A re-examination of the Ghrelin and Ghrelin receptor genes

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

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.

Resolving painful emotional experience during psychodrama

Unresolved painful emotional experiences such as bereavement, trauma and disturbances in core relationships, are common presenting problems for clients of psychodrama or psychotherapy more generally. Emotional pain is experienced as a shattering of the sense of self and disconnection from others and, when unresolved, produces avoidant responses which inhibit the healing process. There is agreement across therapeutic modalities that exposure to emotional experience can increase the efficacy of therapeutic interventions. Moreno proposes that the activation of spontaneity is the primary curative factor in psychodrama and that healing occurs when the protagonist (client) engages with his or her wider social system and develops greater flexibility in response to that system. An extensive case-report literature describes the application of the psychodrama method in healing unresolved painful emotional experiences, but there is limited empirical research to verify the efficacy of the method or to identify the processes that are linked to therapeutic change. The purpose of this current research was to construct a model of protagonist change processes that could extend psychodrama theory, inform practitioners’ therapeutic decisions and contribute to understanding the common factors in therapeutic change. Four studies investigated protagonist processes linked to in-session resolution of painful emotional experiences. Significant therapeutic events were analysed using recordings and transcripts of psychodrama enactments, protagonist and director recall interviews and a range of process and outcome measures. A preliminary study (3 cases) identified four themes that were associated with helpful therapeutic events: enactment, the working alliance with the director and with group members, emotional release or relief and social atom repair. The second study (7 cases) used Comprehensive Process Analysis (CPA) to construct a model of protagonists’ processes linked to in-session resolution. This model was then validated across four more cases in Study 3. Five meta-processes were identified: (i) a readiness to engage in the psychodrama process; (ii) re-experiencing and insight; (iii) activating resourcefulness; (iv) social atom repair with emotional release and (v) integration. Social atom repair with emotional release involved deeply experiencing a wished-for interpersonal experience accompanied by a free flowing release of previously restricted emotion and was most clearly linked to protagonists’ reports of reaching resolution and to post session improvements in interpersonal relationships and sense of self. Acceptance of self in the moment increased protagonists’ capacity to generate new responses within each meta-process and, in resolved cases, there was evidence of spontaneity developing over time. The fourth study tested Greenberg’s allowing and accepting painful emotional experience model as an alternative explanation of protagonist change. The findings of this study suggested that while the process of allowing emotional pain was present in resolved cases, Greenberg’s model was not sufficient to explain the processes that lead to in-session resolution. The protagonist’s readiness to engage and activation of resourcefulness appear to facilitate the transition from problem identification to emotional release. Furthermore, experiencing a reparative relationship was found to be central to the healing process. This research verifies that there can be in-session resolution of painful emotional experience during psychodrama and protagonists’ reports suggest that in-session resolution can heal the damage to the sense of self and the interpersonal disconnection that are associated with unresolved emotional pain. A model of protagonist change processes has been constructed that challenges the view of psychodrama as a primarily cathartic therapy, by locating the therapeutic experience of emotional release within the development of new role relationships. The five meta-processes which are described within the model suggest broad change principles which can assist practitioners to make sense of events as they unfold and guide their clinical decision making in the moment. Each meta-process was linked to specific post-session changes, so that the model can inform the development of therapeutic plans for individual clients and can aid communication for practitioners when a psychodrama intervention is used for a specific therapeutic purpose within a comprehensive program of therapy.

