Elevated concentration of TNF-a induces trophoblast differentiation in mouse blastocyst outgrowths

Elevated expression of tumour necrosis factora (TNF-a) is associated with adverse pregnancy outcome. This study has examined the expression of TNF-a and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF-a on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RTPCR demonstrated TNF-a mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF-a expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF-a (100 ng/ml) for 72 h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF-a-treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean € SD 23.90€10.42 vs 9.37€7.45, t-test, P<0.0001) with no significant change in the numbers of trophoblast cells (19.97€8.14 vs 21.73€7.79, t-test, P=0.39). Within the trophoblast cell population, the TNF-a-treated outgrowths exhibited a significant increase in multinucleated cells (14.10€5.53 vs 6.37€5.80, t-test, P<0.0001) and a corresponding significant decrease in mononucleated cells (5.87€3.60 vs 15.37€5.87, t-test, P<0.0001). In summary, this study describes the expression of TNF-a and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF-a restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased

Vaughan, Suzi and Armstrong, Wendy (2009) Threads of tension. In: Vaughan, Suzi and Schmidt, Christine Margaret, (eds.) Five Fashion Musings. Post Pressed, Brisbane, Qld, pp. 9-15.

five:fashion musings is an upcoming 2009 publicaton to celebrate the fashion discipline's five-year milestone at the Queensland Unviesity of Technology. it represents a body of work by fashion practitioners, aceademic and educators commissioned to explore their research in fashion theory, practice and pedogogy through five key themes.

Children's Fiction About 9/11: Ethnic, Heroic and National Identities

In this study, Lampert examines how cultural identities are constructed within fictional texts for young people written about the attacks on the Twin Towers. It identifi es three significant identity categories encoded in 9/11 books for children:ethnic identities, national identities, and heroic identities,arguing that the identities formed within the selected children’s texts are in flux, privileging performances of identities that are contingent on post-9/11 politics. Looking at texts including picture books, young adult fiction, and a selection of DC Comics, Lampert finds in post-9/11 children’s literature a co-mingling of xenophobia and tolerance; a binaried competition between good and evil and global harmony and national insularity; and a lauding of both the commonplace hero and the super-human. The shifting identities evident in texts that are being produced for children about 9/11 offer implicit and explicit accounts of what constitutes good citizenship, loyalty to nation and community, and desirable attributes in a Western post-9/11 context. This book makes an original contribution to the field of children’s literature by providing a focused and sustained analysis of how texts for children about 9/11 contribute to formations of identity in these complex times of cultural unease and global unrest.

Key performance indicators for innovation implementation : perception vs. actual usage

This paper aimed to explore the proportion associated with the perceived importance and the actual use of performance indicators from manufacturing and non manufacturing industries. The sample was 86 small and medium sized-organizations in Thailand. The perceived importance and the actual use of financial and non financial indicators were found to be significantly related among manufacturing and non manufacturing industries. KPI 3, 9, and 12 (i.e. sales and sales growth; quality of products and /or services; and process time) were perceived the most importance among manufacturing managers (85.3%, 79.4% and 76.5% respectively). While KPI 6, 9, and 12 (i.e. customer satisfaction, quality of products and /or services; and process time) were perceived the most importance among non manufacturing managers (84.8%, 93.5%, and 84.8% respectively). Interestingly, the most used KPIs for manufacturing were sales and sales growth (64.7%); profit margins (61.8%); and customer satisfaction (84.8) while non manufacturing used quality products/services (60.9%); sales and sales growth (54.3%) and employee development (54.3%) respectively. Limitation and implication were also discussed.

(-)-CGP12177 increases contractile force through beta1l-adrenoceptors but not through beta3-adrenoceptors in human right atrial myocardium

