Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Electronic states of trans-polyacetylene, poly(p-phenylene vinylene) and sp-hybridised carbon species in amorphous hydrogenated carbon probed by resonant Raman scattering

Inclusions of sp-hybridised, trans-polyacetylene [trans-(CH)x] and poly(p-phenylene vinylene) (PPV) chains are revealed using resonant Raman scattering (RRS) investigation of amorphous hydrogenated carbon (a-C:H) films in the near IR – UV range. The RRS spectra of trans-(CH)x core Ag modes and the PPV CC-H phenylene mode are found to transform and disperse as the laser excitation energy ћωL is increased from near IR through visible to UV, whereas sp-bonded inclusions only become evident in UV. This is attributed to ћωL probing of trans-(CH)x chain inhomogeneity and the distribution of chains with varying conjugation length; for PPV to the resonant probing of phelynene ring disorder; and for sp segments, to ћωL probing of a local band gap of end-terminated polyynes. The IR spectra analysis confirmed the presence of sp, trans-(CH)x and PPV inclusions. The obtained RRS results for a-C:H denote differentiation between the core Ag trans-(CH)x modes and the PPV phenylene mode. Furthermore, it was found that at various laser excitation energies the changes in Raman spectra features for trans-(CH)x segments included in an amorphous carbon matrix are the same as in bulk trans-polyacetylene. The latter finding can be used to facilitate identification of trans-(CH)x in the spectra of complex carbonaceous materials.

Applying an equity lens to tobacco-control policies and their uptake in six western-european countries

We identified policies that may be effective in reducing smoking among socioeconomically disadvantaged groups, and examined trends in their level of application between 1985 and 2000 in six western-European countries (Sweden, Finland, the United Kingdom, the Netherlands, Germany, and Spain). We located studies from literature searches in major databases, and acquired policy data from international data banks and questionnaires distributed to tobacco policy organisations/researchers. Advertising bans, smoking bans in workplaces, removing barriers to smoking cessation therapies, and increasing the cost of cigarettes have the potential to reduce socioeconomic inequalities in smoking. Between 1985 and 2000, tobacco control policies in most countries have become more targeted to decrease the smoking behaviour of low-socioeconomic groups. Despite this, many national tobacco-control strategies in western-European countries still fall short of a comprehensive policy approach to addressing smoking inequalities.

Business process modeling : perceived benefits

The process-centered design of organizations and information systems is globally seen as an appropriate response to the increased economic pressure on organizations. At the methodological core of process-centered management is process modeling. However, business process modeling in large initiatives can be a time-consuming and costly exercise, making it potentially difficult to convince executive management of its benefits. To date, and despite substantial interest and research in the area of process modeling, the understanding of the actual benefits of process modeling in academia and practice is limited. To address this gap, this paper explores the perception of benefits derived from process modeling initiatives, as reported through a global Delphi study. The study incorporates the views of three groups of stakeholders – academics, practitioners and vendors. Our findings lead to the first identification and ranking of 19 unique benefits associated with process modeling. The study in particular found that process modeling benefits vary significantly between practitioners and academics. We argue that these variations may point to a dangerous disconnect between research projects and practical demands.

The synthesis and application of novel profluorescent nitroxides as probes for polymer degradation

