Student Number : 0213014A - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cytosolic glutathione transferases (GSTs) are obligate stable homo- and heterodimers comprising two GST subunits. Interactions across the subunit interface play an important role in stabilising the subunit tertiary structure and maintain the dimeric structure required for activity. The crystal structure of a rat Mu class GST consisting of two type one subunits (rGST M1-1) reveals a lock-and-key motif and a mixedcharge cluster at the subunit interface. Previous investigations revealed the lock-andkey motif was not essential for dimerisation. It was therefore postulated that the mixed-charge cluster at the dimer interface is primarily responsible for subunit association. Statistical analyses of individual rGST M1-1 chains did not predict the presence of any charge clusters. This suggests that the mixed-charge cluster forms only upon dimerisation and reinforces the probability that quaternary structure stabilisation is a major role of the mixed-charge cluster. Arginine 81 (Arg-81), a structurally conserved residue in the GST family involved in the mixed-charge cluster, was mutated to alanine. Phenylalanine 56 (Phe-56), the ‘key’ residue in the lock-and-key motif, was mutated to serine. These changes were engineered to disrupt the mixed-charge cluster and the lock-and-key motif situated at the dimer interface of rGST M1-1. Sizing by gel filtration chromatography of the mutant GST identified that these engineered amino acids resulted in a stable monomeric protein (F56S/R81A rGST M1). The F56S/R81A rGST M1 displayed almost no catalytic activity, suggesting perturbations of the active site or substrate binding sites. Structural investigations of the monomer by far- and near-UV circular dichroism revealed a similar secondary structural content to the wild-type. However, the tryptophan fluorescence properties suggested the tryptophans were situated in more hydrophilic environments than in the wild-type. ANS binding studies indicated a large increase in the accessible hydrophobic surface area of the monomer. Ureainduced equilibrium unfolding of F56S/R81A rGST M1 follows a cooperative twostate unfolding model. The unfolding data indicates decreased conformational stability and a large increase in the solvent exposed surface area of the monomer. In conclusion, the mixed-charge cluster at the dimer interface of rGST M1-1 is essential for monomeric association, which subsequently contributes to catalytic activity of the dimer and the stabilities of individual rGST M1-1 subunits.

This thesis analyses the Australian approach to taxation of goodwill and related intangibles. It asks the questions: 'Is the current Australian approach to taxation of goodwill coherent?'; and 'Could a different approach minimise any distortions?' The thesis identifies the increasing importance of goodwill and other intangible property in a modern information-based economy. It identifies benchmarks for a 'good' tax system ??? such as efficiency, simplicity, and equity. It emphasises the criteria of simplicity and efficiency but includes other criteria and specifically considers the issue of alignment of accounting and legal concepts. It concludes that the current misalignment makes it difficult for the tax system to deal with goodwill coherently. The thesis criticises the treatment of goodwill under various Australian taxes, including stamp duty; Goods and Services Tax; taxation of capital gains; and income tax. It specifically considers the treatment of intangible sources of goodwill and their relationship with goodwill itself. The discussion of income tax pays particular attention to the role of goodwill and other intangibles in international transfer pricing. The thesis draws conclusions about the treatment of goodwill in Australia and whether the Australian approach meets the benchmarks established at the outset. The thesis demonstrates that the current Australian approach leads, inter alia, to tax avoidance. The current approach also offends a number of other criteria of a 'good' system. The thesis considers the UK tax treatment of intangibles held by resident companies and considers this model for Australia. It also considers the abandoned 'Tax Value Method' previously proposed for Australia. From this and other material, it suggests possible new directions and an alternative approach to taxing goodwill in Australia. These include a consistent and coherent definition of goodwill for tax that is compatible with law and accounting. The thesis also urges the development of a consistent approach to taxing goodwill at both the state and federal levels; and suggests greater reliance on the existence of goodwill as a means to establish jurisdiction to impose tax in international tax situations.