(-)-CGP12177 is a non-conventional partial agonist that causes modest and transient increases of contractile force in human atrial trabeculae (Kaumann and Molenaar, 2008). These effects are markedly increased and maintained by inhibition of phosphodiesterase PDE3. As verified with recombinant receptors, the cardiostimulant effect of (-)-CGP12177 is mediated through a site at the beta1-adrenoceptor with lower affinity (beta1LAR) compared to the site through which (-)-CGP12177 antagonizes the effects of catecholamines (beta1HAR). However, in a recent report it was proposed that the positive inotropic effects of CGP12177 are mediated through beta3-adrenoceptors (Skeberdis et al 2008). We therefore investigated whether the effects of (-)-CGP12177 on human atrial trabeculae are antagonized by the beta3-adrenoceptor-selective antagonist L-748,337 (1 microM). (-)-CGP12177 (200 nM) caused a stable increase in force which was significantly reduced by the addition of (-)-bupranolol (1 microM), P = 0.002, (basal 4.45 ± 0.78 mN, IBMX (PDE inhibitor) 5.47 ± 1.01 mN, (-)-CGP12177 9.34 ± 1.33 mN, (-)-bupranolol 5.79 ± 1.08 mN, n = 6) but not affected by the addition of L-748,337 (1 microM), P = 0.12, (basal 4.48 ± 1.32 mN, IBMX 7.15 ± 2.28 mN, (-)-CGP12177 12.51 ± 3.71 mN, L-748,337 10.90 ± 3.49 mN, n = 6). Cumulative concentration-effect curves for (-)-CGP12177 were not shifted to the right by L-748,337 (1 microM). The –logEC50M values of (-)-CGP12177 in the absence and presence of L-748,337 were 7.21±0.09 and 7.41±0.13, respectively (data from 25 trabeculae from 8 patients, P=0.2) The positive inotropic effects of (-)-CGP12177 (IBMX present) were not antagonized by L-748,337 but were blunted by (-)-bupranolol (1 microM). The results rule out an involvement of beta3-adrenoceptors in the positive inotropic effects (-)-CGP12177 in human right atrial myocardium and are consistent with mediation through beta1LAR. Kaumann A and Molenaar P (2008) Pharmacol Ther 118, 303-336 Skeberdis VA et al (2008) J Clin Invest, 118, 3219-3227

Limits of spherical blur determined with an adaptive optics mirror

We extended an earlier study (Vision Research, 45, 1967–1974, 2005) in which we investigated limits at which induced blur of letter targets becomes noticeable, troublesome and objectionable. Here we used a deformable adaptive optics mirror to vary spherical defocus for conditions of a white background with correction of astigmatism; a white background with reduction of all aberrations other than defocus; and a monochromatic background with reduction of all aberrations other than defocus. We used seven cyclopleged subjects, lines of three high-contrast letters as targets, 3–6 mm artificial pupils, and 0.1–0.6 logMAR letter sizes. Subjects used a method of adjustment to control the defocus component of the mirror to set the 'just noticeable', 'just troublesome' and 'just objectionable' defocus levels. For the white-no adaptive optics condition combined with 0.1 logMAR letter size, mean 'noticeable' blur limits were ±0.30, ±0.24 and ±0.23 D at 3, 4 and 6 mm pupils, respectively. White-adaptive optics and monochromatic-adaptive optics conditions reduced blur limits by 8% and 20%, respectively. Increasing pupil size from 3–6 mm decreased blur limits by 29%, and increasing letter size increased blur limits by 79%. Ratios of troublesome to noticeable, and of objectionable to noticeable, blur limits were 1.9 and 2.7 times, respectively. The study shows that the deformable mirror can be used to vary defocus in vision experiments. Overall, the results of noticeable, troublesome and objectionable blur agreed well with those of the previous study. Attempting to reduce higher-order aberrations or chromatic aberrations, reduced blur limits to only a small extent.

Measuring factors that influence the utilisation of preventive care services provided by general practitioners in Australia

Background: Relatively little research attention has been given to the development of standardised and psychometrically sound scales for measuring influences relevant to the utilisation of health services. This study aims to describe the development, validation and internal reliability of some existing and new scales to measure factors that are likely to influence utilisation of preventive care services provided by general practitioners in Australia.----- Methods: Relevant domains of influence were first identified from a literature review and formative research. Items were then generated by using and adapting previously developed scales and published findings from these. The new items and scales were pre-tested and qualitative feedback was obtained from a convenience sample of citizens from the community and a panel of experts. Principal Components Analyses (PCA) and internal reliability testing (Cronbach's alpha) were then conducted for all of the newly adapted or developed scales utilising data collected from a self-administered mailed survey sent to a randomly selected population-based sample of 381 individuals (response rate 65.6 per cent).----- Results: The PCA identified five scales with acceptable levels of internal consistency were: (1) social support (ten items), alpha 0.86; (2) perceived interpersonal care (five items), alpha 0.87, (3) concerns about availability of health care and accessibility to health care (eight items), alpha 0.80, (4) value of good health (five items), alpha 0.79, and (5) attitudes towards health care (three items), alpha 0.75.----- Conclusion The five scales are suitable for further development and more widespread use in research aimed at understanding the determinants of preventive health services utilisation among adults in the general population.

