Fingerprinting of complex mixtures with the use of high performance liquid chromatography, inductively coupled plasma atomic emission spectroscopy and chemometrics

The molecular and metal profile fingerprints were obtained from a complex substance, Atractylis chinensis DC—a traditional Chinese medicine (TCM), with the use of the high performance liquid chromatography (HPLC) and inductively coupled plasma atomic emission spectroscopy (ICP-AES) techniques. This substance was used in this work as an example of a complex biological material, which has found application as a TCM. Such TCM samples are traditionally processed by the Bran, Cut, Fried and Swill methods, and were collected from five provinces in China. The data matrices obtained from the two types of analysis produced two principal component biplots, which showed that the HPLC fingerprint data were discriminated on the basis of the methods for processing the raw TCM, while the metal analysis grouped according to the geographical origin. When the two data matrices were combined into a one two-way matrix, the resulting biplot showed a clear separation on the basis of the HPLC fingerprints. Importantly, within each different grouping the objects separated according to their geographical origin, and they ranked approximately in the same order in each group. This result suggested that by using such an approach, it is possible to derive improved characterisation of the complex TCM materials on the basis of the two kinds of analytical data. In addition, two supervised pattern recognition methods, K-nearest neighbors (KNNs) method, and linear discriminant analysis (LDA), were successfully applied to the individual data matrices—thus, supporting the PCA approach.

Small molecule–biopolymer interactions : Ultraviolet–visible and fluorescence spectroscopy and chemometrics

Interactions between small molecules with biopolymers e.g. the bovine serum albumin (BSA protein), are important, and significant information is recorded in the UV–vis and fluorescence spectra of their reaction mixtures. The extraction of this information is difficult conventionally and principally because there is significant overlapping of the spectra of the three analytes in the mixture. The interaction of berberine chloride (BC) and the BSA protein provides an interesting example of such complex systems. UV–vis and fluorescence spectra of BC and BSA mixtures were investigated in pH 7.4 Tris–HCl buffer at 37 °C. Two sample series were measured by each technique: (1) [BSA] was kept constant and the [BC] was varied and (2) [BC] was kept constant and the [BSA] was varied. This produced four spectral data matrices, which were combined into one expanded spectral matrix. This was processed by the multivariate curve resolution–alternating least squares method (MCR–ALS). The results produced: (1) the extracted pure BC, BSA and the BC–BSA complex spectra from the measured heavily overlapping composite responses, (2) the concentration profiles of BC, BSA and the BC–BSA complex, which are difficult to obtain by conventional means, and (3) estimates of the number of binding sites of BC.

When 'listeners can't talk' : comparing active listening in opening sequences of telephone and online counselling

While there is growing use of online counselling, little is known about its interactional organisation and how it compares to telephone counselling. This is despite past research suggesting that both counsellors and clients report the impact of the different modalities on the presentation and management of the counselling interaction. This paper compares the interactional affordances of telephone and online web counselling in opening sequences on Kids Help Line, a 24-hour Australian counselling service for children and young people up to the age of 25. We examine two ways that counsellors show active listening through response tokens and formulations. The analysis describes how counsellors’ use of minimal response tokens facilitate the clients’ problem presentation and are used in the management of turn taking and sequence organisation. For example, counsellors use the response token Mm hm to show that they understand that the client’s unit of talk to is not yet complete, and to affirm or invite the client to continue speaking. Formulations in phone and web counselling are another way that counsellors display active listening to re-present stretches of the clients’ preceding talk. In phone and web counselling, however, the respective modalities can complicate matters of turn transition and sequence organisation. By examining actual phone and online counselling sessions, this paper offers empirical demonstrations of the interactional affordances of phone and online counselling, and shows how the institutional practice of active listening is accomplished across different counselling modalities

Parent and child experiences of childhood cancer : an interpretative phenomenological analysis approach