Towards a new employment relationship model

Purpose – The purpose of this study is to examine and extend Noer’s theoretical model of the new employment relationship. Design/methodology/approach – Case study methodology is used to scrutinise the model. The results of a literature-based survey on the elements underpinning the five values in the model are analysed from dual perspectives of individual and organization using a multi-source assessment instrument. A schema is developed to guide and inform a series of focus group discussions from an analysis of the survey data. Using content analysis, the transcripts from the focus group discussions are evaluated using the model’s values and their elements. The transcripts are also reviewed for implicit themes. The case studied is Flight Centre Limited, an Australian-based international retail travel company. Findings – Using this approach, some elements of the five values in Noer’s model are identified as characteristic of the company’s psychological contract. Specifically, to some extent, the model’s values of flexible deployment, customer focus, performance focus, project-based work, and human spirit and work can be applied in this case. A further analysis of the transcripts validates three additional values in the psychological contract literature: commitment; learning and development; and open information. As a result of the findings, Noer’s model is extended to eight values. Research limitations/implications – The study offers a research-based model of the new employment relationship. Since generalisations from the case study findings cannot be applied directly to other settings, the opportunity to test this model in a variety of contexts is open to other researchers. Originality/value – In practice, the methodology used is a unique process for benchmarking the psychological contract. The process may be applied in other business settings. By doing so, organization development professionals have a consulting framework for comparing an organization’s dominant psychological contract with the extended model presented here.

Developing consistency in teacher judgment formation through online meeting centres

This paper investigates the complex interactions that occur as teachers meet online to justify and negotiate their assessment judgments of student work across relatively large and geographically dispersed populations. Drawing from sociocultural theories of learning and technology, the technology is positioned as playing a role in either supporting or hindering teachers reaching a common understanding of assessment standards. Meeting transcripts and interviews with the teachers have been qualitatively analysed in terms of the interactions that occurred and teachers’ perceptions of these interactions. While online meetings offer a partial solution to address the current demands of assessment in education, they also present new challenges as teachers meet, in an unfamiliar environment, to discuss student work.

In-session processes in online counselling with young people : an exploratory approach

Aims : The aim of this study was to conduct an exploratory investigation into the in-session processes and behaviours that occur between therapists and young people in online counseling. Method: The Consensual Qualitative Research method was employed to identify in-session behaviours and a coding instrument was developed to determine their frequency of use and assess whether nuances carried in the meaning of text messages have an influential effect during sessions. Eighty-five single-session transcripts were examined in total by two independent coders. Results: Sample statistics revealed that, on average, rapport-building processes were used more consistently across cases with both types of processes having a moderately strong positive effect on young people. However, closer examination of these processes revealed weaker positive effects for in-session behaviours that rely more heavily on verbal and non-verbal cues to be accurately interpreted. Implications for Practice and Future Research: These findings imply that therapists may focus more on building rapport than accomplishing tasks with young people during online counselling sessions due to the absence of verbal and non-verbal information when communicating via text messages.

The strategic mindset of Australian manufacturing managers : some missing links

Manufacturing managers have a measurable mindset (or frame) that structures their response to the manufacturing environment. Most importantly, this frame represents a set of assumptions about the relative prominence of concepts in the manufacturing domains, about the nature of people, and about the sensemaking processes required to understand the nature of the manufacturing environment as seen through the eyes of manufacturing managers. This paper uses work in the area of text analysis and extends the scope of a methodology that has been approached from two different directions by Carley ( Journal of Organizational Behavior , 18 (51), 533-558, 1997) and Gephart ( Journal of Organizational Behavior , 18 (51), 583-622, 1997). This methodology is termed collocate analysis. Based on the analysis of transcripts of interviews of Australian manufacturing managers mind maps of the concepts used by these managers have been constructed. From an analysis of these mind maps it is argued that strategy plays a minor role in their thinking second only to the improvement domain, whereas design and related concepts play a dominant role in their day-to-day thinking.

Caregivers' beliefs about practice in infant child care programmes

Infant caregivers in centre-based child care were videotaped as they interacted with the children during routine and non-routine activities. During a subsequent interview, the video provided a stimulus for discussion and reflection on practices. Caregivers were also asked to write about their beliefs on good practice in caring for infants. Transcripts of the interviews and the written statements were then analysed for evidence of nave and informed beliefs about caregiving. Most caregivers held nave beliefs and only one caregiver had an informed understanding of professional practice with infants. The usefulness of the analytical framework used in this research is discussed as a means for understanding caregiving practices. It has important implications for approaches to initial professional education of early childhood teachers and for professional development programmes.

