Honours in Biochemistry/Biotechnology/Chemistry

Grain Softness Protein-1 in Durum Wheat
Peter Gollan 
Grain hardness in wheat is largely determined by a protein composite that includes the puro-indoline proteins (PINa and PINb) and the closely related Grain Softness Protein-1 (GSP-1). While a role for the puroindolines in influencing endosperm texture has been identified, a role for GSP-1 remains largely undetermined. The Gsp-1 genes, thought to occur as multigenes, have demonstrated a higher level of sequence mutations than the pinA and pinB genes. This study involved an investigation into the sequence variation of Gsp-1 genes that were isolated from diploid, durum and common wheat lines. RFLP analysis identified novel restriction site polymorphisms in Gsp-1. An RFLP-type model was developed and each Gsp-1 gene analysed was classified according to the model. As a result, multiple Gsp-1 types were observed within some wheat accessions. DNA sequencing of selected Gsp-1 clones further investigated the extent of sequence mutations, as well as the effects of these on amino acid characteristics of GSP-1. The evolutionary relationship between each Gsp-1 variant was estimated and a theoretical model was developed to explain the multiple copies and sequence variations found in Gsp-1.
               
Characterisation of organelle-localized cyclophilins in wheat
Emma Wensley
Common wheat is an important grain for a large proportion of the world’s population. The quality of the wheat grain is influenced by the storage proteins that are accumulated during the development of the wheat endosperm. Investigation of the complex processes that are involved in the formation of storage proteins will allow a greater understanding of the factors that influence grain quality. These traits may be linked to specific gene polymorphisms, enabling the development of molecular markers that are used by the wheat industry breeding programs.
The cyclophilins, a class of peptidyl-prolyl cis-trans isomerases, are thought to play an important role in the folding and assembly of wheat storage proteins. Cytoplasmic cyclophilins from T. aestivum had been characterized and several tentative and cDNA sequences, possibly encoding products that are localized to specific organelles, had been identified. The gene sequences of these organelle-localized cyclophilins were the focus of this investigation. The aims included the isolation and characterization of these genes to allow further investigation into their role within the wheat endosperm, and to identify polymorphic regions that could be used for future development of molecular markers.
                           
Studies of Copper-responsive promoters
Glenn Anderson
The construction of a biosensor in the two promoterless vectors pMU2385 and pCB182 using the pAeco and pEeco copper inducible promoterless vectors of PpA87 as well as the isolated and identified pAalc and pEalc promoters of Alcaliges ssp. AO22 vectors pMU2385 and pCB182 will be presented. It was shown by β-galactosidase assays the pEeco copper inducible promoter initiated the transcription of over 100 times the units/mg of protein (β-galactosidase) when compared to pAeco of the same pco operon at a concentration of 0.4mM copper. The pAalc and pEalc promoters identified in this study were successfully cloned into the pMU2385 promoterless vector although due to time constraints β-galactosidase assays were unable to be conducted. The pEeco promoter of PpA87 and the pEalc promoters amplified using primers designed with restriction enzyme sites were cloned into the promoterless pCB182 vector directionally and non-directionally although there was no β-galactosidase activity could be recorded. The major findings of this research will be presented in the presentation.

Variation of rotavirus electropherotypes and genetic diversity of gene 1
Yvonne Germantis
Rotavirus-positive faecal specimens from patients who visited the Monash Medical Centre in Melbourne, from July 2001 to December 2002, were investigated by polyacrylamide gel electrophoresis. A total of 106 (83%) specimens contained sufficient RNA to produce visible electropheretic patterns. The electropherotypes were designated L and S for long and short patterns, respectively. 95% were identified as strains belonging to subgroup 2 (long patterns) and 3% were identified as subgroup 1 (short). The short electropherotype showed less variability, with only one type identified (S1), whereas the long profiles exhibited nine variations (L1-9). One electropherotype dominated each year and other less common types were also present. The L2 electropherotype was dominant in 2001 and L1 was prevalent in 2002. Genetic diversity of gene 1 was observed between different electropherotypes and within identical electropheretic profiles. Further investigation into the prevalence and emergence of rotavirus strains, as well as genetic variations is required for the study of vaccination for rotavirus disease.

Exploration of the cerate deposition process and Characterisation of cerium species formed in solution
Cassia Soste
Cerium conversion coatings have been developed as an alternative for the use  of chromate in the passivation of aluminium alloys. Relatively little is  known about the details of the deposition process of these coatings and as  such, this study is aimed at investigating this phenomenon. The scope of  this report will provide an introduction to aluminium as a metal and its use  in industry as an alloy. The effects of corrosion on aluminium alloys will  be discussed along with the passivating techniques used in the past to combat this problem. Finally, the studies carried out with the aim of simulating the deposition process will be reported. The scope of this report encompasses the formation of cerium species with pH, via pH rise titrations, and analysis of the resultant products formed. The precipitates formed are identified by a combination of Raman spectroscopy, fourier transform infra-red spectroscopy, X-ray diffraction and thermogravimetric analysis techiques.Analaysis of the cerate coated  aluminium samples allows them to be compared with the results obtained from  the coating simulation experiments.

Study of hydrocarbon degradation in Ultra Low Sulphur Diesel fuel
Christy-Anne Stansfield
The main aim of the project was to develop a method to extract and characterise hydroperoxide species from the non-polar (diesel fuel) matrix. This entailed thermal degradation of model compounds representative of hydrocarbons in the range of C-10 to C-22, which are found in middle distillate fuels. In this study emphasis was placed on tetralin degradation as tetralin is a known contributor to fuel instability.
Diesel fuel is a complex mixture of hydrocarbons varying in size and polarity, consequently development of an extraction method was essential to isolate the oxygenated hydrocarbon species. Liquid-liquid  and solid phase extractions of hydroperoxides were assessed as possible methods for sufficient extraction of the polar species from diesel. Cumene hydroperoxide was utilised as a model compound to determine the most suitable extraction method for isolation. Silation with BSA was used for the determination of the oxygenated degradation products produced and analysed by Gas Chromatography Mass Spectroscopy (GC-MS) and Gas Chromatography Atomic Emission Detection (GC-AED). Results indicate that indene and tetralin are of great concern for fuel stability. The formation of considerable amounts of oxidised species was confirmed by high peroxide values and gas chromatography analyses.

Detection and Identification of Bacteria in Melbourne's Drinking Water
Erin Brady
Melbourne is one of only a few cities in the world to supply unfiltered drinking water to the general population.  Filtration is considered unneeded due to the high quality of the water collected in protected catchments.  Concerns have been raised over the possibility of bacteria in the reservoirs becoming particle-associated, thus acquiring increased resistance to disinfection.  There is also concern that bacteria forming biofilms increases resistance to disinfection via chlorination.  Both of these scenarios may potentially lead to viable bacteria passing into the distribution system without filtration.  Untreated water samples from Silvan Reservoir were cultured and bacterial colonies isolated and identified.  As expected, only low numbers of bacteria were detected, however a number of opportunistic human pathogens were identified.  Isolated bacteria were tested for the ability to form biofilms, with many isolates showing positive growth, including the antibiotic resistant opportunistic pathogen Pseudomonas aeruginosa.  Due to zero colonies growing from treated water samples, the small bacterial counts in untreated samples, and the lack of historical drinking-water-related outbreaks in Melbourne, the current Melbourne Water system appears adequate.  More research into particle-associated-bacteria and the chlorination process is being continued by Swinburne University PhD students.