Nodularia spumigena is an estuarine cyanobacteria that produces the toxin nodularin. This toxic cyanobacteria is known to have caused death to domestic and wild animals and is recognised as dangerous to human health. N. spumigena causes harmful algal blooms in many parts of the world including Australia. The toxic solutes of N. spumigena are potentially dangerous when contact is made to contaminated water bodies or is ingested by primary consumers. In Australia blooms of N. spumigena are common in the Gippsland Lakes in South-eastern Victoria and cause socio - economic hardships to the local communities. This PhD investigates the toxic effects of N. spumigena and its solutes to a range of aquatic life. A method known as SPME - HPLC showed promise in environmental monitoring of N. spumigena toxins by measuring nodularin from water samples. Other research presented study into the lethal and sublethal effects of on an extract from N. spumigena to aquatic fauna. Resu lts showed the N. spumigena extract was not lethal to many aquatic fauna although zooplankton from the Gippsland Lakes showed mortality at environmental relevant levels. Biochemical studies focusing on animal detoxification and antioxidation enzymes and DNA integrity showed sublethal effects to the N. spumigena extract. Results presented in this thesis show that an extract of N. spumigena elicited detoxification and antioxidation responses in animals tested. Furthermore, the use of the COMET assay showed increased damage to DNA of animals tested. Results also showed that different organs in animals tested responded differently to the aqueous extract, suggesting mode of uptake maybe important in toxicosis. Further, feeding studies with N. spumigena help elucidate mode of uptake using enzyme response biomarkers. The overall results of this research provided an assessment of the toxic affects of N. spumigena on aquatic fauna with special reference to the Gippsland Lakes, Victoria, Australia.

No sabem quin nom serà emprat en la posteritat per a caracteritzar la nostra era però amb tota seguretat se la podria nomenar l'Era química, tal és el nombre de molècules sintetitzades per l'home que impregnen l'ambient que ens envolta en la nostra vida diària. Per això el nostre concepte de civilització moderna és inconcebible sense un entorn químic superimpost a l'entorn natural. Si la vida humana és millor gràcies a les nostres creacions químiques, o bé serà destruïda per elles, avui més que mai és una qüestió sense resposta (E. Pellegrino, 1976). Amb aquestes declaracions de Pellegrino, es posa de relleu que el propi home és capaç de provocar, a vegades inconscientment, canvis importants en el Medi Am bient, que alteren les repercussions que podríem conside rar "naturals"i els beneficis perseguits es converteixen moltes vegades en perjudicis. Entre ells, l'anomenada "contaminació ambiental" és la causa de trastorns sobre la nostra salut i origen de noves malalties. En aquesta Tesi s'estudien els efectes sobre l'organisme humà d'alguns d'aquestos agents contaminants, els nomenats electrofílics, que es troben abundantment repartits a l'atmosfera de les àrees industrials i urbanes, en molts ambients laborals, i sobre tot al fum del tabac. Aquestos compostos electrofílics són capaços, una vegada ingressats al nostre organisme, de formar enllaços covalents amb les cèl·lules i provocar en elles lesions irreversibles com a mutagènesi i carcinogènesi. Per agreujar més la situació, l'home disposa de sistemes enzimàtics capaços d'augmentar la toxicitat d'alguna d'aquestes substàncies, o inclus de convertir en tòxiques algunes que d'antuvi no ho eren.Sortosament el nostre organisme compta amb diferents mètodes de defensa contra aquestes agressions, entre els que s'hi troben els sistemes de metabolització que permeten eliminar aquestes substàncies el més ràpidament possible. Els compostos electrofílies segueixen majoritàriament la via metabòlica de la conjugació amb el glutation (GSH), mitjançant l'acció de les Glutation S-Transferasa (GST), la qual cosa priva la toxicitat d'aquests agents i facilita la seva excreció. Així doncs, aquest sistema Glutation-Glutation S-Transferasa té un paper clarament detoxificador i pro tector davant d'aquests compostos. El producte final d'aquesta conjugació és un tioèter inactiu, que s'elimina fàcilment per l'orina. Els tioèters seran, per tant, un reflex del grau d'exposició de l'organisme als compostos electrofílics però, a més, ens permetran diferenciar, a igualtat d'ex posició, la millor o pitjor capacitat de detoxificació de cada individu. De tot això es dedueix que, per a valorar la importància d'un contaminant, no és suficient quantificar la seva concentració ambiental, que és el que podríem nomenar "grau de contaminació externa", sinó que s'han de tenir en compte també les interaccions amb els sistemes biològics (metabolització) que poden modificar el compost inicial. D'aquestes interaccions s'obté el que coneixem com a "grau de contaminació interna" o real, que serà molt variable per a cada individu, i no necessàriament coincidirà amb la contaminació externa. Es per tot això que adquireixen tant de valor els no menats indicadors biològics, com a expressió molt fiable de la nomenada Exposició Interna. Els tioèters urinaris han de considerar-se com a indicadors biològics i incluir-se en els plans de monitorització de Salut Pública, com de fet ja passa a d'altres països. En primer lloc, caldrà una coordinació de les funcions de tots els professionals, com són: químics, fisiòlegs, farmacèutics, farmacòlegs, etc., sobrepassant inclus els límits de competències tradicionalment impostes. Existeixen diferents nivells d'actuació i distintes tasques per a tots, i només petits conflictes territorials i jurisdiccionals poden entorpir la bona coordinació d'aquests especialistes en el gran paper que els té reservat la Salut Pública. El farmacòleg, a part de la funció clàssica de descobrir i desenvolupar nous productes, ha de tenir també un paper preponderant en el control d'agents químics em prats en la ramaderia, agricultura i indústria. Ha de dependre d'ells també l'ensenyar i portar a terme treballs d'investigació bàsica, que permetin després, establir sistemes de vigilància i informació, a fi de què la utilització dels agents químics sigui racional. Per tant, allà on existeixi un problema d'interacció d'agents químics amb la vida, la presència del farmacòleg es fa indispensable i adquireix una gran significació social. (E. Pellegrino, 1976).

