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Characterisation of Newly Discovered Human Polyomaviruses WU and KI

Seweryn Bialasiewicz — 2010-11-22T00:00:00Z

Background: Until recently, only two polyomaviruses, JC virus (JCV) and BK virus (BKV), were known to commonly infect humans. In 2007, two new human polyomaviruses, KI polyomavirus (KIV), and WU polyomavirus (WUV) were discovered in respiratory samples of children suffering from acute respiratory tract disease. Due to their relative novelty, many details about KIV and WUV’s viral infectious cycles, pathobiology, and epidemiology could only be assumed by parallel comparison with other polyomaviruses. Thus, further studies were needed to elucidate the epidemiology, and pathogenesis of KIV and WUV. Methods: Epidemiology: Nearly 3000 respiratory samples collected over an entire year were screened for WUV and KIV by PCR, along with 8 other common respiratory viruses. An encapsidation assay was used to determine if whole viral particles were present in the respiratory tract of patients. Detection methods: Two KIV and six WUV real-time PCR (rtPCR) assays were designed and evaluated against a panel of other respiratory and blood pathogens, and clinical samples to establish sensitivity and specificity. Plasmid controls were created to establish analytical sensitivity. Tissue tropism: Collections of blood, urine, upper and lower respiratory tract secretions, faeces, and cerebrospinal fluid from both immunocompetent and immunocompromised adults and children were screened for WUV, KIV, JCV and BKV, and detection rates were compared. Representative WUV and KIV isolates had their VP1 and non-coding control regions (NCCR) sequenced, and analysed for changes respective to their site of detection. Transcription factor binding sites (TFBS) were predicted and compared to those of JCV and BKV. Global WUV diversity and genotyping: Forty nine WUV isolates collected across four continents had their genomes fully sequenced and analysed for levels of diversity, existence of genotypes, impact on predicted protein functional sites and association with disease or patient demographics. A genotyping scheme was also evaluated for fidelity against the whole-genome phylogenetic tree. Results: KIV and WUV showed a 2.6% and 4.5% average annual prevalence, respectively, in the study population. Predominance of WUV (93%) and KIV (78.6%) detections were in patients under the age of five. There was no significant seasonal variation of KIV, or WUV. The level of co-infection of KIV or WUV with other respiratory viruses was 74.7% and 79.7%, respectively. Respiratory symptoms were indistinguishable from those of other common respiratory infections. The encapsidation assay demonstrated that WUV DNA was fully protected from digestion in a respiratory sample with high WUV load. Seven rtPCR assays did not cross react with unrelated organisms, and all could reliably detect 10 copies of genomic DNA equivalents per reaction, which was more 10x more sensitive than conventional methods. Compared to the conventional PCR assays, clinical sensitivity was 100% in one of the KI and two of the WUV rtPCR assays. Clinical specificity was higher than 95% in four of the assays; the calculations suffered from poor sensitivity in the gold standard however. WUV and KIV were predominantly detected in upper and lower respiratory tract specimens, and faeces from paediatric patients. JCV and BKV were primarily detected in blood, urine and faeces from adult patients. WUV and KIV NCCR/VP1 sequence similarity ranged from 99.5-100% and 97.5-100%, respectively. No substantial difference was noted between the TFBS of JCV, BKV and WUV and KIV. Three main WUV genotypes and five subtypes were identified, provisionally termed Ia, Ib, Ic, II, IIIa and IIIb. Overall nucleotide variation was low (0-1.2%), however individual protein sequence variation between subtypes was as high as 3.0%, with VP1 being the most variable gene, indicating a possibility of multiple serotypes. A general geographical association was also noted. The discriminatory power of the previous VP2 fragment typing method was found to be limited and a new larger genotyping region within the VP1 was proposed. Conclusions: Both of the newly-discovered human polyomaviruses are frequently present in the respiratory samples of children suffering from upper and lower respiratory tract disease. The detection peaks for both WUV and KIV indicates an early age of primary exposure followed by reactivation in older age and under immunosuppression, similar to that seen in JCV and BKV. Clinical symptoms were indistinguishable from other respiratory infections, and the high rate of co-infections confounded any direct association with disease, however evidence of active WUV replication was observed. Multiple real-time PCR assays were developed which were sensitive and specific for use in the detection of WUV and KIV. Both novel viruses have a more restricted tissue tropism range in comparison to JCV and BKV, but VP1/NCCR sequences alone do not appear to offer suggestions as to why. The first extensive genomic survey of global WUV diversity revealed multiple genotypes which were more associated with geo-ethnicity rather than clinical disease. Overall, this series of studies has contributed substantial knowledge to the epidemiology and pathobiology of WUV and KIV, and has created validated tools to facilitate further studies into both of these viruses.

Characterisation of novel Australian rhabdoviruses isolated from vertebrates and insects

Aneta Gubala — 2010-02-03T00:00:00Z

As an outcome of very active arbovirus monitoring programs that began in Australia in the 1950s, some of the most diverse and unusual rhabdoviruses in the world have been isolated from this continent. These novel rhabdoviruses represent an important and valuable pool of highly diverse viruses; however, most of them have remained poorly characterised. In light of the significant disease potential of numerous rhabdoviruses, the characterisation of novel rhabdoviruses is indispensable for threat assessment to livestock, wildlife and humans and preparedness for outbreaks. The genetic characterisation of novel viruses is also an essential step for the development of molecular detection assays for improved monitoring and investigations into unidentified disease cases. In this study, the complete genomes of four novel rhabdoviruses have been sequenced and a fifth is close to completion. The substantial new data generated has significantly extended the understanding of the biology and evolution of the Rhabdoviridae. Wongabel virus (WONV), isolated from the biting midge Culicoides austropalpalis, was found to contain a unique genome structure encoding ten genes, including five novel genes (Chapter 2). Analysis by western blotting suggested that four out of the five novel genes were expressed in infected cell cultures. Ngaingan virus (NGAV), isolated from Culicoides brevitarsis, was found to have the largest genome of any rhabdovirus sequenced to date, and with thirteen genes has the largest number of genes of any (-) ssRNA virus sequenced to date (Chapter 3). Seven of the thirteen genes are novel. Similar to viruses in the genus Ephemerovirus (bovine ephemeral fever virus and Adelaide River virus), NGAV contains a second glycoprotein with an unknown function. Phylogenetic analysis places this virus alongside WONV and the north-American bird and mosquito-associated Flanders virus within the Hart Park group that remains to be classified by the ICTV. Screening of various wildlife and livestock sera collected in northern Australia indicated a strong association of NGAV with macropods. Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle and Culicoides brevitarsis (TIBV). Past serological surveys reported both viruses to be highly prevalent in cattle in northern Australia and demonstrated that the two viruses share a relatively close relationship at the antigenic level. The genomic analyses revealed that these two viruses have a unique genome organization, with three additional genes (Chapter 4). These additional genes are highly diverged at the sequence level but the encoded putative proteins share a significant conservation of secondary structure elements. The sequencing of these two related viruses has provided a unique opportunity to gain insights into the characteristics and evolution of novel proteins in two different rhabdoviruses. Phylogenetic analyses showed that TIBV and CPV form an independent cluster which does not appear to belong to any of the current genera, but which is most closely related to the genus Ephemerovirus based on N protein analysis. Although neither virus has been associated with disease, a serological survey of various animal sera collected in northern Australia showed that these viruses are currently highly prevalent in sentinel cattle and buffalo. Oak Vale virus (OVRV) was isolated from mosquitoes, Culex edwardsi and Ochlerotatus vigilax, from two geographically diverse regions of Australia located approximately 3000 km apart. The genome of OVRV was found to contain only one novel gene (Chapter 5). Comparatively, the genome of this virus is much less complex than the others in this study, but this virus displays considerable divergence from all other rhabdoviruses. A high seroprevalence for this virus was found in the feral pig population in northern Australia. The data generated from this study represents a considerable increase in the quantity of genetic data available for this viral family, and has revealed the existence of a large number of previously unidentified genes, highlighting that that the potential for complexity within the prototype genomic model of a rhabdovirus is much greater than previously thought. The novel nature of the additional genes provides grounds for further research into rhabdovirus evolution. Analysis of this new data suggests that these viruses cannot be classified into existing genera under the current criteria and it is clear that the taxonomy of the Rhabdoviridae requires revision. The observation that these viruses are currently circulating in livestock and wildlife in northern Australia accentuates the need for closer monitoring of animals and the need for further study of this diverse and fascinating group of viruses.

Characterisation of Novel Starch Materials: Structure-Functionality Relationship

Tan, Ihwa — 2008-11-21T00:00:00Z

Starch is an attractive raw material for biodegradable plastic applications due to its low cost, its availability in large quantities and its excellent thermal process-ability using conventional plastic processing equipments. Despite its attractive potential as a biopolymer material, the use of starch in biodegradable plastic applications is yet limited by its structural and functional properties, which are dictated by its genetic make up. This dissertation involves in-depth characterisations of a range of biotechnologically derived novel starches from different cereal sources to elucidate the relationship between starch structure and functionality. The importance of understanding starch structure-functionality relationship to further the development of starch biodegradable plastics are discussed to identify the research questions, which underlie the motivation of this dissertation and to contextualize the objectives of this dissertation. Diversities in starch macromolecular properties namely the amylose content and amylopectin chain length distribution are evident in these novel starches. The variation in amylopectin structure in these novel starches is explicable by considering the particular inhibition of starch biosynthesis gene expression in the generation of these starch mutants. Amylose content and amylopectin chain length distribution are two separate structural parameters in starch, which influence the granular and functional properties of starch. An improved method to analyse the 13C solid state NMR spectra for native starches was developed in this dissertation and provides the first elucidation on the occurrence of V-type polymorph, which is significant in high amylose starches. An increase in starch amylose content (or decrease in amylopectin content) leads to a decrease in the double helix content and crystallinity. A transition in the double helical packing arrangement of amylopectin side chains from A-type to B-type polymorph is noted for high amylose starches. This can be attributed to the changes in their amylopectin chain length distribution, which leads to the tendency of the glucan chains to form the B-type polymorph during crystallisation from thermodynamic considerations. The application of MTDSC provides the first elucidation on the step transition or heat capacity change, which is noted to occur within the gelatinisation endotherm for all starches. The use of Rheoscope, which allows for simultaneous monitoring of the changes in starch granular and rheological properties during gelatinisation, reveals that the manifested changes in viscosity can be attributed to the increase in the granules size as a result of swelling, the change in granules properties from rigid to more deformable granules due to water penetration and the increase in the viscosity of the continuous phase due to leaching of amylose. The variation in starch gelatinisation thermal properties namely the onset temperature, enthalpy and heat capacity change can be attributed to the variation in amylopectin chain length distribution, amylose content and the amount of starch structural order. A reduction in swelling power with increasing amylose content is consistently noted for all starch types. The variation in starch rheological responses during gelatinisation can be mainly attributed to the swelling ability of starch granules and their granule size distribution (to a lesser extent). Further MTDSC investigations on starch gelatinisation in the presence of water and glycerol with different concentrations indicate that plasticisation of starch granules prior to gelatinisation does not occur. The observed mid-temperature of the step transition (heat capacity change) is more likely due to a change in state of the starch macromolecules from being highly restrained within the granular packing to entangled macromolecules (as the order to disorder transition occurs) rather than due to glass transition. The addition of glycerol promotes starch gelatinisation in a similar way as the addition of water, which suggests that the same structural changes occur during gelatinisation regardless of the solvents used. In summary, the following starch structure-functionality relationships are deduced. The variation in starch macromolecular properties can be attributed to their corresponding mutation of starch biosynthetic genes expression. The variation in starch amylose content affects the extent of structural order inside the granules while the double helix packing arrangement is influenced by the amylopectin chain length distribution. Starch gelatinisation thermal properties are mainly influenced by the amylopectin chain length distribution while the swelling power and rheological properties are mainly affected by the amylose content.

