Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

Development of a DNA based aging technique for use in fisheries assessments. Final report, Australian Fisheries Research and Development Corporation Project 2007/033.

The productivity of a fisheries resource can be quantified from estimates of recruitment, individual growth and natural and fisheries-related mortality, assuming the spatial extent of the resource has been quantified and there is minimal immigration or emigration. The sustainability of a fisheries resource is facilitated by management controls such as minimum and maximum size limits and total allowable catch. Minimum size limits are often set to allow individuals the opportunity to reproduce at least once before the chance of capture. Total allowable catches are a proportion of the population biomass, which is estimated based on known reproduction, recruitment, mortality and growth rates. In some fisheries, however, management actions are put in place without quantification of the resource through the stock assessment process. This occurs because species-specific information, for example individual growth, may not be available. In these circumstances, management actions need to be precautionary to protect against future resource collapse, but this often means that the resource is lightly exploited. Consequently, the productivity of the resource is not fully realised. Australia’s most valuable fisheries are invertebrate fisheries (Australian Department of Agriculture Fisheries and Forestry, 2008). For example, Australian fisheries (i.e. excluding aquaculture) production of crustaceans (largely prawns, rock lobster and crab) was 41,000 tonnes in 2006/7, worth $778 million. Production from mollusc (largely abalone, scallops, oysters and squid) fisheries was 39,000 tonnes, worth $502 million. Together, in 2006/7 crustacean and mollusc fisheries represented 58% of the total value of Australian wild fisheries production. Sustainable management of Australia’s invertebrate fisheries is frustrated by the lack of data on species-specific growth rates. This project investigated a new method to estimate age, and hence individual growth rates, in invertebrate fisheries species. The principle behind the new aging method was that telomeres (i.e. DNA end-caps of chromosomes) get shorter as an individual gets older. We studied commercial crustacean and molluscan species. A vertebrate fish species (silver perch, Bidyanus bidyanus) was used as a control to standardise our work against the literature. We found a clear relationship between telomere length and shell size for temperate abalone (Haliotis rubra). Further research is recommended before the method can be implemented to assist management of wildharvested abalone populations. Age needs to be substituted for shell size in the relationship and it needs to be studied for abalone from several regions. This project showed that telomere length declined with increasing age in Sydney rock oysters (Saccostrea glomerata) and was affected by regional variation. A relationship was not apparent between telomere length and age (or size as a surrogate for age) for crustacean species (school prawns, Metapenaeus macleayi; eastern rock lobster, Sagmariasus verreauxi; southern rock lobster, Jasus edwardsii; and spanner crabs, Ranina ranina). For school prawns, there was no difference between telomere length in males and females. Further research is recommended, however, as telomeric DNA from crustaceans was difficult to analyse using the terminal restriction fragment (TRF) assay. Telomere lengths of spanner crabs and lobsters were at the upper limit of resolution of the assay used and results were affected by degradation and possible contamination of telomeric DNA. It is possible that telomere length is an indicator of remaining lifespan in molluscan and crustacean individuals, as suggested for some vertebrate species (e.g. Monaghan, 2010). Among abalone of similar shell size and among lobster pueruli, there was evidence of individuals having significantly longer or shorter telomeres than the group average. At a population level, this may be a surrogate for estimates of future natural mortality, which may have usefulness in the management of those populations. The method used to assay telomere length (terminal restriction fragment assay) performed adequately for most species, but it was too expensive and time-consuming to be considered a useful tool for gathering information for fisheries management. Research on alternative methods is strongly recommended.

IUCN classification zones concord with, but underestimate, the population genetic structure of the zebra shark Stegostoma fasciatum in the Indo-West Pacific

The Indo-West Pacific (IWP), from South Africa in the western Indian Ocean to the western Pacific Ocean, contains some of the most biologically diverse marine habitats on earth, including the greatest biodiversity of chondrichthyan fishes. The region encompasses various densities of human habitation leading to contrasts in the levels of exploitation experienced by chondrichthyans, which are targeted for local consumption and export. The demersal chondrichthyan, the zebra shark, Stegostoma fasciatum, is endemic to the IWP and has two current regional International Union for the Conservation of Nature (IUCN) Red List classifications that reflect differing levels of exploitation: ‘Least Concern’ and ‘Vulnerable’. In this study, we employed mitochondrial ND4 sequence data and 13 microsatellite loci to investigate the population genetic structure of 180 zebra sharks from 13 locations throughout the IWP to test the concordance of IUCN zones with demographic units that have conservation value. Mitochondrial and microsatellite data sets from samples collected throughout northern Australia and Southeast Asia concord with the regional IUCN classifications. However, we found evidence of genetic subdivision within these regions, including subdivision between locations connected by habitat suitable for migration. Furthermore, parametric FST analyses and Bayesian clustering analyses indicated that the primary genetic break within the IWP is not represented by the IUCN classifications but rather is congruent with the Indonesian throughflow current. Our findings indicate that recruitment to areas of high exploitation from nearby healthy populations in zebra sharks is likely to be minimal, and that severe localized depletions are predicted to occur in zebra shark populations throughout the IWP region.