A diagnosis of cancer represents a significant crisis for the child and their family. As the treatment for childhood cancer has improved dramatically over the past three decades, most children diagnosed with cancer today survive this illness. However, it is still an illness which severely disrupts the lifestyle and typical functioning of the family unit. Most treatments for cancer involve lengthy hospital stays, the endurance of painful procedures and harsh side effects. Research has confirmed that to manage and adapt to such a crisis, families must undertake measures which assist their adjustment. Variables such as level of family support, quality of parents’ marital relationship, coping of other family members, lack of other concurrent stresses and open communication within the family have been identified as influences on how well families adjust to a diagnosis of childhood cancer. Theoretical frameworks such as the Resiliency Model of Family Adjustment and Adaptation (McCubbin and McCubbin, 1993, 1996) and the Stress and Coping Model by Lazarus and Folkman (1984) have been used to explain how families and individuals adapt to crises or adverse circumstances. Developmental theories have also been posed to account for how children come to understand and learn about the concept of illness. However more descriptive information about how families and children in particular, experience and manage a diagnosis of cancer is still needed. There are still many unanswered questions surrounding how a child adapts to, understands and makes meaning from having a life-threatening illness. As a result, developing an understanding of the impact that such a serious illness has on the child and their family is crucial. A new approach to examining childhood illness such as cancer is currently underway which allows for a greater understanding of the experience of childhood cancer to be achieved. This new approach invites a phenomenological method to investigate the perspectives of those affected by childhood cancer. In the current study 9 families in which there was a diagnosis of childhood cancer were interviewed twice over a 12 month period. Using the qualitative methodology of Interpretative Phenomenological Analysis (IPA) a semi-structured interview was used to explicate the experience of childhood cancer from both the parent and child’s perspectives. A number of quantitative measures were also administered to gather specific information on the demographics of the sample population. The results of this study revealed a number of pertinent areas which need to be considered when treating such families. More importantly experiences were explicated which revealed vital phenomena that needs to be added to extend current theoretical frameworks. Parents identified the time of the diagnosis as the hardest part of their entire experience. Parents experienced an internal struggle when they were forced to come to the realization that they were not able to help their child get well. Families demonstrated an enormous ability to develop a new lifestyle which accommodated the needs of the sick child, as the sick child became the focus of their lives. Regarding the children, many of them accepted their diagnosis without complaint or question, and they were able to recognise and appreciate the support they received. Physical pain was definitely a component of the children’s experience however the emotional strain of loss of peer contact seemed just as severe. Changes over time were also noted as both parental and child experiences were often pertinent to the stage of treatment the child had reached. The approach used in this study allowed for rich and intimate detail about a sensitive issue to be revealed. Such an approach also allowed for the experience of childhood cancer on parents and the children to be more fully realised. Only now can a comprehensive and sensitive medical and psychosocial approach to the child and family be developed. For example, families may benefit from extra support at the time of diagnosis as this was identified as one of the most difficult periods. Parents may also require counselling support in coming to terms with their lack of ability to help their child heal. Given the ease at which children accepted their diagnosis, we need to question whether children are more receptive to adversity. Yet the emotional struggle children battled as a result of their illness also needs to be addressed.

Establishment of a mouse model of colitis and its use to evaluate the anti-inflammatory effects of two ghrelin peptides

Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.

Robot navigation in sensor space

This thesis investigates the problem of robot navigation using only landmark bearings. The proposed system allows a robot to move to a ground target location specified by the sensor values observed at this ground target posi- tion. The control actions are computed based on the difference between the current landmark bearings and the target landmark bearings. No Cartesian coordinates with respect to the ground are computed by the control system. The robot navigates using solely information from the bearing sensor space. Most existing robot navigation systems require a ground frame (2D Cartesian coordinate system) in order to navigate from a ground point A to a ground point B. The commonly used sensors such as laser range scanner, sonar, infrared, and vision do not directly provide the 2D ground coordi- nates of the robot. The existing systems use the sensor measurements to localise the robot with respect to a map, a set of 2D coordinates of the objects of interest. It is more natural to navigate between the points in the sensor space corresponding to A and B without requiring the Cartesian map and the localisation process. Research on animals has revealed how insects are able to exploit very limited computational and memory resources to successfully navigate to a desired destination without computing Cartesian positions. For example, a honeybee balances the left and right optical flows to navigate in a nar- row corridor. Unlike many other ants, Cataglyphis bicolor does not secrete pheromone trails in order to find its way home but instead uses the sun as a compass to keep track of its home direction vector. The home vector can be inaccurate, so the ant also uses landmark recognition. More precisely, it takes snapshots and compass headings of some landmarks. To return home, the ant tries to line up the landmarks exactly as they were before it started wandering. This thesis introduces a navigation method based on reflex actions in sensor space. The sensor vector is made of the bearings of some landmarks, and the reflex action is a gradient descent with respect to the distance in sensor space between the current sensor vector and the target sensor vec- tor. Our theoretical analysis shows that except for some fully characterized pathological cases, any point is reachable from any other point by reflex action in the bearing sensor space provided the environment contains three landmarks and is free of obstacles. The trajectories of a robot using reflex navigation, like other image- based visual control strategies, do not correspond necessarily to the shortest paths on the ground, because the sensor error is minimized, not the moving distance on the ground. However, we show that the use of a sequence of waypoints in sensor space can address this problem. In order to identify relevant waypoints, we train a Self Organising Map (SOM) from a set of observations uniformly distributed with respect to the ground. This SOM provides a sense of location to the robot, and allows a form of path planning in sensor space. The navigation proposed system is analysed theoretically, and evaluated both in simulation and with experiments on a real robot.