Characterisation of photo-oxo-degradable polyethylene films

Yu-Chieh Hsu — 2012-03-21T00:00:00Z

crop propagation films - a tool to enhance crop growing conditions by minimising the effects of climate fluctuation. It is an economical method to reduce the use of irrigation, pesticide, and fertiliser and to increase crop yield. However, the removal of non-degradable PE films is laborious and the remaining films become a waste problem. A potential solution, such as photo-oxo-degradable PE films, is available but their rate of degradation is not well controlled and would not be suitable for varying crop cycle lengths and farm sites. PE degradation is affected by many factors - the primary structure of PE, type and concentration of pro-degradants used, film processing method and processing conditions, cold drawing, and ageing method (accelerated or natural ageing). One of the key tasks of developing a time-controlled PE degradable film is to understand how various factors play a role in the rate of PE degradation and to understand the change in film properties as the film becomes brittle - termed the embrittlement point. This is the critical feature for crop propagation films where PE transitions from being ductile to brittle, allowing the crop to penetrate and grow through the film. This project identified the dominant factors that influence the degradability of PE - by studying a commercial linear low density PE (LLDPE) grade, Dowlex 2045G catalysed by a Ziegler-Natta catalyst (1-octene/ethylene co-polymer) and a commercial pro-degradant, Ampacet 30091, an iron-stearate based pro-degradant. The Dowlex-Ampacet system was compared with an Elite-Ampacet system to understand the effect of short-chain branching distribution (SCBD), where Elite 5400, is also a 1-octene/ethylene LLDPE catalysed by a metallocene catalyst but with a narrower SCBD than Dowlex. The effects of processing method and cold drawing were also studied. Films were cast extruded and aged in an accelerated ultraviolet light device, QUV, until embrittled. Films were characterised at various stages of degradation by evaluating their mechanical properties, molecular mass, oxidative products, crystal morphology and surface properties. From these results, a conceptual model was developed to understand how PE degraded and became brittle. In a semi-crystalline system, it is well established that the amorphous phase and tie chain molecules are the most susceptible to photo-oxo-degradation, compared to the crystalline phase that is too dense for oxygen diffusion. When PE with added pro-degradant was exposed to UV, free radicals were generated from the decomposition of hydroperoxides initiating chain scission reactions, along with some crosslinking and oxidation. Over time, small chain fragments formed from chain scission diffused together, where this process is known as recrystallisation. Results demonstrated that film embrittlement had little correlation with its oxidative products (as indicated by the carbonyl index calculated from infrared spectroscopy spectra) but correlated well with its crystal morphological properties (from differential scanning calorimetry). Further studies via small angle and wide angle X-ray diffraction showed the inter-lamellar distance decreased significantly as the system became denser, with the critical inter-lamellar spacing reaching about 5 to 6 nm when the film became brittle, regardless of Ampacet pro-degradant concentration. This result correlated well with the changes in double yield points seen in the tensile data, where the absence of the second yield point signified that the tie molecules at the lamellar interface underwent chain scission and could no longer transfer the tensile stress to reach c-axis slip of the lamellar crystals. The key factor that influenced the rate of PE degradation was Ampacet, followed by the initial degree of crystallinity, crystal morphology, chain mobility and regularity of the small chain fragments (formed from chain scission). The main factors affecting the crystal morphology were co-monomer concentration, SCBD, processing method, and cold drawing. This fundamental study on a commercial grade of PE with Ampacet pro-degradant has allowed to gain a better understanding of PE photo-oxo-degradation. This knowledge could assist to design a time-controlled crop propagation PE film suitable for varying crop cycle lengths and for different crop sites across Australia. Not only that, the knowledge gained from this project could be transferred to develop other degradable PE film products such as plastic bags, magazine wraps, and other packaging products.

Characterisation of polarised macrophages and their influence on hepatocyte phenotype and function

Walker, Gene — 2012-07-27T00:00:00Z

Characterisation of samples of ore particles using x-ray micro-tomography

Cakici, Murat — 2010-04-14T00:00:00Z

The degree of mineral liberation is important for the efficiency of subsequent physical separation processes such as froth flotation. Mineral liberation studies involve determining the volumetric abundance or volumetric grade distribution of a specific mineralogical phase in a particular mineral. Currently, methodologies for assessing mineral liberation are laborious regarding sample preparation, analysis time (from weeks to months), and the need for stereological correction. These constraints can be eliminated by using X-ray CT which gives the cross-sections directly from three-dimensional data in shorter time (from ten minutes to hours) with minimal sample preparation. X-ray computed tomography (CT) is a non-destructive technique which allows three-dimensional visualisation of inner structures of an object based on the variations in density and atomic composition. Initially, it was developed as a medical tool for imaging soft tissue and bone. During the last decade, the number of X-ray CT applications in engineering and geology has steadily increased, with the improvements in performance and imaging capabilities. The aim of the present work is to apply X-ray CT technique for finely divided ore samples and to study the relationship between mineral liberation and CT results. Four different ore types were used in this study: Northparkes ore (Australia), Ernest Henry ore (Australia), Keetac ore (USA) and Cannington ore (Australia). Different settings of the desktop X-ray CT technique were applied for each particular ore sample for several ore liberation (particle size distribution) properties. Two dimensional CT images were reconstructed from the three-dimensional X-ray CT data. It was found that the settings for CT technique were a function of the ore type. Particularly in the case of Cannington (high density ore) the best setting conditions split from the rest of the ores tested. The appearance of different artifacts occurring during the analysis were studied and kept to the minimum. A functionality between mineral liberation and CT results was found. The variables affecting the most the results were the Voltage and Minimum Intensity Percentage. Contrary to the expected trends, variables having a negligible effect on the results were found to be exposure time / equivalent Al filter thickness.

Characterisation of shale ash packed-column biofilm reactor for shale wastewater treatment

Ho, Ngai Man — 2013-02-05T09:43:18Z

Characterisation of the epidemiology and molecular biology of Koala retrovirus (KoRV)

Tarlinton, R. E. — 2008-11-21T00:00:00Z

Gammaretroviruses are known to cause leukaemia, lymphoma and immunosuppression in many species and the original isolation of KoRV (koala retrovirus) (Hanger et al 2000) was made as part of a study into the cause of the high rate of leukaemia and lymphoma in koalas. The virus however displayed some unusual features. While classified as an endogenous (inherited) gammaretrovirus it was unusually active. While endogenous viruses are very common with up to 8% of the human genome consisting of retroviral material they are usually mutated and inactive (Gifford and Tristem 2002). KoRV possessed a full length replication competent genome, was actively transcribed and produced viral particles. KoRV was also very closely related to the exogenous (horizontally transmitted) retrovirus of Gibbons (Gibbon ape leukaemia virus or GaLV). Normally closely related viruses are found in closely related species. This unusual similarity between KoRV and GaLV raised the possibility of a recent host species jump (Hanger et al 2000). This thesis makes a case for the recent introduction of KoRV into the koala genome and ongoing endogenisation into the koala genome. KoRV is demonstrated definitively as an endogenous virus but present at a mixed prevalence within the Australian koala population. The thesis also further characterizes KoRVs activity and establishes an association between KoRV and disease in koalas. It is difficult to prove that a virus present in all animals in a population (as is the case for most endogenous viruses) is the cause of a particular disease syndrome. Here real time PCR was used to quantify the level of viraemia and proviral DNA load in the blood of individuals. A significant association between high viral load and leukaemia and lymphoma was demonstrated. This was further confirmed in a follow up study of a group of animals where those with a high viral RNA level were at a greater risk of dying from neoplasia. The real time PCR study also indicated that animals suffering from clinical chlamydiosis had higher viral RNA levels than their healthy counterparts though this was not significant. This disease association was further strengthened when KoRV free populations were identified and were shown to have lower incidences of these diseases. The real time PCR studies demonstrated a considerable variation in the proviral copy number in individual animals. With most endogenous retroviruses the proviral copy number is fixed within the species or population (Boeke and Stoye 1997). Southern blotting was used to confirm this variation in copy number and also demonstrated variation in the pattern of these proviral inserts between unrelated animals. The random insertion pattern of KoRV provirus loci was further confirmed using cytogenetics. Sequencing of the KoRV envelope gene revealed marked variation in sequence within individual animals. These sequences despite considerable mutation in some cases were all potentially functional and indicate positive selection pressure for active virus within the individual animals. Inverse PCR was used to identify the koala genomic sequence interrupted by the proviral loci. While koala genomic sequence was successfully amplified no potential oncogenes were found. This study did however demonstrate the presence of truncated KoRV sequences missing a large part of the gagpro- pol gene. Electron microscopy also demonstrated KoRV viral particles in the bone marrow of a leukaemic animal. KoRV had originally been classified as an endogenous virus based on the fact that it was present in all animals tested and in all tissues of individual animals. However the accepted definition of an endogenous virus is one that is present in the germ line DNA and is inherited over several generations (Boeke and Stoye 1997). Single cell PCR of koala sperm was used to demonstrate that KoRV is present in germ line DNA. This was confirmed using fluorescent in situ hybridization (FISH) techniques. Southern blotting of blood obtained from a group of related captive animals showed the inheritance of proviral loci over several generations. To determine whether all koalas carried KoRV loci, samples from geographically diverse populations were obtained. As expected all animals in Queensland were positive for KoRV using PCR techniques. Unexpectedly animals from Kangaroo Island in South Australia were free of KoRV. Samples obtained from Victorian animals demonstrated a mixed KoRV status. The animals on Kangaroo Island have been isolated from other populations since the 1920s and this raises the possibility that KoRV has integrated into the mainland koala population during the last 100 years. This is unprecedented for an endogenous virus with the most recent estimate for integration in other species being the type C viruses of pigs 5000 years ago (Mang et al 2001). The mixed prevalence of KoRV and its high activity indicate that this virus is still undergoing the process of endogenisation and provides a unique opportunity to study this process in a wild animal population.