An evaluation of electrofishing as a control measure for an invasive tilapia (Oreochromis mossambicus) population in northern Australia.

Combating the spread of invasive fish is problematic, with eradication rarely possible and control options varying enormously in their effectiveness. In two small impoundments in north-eastern Australia, an electrofishing removal program was conducted to control an invasive tilapia population. We hypothesised that electrofishing would reduce the population density of Oreochromis mossambicus (Mozambique tilapia), to limit the risk of downstream spread into areas of high conservation value. We sampled by electrofishing monthly for 33 months. Over this period, there was an 87% decline in catch per unit effort (CPUE) of mature fish, coupled with a corresponding increase of 366% in the number of juveniles, suggesting a density-dependent response in the stock-recruitment relationship for the population. Temperature was inversely related to CPUE (r=0.43, lag=10 days), implying greater electrofishing efficiency in cooler months. The reduction in breeding stock is likely to reduce the risk of spread and render the population vulnerable to other control measures such as netting and/or biological control. Importantly, the current study suggests routine electrofishing may be a useful control tool for invasive fish in small impoundments when the use of more destructive techniques, such as piscicides, is untenable.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Assessing the biological control potential of an adventitiously-established "pest", Scirtothrips aurantii (Faure), on a weed, Bryophyllum delagoense (Eckl. & Zeyh.), in Queensland

South African citrus thrips (Scirtothrips aurantii) established adventitiously in Australia. Although it is a major horticultural pest in Africa, it is now advocated as a possible biological control agent against Bryophyllum delagoense Eckl. & Zeyh. (Crassulaceae). To evaluate the biocontrol potential of S. aurantii a two year field study was conducted on the western Darling Downs of southern Queensland. Imidacloprid insecticide was applied to two quadrats at each of 18 field sites to assess, in the absence of S. aurantii, the persistence of individual plants and to quantify propagule production and recruitment by this declared weed. A third quadrat was left, as a control, to be infested naturally by S. aurantii. When released from herbivory by thrips in the field, plants grew significantly more, flowered more, and were significantly more fecund than plants in the quadrats with S. aurantii. Increases in growth and fecundity translated into significantly increased plant numbers but not increased recruitment. Recruitment even declined in experimental quadrats, through the indirect effects of releasing plants from herbivory. Field sampling also revealed that S. aurantii may be sensitive to seasonal climatic fluctuations. These and other local climatic influences may limit the biological control potential of the insect.

Evidence-based perspective on CP rehabilitation : Reviews on physiotherapy, physiotherapy-related motor-based interventions and orthotic devices