Rough set-based reasoning and pattern mining for information filtering

An information filtering (IF) system monitors an incoming document stream to find the documents that match the information needs specified by the user profiles. To learn to use the user profiles effectively is one of the most challenging tasks when developing an IF system. With the document selection criteria better defined based on the users’ needs, filtering large streams of information can be more efficient and effective. To learn the user profiles, term-based approaches have been widely used in the IF community because of their simplicity and directness. Term-based approaches are relatively well established. However, these approaches have problems when dealing with polysemy and synonymy, which often lead to an information overload problem. Recently, pattern-based approaches (or Pattern Taxonomy Models (PTM) [160]) have been proposed for IF by the data mining community. These approaches are better at capturing sematic information and have shown encouraging results for improving the effectiveness of the IF system. On the other hand, pattern discovery from large data streams is not computationally efficient. Also, these approaches had to deal with low frequency pattern issues. The measures used by the data mining technique (for example, “support” and “confidences”) to learn the profile have turned out to be not suitable for filtering. They can lead to a mismatch problem. This thesis uses the rough set-based reasoning (term-based) and pattern mining approach as a unified framework for information filtering to overcome the aforementioned problems. This system consists of two stages - topic filtering and pattern mining stages. The topic filtering stage is intended to minimize information overloading by filtering out the most likely irrelevant information based on the user profiles. A novel user-profiles learning method and a theoretical model of the threshold setting have been developed by using rough set decision theory. The second stage (pattern mining) aims at solving the problem of the information mismatch. This stage is precision-oriented. A new document-ranking function has been derived by exploiting the patterns in the pattern taxonomy. The most likely relevant documents were assigned higher scores by the ranking function. Because there is a relatively small amount of documents left after the first stage, the computational cost is markedly reduced; at the same time, pattern discoveries yield more accurate results. The overall performance of the system was improved significantly. The new two-stage information filtering model has been evaluated by extensive experiments. Tests were based on the well-known IR bench-marking processes, using the latest version of the Reuters dataset, namely, the Reuters Corpus Volume 1 (RCV1). The performance of the new two-stage model was compared with both the term-based and data mining-based IF models. The results demonstrate that the proposed information filtering system outperforms significantly the other IF systems, such as the traditional Rocchio IF model, the state-of-the-art term-based models, including the BM25, Support Vector Machines (SVM), and Pattern Taxonomy Model (PTM).

High quality/high equity systems of education: Curriculum, teachers and reform in the search for equitable outcomes for all students

The issue of what an effective high quality / high equity education system might look like remains contested. Indeed there is more educational commentary on those systems that do not achieve this goal (see for example Luke & Woods, 2009 for a detailed review of the No Child Left Behind policy initiatives put forward in the United States under the Bush Administration) than there is detailed consideration of what such a system might enact and represent. A long held critique of socio cultural and critical perspectives in education has been their focus on deconstruction to the supposed detriment of reconstructive work. This critique is less warranted in recent times based on work in the field, especially the plethora of qualitative research focusing on case studies of ‘best practice’. However it certainly remains the case that there is more work to be done in investigating the characteristics of a socially just system. This issue of Point and Counterpoint aims to progress such a discussion. Several of the authors call for a reconfiguration of the use of large scale comparative assessment measures and all suggest new ways of thinking about quality and equity for school systems. Each of the papers tackles different aspects of the problematic of how to achieve high equity without compromising quality within a large education system. They each take a reconstructive focus, highlighting ways forward for education systems in Australia and beyond. While each paper investigates different aspects of the issue, the clearly stated objective of seeking to delineate and articulate characteristics of socially just education is consistent throughout the issue.