Characterisation of the epidermal differentiation complex (EDC) on mouse chromosome 3 and human chromosome 1q21 : characterisation of Flg-2, a novel epidermal protein that is abundantly expressed in mammalian epidermis and functionally related to filaggr

Listwan, Pawel — 2007-08-24T00:00:00Z

Characterisation of the G3BP family of proteins

French, Juliet Danielle. — 2007-08-24T00:00:00Z

Characterisation of the Host Immune Response in Cystic Fibrosis Mice

Palmer, James — 2008-11-21T00:00:00Z

Lung disease is the major cause of mortality in cystic fibrosis patients. Lack of functional cystic fibrosis conductance transmembrane conductance regulator (CFTR) in airway epithelia alters the environment of the lung, predisposing patients to chronic infections predominated by the opportunistic pathogen Pseudomonas aeruginosa. Such infections cause an exaggerated host inflammatory response characterised by a sustained neutrophilic influx which ultimately destroys the lung tissue resulting in respiratory failure. In addition to the inability for CF patients to effectively clear airway infections evidence exists suggesting an innate defect in inflammatory signalling pathways due to a lack of CFTR, priming the CF lung for a heightened state of inflammation even in the absence of detectable infection. In order to develop effective strategies for dealing with CF lung disease a greater understanding is required of the dysregulation in the molecular signalling pathways that leads to the uncontrolled state of inflammation in the CF lung. The G551D CF mouse previously generated in our laboratory exhibits characteristic CF phenotypes including altered chloride conduction in the airways, reduced ability for bacterial clearance and a dysregulated lung inflammatory response upon bacterial challenge. The work presented in this study further characterises the response in these animals upon stimulation with an intratracheal dose of bacterial lipopolysaccharide (LPS). This treatment regime was sufficient to initiate an acute inflammatory response in the lungs of both WT and CF mice, from which animals of both genotypes routinely recovered. G551D animals consistently lost a greater amount of weight than their WT counterparts and exhibited enhanced recruitment of inflammatory cells, predominantly neutrophils, to the lungs. To profile transcriptional responses occurring in the lung after exposure to inflammatory stimulus both CF and WT animals were treated with LPS and sacrificed six hours post-treatment. Six hours marks a point where no discernable difference existed between the two genotypes, as measured by cellular infiltrate and cytokine levels in the airways. cDNA and oligonucleotide microarrays were utilised to determine relative differences in transcriptional activity between CF and WT mice. A series of comparisons was performed to incorporate potential differences in gene expression levels between the genotypes in the absence of stimulus. A robust transcriptional response was detected in both WT and CF animals in response to LPS. The majority of genes were similarly regulated in both genotypes, leaving overall levels of transcript comparable after stimulus. Similarly, few differences were detected between CF and WT mice not exposed to LPS. Analysis of microarrays directly comparing transcript levels in the lungs of WT and CF mice exposed to LPS revealed a number of genes expressed at lower levels in the CF samples which shared the common feature of being known targets of interferon signalling. Independent validation of a series of molecules integral to interferon beta signalling confirmed genes downstream of interferon beta, namely Stat1, Irf7 and Tap2, were consistently expressed at lower levels in the CF lung compared to WT after exposure to LPS. While all three of these genes were transcriptionally upregulated by LPS in both genotypes the response was diminished in the CF lung. Whether or not this perturbation in signalling stems from a deficiency in production of interferon beta itself or is a result of impaired downstream events remains to be elucidated. Nevertheless, with increasing evidence promoting interferon beta as an important molecule in inflammatory regulation the data presented in this thesis suggests this as a worthy point of intervention in managing the persistent deleterious effects associated with CF lung disease.

Characterisation of the immunopathology associated with cerebral malaria

Louise Randall — 2009-03-02T00:00:00Z

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children in sub-Saharan Africa. Patients with CM are comatose and often convulse, develop retinal haemorrhages and motor abnormalities. Recent histological studies on brain tissue obtained from patients who have died from CM have identified heterogeneity in brain pathology. As a result, CM is considered to be a complex disease that may be comprised of a number of syndromes. Patients admitted to hospital with CM are treated with anti-malaria drugs; however, even in the best equipped hospitals, a large number of CM patients die within the first 24-hours following hospital admission before the anti-malarial treatment can have an effect. For this reason, it is critical that the mechanisms leading to CM are elucidated in order to develop effective adjunct therapies. Experimental cerebral malaria (ECM) caused by P. berghei ANKA (PbA) infection of susceptible mice displays many features of human CM. A key feature of this model is the pivotal role of the host immune response in pathogenesis, particularly the involvement of T cells. Evidence, predominantly from ECM studies, suggests that tumour necrosis factor (TNF) superfamily (TNFSF) members play critical roles in the immunopathology associated with CM. The first hypothesis investigated in this thesis was that key immune response pathways contribute to the development of CM and, despite the heterogeneity observed between CM patients, common pathways exist that may be targeted to prevent CM. The second hypothesis tested was that members of the TNF superfamily modulate the immune response to infection and are involved in the development of pathology observed in severe malaria (SM). In order to investigate the above hypotheses, three projects were carried out. First, we examined the great heterogeneity in brain expression profiles between ECM-susceptible CBA/CaH (CBA) and C57BL/6 (B6) mice at the peak of disease, as well as the significant differences in circulating cytokine expression and expansion of microglia in brain tissue. We found that, despite these differences, common therapeutic and preventative strategies existed to disrupt the development of ECM in the two ECM-susceptible mouse strains. Second, studies in ECM mice have identified T cells and TNFSF members, TNF and lymphotoxin (LT)-a, as critical mediators of ECM pathology. We extend these studies to examine the role of the TNFSF member LIGHT in ECM. Specific blockade of LIGHT signalling through its receptor, LTβR, in PbA-infected B6 mice abrogated the hallmark features of ECM brain pathology and improved the control of parasite growth. Importantly, specific blockade of LIGHT-LTβR signalling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process antigen during infection. Together, this study discovered a novel pathogenic role for LIGHT and LTβR in ECM and identified this TNF family receptor-ligand interaction as a potential target for therapeutic intervention in SM. Finally, we investigated the role of LTa in human SM and, more specifically, CM. We tested whether the polymorphisms within the gene encoding LTa (LTA) were associated with susceptibility to SM in Papuan Highland children and adults who had migrated from an area without malaria pressure to a region where malaria is endemic. Despite a lack of association between single nucleotide polymorphisms (SNPs) in the LTA/TNF locus and susceptibility to SM in Papuan Highland children and adults, we found a significant association between a SNP in the LTa-related gene encoding galactin-2 (LGALS2) and susceptibility to CM in children, but not adults in this study population. Interestingly, no association was found between this SNP and susceptibility to CM in Tanzanian children originating from and living in a malaria endemic region. These results suggest that there may be differences in the mechanisms leading to CM in adults and children, as well as between individuals from malaria endemic and non-endemic areas. Together, the findings outlined in this thesis are important to both the understanding of the underlying mechanisms leading to CM and to the development of improved interventions and adjunct therapies.

Characterisation of the mechanical properties of thin solid films

Liu, Sheng — 2013-01-15T15:19:52Z

Characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms

Hatfield, Jodie — 2008-11-21T00:00:00Z

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 gene participates in at least six cellular processes: telomere biogenesis and maintenance, packaging of nascent premRNAs, alternative splicing, mRNA stability, mRNA cytoplasmic trafficking and translation initiation. At the start of this project the mouse gene had not yet been described, however, the human gene was known to produce two protein isoforms, A2 and B1, but the functional boundaries between these two proteins were ill defined. The characterisation of the mouse hnRNP A2/B1 gene and its protein isoforms was, therefore, undertaken to facilitate research in mouse which would help to define functional differences between the protein isoforms. The mouse gene was found to be a 14 kb, single-copy gene located on chromosome 6 B3, comprised of 12 exons and 11 introns. This gene was regulated by a housekeeping-style promoter which was predicted to be affected by processes such as cell differentiation and maturation, cell cycle progression and stress responses. Transcription from this gene, and the human orthologue, gave rise to a number of RNAs that encoded four protein isoforms through alternative splicing of exons 2 and 9: B1, A2, B1b and A2b. In mouse, the expression and alternative splicing of the hnRNP A2/B1 transcripts were influenced by tissue-type and age. A subset of these transcripts also exhibited alterations to their 3 UTRs such as retention of the 11th intron, alternative polyadenylation and 3 UTR extension. The extended 3 UTR region, intron 11 and sequence flanking the alternatively spliced exons were highly conserved between mouse and human (over 90%) and were predicted to contain regulatory motifs that governed post-transcriptional modifications to the RNA messages. The mouse genome was also shown to contain at least six processed pseudogenes derived from the hnRNP A2/B1 gene, five of which were unlikely to be functional due to frequent variations from the real gene sequence, including insertions, deletions and rearrangements. The remaining pseudogene, pseudogene 1, could give rise to an independent source of A2 proteins and its status is yet to be determined. To date, it is unclear what role each of the four hnRNP A2/B1 protein isoforms play in the cell. It was proposed that protein isoforms with a unique functional role would exhibit differences in subcellular localisation and expression patterns. In order to test this hypothesis, a number of biological tools were developed to distinguish between the highly similar proteins. In addition to two rabbit polyclonal antibodies previously raised in our lab that recognised all four protein isoforms (HA2) and isoforms B1 and B1b (HB1), another four polyclonal antibodies were raised in rabbits and chickens. These antibodies were raised to recognise the following combinations: all four proteins (chicken HA2), A2 and A2b (chicken Hx1/3), B1 and A2 (rabbit Hx9) and A2b and B1b (rabbit Hx8/10). These antibodies were demonstrated to be suitable for Western analyses and immunostaining studies, although antibodies Hx1/3, Hx8/10 and chicken HA2 will require further purification to reduce nonspecific signals seen in immunostaining experiments. A number of fluorescently tagged fusion proteins were also engineered for each of the A2/B1 protein isoforms. Western analyses showed that in mouse tissues and a number of immortalised cell lines, A2 and B1 were the major hnRNP A-type proteins present. Their abundance varied between tissue- and cell-type, but both were constitutively expressed. In contrast, A2b was only present in younger mouse tissues and was absent from most immortalised cell lines, while B1b was not detected in the samples examined. Localisation studies showed that in the immortalised cell lines, HaCaT and PC12, the expressed protein isoforms and related fusion proteins localised to the cell nucleus but were excluded from the nucleoli and exhibited distinct perinucleolar staining. Little difference was observed between the localisation patterns of the protein isoforms A2 and B1 in these cell types. Interestingly in oligodendrocytes, a cell type known to carry out A2-dependent mRNA trafficking, B1 was localised to the nucleus, A2 was predominantly nuclear but also exhibited some cytoplasmic localisation and A2b was distributed almost evenly between the nucleus and cytoplasm (M. Maggipinto and R. Smith, personal communication). Hence, differences were observed in the localisation of these proteins but were cell-type dependent. In conclusion, the hnRNP A2/B1 splice variants and their protein products were differentially expressed temporally and spatially, suggesting that these highly similar protein isoforms were likely to possess unique functional roles. Future research using the biological tools developed and described in this thesis, will be directed towards further defining the different roles each of the protein isoforms may have in the cellular processes associated with the hnRNP A2/B1 gene. Lastly, the hnRNP A2/B1 gene belongs to a sub-family of hnRNP genes that have arisen through chromosomal duplication events and include hnRNPs A1 and A3. Comparisons of their gene structures and subsequent RT-PCR assays have revealed that, in addition to the two previously described splice variants of the hnRNP A3 gene, A3a and A3b, the eighth exon of the A3 transcript was also alternatively spliced in humans and gave rise to two new variants termed A3c and A3d.