This thesis utilises an evidence-based approach to critically evaluate and summarize effectiveness research on physiotherapy, physiotherapy-related motor-based interventions and orthotic devices in children and adolescents with cerebral palsy (CP). It aims to assess the methodological challenges of the systematic reviews and trials, to evaluate the effectiveness of interventions in current use, and to make suggestions for future trials Methods: Systematic reviews were searched from computerized bibliographic databases up to August 2007 for physiotherapy and physiotherapy-related interventions, and up to May 2003 for orthotic devices. Two reviewers independently identified, selected, and assessed the quality of the reviews using the Overview Quality Assessment Questionnaire complemented with decision rules. From a sample of 14 randomized controlled trials (RCT) published between January 1990 and June 2003 we analysed the methods of sampling, recruitment, and comparability of groups; defined the components of a complex intervention; identified outcome measures based on the International Classification of Functioning, Disability and Health (ICF); analysed the clinical interpretation of score changes; and analysed trial reporting using a modified 33-item CONSORT (Consolidated Standards of Reporting Trials) checklist. The effectiveness of physiotherapy and physiotherapy-related interventions in children with diagnosed CP was evaluated in a systematic review of randomised controlled trials that were searched from computerized databases from January 1990 up to February 2007. Two reviewers independently assessed the methodological quality, extracted the data, classified the outcomes using the ICF, and considered the level of evidence according to van Tulder et al. (2003). Results: We identified 21 reviews on physiotherapy and physiotherapy-related interventions and five on orthotic devices. These reviews summarized 23 or 5 randomised controlled trials and 104 or 27 observational studies, respectively. Only six reviews were of high quality. These found some evidence supporting strength training, constraint-induced movement therapy or hippotherapy, and insufficient evidence on comprehensive interventions. Based on the original studies included in the reviews on orthotic devices we found some short-term effects of lower limb casting on passive range of movement, and of ankle-foot orthoses on equinus walk. Long term effects of lower limb orthoses have not been studied. Evidence of upper limb casting or orthoses is conflicting. In the sample of 14 RCTs, most trials used simple randomisation, complemented with matching or stratification, but only three specified the concealed allocation. Numerous studies provided sufficient details on the components of a complex intervention, but the overlap of outcome measures across studies was poor and the clinical interpretation of observed score changes was mostly missing. Almost half (48%) of the applicable CONSORT-based items (range 28 32) were reported adequately. Most reporting inadequacies were in outcome measures, sample size determination, details of the sequence generation, allocation concealment and implementation of the randomization, success of assessor blinding, recruitment and follow-up dates, intention-to-treat analysis, precision of the effect size, co-interventions, and adverse events. The systematic review identified 22 trials on eight intervention categories. Four trials were of high quality. Moderate evidence of effectiveness was established for upper extremity treatments on attained goals, active supination and developmental status, and of constraint-induced therapy on the amount and quality of hand use and new emerging behaviours. Moderate evidence of ineffectiveness was found for strength training's effect on walking speed and stride length. Conflicting evidence was found for strength training's effect on gross motor function. For the other intervention categories the evidence was limited due to the low methodological quality and the statistically insignificant results of the studies. Conclusions: The high-quality reviews provide both supportive and insufficient evidence on some physiotherapy interventions. The poor quality of most reviews calls for caution, although most reviews drew no conclusions on effectiveness due to the poor quality of the primary studies. A considerable number of RCTs of good to fair methodological and reporting quality indicate that informative and well-reported RCTs on complex interventions in children and adolescents with CP are feasible. Nevertheless, methodological improvement is needed in certain areas of the trial design and performance, and the trial authors are encouraged to follow the CONSORT criteria. Based on RCTs we established moderate evidence for some effectiveness of upper extremity training. Due to limitations in methodological quality and variations in population, interventions and outcomes, mostly limited evidence on the effectiveness of most physiotherapy interventions is available to guide clinical practice. Well-designed trials are needed, especially for focused physiotherapy interventions.

Multiple socioeconomic circumstances and health : associations and explanations among Finnish and British public sector employees

Socioeconomic health inequalities have been widely documented, with a lower social position being associated with poorer physical and general health and higher mortality. For mental health the results have been more varied. However, the mechanisms by which the various dimensions of socioeconomic circumstances are associated with different domains of health are not yet fully understood. This is related to a lack of studies tackling the interrelations and pathways between multiple dimensions of socioeconomic circumstances and domains of health. In particular, evidence from comparative studies of populations from different national contexts that consider the complexity of the causes of socioeconomic health inequalities is needed. The aim of this study was to examine the associations of multiple socioeconomic circumstances with physical and mental health, more specifically physical functioning and common mental disorders. This was done in a comparative setting of two cohorts of white-collar public sector employees, one from Finland and one from Britain. The study also sought to find explanations for the observed associations between economic difficulties and health by analysing the contribution of health behaviours, living arrangements and work-family conflicts. The survey data were derived from the Finnish Helsinki Health Study baseline surveys in 2000-2002 among the City of Helsinki employees aged 40-60 years, and from the fifth phase of the London-based Whitehall II study (1997-9) which is a prospective study of civil servants aged 35-55 years at the time of recruitment. The data collection in the two countries was harmonised to safeguard maximal comparability. Physical functioning was measured with the Short Form (SF-36) physical component summary and common mental disorders with the General Health Questionnaire (GHQ-12). Socioeconomic circumstances were parental education, childhood economic difficulties, own education, occupational class, household income, housing tenure, and current economic difficulties. Further explanatory factors were health behaviours, living arrangements and work-family conflicts. The main statistical method used was logistic regression analysis. Analyses were conducted separately for the two sexes and two cohorts. Childhood and current economic difficulties were associated with poorer physical functioning and common mental disorders generally in both cohorts and sexes. Conventional dimensions of socioeconomic circumstances i.e. education, occupational class and income were associated with physical functioning and mediated each other’s effects, but in different ways in the two cohorts: education was more important in Helsinki and occupational class in London. The associations of economic difficulties with health were partly explained by work-family conflicts and other socioeconomic circumstances in both cohorts and sexes. In conclusion, this study on two country-specific cohorts confirms that different dimensions of socioeconomic circumstances are related but not interchangeable. They are also somewhat differently associated with physical and mental domains of health. In addition to conventionally measured dimensions of past and present socioeconomic circumstances, economic difficulties should be taken into account in studies and attempts to reduce health inequalities. Further explanatory factors, particularly conflicts between work and family, should also be considered when aiming to reduce inequalities and maintain the health of employees.