SCREENING FOR ANTIOXIDANT ACTIVITIES OF SEVERAL MEDICINAL PLANT EXTRACTS AND THEIR EFFECTS ON GLUTATHIONE-S-TRANSFERASE ACTIVITY ABSTRACT Sagdi&ccedil / oglu Celep, A. G&uuml / l&ccedil / in Ph.D., Department of Biochemistry Supervisor: Assoc. Prof. Nursen &Ccedil / oruh May 2005, 154 pages The consumption of fresh fruits, vegetables, and medicinal plants are known to be associated with a long life span and low incidence of oxidative stress related diseases such as Alzheimer&amp / #8217 / s, Parkinson&amp / #8217 / s, cancer, aging and cardiovascular diseases. Fitotherapeutic effects of medicinal plants is virtually attributable to their phenolic compounds with low cytotoxicity. In this study, plants used in Anatolian folk medicine for their effects such as antiinflammatory, antiulcer, antipyretic, fertility, analgesic and aphrodisiac, namely Aesculus hippocastanum L., Papaver bracteatum L., Urtica urens L., Gundelia tournefortii L., Prangos ferulacea L., Chaerophyllum macropodum Boiss., Heracleum persicum Desf., Allium vineale L., Aconitum cochleare Woroschin, Rheum ribes L., Ferula rigidula DC., Rosa heckeliana Tratt, were screened for their antioxidative effects. Antioxidant characteristics of the specified plants were studied using lipid peroxidation inhibiton and DPPH radical scavenging methods. Total phenolics content and their effects on glutathione-S-transferase activity of the plants were further investigated. Rheum ribes L, Ferula rigidula DC, Rosa heckeliana Tratt., Prangos ferulacea L. were found to be very effective antioxidants and also effective inhibitors for glutathione-S-transferase activities among the plants. Rosa heckeliana Tratt. root extracts exhibited very high total phenolics content (0.7 mg/mg of extract) and antioxidant activity with IC50 values of 11.2 &micro / g/mL and 5.1 &micro / g/mL for DPPH scavenging and lipid peroxidation inhibition, respectively. Ferula rigidula DC was identified as the most potent inhibitor for glutathione-S-transferase activity, with IC50 values of 49 &micro / g/mL.