Characterisation of White Patch Syndrome, a putative disease affecting corals in the genus Porites on the Great Barrier Reef

Davy, Joanne — 2008-11-21T00:00:00Z

Coral reefs around the globe are increasingly threatened by a number of natural and anthropogenic factors, and coral cover on many reefs has already been greatly reduced due to mortality associated with bleaching events and coral disease outbreaks. Understanding the potential effects of disease is, in most instances, hampered by a lack of knowledge. The pathogens causing the majority of coral diseases have yet to be identified and little is known about the effects of environmental factors on disease initiation and progression. In general, reefs in the Caribbean and Western Atlantic have suffered the greatest losses due to coral disease and the majority of research has focused on these regions. However, reports of disease outbreaks in the Indo-Pacific have increased in recent years and the need to understand the potential risks to reefs in this region is no less important. The aim of this research was to characterise a newly discovered disease-like state affecting corals in the genus Porites at Heron and Wistari reefs, at the southern end of the Great Barrier Reef (GBR). Results of the study revealed that this disease is dissimilar to other coral diseases in several respects. Named White Patch Syndrome (WPS), it manifests as discrete bleached foci on colonies of the common massive corals Porites australiensis, P. lobata and P. lutea. The results of a long-term monitoring programme of tagged corals indicated a discrete and dynamic (yet persistent) pattern of lesion formation that could suggest a chronic, recurring infection, but which importantly was not observed to result in colony mortality. The erratic pattern of lesion progression and regression was not linked to seawater temperature fluctuations, and colonies in close proximity to each other showed markedly different patterns of disease occurrence. Analysis of physiological variables, such as host protein content (i.e. tissue biomass), Symbiodinium density and Symbiodinium photosynthetic capacity was performed to clarify how the host and symbionts differed in the lesion tissue as compared to the healthy tissue surrounding it. Symbiont density was found to be reduced by approximately two thirds in lesion tissue when compared to the visibly healthy tissue, but symbiont cells remaining within WPS lesions photosynthesised efficiently and no host tissue loss was evident. Furthermore, there was no difference in the sub-clade of Symbiodinium sp. contained within lesion or visibly healthy tissue. Microscopic examination of diseased, visibly healthy and healthy control tissues, using a range of microscopic techniques, was carried out to further clarify if and how both partners in the symbiosis were affected by WPS. A bacterial role in the disease was not evident using histopathology or fluorescence in situ hybridisation (FISH). However, transmission electron microscopy (TEM) revealed the presence of virus-like particles (VLPs) in both visibly healthy and diseased tissue. TEM also revealed that both apoptosis and necrosis were occurring at a low level in both the host and symbiont. TEM analysis of VLPs associated with the coral surface microlayer (CSM) of visibly healthy and diseased tissues showed that while a single viral morphotype was present within the coral tissue more than 14 morphotypes were identifiable in the CSM. No discernible difference between the viral communities of the CSM of diseased and visibly healthy tissues was apparent, however. A methodology combining density gradient centrifugation and TEM was established to isolate the DNA from VLPs in the tissues of Porites corals. Preliminary phylogenetic analysis of the protein sequence of a fragment of the ribonucleoside reductase (small sub-unit) gene (rr2) indicated that the virus may be unique, but further characterisation is needed to confirm this. Important aspects of a previously undocumented disease affecting a major genus of reefbuilding coral on the GBR were determined through the course of this study. Most notably, the disease does not exhibit temporal changes that are related to seawater temperature, which is in contrast to the majority of other coral diseases, nor does it result in coral colony mortality. The discovery of a putative virus within tissues of Porites colonies affected by WPS could be indicative of a chronic viral infection, and this may or may not be related to the appearance of the bleached foci. The discovery of a coral tissue-associated VLP represents the first such discovery for wild corals. Further research is now required to complete the molecular characterisation of the Porites virus and to establish what role, if any, it plays in WPS.

Characterisation, optimisation and application of banana streak virus (BSV) promoters

Remans, Tony. — 2011-09-15T00:00:00Z

Characterising and Removing Dissolved Organic Nitrogen and Colour from Wastewater Containing Melanoidin

Jason Ian Dwyer — 2008-10-03T00:00:00Z

Characterising - (an) Education Policy

Heimans, Stephen — 2013-04-09T15:20:29Z

Characterising Crim1 in Vertebrate Development

Genevieve Kinna — 2009-11-29T00:00:00Z

This thesis investigates the role of Crim1, a transmembrane protein that is expressed in a number of areas in the vertebrate embryo including the developing kidney, eye, testis and spinal cord, which we believe may be a regulator of vertebrate tissue development. To dissect the function of Crim1 in normal mammalian development, two vertebrate models were used, zebrafish and mice. The results show that in zebrafish, crim1 is expressed early in development from the 16-cell stage through to 30 hours post fertilisation (Chapter 3). At 24 hours post fertilisation crim1 is expressed in the intermediate cell mass (icm), the site of haemangioblast development. Haemangioblasts are precursor cells that contribute to the formation of the blood and endothelial cell lineages. Injection of crim1 antisense oligonucleotides into zebrafish embryos (crim1 morphants) lead to an expansion of the icm and defects in the trunk, tail, somites and vasculature. The injection of crim1 antisense oligonucleotides into transgenic fli:GFP zebrafish revealed defects in the intersegmental, dorsal longitudinal anastomotic and parachordal vessels. Although crim1 is expressed during haemagiogensis the primary defect in the crim1 morphant zebrafish appears to be vascular. Further experiments used a ‘knock-in’ mouse, Crim1KST264, in which a loss of functional Crim1 leads to defects in limb (syndactyly), skeleton, eye, vascular, kidney and placental development. Analysis of the kidney phenotype in the embryonic Crim1KST264 homozygotes showed that a loss of Crim1 affects ERK1/2 and phosphorylated-Smad1/5/8 protein expression, although has no direct effect on BMP or TGFβ protein expression (Chapter 4). Analysis of the adult Crim1 outbred kidneys revealed they have albuminuria and leaky vasculature. The complex phenotype presented by the Crim1KST264 homozygote kidneys suggests Crim1 may be regulating multiple growth factor pathways. As Crim1 was shown to be expressed in the placenta, we characterised the role of Crim1 in placental development using the Crim1KST264 mouse (Chapter 5). Crim1KST264 homozygote placentas and embryos are smaller than their wild-type littermates. Our investigations revealed that Crim1 is expressed in trophoblast giant cells and in spongiotrophoblasts. A loss of Crim1 causes a developmental defect in that the junctional zone (region of the placenta containing spongiotrophoblasts and glycogen cells) is expanded, although this phenotype does not appear to be due to a defect in proliferation or apoptosis. Further analysis of E15.5 Crim1KST264 homozygote placentas revealed there was a reduction in the number of labyrinth trophoblast gaint cells. Thus, by using zebrafish and mouse as two model organisms of vertebrate development, this thesis has showed that Crim1 is clearly important for normal embryonic development. To dissect the complex phenotype presented by the Crim1KST264 mouse, further studies of Crim1 and its interaction with other growth factor pathways is needed to elucidate how and to what extent they interact with Crim1 to determine its biological effect on vertebrate tissue.

Characterising the Biophysical Properties of a Mangrove Forest to Inform Mosquito Control

Jonathan Mark Knight — 2008-06-06T00:00:00Z

Characterising the multi-component sources of subduction zone magmas: A geochemical study of submarine volcanoes and active spreading centres, Vanuatu

Zarah Heyworth — 2011-07-01T00:00:00Z

The currently accepted model for subduction zone magmatism involves metasomatism of the mantle wedge by slab-derived components and then subsequent partial melting of this source. However, there is no consensus on whether the slab-derived component is transferred as a hydrous fluid, a silicate melt, or both. Whether these fluids or melts are derived from the altered oceanic crust, the subducted sediment, or both is also debated. Constraining the nature of the subduction component has important implications for solid-fluid-melt partitioning and slab dehydration depths, particularly elemental budgets and recycling of elements into the deep mantle. Vanuatu is an intra-oceanic subduction zone in the SW Pacific where recent scientific cruises have mapped and dredged submarine lavas that extend the sample coverage of the Vanuatu arc. Major and trace elements and radiogenic isotopes are used to identify the source and transfer mechanism of slab-derived components in the Vanuatu subduction zone. Mixing calculations estimate the mantle wedge, sediment and fluid contributions to Vanuatu arc and backarc lavas. New geochemical results are integrated with the large database of published results for Vanuatu, to investigate the along- and across-arc variation in the slab component and mantle domains. Lavas from the Vanuatu backarc have Indian-like Pb isotope ratios, similar to the Indian-type mantle signatures of central arc volcanoes, but distinct from adjacent southern arc volcanoes with Pacific-like Pb isotope ratios. The Pb isotopic anomaly (∆8/4) in the Vanuatu subduction zone decreases from the central arc segment to the southern backarc, implying trench-parallel flow of Indian-type mantle to the south. The spatial distribution of mantle domains implies that mantle flow patterns in southern Vanuatu are dominated by southward trench-parallel flow, rather than pure corner flow induced by viscous drag along the upper surface of the subducting plate. This southward flow appears to be related to pressure gradients developed during asymmetric rollback of the Australian plate. Despite these unusual mantle dynamics in southern Vanuatu, a subduction component is detectable in the across-arc and backarc lavas. In the southern Vanuatu backarc, the average amount of sediment addition decreases from ~1.2% in the north trough to ~0.3% in the central trough. The average amount of altered oceanic crust fluid addition also decreases from ~0.3% in the northern trough to ~0.1% in the central trough. The southern backarc trough lavas are not enriched in large ion lithophile elements and have light rare earth element depletions, suggesting that these lavas do not have a significant contribution from slab-derived components. In the across-arc volcanoes, the average amount of sediment addition decreases from ~2% in the volcanic front to ~0.5% in the easternmost across-arc volcano. The average amount of altered oceanic crust fluid addition also decreases from ~0.9% in the volcanic front to ~0.3% in the easternmost across-arc volcano. For the backarc and across-arc lavas, sediment and fluid contributions progressively decrease with distance from the trench. Fluid-mobile/fluid-immobile trace element ratios, such as Ba/Yb and Ba/La ratios also decrease with distance from the trench and support progressive distillation of the subducting slab. Enrichment of fluid mobile elements in the southern across-arc volcanoes reflects the addition of hydrous fluids to slab depths of at least 200 km. These constraints on slab dehydration signify that not all fluid mobile elements are recycled at shallow depths. The degree of melting in the across-arc volcanoes progressively decreases with depth to the slab, demonstrating that the degree of melting is not solely controlled by the melt column height. Smaller degree melts from southern Vanuatu have a smaller contribution from slab-derived components. These constraints argue for fluid-fluxed melting, where the extent of melting progressively decreases as the amount of slab-derived fluid decreases. This positive wet melting function contrasts with that observed at mid ocean ridges, where increasing water content of the mantle source is associated with decreasing extents of melting.