Year 8, 9 and 10 students’ understanding and access of percent knowledge

This paper reports on Years 8, 9 and 10 students’ knowledge of percent problem types, use of diagrams, and type of solution strategy. Non- and semi-proficient students displayed the expected inflexible formula approach to solution but proficient students used a flexible mixture of estimation, number sense and trial and error instead of expected schema based methods.

The population approach to falls injury prevention in older people : findings of a two community trial

Background: There is a sound rationale for the population-based approach to falls injury prevention but there is currently insufficient evidence to advise governments and communities on how they can use population-based strategies to achieve desired reductions in the burden of falls-related injury.---------- Aim: To quantify the effectiveness of a streamlined (and thus potentially sustainable and cost-effective), population-based, multi-factorial falls injury prevention program for people over 60 years of age.---------- Methods: Population-based falls-prevention interventions were conducted at two geographically-defined and separate Australian sites: Wide Bay, Queensland, and Northern Rivers, NSW. Changes in the prevalence of key risk factors and changes in rates of injury outcomes within each community were compared before and after program implementation and changes in rates of injury outcomes in each community were also compared with the rates in their respective States.---------- Results: The interventions in neither community substantially decreased the rate of falls-related injury among people aged 60 years or older, although there was some evidence of reductions in occurrence of multiple falls reported by women. In addition, there was some indication of improvements in fall-related risk factors, but the magnitudes were generally modest.---------- Conclusion: The evidence suggests that low intensity population-based falls prevention programs may not be as effective as those are intensively implemented.

Does research into sensitive areas do harm? Experiences of research participation after a child’s diagnosis with Ewing’s Sarcoma

Objective: To investigate family members’ experiences of involvement in a previous study (conducted August 1995 to June 1997) following their child’s diagnosis with Ewing’s sarcoma. Design: Retrospective survey, conducted between 1 November and 30 November 1997, using a postal questionnaire. Participants: Eighty-one of 97 families who had previously completed an in-depth interview as part of a national case–control study of Ewing’s sarcoma. Main outcome measures: Participants’ views on how participation in the previous study had affected them and what motivated them to participate. Results: Most study participants indicated that taking part in the previous study had been a positive experience. Most (n = 79 [97.5%]) believed their involvement would benefit others and were glad to have participated, despite expecting and finding some parts of the interview to be painful. Parents whose child was still alive at the time of the interview recalled participation as more painful than those whose child had died before the interview. Parents who had completed the interview less than a year before our study recalled it as being more painful than those who had completed it more than a year before. Conclusions: That people suffering bereavement are generally eager to participate in research and may indeed find it a positive experience is useful information for members of ethics review boards and other “gatekeepers”, who frequently need to determine whether studies into sensitive areas should be approved. Such information may also help members of the community to make an informed decision regarding participation in such research.

The National Aboriginal and Torres Strait Islander Women's Health Strategy on Talkin with Tiga Bayles, 98.9 AM National Indigenous Radio Service (NIRS), Wednesday 21 July 2010. (9.00 - 10.00am).

Radio Program. Talkin with Tiga Bayles, 98.9 AM National Indigenous Radio Service (NIRS), 9.00-10.00am, Wednesday 21 July 2010. (1 hour program).----- Bronwyn Fredericks discssed the National Aboriginal and Torres Strait Islander Women’s Health Strategy was launched at the Australian Women’s Health Network (AWHN) National Conference in Hobart on the 19 May 2010. Within this radio interview the background of the Strategy is discussed, funding, who did the consultations and the writing. In the interview Bronwyn Fredericks outlines the process of the Strategy’s development and its uses for the future.----- It is important to note that this Strategy does not replace other national or State and Territory documents which identify priorities and needs. The aim is to supplement existing work.

