The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Development and evaluation of hydrocarbon sorbent materials with the aid of chemometrics

Hydrocarbon spills on roads are a major safety concern for the driving public and can have severe cost impacts both on pavement maintenance and to the economy through disruption to services. The time taken to clean-up spills and re-open roads in a safe driving condition is an issue of increasing concern given traffic levels on major urban arterials. Thus, the primary aim of the research was to develop a sorbent material that facilitates rapid clean-up of road spills. The methodology involved extensive research into a range of materials (organic, inorganic and synthetic sorbents), comprehensive testing in the laboratory, scale-up and field, and product design (i.e. concept to prototype). The study also applied chemometrics to provide consistent, comparative methods of sorbent evaluation and performance. In addition, sorbent materials at every stage were compared against a commercial benchmark. For the first time, the impact of diesel on asphalt pavement has been quantified and assessed in a systematic way. Contrary to conventional thinking and anecdotal observations, the study determined that the action of diesel on asphalt was quite rapid (i.e. hours rather than weeks or months). This significant finding demonstrates the need to minimise the impact of hydrocarbon spills and the potential application of the sorbent option. To better understand the adsorption phenomenon, surface characterisation techniques were applied to selected sorbent materials (i.e. sand, organo-clay and cotton fibre). Brunauer Emmett Teller (BET) and thermal analysis indicated that the main adsorption mechanism for the sorbents occurred on the external surface of the material in the diffusion region (sand and organo-clay) and/or capillaries (cotton fibre). Using environmental scanning electron microscopy (ESEM), it was observed that adsorption by the interfibre capillaries contributed to the high uptake of hydrocarbons by the cotton fibre. Understanding the adsorption mechanism for these sorbents provided some guidance and scientific basis for the selection of materials. The study determined that non-woven cotton mats were ideal sorbent materials for clean-up of hydrocarbon spills. The prototype sorbent was found to perform significantly better than the commercial benchmark, displaying the following key properties: • superior hydrocarbon pick-up from the road pavement; • high hydrocarbon retention capacity under an applied load; • adequate field skid resistance post treatment; • functional and easy to use in the field (e.g. routine handling, transportation, application and recovery); • relatively inexpensive to produce due to the use of raw cotton fibre and simple production process; • environmentally friendly (e.g. renewable materials, non-toxic to environment and operators, and biodegradable); and • rapid response time (e.g. two minutes total clean-up time compared with thirty minutes for reference sorbents). The major outcomes of the research project include: a) development of a specifically designed sorbent material suitable for cleaning up hydrocarbon spills on roads; b) submission of patent application (serial number AU2005905850) for the prototype product; and c) preparation of Commercialisation Strategy to advance the sorbent product to the next phase (i.e. R&D to product commercialisation).

Real-time performance modelling of a sustained attention to response task

Vigilance declines when exposed to highly predictable and uneventful tasks. Monotonous tasks provide little cognitive and motor stimulation and contribute to human errors. This paper aims to model and detect vigilance decline in real time through participant’s reaction times during a monotonous task. A lab-based experiment adapting the Sustained Attention to Response Task (SART) is conducted to quantify the effect of monotony on overall performance. Then relevant parameters are used to build a model detecting hypovigilance throughout the experiment. The accuracy of different mathematical models are compared to detect in real-time – minute by minute - the lapses in vigilance during the task. We show that monotonous tasks can lead to an average decline in performance of 45%. Furthermore, vigilance modelling enables to detect vigilance decline through reaction times with an accuracy of 72% and a 29% false alarm rate. Bayesian models are identified as a better model to detect lapses in vigilance as compared to Neural Networks and Generalised Linear Mixed Models. This modelling could be used as a framework to detect vigilance decline of any human performing monotonous tasks.

The naked audience : audience performance at a recent staging of Gibson’s "the Miracle Worker"

Contemporary mainstream theatre audiences observe etiquette strictures that regulate behaviour. As Baz Kershaw argues, “the idea of the passive audience for performance has been associated usually with mainstream theatre.” This paper explores a mainstream event where the extant contract of audience silence was replaced with a raw, emotional audience response that continued into the post-performance discussion. William Gibson’s The Miracle Worker was performed by Crossbow Productions at the Brisbane Powerhouse to an audience made up of mainstream theatre patrons and people living with hearing and visual impairment. Various elements such as shadow signing and tactile tours worked metatheatrically and self-referentially to heighten audience awareness. During the performances the verbal and non-verbal responses of the audience were so pervasive that the audience became not only co-creators of the performance text but performers of a rich audience text that had a dramatic impact on the theatrical experience for audience and actors alike. During the post-performance discussion the audience performers spilled onto the stage interacting with the actors, extending the pleasure of the experience. This paper discusses how in privileging the audience as co-creators and performers, the chasm between stage and audience was bridged. The audiences’ performance changed, enriched and created new meanings for each performance.