User behaviours and intentions in digital media in Australia

This paper reports findings from a study of user behaviours and intentions towards online news and information in Australia, undertaken by the Queensland University of Technology Creative Industries Faculty and the Smart Services Cooperative Research Centre. It has used a literature review, online survey, focus groups and interviews to explore attitudes and behaviours towards online news and information. The literature review on consumer user of online media highlighted emerging technical opportunities, and flagged existing barriers to access experienced by consumers in the Australian digital media sector. The literature review highlighted multiple disconnects between consumer interests in online news and their ability to fulfil them. This presents an opportunity for news entities to appraise and resolve. Doing so may enhance their service offering, attract consumers and improve loyalty. These themes were further explored by the survey. The survey results revealed three typologies of user, described as ‘convenience’, ‘loyal’ and ‘customising’. Convenience users tend to access news by default, for example when they log out of email. Loyal users seek out a trusted brand such as mainstream news mastheads. Customising users tend to tailor news to their preferences, and be the first to use leading edge media. Respondents to the survey were then invited to participate in focus groups, which aimed to test the survey results. Consumer perceptions and attitudes are important factors in progression towards an information economy, because ultimately consumers are customers. By segmenting the online news market according to customer typology, media providers may identify new opportunities to attract and retain customers.

Practice as method : the ex/centric fixations project

In the past decade, scholars have proposed a range of terms to describe the relationship between practice and research in the creative arts, including increasingly nuanced definitions of practice-based research, practice-led research and practice-as-research. In this paper, I consider the efficacy of creative practice as method. I use the example of The Ex/Centric Fixations Project – a project in which I have embedded creative practice in a research project, rather than embedding research in a creative project. The Ex/Centric Fixations project investigates the way spectators interpret human experiences – especially human experiences of difference, marginalisation or discrimination – depicted onstage. In particular, it investigates the way postmodern performance writing strategies, and the presence of performing bodied to which the experience depicted can be attached, impacts on interpretations. It is part of a broader research project which examines the performativity of spectatorship, and intervenes in emergent debates about performance, ethics and spectatorship in the context of debate about whether live performance is a privileged site for the emergence of an ethical face-to-face encounter with the Other. Using the metaphor of the Mobius strip, I examines the way practice – as a method, rather than an output – has informed, influenced and problematised the broader research project.

Celebrating difference/censoring difference? : Marie Chouinard’s bODY rEMIX

This paper examines performances that defy established representations of disease, deformity and bodily difference. Historically, the ‘deformed’ body has been cast – onstage and in sideshows – as flawed, an object of pity, or an example of the human capacity to overcome. Such representations define the boundaries of the ‘normal’ body by displaying its Other. They bracket the ‘abnormal’ body off as an example of deviance from the ‘norm’, thus, paradoxically, decreasing the social and symbolic visibility (and agency) of disabled people. Yet, in contemporary theory and culture, these representations are reappropriated – by disabled artists, certainly, but also as what Carrie Sandahl has called a ‘master trope’ for representing a range of bodily differences. In this paper, I investigate this phenomenon. I analyse French Canadian choreographer Marie Chouinard’s bODY rEMIX/gOLDBERG vARIATIONS, in which 10 able-bodied dancers are reborn as bizarre biotechnical mutants via the use of crutches, walkers, ballet shoes and barres as prosthetic pseudo-organs. These bodies defy boundaries, defy expectations, develop new modes of expression, and celebrate bodily difference. The self-inflicted pain dancers experience during training is cast as a ‘disablement’ that is ultimately ‘enabling’. I ask what effect encountering able bodies celebrating ‘dis’ or ‘diff’ ability has on audiences. Do we see the emergence of a once-repressed Other, no longer silenced, censored or negated? Or does using ‘disability’ to express the dancers’ difference and self-determination usurp a ‘trope’ by which disabled people themselves might speak back to the dominant culture, creating further censorship?

Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker.

BACKGROUND: Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. METHODS AND RESULTS: Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. CONCLUSIONS: Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Safeguarding Rural Australia: Data Report No. 3 - Unintentional Violence

This report focuses on our examination of extant data which have been sourced with respect to unintentional serious and violent injuries to males living in regional and remote Australia. Such injuries typically might be caused by, for example, transport accidents, occupational exposures and hazards, burns and so on. Thus unintentional violent incidents cause physical trauma the consequences of which can sometimes lead to chronic conditions including psychological harm or substance abuse. Additional commentary resulting from exploration, examination and analyses of secondary data is published online in complementary reports in this series.

Conceptions of school based youth health nursing : a phenomenographic study

The School Based Youth Health Nurse Program was established in 1999 by the Queensland Government to fund school nurse positions in Queensland state high schools. Schools were required to apply for a School Based Youth Health Nurse during a five-phase recruitment process, managed by the health districts, and rolled out over four years. The only mandatory selection criterion for the position of School Based Youth Health Nurse was registration as a General Nurse and most School Based Youth Health Nurses are allocated to two state high schools. Currently, there are approximately 115 Full Time Equivalent School Based Youth Health Nurse positions across all Queensland state high schools. The literature review revealed an abundance of information about school nursing. Most of the literature came from the United Kingdom and the United States, who have a different model of school nursing to school based youth health nursing. However, there is literature to suggest school nursing is gradually moving from a disease-focused approach to a social view of health. The noticeable number of articles about, for example, drug and alcohol, mental health, and contemporary sexual health issues, is evidence of this change. Additionally, there is a significant the volume of literature about partnerships and collaboration, much of which is about health education, team teaching and how school nurses and schools do health business together. The surfacing of this literature is a good indication that school nursing is aligning with the broader national health priority areas. More particularly, the literature exposed a small but relevant and current body of research, predominantly from Queensland, about school based youth health nursing. However, there remain significant gaps in the knowledge about school based youth health nursing. In particular, there is a deficit about how School Based Youth Heath Nurses understand the experience of school based youth health nursing. This research aimed to reveal the meaning of the experience of school based youth health nursing. The research question was How do School Based Youth Health Nurses’ understand the experience of school based youth health nursing? This enquiry was instigated because the researcher, who had a positive experience of school based youth health nursing, considered it important to validate other School Based Youth Health Nurses’ experiences. Consequently, a comprehensive use of qualitative research was considered the most appropriate manner to explore this research question. Within this qualitative paradigm, the research framework consists of the epistemology of social constructionism, the theoretical perspective of interpretivism and the approach of phenomenography. After ethical approval was gained, purposeful and snowball sampling was used to recruit a sample of 16 participants. In-depth interviews, which were voluntary, confidential and anonymous, were mostly conducted in public venues and lasted from 40-75 minutes. The researcher also kept a researchers journal as another form of data collection. Data analysis was guided by Dahlgren and Fallsbergs’ (1991, p. 152) seven phases of data analysis which includes familiarization, condensation, comparison, grouping, articulating, labelling and contrasting. The most important finding in this research is the outcome space, which represents the entirety of the experience of school based youth health nursing. The outcome space consists of two components: inside the school environment and outside the school environment. Metaphorically and considered as whole-in-themselves, these two components are not discreet but intertwined with each other. The outcome space consists of eight categories. Each category of description is comprised of several sub-categories of description but as a whole, is a conception of school based youth health nursing. The eight conceptions of school based youth health nursing are: 1. The conception of school based youth health nursing as out there all by yourself. 2. The conception of school based youth health nursing as no real backup. 3. The conception of school based youth health nursing as confronted by many barriers. 4. The conception of school based youth health nursing as hectic and full-on. 5. The conception of school based youth health nursing as working together. 6. The conception of school based youth health nursing as belonging to school. 7. The conception of school based youth health nursing as treated the same as others. 8. The conception of school based youth health nursing as the reason it’s all worthwhile. These eight conceptions of school based youth health nursing are logically related and form a staged hierarchical relationship because they are not equally dependent on each other. The conceptions of school based youth health nursing are grouped according to negative, negative and positive and positive conceptions of school based youth health nursing. The conceptions of school based youth health nursing build on each other, from the bottom upwards, to reach the authorized, or the most desired, conception of school based youth health nursing. This research adds to the knowledge about school nursing in general but especially about school based youth health nursing specifically. Furthermore, this research has operational and strategic implications, highlighted in the negative conceptions of school based youth health nursing, for the School Based Youth Health Nurse Program. The researcher suggests the School Based Youth Health Nurse Program, as a priority, address the operational issues The researcher recommends a range of actions to tackle issues and problems associated with accommodation and information, consultations and referral pathways, confidentiality, health promotion and education, professional development, line management and School Based Youth Health Nurse Program support and school management and community. Strategically, the researcher proposes a variety of actions to address strategic issues, such as the School Based Youth Health Nurse Program vision, model and policy and practice framework, recruitment and retention rates and evaluation. Additionally, the researcher believes the findings of this research have the capacity to spawn a myriad of future research projects. The researcher has identified the most important areas for future research as confidentiality, information, qualifications and health outcomes.