The effect of graded levels of exercise on energy intake and balance in free-living men, consuming their normal diet

Objective: To assess the effect of graded increases in exercised-induced energy expenditure (EE) on appetite, energy intake (EI), total daily EE and body weight in men living in their normal environment and consuming their usual diets. Design: Within-subject, repeated measures design. Six men (mean (s.d.) age 31.0 (5.0) y; weight 75.1 (15.96) kg; height 1.79 (0.10) m; body mass index (BMI) 23.3(2.4) kg/m2), were each studied three times during a 9 day protocol, corresponding to prescriptions of no exercise, (control) (Nex; 0 MJ/day), medium exercise level (Mex; ~1.6 MJ/day) and high exercise level (Hex; ~3.2 MJ/day). On days 1-2 subjects were given a medium fat (MF) maintenance diet (1.6 ´ resting metabolic rate (RMR)). Measurements: On days 3-9 subjects self-recorded dietary intake using a food diary and self-weighed intake. EE was assessed by continual heart rate monitoring, using the modified FLEX method. Subjects' HR (heart rate) was individually calibrated against submaximal VO2 during incremental exercise tests at the beginning and end of each 9 day study period. Respiratory exchange was measured by indirect calorimetry. Subjects completed hourly hunger ratings during waking hours to record subjective sensations of hunger and appetite. Body weight was measured daily. Results: EE amounted to 11.7, 12.9 and 16.8 MJ/day (F(2,10)=48.26; P<0.001 (s.e.d=0.55)) on the Nex, Mex and Hex treatments, respectively. The corresponding values for EI were 11.6, 11.8 and 11.8 MJ/day (F(2,10)=0.10; P=0.910 (s.e.d.=0.10)), respectively. There were no treatment effects on hunger, appetite or body weight, but there was evidence of weight loss on the Hex treatment. Conclusion: Increasing EE did not lead to compensation of EI over 7 days. However, total daily EE tended to decrease over time on the two exercise treatments. Lean men appear able to tolerate a considerable negative energy balance, induced by exercise, over 7 days without invoking compensatory increases in EI.

Merging fact with fiction : a study of year 9 students' developing scientific literacy through the writing of hybridised scientific narratives on a socioscientific issue

International assessments of student science achievement, and growing evidence of students' waning interest in school science, have ensured that the development of scientific literacy continues to remain an important educational priority. Furthermore, researchers have called for teaching and learning strategies to engage students in the learning of science, particularly in the middle years of schooling. This study extends previous national and international research that has established a link between writing and learning science. Specifically, it investigates the learning experiences of eight intact Year 9 science classes as they engage in the writing of short stories that merge scientific and narrative genres (i.e., hybridised scientific narratives) about the socioscientific issue of biosecurity. This study employed a triangulation mixed methods research design, generating both quantitative and qualitative data, in order to investigate three research questions that examined the extent to which the students' participation in the study enhanced their scientific literacy; the extent to which the students demonstrated conceptual understanding of related scientific concepts through their written artefacts and in interviews about the artefacts; and the extent to which the students' participation in the project influenced their attitudes toward science and science learning. Three aspects of scientific literacy were investigated in this study: conceptual science understandings (a derived sense of scientific literacy), the students' transformation of scientific information in written stories about biosecurity (simple and expanded fundamental senses of scientific literacy), and attitudes toward science and science learning. The stories written by students in a selected case study class (N=26) were analysed quantitatively using a series of specifically-designed matrices that produce numerical scores that reflect students' developing fundamental and derived senses of scientific literacy. All students (N=152) also completed a Likert-style instrument (i.e., BioQuiz), pretest and posttest, that examined their interest in learning science, science self-efficacy, their perceived personal and general value of science, their familiarity with biosecurity issues, and their attitudes toward biosecurity. Socioscientific issues (SSI) education served as a theoretical framework for this study. It sought to investigate an alternative discourse with which students can engage in the context of SSI education, and the role of positive attitudes in engaging students in the negotiation of socioscientific issues. Results of the study have revealed that writing BioStories enhanced selected aspects of the participants' attitudes toward science and science learning, and their awareness and conceptual understanding of issues relating to biosecurity. Furthermore, the students' written artefacts alone did not provide an accurate representation of the level of their conceptual science understandings. An examination of these artefacts in combination with interviews about the students' written work provided a more comprehensive assessment of their developing scientific literacy. These findings support extensive calls for the utilisation of diversified writing-to-learn strategies in the science classroom, and therefore make a significant contribution to the writing-to-learn science literature, particularly in relation to the use of hybridised scientific genres. At the same time, this study presents the argument that the writing of hybridised scientific narratives such as BioStories can be used to complement the types of written discourse with which students engage in the negotiation of socioscientific issues, namely, argumentation, as the development of positive attitudes toward science and science learning can encourage students' participation in the discourse of science. The implications of this study for curricular design and implementation, and for further research, are also discussed.

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.