Adaptation of Sudanese refugees in an Australian context : investigating helps and hindrances

The present study investigates the experiences of Sudanese refugees by exploring the themes that characterise participants’ experiences in Sudan, en route, and at their Australian destination. In particular, the research identifies several factors that may be seen as ‘helps’ or ‘hindrances’ to Sudanese refugees’ adaptation. Participants were 12 Sudanese refugees aged between 19 and 40 years who had been residing in Australia for five years or less. A qualitative phenomenological approach to data collection and analysis was employed. Examination of the interview transcripts revealed that all participants identified both ‘hindrances’ and ‘helps’ toward adaptation and indicated that positive adaptation is not only possible, but probable for Sudanese refugees in spite of their past experiences of trauma and present resettlement difficulties. Several practical implications were elicited from the research including a need for programs that actively promote refugees’ adaptation by encouraging the broadening of social networks.

How to be masculine in the block area

This article focuses on the social interactions of several boys aged 3-5 years in the block area of a preschool classroom in a childcare setting. Using transcripts of video segments showing these boys engaged in daily play and interactions, the article analyses two episodes that occurred in the first weeks of the school year. At first glance, both episodes appear chaotic, with little appearance of order among the players. A closer analysis reveals a finely organized play taking place, with older boys teaching important lessons to the newcomers about how to be masculine in the block area. These episodes illustrate that masculinity is not a fixed character trait, but is determined through practice and participation in the activities of masculinity. Play and conflict are the avenues through which this occurs.

Eucharistic rituals in Catholic primary schools in the Archdiocese of Brisbane : a study of administrators' conceptions

The focus of this study is the celebration of Eucharist in Catholic primary schools within the Archdiocese of Brisbane. The context of the contemporary Australian Catholic primary school embodies certain 'problematical realities' in relation to the time-honoured way in which school Eucharistic rituals have been celebrated. These contemporary realities raise a number of issues that impact on school celebrations of Eucharist. The purpose of this study is to explore administrators' differing conceptions of school Eucharistic rituals in an attempt to investigate some of these issues and assist members of individual school communities as they strive to make celebrations of Eucharist appropriate and meaningful for the group gathered. The phenomenographic research approach was adopted, as it is well suited to the purpose of this study and the nature of the research question. Phenomenography is essentially a study of variation. It attempts to map the 'whole' phenomenon under investigation by describing on equal terms all conceptions of the phenomenon and establishing an ordered relationship among them. The purpose of this study and the nature of the research question necessitate an approach that allows the identification and description of the different ways in which administrators' experience school Eucharistic rituals. Accordingly, phenomenography was selected. Members of the Administration Team, namely the principal, the APRE (Assistant to the Principal Religious Education) and, in larger primary schools, the AP A (Assistant to the Principal Administration) share responsibility for leading change in Catholic primary schools in the Archdiocese of Brisbane. In practice, however, principals delegate the role of leading the development of the school's religion program and providing leadership in the religious life of the school community to the APRE (Brisbane Catholic Education, 1997). Informants in this study are nineteen APREs from a variety of Catholic primary schools in the Archdiocese of Brisbane. These APREs come from schools across the archdiocese, rather than from within one particular region. Several significant findings resulted from this study. Firstly, the data show that there are significant differences in how APREs' experience school Eucharistic rituals, although the number of these qualitatively different conceptions is quite limited. The study identifies and describes six distinct yet related conceptions of school Eucharistic rituals. The logical relationship among these conceptions (the outcome space) is presented in the form of a diagram with accompanying explication. The variation among the conceptions is best understood and described in terms of three dimensions of the role of Eucharist in the Catholic primary school and is represented on the model of the outcome space. Individual transcripts suggest that individual APREs tend to emphasise some conceptions more than others. It is the contention of the present study that change in the practice of school Eucharistic rituals is unlikely to occur until all of a school community's conceptions are brought out into the open and articulated. As leaders of change, APREs need to be alerted to their own biases and become aware of alternative ways of conceiving school Eucharistic ritual. It is proposed that the different categories of description and dimensions, represented by the model of the outcome space, can be used to help in the process of articulating a school community's conceptions of Eucharist, with the APRE as facilitator of this process. As a result, the school community develops a better understanding of why their particular school does what it does in relation to school Eucharistic rituals.