Investigation of molecular mechanisms regulating biomineralization of pearl oyster Pinctada maxima

Biomineralization is a process encompassing all mineral containing tissues produced within an organism. The most dynamic example of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this remarkable architecture. Subsequently, for the past decade considerable research have been undertaken to identify and characterize the protein components involved in biomineralization. Despite these efforts the general understanding of the process remains ambiguous. This study employs a novel molecular approach to further the elucidation of the shell biomineralization. A microarray platform has been custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from the mantle, an organ involved in shell formation. This microarray has been used as the primary tool for three separate investigations in an effort to associate transcriptional gene expression from P. maxima to the process of shell biomineralization. The first investigation analyzes the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and each analyzed for gene expression with PmaxArray 1.0. Over 2000 ESTs were differentially expressed among the tissue sections, identifying five major expression regions. Three of these regions have been proposed to have shell formation functions belonging to nacre, prismatic calcite and periostracum. The spatial gene expression map was confirmed by in situ hybridization, localizing a subset of ESTs from each expression region to the same mantle area. Comparative sequence analysis of ESTs expressed in the proposed shell formation regions with the BLAST tool, revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell formation genes. The second investigation correlates temporal EST expression during P. maxima larval ontogeny with transitions in shell mineralization during the same period. A timeline documenting the morphologicat microstructural and mineralogical shell characteristics of P. maxima throughout larval ontogeny has been established. Three different shell types were noted based on the physical characters and termed, prodissoconch I, prodissoconch 11 and dissoconch. PmaxArray 1.0 analyzed ESTs expression of animals throughout the larval development of P. maxima, noting up-regulation of 359 ESTs in association with the shell transitions from prodissoconch 1 to prodissoconch 11 to dissoconch. Comparative sequence analysis of these ESTs indicates a number of the transcripts are novel as well as showing significant sequence similarities between ESTs and known shell matrix associated genes and proteins. These ESTs are discussed in relation to the shell characters associated with their temporal expression. The third investigation uses PmaxArray 1.0 to analyze gene expression in the mantle tissue of P. maxima specimens exposed to sub-lethal concentrations of a shell-deforming toxin, tributyltin (TBT). The shell specific effects of TBT are used in this investigation to interpret differential expression of ESTs with respect to shell formation functions. A lethal and sublethal TBT concentration range was established for P. maxima, noting a concentration of 50 ng L- 1 TBT as sub-lethal over a 21 day period. Mantle tissue from P. maxima animals treated with 50 ng L- 1 TBT was assessed for differential EST expression with untreated control animals. A total of 102 ESTs were identified as differentially expressed in association with TBT exposure, comparative sequence identities included an up-regulation of immunity and detoxification related genes and down-regulation of several shell matrix genes. A number of transcripts encoding novel peptides were additionally identified. The potential actions of these genes are discussed with reference to TBT toxicity and shell biomineralization. This thesis has used a microarray platform to analyze gene expression in spatial, temporal and toxicity investigations, revealing the involvement of numerous gene transcripts in specific shell formation functions. Investigation of thousands of transcripts simultaneously has provided a holistic interpretation of the organic components regulating shell biomineralization.