Characteristics of Phonetics and Lexis of the Vietnamese language in Australia

Dao, Dich Muc — 2013-04-05T09:56:09Z

Characteristics of spinal manual therapy induced hypoalgesia

Souvlis, Tina. — 2007-08-24T00:00:00Z

Characterization and discovery of small RNAs in metazoa

Fernandez Valverde, Selene Lizbeth — 2013-01-11T11:09:23Z

Characterization and knockdown of zebrafish hnRNP A1

Mr Imam Cartealy — 2008-10-23T00:00:00Z

Characterization and SAR studies of Novel and Selected α- and μ- Conotoxins

Kalyana Bharati Akondi — 2012-02-22T00:00:00Z

Conotoxins are small disulfide rich peptides present in cone snail venom. They target a wide array of receptors and ion-channels and exhibit extensive pharmacological diversity. The huge biodiversity displayed by these venom peptides holds considerable promise for novel ligand discovery. Conotoxins targeting nicotinic acetylcholine receptors (nAChRs) and voltage gated sodium (Nav) channels have received particular interest in view of their research and therapeutic potential. This thesis specifically focuses on the synthesis and structural and functional characterization of novel peptides from the α- and μ-conotoxin families. The α-conotoxins target a wide array of nAChR subtypes whereas the μ-conotoxins are Nav channel blockers. Chapter 1 includes a general introduction of conotoxins and the current status of conotoxin research. All of the peptides in this thesis were synthesized using Boc or Fmoc solid phase peptide chemistry. A brief overview of solid phase peptide synthesis along with the materials and general methods used for conotoxin structural and functional characterization throughout this thesis are described in Chapter 2. The process of identifying novel conotoxin sequences has accelerated significantly with the advent of DNA-based technology and MS/MS techniques. Though many conotoxin primary sequences are available, their pharmacological targets and structures still remain to be characterized. This issue is partially addressed in Chapter 3, which focuses on the characterization of novel α-conotoxins. The α4/3 subfamily of conotoxins were specifically selected as only three peptides have been characterized in this interesting subfamily to date. The characterization of α4/3 conotoxins isolated from C. regius not only aided in expanding the repertoire of this subfamily but also identified α4/3 RegIA to be the most potent α9α10 targeting α-conotoxin known. In addition another conotoxin RegIIA was characterized, which exhibited the highest potency towards the α3β4 nAChR subtype in the α4/7 subfamily. Chapter 4 focuses on a novel α4/7 conotoxin LtIA, which lacks the conserved Ser-X-Pro motif in loop 1. Despite the lack of the conserved motif, LtIA was found to potently and selectively block the α3β2 nAChR. Structural characterization showed that the peptide was flexible with multiple conformations in solution. Engineering the conserved motif back into LtIA caused the nascent helix to stabilize indicating its importance in maintaining conotoxin structural integrity. Receptor mutagenesis studies revealed that LtIA establishes a unique set of interactions at the receptor binding site suggesting it targets a novel microsite in the nAChR. Structure activity relationship (SAR) studies on the analgesic α4/6 conotoxin AuIB using alanine scan mutagenesis were carried out in Chapter 5. The interest in this peptide heightened with the recent finding that α-conotoxin AuIB that targets the α3β4 nAChR also inhibits GABAB receptor induced N-type Ca2+ channel currents similar to the therapeutically important α-conotoxins Vc1.1 and RgIA. The affect of each mutation on conotoxin structure and function was examined in order to identify residues of structural and functional importance. Electrophysiological analysis identified Pro 6 and Phe 9 residues to be crucial for α3β4 subtype activity. NMR data indicated that replacement of the conserved Pro 6 residue destabilized the structural integrity of AuIB thus leading to a loss of activity. The Phe 9 to Ala mutation however, did not impact the conotoxin structure, but significantly lowered α3β4 subtype potency. This indicates that Phe 9 residue is involved in crucial toxin-α3β4 receptor binding site interactions. Interestingly, the selectivity profile of this mutant also shifted towards recognition of the α9α10 nAChR subtype. Finally, in Chapter 6, a truncation mutagenesis strategy was used to identify the minimum functional scaffold of the μ-conotoxin SIIIA. Previous studies have shown that loop two and loop three residues in SIIIA are important for maintaining potency at Nav channels. We demonstrated that loop 1 truncation yielded a structurally minimized SIIIA scaffold that retains potency equivalent to that of the native peptide. The study also showed that SIIIA 3-20 N5K D15A which has the neutral Asn residue in loop 1 replaced with a positively charged residue had a significant 13.7-fold increase in potency towards the rat Nav 1.2 subtype when compared to native SIIIA. Also, the truncation mutants with only a single residue in loop 1 (SIIIA 3-20 desN5,G6;D15A) were found to retain potency equal to that of the native peptide. Thus, this study generated a synthetic SIIIA analog with enhanced potency, in addition to identifying the minimum SIIIA scaffold required to maintain near native binding potency at rat Nav1.2 subtypes. The work in this thesis has contributed towards expanding the characterized conotoxin repertoire and bridging the gap between identified conotoxin sequences and those that have actually been structurally and pharmacologically characterized. It has advanced our understanding of structure activity relationships of selected novel and therapeutically significant α- and μ-conotoxins. In the process, several α-conotoxins showing the highest known nAChR subtype potencies have been characterized. The thesis also provides advanced SAR information on μ-conotoxin SIIIA and identified analogs exhibiting enhanced Nav channel potencies.

Characterization of Alu element expression and A-to-I RNA editing in mammals

Cattenoz, M. Pierre Barnabe — 2012-11-19T23:45:41Z

Characterization of a Newly Identified Human Rhinovirus: HRV-QPM

Mr Peter Mcerlean — 2008-11-21T00:00:00Z

Characterization of a reciprocal-like translocation involving 6q in a melanoma cell line

Ms Jackie Fung — 2009-04-29T00:00:00Z

Deletion of the long arm of chromosome 6 is one of the most common genetic alterations in human malignant melanoma. Recently, a reciprocal translocation between chromosomes 6q and 17p was detected in a melanoma cell line, UACC-930, using arm painting probes of 6p and 6q. Reciprocal translocation is seldom observed in solid tumors. Upon further characterization of the translocation marker using techniques such as Southern blotting, genomic library screening and DNA sequencing, a complex rearrangement including two inversions of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q21q22)(q22q27)t(6;17)(q27;p13)], was detected. An NCBI blast search revealed 3 genes being interrupted by the breakpoints: prenyl diphosphate synthase subunit 2 (PDSS2) at 6q21, Parkin at 6q27 and p53 at 17p13. Down-regulation of PDSS2 was commonly observed in 59/87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7/66, 10.6%, p<0.001), indicating the tumor-suppressive potential of PDSS2 in melanoma development. To characterize the function of PDSS2 in tumorigenesis, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was demonstrated by both in vitro and in vivo assays. The results showed that PDSS2 could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. PDSS2 is the first enzyme involved in the CoQ10 biosynthesis pathway. Other studies have demonstrated PDSS2 mutations can cause severe CoQ10 deficiency and markedly reduced ATP production because of respiratory chain dysfunction. Interestingly, proteomics analysis revealed 7 out of 11 identified proteins (HSPA8, GAPDHS, TPI1, HSPA5, PGK1, ENO1, and ATP5B) differentially expressed in PDSS2-overexpressing cells were related to energy metabolism. Further studies are required to determine how PDSS2 could alter the energy supply in tumor cells. Taken together, these results support the proposal that PDSS2 is a novel tumor suppressor gene which may play an important role in the development of malignant melanoma via altering tumor metabolism.

Characterization of glioma initiating cells and its role in radiation response

Lim, Yi Chieh — 2012-10-30T09:19:58Z

Characterization of hot-carrier induced degradation via small-signal characteristics in mosfets

Lau, Mei Po Mabel — 2007-08-24T17:51:41Z

Characterization of hot-carriers induced degradation in mosfets through gate capacitances measurement at room and cryogenic temperatures

Hsu, Clement Che Ta. — 2007-08-24T17:51:39Z

Characterization of interactions formed by the SNARE Syntaxin4 protein and the Sec/Munc protein Munc18c

Michelle Christie — 2010-11-22T00:00:00Z

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a family of proteins essential for vesicle fusion. SNAREs mediate fusion by the formation of the SNARE ternary complex whereby SNARE motifs of cognate SNARE proteins interact. Central to the work described in this thesis is the SNARE protein, Syntaxin (Sx). Most Sxs can exist in two different conformations: a fusion competent open conformation and a closed conformation that prevents the interaction of Syntaxin with its SNARE partners to form the ternary complex. SNARE proteins alone do not mediate vesicle fusion; many other proteins are involved in fusion regulation. The SM family of proteins are a group of regulatory proteins that regulate fusion, at least in part, via their interaction with Sxs. Several binding modes for the SM:Sx interaction have been reported: binding to Sx in the closed conformation; binding to the N-terminus of Sx; or binding to assembled SNARE ternary complexes. SM proteins act as negative regulators when bound to Sx in the closed conformation, whereas binding to the N-terminal 10 residues (the N-terminal peptide) of Sx is thought to facilitate SNARE complex formation. In the case of M18c and Sx4, M18c has been shown to be a positive regulator of fusion by up regulating SNARE complex assembly through its interaction with Sx4. A structure for M18c in complex with full length Sx4 has not been previously reported. Small angle scattering experiments were undertaken to obtain a model for this protein complex. Neutron contrast variation experiments were undertaken to determine the conformation of Sx4 whether open or closed, in the complex. As part of this work, I established a protocol for the expression and purification of deuterated Sx4 (DSx4). Sx4 obtained by this method was shown to be 87% deuterated by mass spectrometry analysis. Scattering experiments along with cross-linking studies were used to obtain a model for M18c:Sx4 complex. The model showed M18c bound to Sx4 in an open conformation. In contrast, M18a, the neuronal homolog of M18c has been shown to bind to its cognate Sx, Sx1a, when Sx1a is in a closed conformation. The M18c:Sx4 structure obtained here provides additional evidence to support a positive regulatory role in fusion for M18c. The SM:Sx complex is thought to be highly specific between cognate SM:Sx partners. However recent work on the crystal structure of M18c:Sx4 N-terminal peptide showed that the peptide binding pocket in SM proteins and the residues that interact with it on the Sx N-terminal peptide were highly conserved. Sequence analysis showed that M18a also had the conserved N-terminal peptide binding pocket. Thus M18a and M18c along with their cognate Syntaxin N-terminal peptides, Sx1a and Sx4 were used to investigate the specificity of the interaction using ITC and crystallization experiments. ITC experiments showed that M18c and M18a bind to both cognate and non-cognate Sx N-terminal peptides with micromolar affinity. The affinity of M18c to Sx N-terminal peptide was found to be slightly higher because M18c made more contacts with the peptide. I was also able to obtain crystals grown from M18c:Sx1a1-10 N-terminal peptide and M18a:Sx1a1-10 N-terminal peptide. Some of these crystals diffracted to 4.5 Å. Previous work had shown that Sx4 interacts with metal ions. Pulldown experiments were carried out to confirm the metal binding ability of Sx4 using metal chelated resins (Co2+ and Ni2+). These experiments showed that Sx4 did bind metal ions as previously described. Pulldown experiments to determine the region on Sx4 that binds metal ions showed that full length Sx4 and the SNARE motif bound metal ions. The SNARE motif is therefore likely to be the region that binds to metal chelated resin. However a Sx4 construct with 29 N-terminal residues deleted did not bind metal ions despite containing the SNARE motif. I suggest that Sx4 was in a closed conformation in this construct and that the conformation of Sx4 may play a role in the ability of Sx4 to bind metal ions. Pulldown experiments also showed that Sx1a, the neuronal counterpart of Sx4 did not bind metal ions. Further experiments will be required to confirm these observations. Overall I was able to obtain a structure for M18c:Sx4 full-length complex showing for the first time that an SM protein, M18c can interact with a Sx, (Sx4) in an open conformation. This is consistent with a positive role for M18c in fusion regulation. I was also able to show that the SM:Sx N-terminal peptide interaction does not contribute toward the specificity of SM:Sx interactions and that the SNARE motif of Sx4 interacts with metal ions. Collectively this work has added to the overall knowledge on the mechanism governing GLUT4 transport and sheds light on the interaction between SM:Sx proteins in general.