"Surviving" adolescence : apocalyptic and post-apocalyptic transformations in young adult fiction

This study, entitled "Surviving" Adolescence: Apocalyptic and post-apocalyptic transformations in young adult fiction‖, analyses how discourses surrounding the apocalyptic and post-apocalyptic are represented in selected young adult fiction published between 1997 and 2009. The term ―apocalypse‖ is used by current theorists to refer to an uncovering or disclosure (most often a truth), and ―post-apocalypse‖ means to be after a disclosure, after a revelation, or after catastrophe. This study offers a double reading of apocalyptic and post-apocalyptic discourses, and the dialectical tensions that are inherent in, and arise from, these discourses. Drawing on the current scholarship of children‘s and young adult literature this thesis uses post-structural theoretical perspectives to develop a framework and methodology for conducting a close textual analysis of exclusion, ‗un‘differentiation, prophecy, and simulacra of death. The combined theoretical perspectives and methodology offer new contributions to young adult fiction scholarship. This thesis finds that rather than conceiving adolescence as the endurance of a passing phase of a young person‘s life, there is a new trend emerging in young adult fiction that treats adolescence as a space of transformation essential to the survival of the young adult, and his/her community.

Shoot growth dynamics and photosynthetic response to increased nitrogen availability in the alpine willow salix-glauca

Plants subjected to increases in the supply of resource(s) limiting growth may allocate more of those resources to existing leaves, increasing photosynthetic capacity, and/or to production of more leaves, increasing whole-plant photosynthesis. The responses of three populations of the alpine willow, Salix glauca, growing along an alpine topographic sequence representing a gradient in soil moisture and organic matter, and thus potential N supply, to N amendments, were measured over two growing seasons, to elucidate patterns of leaf versus shoot photosynthetic responses. Leaf-(foliar N, photosynthesis rates, photosynthetic N-use efficiency) and shoot-(leaf area per shoot, number of leaves per shoot, stem weight, N resorption efficiency) level measurements were made to examine the spatial and temporal variation in these potential responses to increased N availability. The predominant response of the willows to N fertilization was at the shoot-level, by production of greater leaf area per shoot. Greater leaf area occurred due to production of larger leaves in both years of the experiment and to production of more leaves during the second year of fertilization treatment. Significant leaf-level photosynthetic response occurred only during the first year of treatment, and only in the dry meadow population. Variation in photosynthesis rates was related more to variation in stomatal conductance than to foliar N concentration. Stomatal conductance in turn was significantly related to N fertilization. Differences among the populations in photosynthesis, foliar N, leaf production, and responses to N fertilization indicate N availability may be lowest in the dry meadow population, and highest in the ridge population. This result is contrary to the hypothesis that a gradient of plant available N corresponds with a snowpack/topographic gradient.

Collaborative research to develop a specialist, post-graduate competency-based education curriculum and continuing professional development structure for health librarians in Australia

Through a grant received from the Australian Library and Information Association (ALIA), members of Health Libraries Australia (HLA) are collaborating with a researcher/educator to conduct a twelve month research project with the goal of developing an educational framework for the Australian health librarianship workforce of the future. The collaboration comprises the principal researcher and a representative group of practitioners from different sectors of the health industry who are affiliated with ALIA in various committees, advisory groups and roles. The research has two main aims: to determine the future skills requirements for the health librarian workforce in Australia; and to develop a structured, modular education framework for specialist post-graduate qualifications together with a structure for ongoing continuing professional development. The paper highlights some of the major trends in the health sector and some of the main environmental influences that may act as drivers for change for health librarianship as a profession, and particularly for educating the future workforce. The research methodology is outlined and the main results are described; the findings are discussed with regard to their implications for the development of a structured, competency-based education framework.

Vulnerability factors for disaster-induced child post-traumatic stress disorder : the case for low family resilience and previous mental illness

Objective: The aim of the present study was to investigate whether parent report of family resilience predicted children’s disaster-induced post-traumatic stress disorder (PTSD) and general emotional symptoms, independent of a broad range of variables including event-related factors, previous child mental illness and social connectedness. ---------- Methods: A total of 568 children (mean age = 10.2 years, SD = 1.3) who attended public primary schools, were screened 3 months after Cyclone Larry devastated the Innisfail region of North Queensland. Measures included parent report on the Family Resilience Measure and Strengths and Difficulties Questionnaire (SDQ)–emotional subscale and child report on the PTSD Reaction Index, measures of event exposure and social connectedness. ---------- Results: Sixty-four students (11.3%) were in the severe–very severe PTSD category and 53 families (28.6%) scored in the poor family resilience range. A lower family resilience score was associated with child emotional problems on the SDQ and longer duration of previous child mental health difficulties, but not disaster-induced child PTSD or child threat perception on either bivariate analysis, or as a main or moderator variable on multivariate analysis (main effect: adjusted odds ratio (ORadj) = 0.57, 95% confidence interval (CI) = 0.13–2.44). Similarly, previous mental illness was not a significant predictor of child PTSD in the multivariate model (ORadj = 0.75, 95%CI = 0.16–3.61). ---------- Conclusion: In this post-disaster sample children with existing mental health problems and those of low-resilience families were not at elevated risk of PTSD. The possibility that the aetiological model of disaster-induced child PTSD may differ from usual child and adolescent conceptualizations is discussed.

Response of cells on surface-induced nanopatterns: Fibroblasts and mesenchymal progenitor cells

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns