evolution of New Zealand alpine plants, in particular the ecological drivers of diversification and the molecular basis of adaptive traits. We use a combination of global gene expression studies, genetic and gene expression studies of candidate genes and native habitat studies. Depending on the project we collaborate with labs specialized in plant systematic (Landcare), plant physiology (Landcare), proteomics (Maquarie University), and metabolomics (MPI Chemical Ecology). Another important aspect of our work is the bioinformatic and statistical analysis of RNA-sequencing data from Next generation sequencing machines, mostly obtained by Massey University’s Genome Analyzer II (Illumina). Project 1: The evolution of adaptive genes in Pachycladon Recent transcriptomics and proteomics experiments identified differentially expressed genes potentially associated with adaptive diversification in the New Zealand alpine genus Pachycladon. One group of genes encodes myrosinase-associated proteins (e.g. ESP) which determine the type of mustard oil formed by a plant and thus directly affect the toxicity and palatability of plants to insect and other herbivores. We are interested in testing the hypothesis that diversification of Pachycladon species was driven by herbivory. We have a student project that involves comparative genetic and gene expression analyses of the ESP gene and its relatives across the genus. During the project the student will develop skills in comparative sequence analysis, gene cloning, PCR and real time PCR. Alternatively, the student could focus on a group of carbonic anhydrase genes, which have been related to differences in water use efficiency and whose expression has been found to differ across Pachycladon species. Project 2: Significance of hybridization in range expansion of North Island Ranunculus Hybridization and thus the shuffling of two divergent genomes is believed to create new evolutionary opportunities. New Zealand R. verticillatus and R. insignis are hypothesized to have hybridized leading to the formation of the allotetraploid R. nivicola. Today the latter is found in habitats not shared with its two progenitors. We are interested in testing the hypothesis that hybridization has led to changes in gene expression which in turn have resulted in new traits that have enabled range expansion. The project can take an analytical or molecular direction; the former would involve comparative analyses of mRNA sequencing data from all three species whereas the latter would require real time PCR analysis of candidate genes. Both projects would be suitable for either a Bsc(Hons) or a Masters degree.

R. insignis

R. nivicolaR. verticillatus

=Project 1: Pachycladon plant eaten by a native grasshopper

Project 2: R. nivicola, a hybrid between R. verticillatus and R. insignis occupies drier habitats than its parents.

Institute of Molecular BioSciencesMassey UniversityPrivate Bag 11 222Palmerston North

Phone: (06) 350 5515Fax: (06) 350 5688Email: imbs@massey.ac.nzWeb: http://imbs.massey.ac.nz