Characterization of mouse frizzled-3 gene and identification of human homolog in keratinocytes

Hung, Betsy Shih-Min — 2007-08-24T00:00:00Z

Characterization of ras isoform activation by ras guanine nucleotide exchange factors

Clyde-Smith, Jodi. — 2011-05-12T00:00:00Z

Characterization of Surface Properties and Attachment of Campylobacter jejuni and Campylobacter coli to Abiotic Surfaces

Vu Tuan Nguyen — 2012-03-29T00:00:00Z

Campylobacter jejuni and C. coli are among the leading causes of human foodborne diarrhoeal diseases. Attachment to food related surfaces have been shown to enhance the movement of foodborne pathogens through food systems to ultimately cause disease. The mechanisms of attachment of C. jejuni and C. coli to abiotic surfaces remain relatively poorly understood. In order to assist in understanding this attachment, this work aimed to investigate the influence of cell surface hydrophobicity (water contact angle), surface charge (zeta potential), prior planktonic (broth) or sessile (agar) mode of growth, environmental temperature, contact time and the presence of cell surface polysaccharides on attachment of 13 C. jejuni strains and 5 C. coli strains to three abiotic surfaces (stainless steel, glass and polyurethane). All strains had different abilities to attach to stainless steel and glass but did not attach to polyurethane with the exception of one strain. The number of cells attaching to stainless steel and glass significantly correlated with water contact angles (P≤0.007), but not with zeta potential (P≥0.507). Cells grown as planktonic and sessile culture generally differed significantly from each other with respect to hydrophobicity and attachment (P<0.05), but not with respect to surface charge (P>0.05). These results suggest that increased cell surface hydrophobicity, but not surface charge, may enhance attachment of C. jejuni and C. coli to abiotic surfaces, while previous mode of growth affects their surface properties and attachment in a strain-dependent manner. Six strains (five C. jejuni strains and one C. coli strain) showing distinct hydrophobicity and ability to attach to abiotic surfaces were found to attach to stainless steel at significantly (P<0.05) lower numbers at 4°C (~ 4 log cells/cm2) than at higher temperatures (~ 5 log cells/cm2 at 55°C) after 30 min of contact, while their probability of detachment significantly (P<0.05) decreased from ≥ 0.219 at 4°C to ≥ 0.111 at 25°C. At 4°C, the numbers of cells attaching to stainless steel increased from ~ 4 to ~ 5 log cell/cm2 while the probability of detachment significantly (P<0.05) decreased from 0.443 to 0.134 after 1 min to 240 min of contact. These results suggest that elevated temperatures and longer contact times may increase the level and strength of attachment of C. jejuni and C. coli to abiotic surfaces. Three out of these five C. jejuni strains attached differently to abiotic surfaces from each other. This data could be interpreted using the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory because these strains showed a significant difference (P<0.05) in contact angle measurement (CAM) data and subsequent acid-base (AB) interactions. However, XDLVO theory failed to explain the attachment of three Salmonella strains due to similar AB interactions, as established by CAM, between them. These findings indicate that the XDLVO theory could only predict bacterial attachment if differences in AB interactions between strains were apparent by CAM. However, CAM may not always reflect AB interactions exactly since microbial adhesion to solvents (MATS) revealed a significant difference (P<0.05) in AB interactions between the three Salmonella strains. The difference in the results from MATS and CAM should be therefore considered when predicting bacterial attachment using the XDLVO theory. Since surface polysaccharides including capsular polysaccharides (CPS) and lipooligosaccharides (LOS) are associated with cell surface hydrophobicity, four highly hydrophobic and adherent strains (three C. jejuni strains and one C. coli strain) were further characterized to investigate the role of CPS and LOS in cell surface hydrophobicity, surface charge, autoagglutination (AAG) activity and their attachment to stainless steel and glass. This was achieved by removal of CPS and truncation of LOS core oligosaccharides by inactivating the kpsE and waaF genes, respectively. There were no significant (P>0.05) differences between the three C. jejuni strains and their ∆kpsE and ∆waaF mutants with respect to all traits tested. However, inactivation of the kpsE gene significantly (P≤0.026) reduced the zeta potential of the C. coli strain from -10 to -6 mV and increased its AAG activity from 0.204 to 0.924 while disruption of the waaF gene significantly (P≤0.037) increased its water contact angles of > 8° and decreased numbers of cells attaching to stainless steel and glass of ~ 0.5 log/cm2. These results suggest that CPS and LOS may play a role in the cell surface properties and attachment to abiotic surfaces for C. coli but not C. jejuni. In conclusion, the initial attachment of C. jejuni and C. coli to food processing surfaces plays an important role in their contamination of food and transmission through food chain to infection. However, most previous studies have focused on their attachment to intestinal cells during infection rather than their initial attachment to abiotic surfaces. This work has made an original contribution to the field of food microbiology by, for the first time, investigating some bacterial surface and environmental factors associated with the attachment of C. jejuni and C. coli to abiotic surfaces commonly used in food processing. These results not only contribute towards a better understanding of the mechanisms of C. jejuni and C. coli attachment to abiotic surfaces but also provide a rationale for investigating the effect of other factors such as substratum properties, O-linked and N-linked glycans that are potentially involved in bacterial attachment. This knowledge is important with respect to food safety and public health because a better understanding of the mechanism of C. jejuni and C. coli attachment to abiotic surfaces may assist the development of anti-attachment interventions to ultimately reduce food contamination and human infections by these pathogens.

Characterization of Temperature Phased Anaerobic Digestion for Organic Solids Stabilization

Huoqing Ge — 2011-06-23T00:00:00Z

Modern wastewater treatment plants now produce more waste activated sludge, with low inherent treatability, due to a shift in focus towards nutrient removal rather than only sanitation. Food industry and abattoirs also produce significant volumes of waste solids. Organics in these solids streams can be readily converted to energy by anaerobic processes, which generate renewable methane. Conventional anaerobic processes require readily degradable substrates for the best outcomes. However, the degradability of long sludge-age activated sludge is normally poor, resulting in long digester hydraulic retention time (HRT), high mixing costs and poor methane production. To enhance the sludge degradability, incorporating a pre-treatment prior to conventional anaerobic digestion has been used and proven as a promising method. Since many long sludge-age activated sludge systems are small-medium scale (<5 dry tonnes solids produced per day), energy and capital intensive options such as thermal hydrolysis may not be viable in these applications. To specifically address the needs of smaller scale solids producers, this research focuses on enhanced performance with moderate thermal pre-treatment, as thermal energy is available for free when methane is converted to electricity. The process is named as “Temperature phased anaerobic digestion (TPAD)”, which consists of a biological pre-treatment digester operated at 50-70°C (2 days HRT) and a following mesophilic (35°C) anaerobic digester, which produces methane. Literature review identified that while improved performance has been observed, improvements have not been quantitatively characterised, and the mechanism by which improvement occurs has not been identified. Two two-stage lab-scale TPAD systems were set up and operated in parallel under various operating conditions with both primary and activated sludges as substrates. One was control, with the first stage at 35°C, while the other was the experiment, with the first stage operated at thermophilic conditions (50-70°C). The experimental system exhibited performance improvements of 10-30% additional VS destruction over the control system. The mechanism was identified by model based analysis, which showed thermophilic pre-treatment increased the degradation rate, but not ultimate extent for both primary and activated sludges. Methane production was also enhanced in the first stage of the thermophilic system, presumptively related to acetate oxidation. A novel two-stage batch test method was also developed and used to find the optimal temperature for the first stage. With a first stage temperature of 65°C, neutral pH and short retention time (1-2 days), the optimal TPAD performance was observed. A relative kinetics analysis using acclimatised inoculum identified that hydrolysis rates of cellulose followed the Arrhenius equation. Based on the results obtained, hydrolysis coefficients will at least triple from 0.2 d-1 to 0.6 d-1 (95% error of ~0.1 d-1), allowing substantial process intensification, positive energy balance, and mitigation of process risk through methanogenesis in the first stage. These results can be used directly by end-users through a set of design curves provided in this thesis.

Characterization of the Role of Peroxisome Proliferator Activator Receptor (PPAR)-Delta in Osteoclast Biology

Joao P. Marcal Fidalgo — 2011-06-02T00:00:00Z

Osteoclasts are the cells responsible for the resorption of bone and their activity is tightly controlled to maintain bone structure and calcium homeostasis. The elucidation of the function of known genes and the identification of novel genes involved with osteoclast biology are both critically important in the development of novel treatments for bone diseases. The objective of this project was to investigate the involvement of PPAR-delta in osteoclast formation and/or function using both in vitro and in vivo models through gene inactivation and gene overexpression/activation approaches. In the RAW/C4 osteoclast cell model, osteoclastogenic treatment with RANKL and CSF-1 induces upregulation of expression of PPAR-delta whereas expression of PPAR-alpha and PPAR-gamma subtypes is downregulated. The pattern of PPAR-delta expression during osteoclast differentiation is similar to typical osteoclast markers that normally show maximal expression levels at about day 5 of osteoclastogenesis. Treatment of cells with GW501516, a powerful and highly specific synthetic PPAR-delta agonist results in an increase in expression of all three osteoclast expression markers that tested: TRAP, NFATc1 and CTR. Over-expression of PPAR-delta in RANKL/CSF-1-stimulated RAW/C4 osteoclast cells markedly up-regulates the expression of osteoclast markers. NFATc1 is an early osteoclast differentiation marker whereas both TRAP and CTR are functional markers expressed at later stages so the impact of PPAR-delta over-expression is observed at both early and later stages of osteoclast differentiation. A moderate increase in the formation of giant multinucleated osteoclasts was detected in PPAR-delta over-expressing cells and also a further increase with GW501516 treatment. Even though PPAR-delta over-expression does not result in a significant increase in osteoclast numbers, an interesting observation was that it markedly induced TRAP enzyme activity in single nucleated cells that did not fuse to form the mature multinucleated osteoclasts, a potential indication of direct regulation by PPAR-delta. Using the Cre/LoxP recombination system, the expression of PPAR-delta in bone-marrow derived osteoclasts was knocked-down by 60% of normal values. Expression profiling of osteoclasts with reduced levels of PPAR-delta transcript revealed that TRAP, CTR and NFATc1 were downregulated in these cells. The PPAR-delta deficient osteoclasts also show reduced potential to differentiate into fully mature osteoclasts under suboptimal RANKL conditions. A mouse model was developed with a conditional knock-out of PPAR-delta specifically in osteoclasts and the resulting bone phenotype was characterized using histology, immunohistochemistry, X-ray, bone densitometry (DXA) and bone tomography (microCT) analysis. There were no skeletal differences in the PPAR-delta deficient animals when compared to normal control mice, and indication that PPAR-delta might not be essential for osteoclast differentiation/function under physiological conditions. The relative importance of the two PPAR-gamma isoforms in osteoclasts was investigated, by over-expressing them in RAW/C4 cells and assessing the effect on osteoclastogenic potential. PPAR-gamma1 expression is markedly upregulated during RANKL/CSF-1-stimulated differentiation whereas PPAR-gamma2 follows an opposite trend being markedly downregulated. Interestingly, a completely opposite expression profile for these two isoforms is obtained in adipocyte differentiation where PPAR-gamma2 showed to be the main molecular switch in that pathway whereas PPAR-gamma1 does not seem to be involved in that process. Over-expression of PPAR-gamma1, but not PPAR-gamma2, also stimulates TRAP expression and activity. Consistent with these results, PPAR-gamma1 overexpression clearly stimulates osteoclast formation, whereas PPAR-gamma2 does not induce any major changes in this process. Furthermore, PPAR-gamma1 overexpression not only significantly increases the total osteoclast number but also markedly increases the number of giant osteoclasts, cells with 25 or more nuclei. The results obtained in this study show that PPAR-delta has a role in in vitro osteoclast biology, possibly by regulating NFATc1. However, the moderate effect of over-expression on the number of osteoclast formed suggests that whilst PPAR-delta is involved in osteoclast differentiation and/or function, parallel pathways to PPAR-delta may be more decisive in osteoclast formation. The over-expression of PPAR-delta in RAW/C4 cells does not seem to take precedence over these pathways by having a potent effect on osteoclast differentiation. The lack of bone phenotype obtained in the PPAR-delta conditional knock-out mice indicates that PPAR-delta might not be essential for osteoclastogenesis under physiological conditions. Nevertheless, the investigation of the importance of the two PPAR-gamma isoforms in osteoclasts allows to speculate that PPAR-delta deficiency can be compensated by PPAR-gamma1 in these animals.