This PhD project has expanded the knowledge in the area of profluorescent nitroxides with regard to the synthesis and characterisations of novel profluorescent nitroxide probes as well as physical characterisation of the probe molecules in various polymer/physical environments. The synthesis of the first example of an azaphenalene-based fused aromatic nitroxide TMAO, [1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl, was described. This novel nitroxide possesses some of the structural rigidity of the isoindoline class of nitroxides, as well as some properties akin to TEMPO nitroxides. Additionally, the integral aromatic ring imparts fluorescence that is switched on by radical scavenging reactions of the nitroxide, which makes it a sensitive probe for polymer degradation. In addition to the parent TMAO, 5 other azaphenalene derivatives were successfully synthesised. This new class of nitroxide was expected to have interesting redox properties when the structure was investigated by high-level ab initio molecular orbitals theory. This was expected to have implications with biological relevance as the calculated redox potentials for the azaphenalene ring class would make them potent antioxidant compounds. The redox potentials of 25 cyclic nitroxides from four different structural classes (pyrroline, piperidine, isoindoline and azaphenalene) were determined by cyclic voltammetry in acetonitrile. It was shown that potentials related to the one electron processes of the nitroxide were influenced by the type of ring system, ring substituents or groups surrounding the moiety. Favourable comparisons were found between theoretical and experimental potentials for pyrroline, piperidine and isoindoline ring classes. Substitution of these ring classes, were correctly calculated to have a small yet predictable effect on the potentials. The redox potentials of the azaphenalene ring class were underestimated by the calculations in all cases by at least a factor of two. This is believed to be due to another process influencing the redox potentials of the azaphenalene ring class which is not taken into account by the theoretical model. It was also possible to demonstrate the use of both azaphenalene and isoindoline nitroxides as additives for monitoring radical mediated damage that occurs in polypropylene as well as in more commercially relevant polyester resins. Polymer sample doped with nitroxide were exposed to both thermo-and photo-oxidative conditions with all nitroxides showing a protective effect. It was found that isoindoline nitroxides were able to indicate radical formation in polypropylene aged at elevated temperatures via fluorescence build-up. The azaphenalene nitroxide TMAO showed no such build-up of fluorescence. This was believed to be due to the more labile bond between the nitroxide and macromolecule and the protection may occur through a classical Denisov cycle, as is expected for commercially available HAS units. Finally, A new profluorescent dinitroxide, BTMIOA (9,10-bis(1,1,3,3- tetramethylisoindolin-2-yloxyl-5-yl)anthracene), was synthesised and shown to be a powerful probe for detecting changes during the initial stages of thermo-oxidative degradation of polypropylene. This probe, which contains a 9,10-diphenylanthracene core linked to two nitroxides, possesses strongly suppressed fluorescence due to quenching by the two nitroxide groups. This molecule also showed the greatest protective effect on thermo-oxidativly aged polypropylene. Most importantly, BTMIOA was found to be a valuable tool for imaging and mapping free-radical generation in polypropylene using fluorescence microscopy.

Reduction of Secondary Degeneration after Spinal Cord Injury by Acute Delivery of Proinflammatory Growth Factors

INTRODUCTION Inflammation is a protective attempt to facilitate the removal of damaged tissue and to initiate the healing response in other tissues. However, after spinal cord injury (SCI), this response is prolonged leading to secondary degeneration and glial scarring. Here, we investigate the potential of sustained delivery of pro-inflammatory factors vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) to increase early inflammatory events and promote inflammatory resolution. Method Animal ethics approval was obtained from the Queensland University of Technology. Adult Wistar-Kyoto rats (12-16 weeks old) were subjected to laminectomies and T10 hemisections. Animals were then randomised to treatment (implantation of osmotic pump (Alzet) loaded with 5ug VEGF & 5 ug PDGF) or control groups (lesion control or lesion plus pump delivering PBS). Rats were sacrificed at one month and the spinal cords were harvested and examined by immunohistology, using anti-neurofilament-200(NF200) and anti- ionized calcium binding adapter molecule 1 (Iba1). One way ANOVA was used for statistic analysis. Results At 1 month, active pump-treated cords showed a high level of axonal filament throughout the defects as compared to the control groups. The mean lesion size, as measured by NF200, was 0.47mm2 for the lesion control, 0.39mm2 for the vehicle control and 0.078mm2 for the active pump group. Significant differences were detected between the active pump group and the two control groups (AP vs LC p= 0.017 AG vs VC p= 0.004). Iba-1 staining also showed significant differences in the post-injury inflammatory response. Discussion We have shown that axons and activated microglia are co-located in the lesion of the treated cord. We hypothesise the delivery of VEGF/PDGF increases the local vessel permeability to inflammatory cells and activates these along with the resident microglia to threshold population, which ultimately resolved the prolonged inflammation. Here, we have shown that maintaining the inflammatory signals for at least 7 days improved the morphology of the injured cord. Conclusion This study has shown that boosting inflammation, by delivery VEGF/PDGF, in the early phase of SCI helps to reduce secondary degeneration and may promote inflammation resolution. This treatment may provide a platform for other neuro-regenrative therapies.

Making sense of working lives and working choices : the role of satirical cartoons

This study examined the tone and content of 107 political, satirical cartoons images published in the popular culture forum of mainstream newspapers. The cartoons illustrated the reform of the industrial relations system in Australia in 2005 and 2006. The images were conveyed in a moderate tone. That is, they were more about poking fun at and questioning authority and power, rather than simply describing the issues on one hand, or demonstrating any revolutionary fervor on the other. The cartoons’ content represented many of the concerns and issues being voiced by employer groups, government, opposition, unions and the media at the time. Themes likely to evoke a strong response from the readership included the importance of a collective response in voicing opposition to the legislation and enacting change, the risks to fundamental working conditions, the stealth and dogma associated with the rollout of the changes and the increasing disparity in wealth and power between employers and workers. The images were an important part of the wider discourse and a mechanism which helped place industrial relations squarely in the minds of working Australians.

The evolution of a community of practice at the Queensland University of Technology for lecturers involved in large first year units

Communities of practice (CoPs) may be defined as groups of people who are mutually bound by what they do together (Wenger, 1998, p. 2), that is, they “form to share what they know, to learn from one another regarding some aspects of their work and to provide a social context for that work” (Nickols, 2000, para. 1). They are “emergent” in that the shape and membership emerges in the process of activity (Lees, 2005, p. 7). People in CoPs share their knowledge and experiences freely with the purpose of finding inventive ways to approach new problems (Wenger & Snyder, 2000, p. 2). They can be seen as “shared histories of learning” (Wenger, 1998, p. 86). For some time, QUT staff have been involved in a number of initiatives aimed at sharing ideas and resources for teaching first year students such as the Coordinators of Large First Year Units Working Party. To harness these initiatives and maximise their influence, the leaders of the Transitions In Project (TIP)1 decided to form a CoP around the design, assessment and management of large first year units.

Getting skilled for education, training and employment : Indigenous Cultural Knowledges and mainstream knowledges of mathematics

This abstract is a preliminary discussion of the importance of blending of Indigenous cultural knowledges with mainstream knowledges of mathematics for supporting Indigenous young people. This import is emphasised in the documents Preparing the Ground for Partnership (Priest, 2005), The Indigenous Education Strategic Directions 2008–2011 (Department of Education, Training and the Arts, 2007) and the National Goals for Indigenous Education (Department of Education, Employment and Work Relations, 2008). These documents highlight the contextualising of literacy and numeracy to students’ community and culture (see Priest, 2005). Here, Community describes “a culture that is oriented primarily towards the needs of the group. Martin Nakata (2007) describes contextualising to culture as about that which already exists, that is, Torres Strait Islander community, cultural context and home languages (Nakata, 2007, p. 2). Continuing, Ezeife (2002) cites Hollins (1996) in stating that Indigenous people belong to “high-context culture groups” (p. 185). That is, “high-context cultures are characterized by a holistic (top-down) approach to information processing in which meaning is “extracted” from the environment and the situation. Low-context cultures use a linear, sequential building block (bottom-up) approach to information processing in which meaning is constructed” (p.185). In this regard, students who use holistic thought processing are more likely to be disadvantaged in mainstream mathematics classrooms. This is because Westernised mathematics is presented as broken into parts with limited connections made between concepts and with the students’ culture. It potentially conflicts with how they learn. If this is to change the curriculum needs to be made more culture-sensitive and community orientated so that students know and understand what they are learning and for what purposes.

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly 3-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 mg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing microcomputed tomography (mCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non- BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by mCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, microcompression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects. Crown Copyright 2008 Published by Elsevier Ltd. All rights reserved.

Exercise for health : a breast cancer recovery program - quality of life benefits [Conference Abstract]