Androgen-regulated genes differentially modulated by the androgen receptor coactivator L-dopa decarboxylase in human prostate cancer cells

Background The androgen receptor is a ligand-induced transcriptional factor, which plays an important role in normal development of the prostate as well as in the progression of prostate cancer to a hormone refractory state. We previously reported the identification of a novel AR coactivator protein, L-dopa decarboxylase (DDC), which can act at the cytoplasmic level to enhance AR activity. We have also shown that DDC is a neuroendocrine (NE) marker of prostate cancer and that its expression is increased after hormone-ablation therapy and progression to androgen independence. In the present study, we generated tetracycline-inducible LNCaP-DDC prostate cancer stable cells to identify DDC downstream target genes by oligonucleotide microarray analysis. Results Comparison of induced DDC overexpressing cells versus non-induced control cell lines revealed a number of changes in the expression of androgen-regulated transcripts encoding proteins with a variety of molecular functions, including signal transduction, binding and catalytic activities. There were a total of 35 differentially expressed genes, 25 up-regulated and 10 down-regulated, in the DDC overexpressing cell line. In particular, we found a well-known androgen induced gene, TMEPAI, which wasup-regulated in DDC overexpressing cells, supporting its known co-activation function. In addition, DDC also further augmented the transcriptional repression function of AR for a subset of androgen-repressed genes. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time RT-PCR. Conclusion Taken together, our results provide evidence for linking DDC action with AR signaling, which may be important for orchestrating molecular changes responsible for prostate cancer progression.

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

The provision of patient personal hygiene in the intensive care unit : a descriptive exploratory study of bed-bathing practice

Background/objectives The provision of the patient bed-bath is a fundamental nursing care activity yet few quantitative data and no qualitative data are available on registered nurses’ (RNs) clinical practice in this domain in the intensive care unit (ICU). The aim of this study was to describe ICU RNs current practice with respect to the timing, frequency and duration of the patient bed-bath and the cleansing and emollient agents used. Methods The study utilised a two-phase sequential explanatory mixed method design. Phase one used a questionnaire to survey RNs and phase two employed semi-structured focus group (FG) interviews with RNs. Data was collected over 28 days across four Australian metropolitan ICUs. Ethical approval was granted from the relevant hospital and university human research ethics committees. RNs were asked to complete a questionnaire following each episode of care (i.e. bed-bath) and then to attend one of three FG interviews: RNs with less than 2 years ICU experience; RNs with 2–5 years ICU experience; and RNs with greater than 5 years ICU experience. Results During the 28-day study period the four ICUs had 77.25 beds open. In phase one a total of 539 questionnaires were returned, representing 30.5% of episodes of patient bed-baths (based on 1767 bed occupancy and one bed-bath per patient per day). In 349 bed-bath episodes 54.7% patients were mechanically ventilated. The bed-bath was given between 02.00 and 06.00 h in 161 episodes (30%), took 15–30 min to complete (n = 195, 36.2%) and was completed within the last 8 h in 304 episodes (56.8%). Cleansing agents used were predominantly pH balanced soap or liquid soap and water (n = 379, 71%) in comparison to chlorhexidine impregnated sponges/cloths (n = 86, 16.1%) or other agents such as pre-packaged washcloths (n = 65, 12.2%). In 347 episodes (64.4%) emollients were not applied after the bed-bath. In phase two 12 FGs were conducted (three FGs at each ICU) with a total of 42 RN participants. Thematic analysis of FG transcripts across the three levels of RN ICU experience highlighted a transition of patient hygiene practice philosophy from shades of grey – falling in line for inexperienced clinicians to experienced clinicians concrete beliefs about patient bed-bath needs. Conclusions This study identified variation in process and products used in patient hygiene practices in four ICUs. Further study to improve patient outcomes is required to determine the appropriate timing of patient hygiene activities and cleansing agents used to improve skin integrity.