Characterizing the operation of a large hydraulic excavator

Hall, Andrew (Andrew Stuart) — 2007-08-24T00:00:00Z

Characterizing the role of Myosin VI at E-cadherin cell-cell adhesions

Sabine Mangold — 2011-08-23T00:00:00Z

Epithelial cadherin (E-cad) cell-cell adhesion junctions play an important role in tissue organization and maintenance of tissue integrity. This is vital for many physiological processes such as morphogenesis during embryonal development, wound healing or tissue turnover (Takeichi, 1991). Disruption of cell-cell adhesion function not only has a negative impact on these essential physiological processes but also contributes to tumorigenesis and can lead to tumor progression into metastasis and invasion (Derksen et al., 2006). Therefore a detailed knowledge of the mechanisms of the formation, maintenance and disruption of E-cadherin adhesion junctions makes a vital contribution both towards the fields of molecular cell biology and cancer research. E-cad is a trans-membrane protein whose ectodomain engages in homophilic ligation and other complex adhesion mechanisms with cadherins on adjacent cells and thus contributes to cell-cell adhesion (Leckband and Prakasam, 2006). However its importance is not only as a structural protein, since it is also known to mediate dynamic processes such as cell polarity formation (Iden et al., 2006), and is able to modulate signaling at cell-cell adhesions (McLachlan and Yap, 2007). Its cytoplasmic tail plays an important part in the communication between adhesion junctions and the cytoskeleton (Goodwin and Yap, 2004; Mege et al., 2006). However the precise mechanism of this interaction is still subject to investigation. Myosin VI (MyoVI) is an unconventional actin-binding motor protein (Wells et al., 1999), which serves a multitude of important roles in the cell, both during development and in the adult organism. We are explicitly interested in the MyoVI pool that interacts with E-cad at cell-cell adhesions. Previous work showed that MyoVI engages physically with the E-cad/-catenin complex and plays an important role during the maturation and stabilization of newly forming contacts (Maddugoda et al., 2007). The physical properties of MyoVI imply that it could act as an anchor protein at adhesion junctions and thus contributes to junction stabilization (Altman et al., 2004; Oguchi et al., 2008). However to date very little is known about the precise mechanisms of MyoVI function or its regulation at E-cad adhesion junctions. The first part of this thesis is dedicated to the investigation of the biochemical properties of the interaction between E-cad and MyoVI. Direct binding experiments of recombinant proteins expressed in Escherichia coli (E. coli), revealed that E-cad and MyoVI interact via direct binding of their tail domains. Truncation analysis of the E-cad tail domain revealed that more than one binding site for MyoVI is likely to exist and that one of the binding sites is located in the membrane proximal 28 amino acids of the E-cad cytoplasmic tail. These results support the hypothesis that MyoVI could act as an anchor protein that is able to directly link the junctional E-cadherin/catenin complex to the actin cytoskeleton. By establishing that E-cad and MyoVI bind directly this study provides a novel insight into the organization of adherens junctions and a valuable basis for future mechanistic investigations. The second part of this thesis focuses on novel findings regarding the regulation and function of junctional MyoVI in Hepatocyte growth factor (HGF) treatment. HGF and its receptor, the tyrosine kinase c-met (Met), are molecular factors involved in Epithelial to mesenchymal transition (EMT), a well-investigated process in which cell-cell adhesions are disrupted and the cells develop a migratory phenotype. The process of EMT is important in embryonic development (Birchmeier and Gherardi, 1998), but also employed in tumor invasion and metastasis (Thiery, 2002; Vergara et al., 2010), which makes HGF induced Met signaling also an important target for cancer drug development (Cecchi et al., 2010; Sattler and Salgia, 2009). Upon HGF treatment I observed that MyoVI was lost from the zonula adherens (ZA) within 15 min. During the same time frame I also saw a dramatic re-arrangement of the apical F-actin cytoskeleton and a change in E-cad morphology. By means of over-expression of a GFP-tagged porcine MyoVI construct I could show that the HGF induced loss of MyoVI from the junctions was causal for the changes in F-actin organization. Fluorescence recovery after photobleaching (FRAP) and studies of a photoactivatable G-actin construct showed no difference in actin turnover dynamics in HGF treated versus untreated cells. Live cell studies however revealed a loss of anchorage of actin filaments at the ZA in HGF treatment, which could be counter-acted by over-expression of GFP-tagged MyoVI. The biochemical interaction between MyoVI and E-cad was reduced upon HGF treatment, which implied that the disruption of their interaction might be causal for the loss of actin filament retention at the ZA. Finally I could show that calcium signaling plays a crucial role in the HGF-induced regulation of junctional MyoVI and actin. Thus this study identifies MyoVI as a novel target of HGF signalling, which regulates actin filament retention at the ZA.

Charged Liposaccharide Based Drug Delivery Systems

Adel Abdel Rahim — 2010-05-24T00:00:00Z

Abstract Many hydrophilic drugs do not easily cross biological membranes. One approach to improve intestinal absorption of hydrophilic compounds is to co-administer these drug molecules with absorption enhancers to optimize their physico-chemical properties. In this project, novel liposaccharides were used to modulate the aqueous solubility as well as increase the lipophilicity, improve the intestinal permeability and hence, oral bioavailability of hydrophilic molecules. However, the solubility of the drug/liposaccharide mixtures in aqueous system might be poor, therefore, introducing a hydrophilic component such as quaternization of amine derivatives or sodium salt of carboxylic acid is required to modulate hydrophilic/lipophilic balance and in the same time and promote surfactant and ion pairing characteristics of these derivatives. This dissertation illustrates the development of novel liposaccharide absorption enhancers to be used for oral delivery of hydrophilic drugs. This research includes chemical synthesis of new ionic liposaccharides, examination of their physico-chemical properties, and their in vitro and in vivo biological examinations. The work consists of two parts; cationic and anionic liposaccharide absorption enhancers. a) Cationic Liposaccharide Absorption Enhancers The focus of this work was on the molecular design, synthesis, and evaluation of novel cationic liposaccharide derivatives as drug delivery agents to improve the oral bioavailability of piperacillin as a model hydrophilic drug. These derivatives were designed to possess surfactant as well as ion pairing properties, and were synthesized from biocompatible and biodegradable materials such as glucose, lipoamino acids and lipophilic amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Minimum inhibitory concentrations of piperacillin/liposaccharide conjugates were studied to investigate the effect of liposaccharides on piperacillin activity. The usefulness of these derivatives as absorption enhancers for the oral delivery of piperacillin was assessed in vitro (Caco-2 cells) and in vivo (rat oral absorption). It was concluded that these derivatives did show hemolytic activity in low concentration (suitable for enhancing activity), while they increased permeability of piperacillin through Caco-2 cells. However, these promising results obtained from in vitro assay were not confirmed in vivo. viii b) Anionic Liposaccharide Absorption Enhancers In this thesis, molecular design, synthesis, and evaluation of novel anionic liposaccharide derivatives as absorption enhancers to improve the intestinal permeability of a model hydrophilic drug such as tobramycin were carried out. These derivatives were designed to have surfactant as well as ion pairing properties, and were synthesized from biocompatible materials such as sugar, lipoamino acids and amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Many absorption enhancers have (to some extent) cytotoxic activity, therefore, cytotoxic activity of the novel anionic liposaccharides were examined. The usefulness of these derivatives as absorption enhancers to increase the lipophilicity of tobramycin and hence, its oral bioavailability, was evaluated through carrying out partition coefficient examination. It was concluded that these derivatives did not show haemolytic and cytotoxic activities in low concentration (suitable for enhancing activity). In addition, the permeability of tobramycin into the organic phase (n-octanol) was increased when liposaccharide derivatives were used. The obtained results are promising and encouraging to continue the work to include Caco-2 cells assay (in vitro assay) and oral absorption in rats (in vivo assay) as a prospective work in the future.

Charge transport and recombination in organic semiconductors and photovoltaic solar cells

Krueger, Karsten Bernd — 2013-04-11T05:42:13Z

CHARGE TRANSPORT IN DYE-SENSITISED SOLAR CELLS

Blake, David — 2008-11-21T00:00:00Z

In this thesis we report a study of the transport dynamics of dye-sensitised solar cells (DSCs). The work is motivated by a desire to better understand the operation of the DSC in order to realise improved power conversion efficiencies. Specifically, we focus on charge transport in these devices and the elements of DSC operation that either assist or impede this transport. We confirm that the transport in the DSC is dispersive under virtually all operating conditions (particularly around the maximum power point). We also describe a new technique for probing transport dynamics - namely transient photocurrent measurements coupled with an adapted equivalent circuit model. This technique has allowed us to calculate for the first time critical parameters such as the equivalent recombination resistance. We have also demonstrated the correspondence between a commonly accepted open circuit photovoltage decay measurement technique and a leading equivalent circuit model. This correspondence validates both our own method and the model from which it extends.

Charge Transport in Eumelanin

Johannes De Boor — 2009-05-25T00:00:00Z

Melanins are a class of bio-macromolecules that are found throughout the biosphere. They fulfill various functions in human beings, which makes them a long studied substance in medicine and biology. Furthermore they possess a set of rare and special physico-chemical properties which include featureless broad band absorption in the UV-Vis spectrum and condensed phase electrical conduction. Many scientists have interpreted their findings in terms of an amorphous semiconductor model, but this was done under the a priori assumption that charge transport in melanin is electronic. However, a very strong dependence of melanin’s electrical properties on its level of hydration has recently led to speculations that the dominant charge carrier for high hydration is of protonic rather than electronic nature. This thesis will present direct evidence for electronic charge transport, found by investigating the influence of different environmental parameters on the conductivity of melanin. It will furthermore be shown that the hydration dependent conductivity of melanin can be understood in terms of a dielectric response model for an amorphous semiconductor. This establishment of the major charge carrier is an important step in the on-going effort to fully map the structure-property relationship of melanin and will help to understand its function in vivo. With the ultimate goal to make use of melanin’s fascinating properties, thin films, a new class of device has been characterized and investigated. These thin films, while exhibiting melanin’s characteristics, show improved mechanical stability, a very uniform morphology and a much faster response than standard pellet samples. They are furthermore applicable to standard polymer processing techniques which brings technological applications within reach.

Charge Transport Properties in Eumelanin: Probing the Effect of Hydration on the Ubiquitous Biomacromolecular Pigment via Conductivity, MuSR and EPR Experiments.

Albertus Mostert — 2011-06-29T00:00:00Z

The brown-black pigment eumelanin is one of the most common bio-macromolecules in the biosphere. The biological functions of eumelanin include protection from harmful UV rays and regulating metal ion concentrations to prevent toxicity effects in the human brain. In addition, eumelanin has potential applications as a bio-electronic material with sensing capabilities. However, it is important to understand the structure-property relationship that controls charge transport in eumelanin in order to tailor its properties for such applications. In this work it will be shown that the current paradigm for understanding charge transport in eumelanin, the Mott-Davis amorphous semiconductor model, does not describe eumelanin's electrical properties. Instead, a new model will be proposed based on chemical principles: the comproportionation reaction. This hypothesis is consistent with the hydration dependent adsorption, conductivity, MuSR and EPR experiments reported here. These data will show that with the addition of water, new charge carriers are generated within eumelanin. This self-doping effect leads naturally to the suggestion that eumelanin may be a mixed protonic/electronic conductor. This suggests new possibilities for applications in bio-compatible devices and solid state organic electrochemical transistors.