Introduction: Five-year survival from breast cancer in Australia is 87%. Hence, ensuring a good quality of life (QOL) has become a focal point of cancer research and clinical interest. Exercise during and after treatment has been identified as a potential strategy to optimise QOL of women diagnosed with breast cancer.----- Methods: Exercise for Health is a randomised controlled trial of an eight-month, exercise intervention delivered by Exercise Physiologists. An objective of this study was to assess the impact of the exercise program during and following treatment on QOL. Queensland women diagnosed with unilateral breast cancer in 2006/07 were eligible to participate. Those living in urban-Brisbane (n=194) were allocated to either the face-to-face exercise group, the telephone exercise group, or the usual-care group, and those living in rural Queensland (n=143) were allocated to the telephone exercise group or the usual-care group. QOL, as assessed by the Functional Assessment of Cancer Therapy-Breast (FACT-B+4) questionnaire, was measured at 4-6 weeks (pre-intervention), 6 months (mid-intervention) and 12 months (three months post-intervention) post-surgery.----- Results: Significant (P<0.01) increases in QOL were observed between pre-intervention and three months post-intervention 12 months post-surgery for all women. Women in the exercise groups experienced greater mean positive changes in QOL during this time (+10 points) compared with the usual-care groups (+5 to +7 points) after adjusting for baseline QOL. Although all groups experienced an overall increase in QOL, approximately 20% of urban and rural women in the usual-care groups reported a decline in QOL, compared with 10% of women in the exercise groups.----- Conclusions: This work highlights the potential importance of participating in physical activity to optimise QOL following a diagnosis of breast cancer. Results suggest that the telephone may be an effective medium for delivering exercise counselling to newly diagnosed breast cancer patients.

Corneal sensitivity and slit scanning in vivo confocal microscopy of the subbasal nerve plexus of the normal central and peripheral human cornea

Purpose: To determine the subbasal nerve density and tortuosity at 5 corneal locations and to investigate whether these microstructural observations correlate with corneal sensitivity. Method: Sixty eyes of 60 normal human subjects were recruited into 1 of 3 age groups, group 1: aged ,35 years, group 2: aged 35–50 years, and group 3: aged .50 years. All eyes were examined using slit-lamp biomicroscopy, noncontact corneal esthesiometry, and slit scanning in vivo confocal microscopy. Results: The mean subbasal nerve density and the mean corneal sensitivity were greatest centrally (14,731 6 6056 mm/mm2 and 0.38 6 0.21 millibars, respectively) and lowest in the nasal mid periphery (7850 6 4947 mm/mm2 and 0.49 6 0.25 millibars, respectively). The mean subbasal nerve tortuosity coefficient was greatest in the temporal mid periphery (27.3 6 6.4) and lowest in the superior mid periphery (19.3 6 14.1). There was no significant difference in mean total subbasal nerve density between age groups. However, corneal sensation (P = 0.001) and subbasal nerve tortuosity (P = 0.004) demonstrated significant differences between age groups. Subbasal nerve density only showed significant correlations with corneal sensitivity threshold in the temporal cornea and with subbasal nerve tortuosity in the inferior and nasal cornea. However, these correlations were weak. Conclusions: This study quantitatively analyzes living human corneal nerve structure and an aspect of nerve function. There is no strong correlation between subbasal nerve density and corneal sensation. This study provides useful baseline data for the normal living human cornea at central and mid-peripheral locations

What determines the health-related quality of life among regional and rural breast cancer survivors?

Objective: To assess the health-related quality of life (HRQoL) of regional and rural breast cancer survivors at 12 months post-diagnosis and to identify correlates of HRQoL. Methods: 323 (202 regional and 121 rural) Queensland women diagnosed with unilateral breast cancer in 2006/2007 participated in a population-based, cross-sectional study. HRQoL was measured using the Functional Assessment of Cancer Therapy, Breast plus arm morbidity (FACT-B+4) self-administered questionnaire. Results: In age-adjusted analyses, mean HRQoL scores of regional breast cancer survivors were comparable to their rural counterparts 12 months post-diagnosis (122.9, 95% CI: 119.8, 126.0 vs. 123.7, 95% CI: 119.7, 127.8; p>0.05). Irrespective of residence, younger (<50 years) women reported lower HRQoL than older (50+ years) women (113.5, 95% CI: 109.3, 117.8 vs. 128.2, 95%CI: 125.1, 131.2; p<0.05). Those women who received chemotherapy, reported two complications post-surgery, had poorer upper-body function than most, reported more stress, reduced coping, who were socially isolated, had no confidante for social-emotional support, had unmet healthcare needs, and low health self-efficacy reported lower HRQoL scores. Together, these factors explained 66% of the variance in overall HRQoL. The pattern of results remained similar for younger and older age groups. Conclusions and Implications: The results underscore the importance of supporting and promoting regional and rural breast cancer programs that are designed to improve physical functioning, reduce stress and provide psychosocial support following diagnosis. Further, the information can be used by general practitioners and other allied health professionals for identifying women at risk of poorer HRQoL.