This thesis analyses the Australian approach to taxation of goodwill and related intangibles. It asks the questions: 'Is the current Australian approach to taxation of goodwill coherent?'; and 'Could a different approach minimise any distortions?' The thesis identifies the increasing importance of goodwill and other intangible property in a modern information-based economy. It identifies benchmarks for a 'good' tax system ??? such as efficiency, simplicity, and equity. It emphasises the criteria of simplicity and efficiency but includes other criteria and specifically considers the issue of alignment of accounting and legal concepts. It concludes that the current misalignment makes it difficult for the tax system to deal with goodwill coherently. The thesis criticises the treatment of goodwill under various Australian taxes, including stamp duty; Goods and Services Tax; taxation of capital gains; and income tax. It specifically considers the treatment of intangible sources of goodwill and their relationship with goodwill itself. The discussion of income tax pays particular attention to the role of goodwill and other intangibles in international transfer pricing. The thesis draws conclusions about the treatment of goodwill in Australia and whether the Australian approach meets the benchmarks established at the outset. The thesis demonstrates that the current Australian approach leads, inter alia, to tax avoidance. The current approach also offends a number of other criteria of a 'good' system. The thesis considers the UK tax treatment of intangibles held by resident companies and considers this model for Australia. It also considers the abandoned 'Tax Value Method' previously proposed for Australia. From this and other material, it suggests possible new directions and an alternative approach to taxing goodwill in Australia. These include a consistent and coherent definition of goodwill for tax that is compatible with law and accounting. The thesis also urges the development of a consistent approach to taxing goodwill at both the state and federal levels; and suggests greater reliance on the existence of goodwill as a means to establish jurisdiction to impose tax in international tax situations.

Philosophiae Doctor - PhD / Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.

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No description available.

CLIC1 is an intracellular membrane protein that is unusual in that it can exist in both a soluble and an integral membrane form. The manner in which this protein inserts into membranes is unknown although it is proposed to undergo a change in structure whereby it initially experiences a degree of unfolding and then refolds into its new membrane-bound conformation. This study focuses on the characterisation of CLIC1 in terms of its secondary, tertiary and quaternary structure, the determination of its conformational stability at equilibrium and the establishment of its unfolding kinetics, all under conditions of varying pH, polarity, redox conditions, temperature and ionic strength. CLIC1 was found to be most stable at pH 7.0 / 20oC. The unfolding process is two-state and cooperative, producing a DG(H2O) of ~10 kcal/mol and a m-value of ~2 kcal/mol per molar urea. A decrease in pH to 5.5 or an increase in temperature to 37oC resulted in the stabilisation of an equilibrium intermediate species under mild denaturing conditions and a destabilisation of the native state. This was further evidenced by an increase in the rate of unfolding of CLIC1 from the native state to the denatured state under these conditions. A state with similar properties to the intermediate species was detected in the absence of urea at pH 5.5 / 37oC and under non-reducing conditions at both pH 7.0 / 20oC and pH 5.5 / 20oC. The intermediate species is more hydrophobic than either the native or denatured state; it is stabilised by salts, has a reduced secondary structure, increased flexibility and a buried Trp35 relative to the native state. The rate of formation of the intermediate species is a slow process which may involve an oligomerisation step. The results from this study provide an interpretation for the structure and mechanism of CLIC1 pore formation in vivo by comparing the effects of the environment on the structure and stability of the protein.