Chemical and Biological Studies of Cyclotides

Plan, Manual Rey Raña — 2008-11-21T00:00:00Z

The cyclotides are an interesting family of naturally occurring macrocyclic peptides that are highly stable and possess a diversity of biological activities, including uterotonic, haemolytic, anti-HIV, neurotensin antagonist, anti-microbial, trypsin inhibitory, anti-tumour/cytotoxic and insecticidal activity. Their chemical stability, as demonstrated by their resistance to proteolytic degradation and capacity to withstand extreme temperature and pH conditions, is attributed to their unique protein structural motif termed the cyclic cystine knot (CCK), which is composed of a cyclised peptide backbone and a knotted disulfide topology, where two disulfide bridges and their connecting backbone segments form an embedded ring that is threaded by a third disulfide bond. In this project, the cyclotides were investigated for their potential use as naturally occurring crop protection agents against a number of economically important agricultural pests. However, the study was broader than this and included the discovery of novel cyclotides, bioassays and NMR studies. Chapters 1 and 2 provide an introduction to the topics and describe the materials and methods, respectively. Chapter 3 describes the complete suite of cyclotides present in the African plant Oldenlandia affinis, characterised by LC-MS, MS-MS, total amino acid analyses and UV-visible scans. This study expanded the total number of known cyclotides in this plant to 17, of which nine new sequences were characterised. In addition, five cyclotide derivatives containing oxidation products of tryptophan were identified, including oxindolylalanine, n-formylkynurenine, and kynurenine derivatives. Furthermore, two linear derivatives of cyclotides were described. These acyclic derivatives have three intact disulfide bonds, and their N- and C- termini coincide with the hypothesised cleavage site from the precursor protein. This work has increased knowledge about sequence variation accommodated by the CCK scaffold, confirms its applicability as a template for drug design, and also shows the first natural degradation pathways for the cyclotides. These degradation pathways have important implications on the half-life of the cyclotides, and hence their persistence and environmental fate if used in agriculture. The The molluscicidal activity of members of the cyclotide family is reported in Chapter 4. Pure and crude cyclotide extracts from O. affinis and the ornamental groundcover Viola odorata were found to have molluscicidal activities against the most important pest in wetland rice production, the golden apple snail (GAS), Pomacea canliculata. Crude cyclotide mixtures from V. odorata and O. affinis both have molluscicidal activities, the former being more potent than the latter. The 24 h LC[subscript]50 for kalata B1, B2 and metaldehyde, a known molluscicide, were determined at 90, 54 and 105 µM, respectively. At 65 µM, pure cyclotides from V. odorata, namely cycloviolacin O1 and kalata S, both caused 100% GAS mortality while kalata B1, B2 and metaldehyde caused 78, 68 and 60% mortality, respectively. Kalata B7 was weakly active, while kalata B8 did not have molluscicidal activity. The possible downstream effects of the cyclotides on non-target freshwater (Nile tilapia) and saltwater (brine shrimp) marine organisms were investigated. The studies showed that kalata B1was only mildly toxic to brine shrimp compared to podophyllotoxin, a known cytotoxic agent. Similarly, kalata B2 was at least three times less toxic to Nile tilapia than rotenone, a known piscicidal agent. Autolytic degradation sites in present cyclotides have been identified in exposed inter-cysteine loops, particularly among the Trp and Gly residues. Knowledge of these autolytic degradation sites gives insights into how the half-life of these novel molluscicidal agents can be manipulated to ensure a balance between efficacy on pests and proper degradation in the environment. In Chapter 5, members of the cyclotide family were tested on three nematode species, Caenorhabditis elegans, Meloidogyne javanica and Radopholus similis, to investigate their potential use as nematicidal agents. The results show that the cyclotides have nematicidal activities comparable to, if not better than, the known nematicidal agent fenamiphos. Although active at hundreds of micromolar concentrations, the mortality rate increased with increasing time of treatment. It was also found that C. elegans, M. javanica and R. similis have different degrees of susceptibility to the peptides. Different cyclotides have varying haemolytic activities, but compared to melittin the cyclotides are only mildly haemolytic. The pesticidal mode of action of the cyclotides is yet to be fully elucidated but appears to involve more than just their capacity to disrupt cell membranes, their hydrophobicity, or their CCK topology. The varying pesticidal potencies of different cyclotides show that their activities are based on their sequence variations. Kalata B2 was not phytotoxic to tomatoes, suggesting that cyclotides can potentially be used as a soil drench or in seed treatment for nematode control without adverse effects on crops. Alternatively, transgenic plants can be created to specifically express cyclotides in their roots to protect them from nematode infestation. In Chapter 6, cyclotides with insecticidal activities were identified and their possible mode of action was elucidated. It was confirmed that fresh O. affinis leaves contain insecticidal agents. Cotton bollworm (Helicoverpa armigera) larvae fed with young fully-expanded leaves did not grow beyond first instar and died three days after treatment. In a search for putative insecticidal peptides in O. affinis, kalata B2 was identified as the major leaf-specific peptide that was expressed in both winter and summer. Kalata B5 was identified as a differentially-expressed peptide in the aerial parts of the plant during winter, when the plant is at its reproductive phase, possibly to protect its flowers and pods from insect attack. Therefore, the insecticidal properties of kalata B2 and B5 were investigated and compared against those of kalata B1. Assays on H. armigera larvae fed on an artificial soya bean diet showed that kalata B5 is a potent insecticidal peptide, killing 100% of the treated population after four days of treatment, while kalata B2 had a similar activity as kalata B1, with 27.5% and 20.0% mortality, respectively. Nevertheless, kalata B1 and B2 inhibited the growth and development of the surviving larvae by 75.4 and 70.8%, respectively. Results from a protein blot assay of the insect frass showed that the larvae did ingest the peptides from the artificial diet and that the peptides were not degraded even after passing through the insect gut. The possible modes of action of kalata B1, as demonstrated in the human body lice (Pedicularis humanus humanus) assays, was mainly systemic- and to some extent contact-, but not repellent-insecticidal. In Chapter 7, the three-dimensional structure of kalata B5 was determined by NMR spectroscopy and was compared to the structures of kalata B1 and B2 in an endeavour to elucidate the observed differences in insecticidal activity reported in Chapter 6. Kalata B5 was also compared to the prototypic bracelet cyclotide, cycloviolacin O1. Detailed structural studies were conducted on all four peptides by subjecting them to various pH and temperature conditions. Kalata B5 adopts a cyclic cystine knot motif. Its solution structure can be described as a well-defined bracelet structure consisting of 30 amino acids with a cyclised peptide backbone, a cystine knot core, a region of β-sheet, a 3[subscript]10 helix, and a number of β-turns. Although kalata B1 and B2 adopt a similar global fold, they lack the 3[subscript]10 helix in loop 3 that is present in kalata B5. Nevertheless, for all four peptides, it was found that residues in loop 3 interacted with the conserved Glu3 residue in loop 1 to stabilise the structure of the peptide during the deprotonation. While the amino acid composition of kalata B5 is significantly different to that of B1 and B2, many of the intra-molecular hydrogen-bonds identified were situated in similar places within the inter-cysteine loops. However, a number of residues in loops 2, 3 and 5 are not hydrogen bonded, and could potentially form hydrogen-bond interactions with a putative receptor. The surface representations of the three peptides highlighted these obvious differences and found that loops 2 and 3 in kalata B5 formed a region of hydrophobic patch, while in kalata B1 and B2 this region is predominantly hydrophilic. In addition, loop 5 in kalata B5 consists of charged residues, while this region is substituted by hydrophobic residues in kalata B1 and B2. Overall, this project has confirmed the potential use of the cyclotides as naturally occurring pesticidal agents against a number of economically important agricultural pests. The project elucidated the gross modes of action, the downstream effects on non-target aquatic organisms, the degradation pathways and the phytotoxic effects of the cyclotides. It also contributed in the discovery of novel cyclotides, including some modified and linear derivatives. Finally, this project resulted in the structure elucidation of kalata B5 and identified the important roles of particular residues in the activity and structural stability of the cyclotides.

Chemical Characterisation of Novel Metabolites from Bioactive Herbs

Julia Martina Ursula Stuthe — 2011-03-28T00:00:00Z

One reality in the pharmaceutical and medical industries is the need to discover new lead compounds to allow for the development of new drug targets. In this endeavour, science is increasingly turning to natural sources in order to increase the pool of active compounds to enter development. Herbal medicine has a long and established history in the treatment of disease, stretching back to very early civilisations. Still utilised today, herbal medicines provide vital health care for much of the world’s population, and are increasingly popular in affluent countries despite access to clinical-based medicine. Herbal medicine utilises active ingredients, typically novel metabolites, from plants to provide clinical effect. This makes the investigation of herbal remedies important for the continued development of Western medicine. In order to prevent the adulteration, misidentification or inactivity of herbal medicines, chemical profiling and identification of the active constituents of these medicines is necessary to guarantee their efficacy and safety. One of the most powerful tools available to chemists undertaking this task is Nuclear Magnetic Resonance Spectroscopy (NMR), which has enabled the identification of increasing numbers of natural products of high complexity. The aims of this project1 were to phytochemically characterise unidentified medicinal plants, with the subsidiary aim of searching for compounds with potential antihelmintic activity. Plants from two different families, Liliaceae and Stemonaceae, were investigated in this study, with the first species Chamaelirium luteum, a member of Liliaceae and the second species, an unknown Stemona species, a member of Stemonaceae. Three structurally unique steroidal saponins 1-3 were isolated and found to be the major constituents of the medicinal herb Chamaelirium luteum. Absolute structural determination of the compounds was accomplished through extensive 1D and 2D NMR experiments, MS spectral analysis and comparison to synthetic structural analogues, which revealed the presence of an unusual erythro diol moiety in Saponins 1 and 2. An investigation of an unknown Stemona species led to the isolation of two previously unreported alkaloids 84-85 and one recently reported Stemona alkaloid, which are a class of plant alkaloids characterised by a pyrrolo[1,2-a]azepine nucleus. Profiling of the total alkaloids present in this species revealed a trace that does not correspond to any Stemona species reported to date. The profiling of the alkaloid content of an Australian species of Stemona was also performed, which indicated that further unreported alkaloids are likely to be contained in this species. As NMR was extensively used as a structural tool in the assignment of the complex natural products isolated in this study, this acquired knowledge was also applied to solve various structural questions in the fields of enzymatic mechanisms and in herbal medicine quality control. These investigations proved fruitful, and allowed certain aspects of the mechanism of cytochrome P450 enzymes to be elucidated through (NMR) identification of the structure of a product from oxidation of a novel mechanistic probe. The use of NMR to identify an unknown adulterant in an herbal extract was also successful, resulting in the identification of the unknown adulterant as Amaranth, a synthetic azo sulfonic acid containing naphthyl dye.

Chemical fingerprinting of the volatile fraction of species-specific floral Australian honeys

Rintoul, Gavin. — 2007-08-24T00:00:00Z

Chemical investigation of marine invertebrates from South East Queensland: secondary metabolites from nudibranchs and sponges

Yong, Ken Wai Lik — 2012-11-15T14:42:21Z

Chemical studies of marine sponges from South-East Queensland

Jumaryatno, Pinus — 2012-10-12T12:16:46Z