Molecular Background of Common Dyslipidemias

The leading cause of death in the Western world continues to be coronary heart disease (CHD). At the root of the disease process is dyslipidemia an aberration in the relevant amounts of circulating blood lipids. Cholesterol builds up in the arterial wall and following rupture of these plaques, myocardial infarction or stroke can occur. Heart disease runs in families and a number of hereditary forms are known. The leading cause of adult dyslipidemia presently however is overweight and obesity. This thesis work presents an investigation of the molecular genetics of common, hereditary dyslipidemia and the tightly related condition of obesity. Familial combined hyperlipidemia (FCHL) is the most common hereditary dyslipidemia in man with an estimated population prevalence of 1-6%. This complex disease is characterized by elevated levels of serum total cholesterol, triglycerides or both and is observed in about 20% of individuals with premature CHD. Our group identified the disease to be associated with genetic variation in the USF1 transcription factor gene. USF1 has a key role in regulating other genes that control lipid and glucose metabolism as well as the inflammatory response all central processes in the progression of atherosclerosis and CHD. The first two works of this thesis aimed at understanding how these USF1 variants result in increased disease risk. Among the many, non-coding single-nucleotide polymorphisms (SNPs) that associated with the disease, one was found to have a functional effect. The risk-enhancing allele of this SNP seems to eradicate the ability of the important hormone insulin to induce the expression of USF1 in peripheral tissues. The resultant changes in the expression of numerous USF1 target genes over time probably enhance and accelerate the atherogenic processes. Dyslipidemias often represent an outcome of obesity and in the final work of this thesis we wanted to address the metabolic pathways related to acquired obesity. It is recognized that active processes in adipose tissue play an important role in the development of dyslipidemia, insulin resistance and other pathological conditions associated with obesity. To minimize the confounding effects of genetic differences present in most human studies, we investigated a rare collection of identical twins that differed significantly in the amount of body fat. In the obese, but otherwise healthy young adults, several notable changes were observed. In addition to chronic inflammation, the adipose tissue of the obese co-twins was characterized by a marked (47%) decrease in amount of mitochondrial DNA (mtDNA) a change associated with mitochondrial dysfunction. The catabolism of branched chain amino acids (BCAAs) was identified as the most down-regulated process in the obese co-twins. A concordant increase in the serum level of these insulin secretagogues was identified. This hyperaminoacidemia may provide the feed-back signal from insulin resistant adipose tissue to the pancreas to ensure an appropriately augmented secretory response. The down regulation of BCAA catabolism correlated closely with liver fat accumulation and insulin. The single most up-regulated gene (5.9 fold) in the obese co-twins was osteopontin (SPP1) a cytokine involved in macrophage recruitment to adipose tissue. SPP1 is here implicated as an important player in the development of insulin resistance. These studies of exceptional study samples provide better understanding of the underlying pathology in common dyslipidemias and other obesity associated diseases important for future improvement of intervention strategies and treatments to combat atherosclerosis and coronary heart disease.

Inflammation and Complement Activation in Intracranial Artery Aneurysms

Intracranial artery aneurysms (IAs) are estimated to be present in 2.3% of the population. A rupture of an IA causes subarachnoid hemorrhage, with up to 50% mortality. The annual low rupture risk of an IA indicates that most IAs never rupture. The current treatment options are invasive and somewhat risky. Thus rupture-prone IAs should be identified and this requires a better understanding of the IA wall pathobiology. Inflammatory cell infiltrations have been found to precede IA rupture, indicating the role of inflammation in IA wall degeneration and rupture. The complement system is a key mediator of inflammation and house-hold processing of injured tissue. This study aimed at identifying the role of complement activation in IA wall degeneration and the complement activators involved and determining how the complement system is regulated in the IA wall. In immunostainings, the end-product of complement activation, the terminal complement complex (TCC), was located mainly in the outer part of the IA wall, in areas that had also sustained loss of cells. In electron microscopy, the area of maximum TCC accumulation contained cellular debris and evidence of both apoptotic and necrotic cell death. Complement activation correlated with IA wall degeneration and rupture, de-endothelialization, and T-cell and CD163-positive macrophage infiltration. The complement system was found to become activated in all IAs by the classical pathway, with recruitment of alternative pathway amplification. Of the potential activators immunoglobulins G and M and oxidatively modified lipids were found in large areas. Lipid accumulation was observed to clearly colocalize with TCC and C-reactive protein. In the luminal parts of the IA wall, complement activation was limited by cellular expression of protectin (CD59) and extracellular matrix-bound inhibitors, C4b binding protein and factor H whereas the outer part of the wall lacked cells expressing protectin as well as matrix-bound factor H. In single nucleotide polymorphism-analysis, age-related macular degeneration-associated factor H Y402H polymorphism did not associate with the presence of IAs or their rupture The data suggest that complement activation and TCC formation are involved in IA wall degeneration and rupture. Complement seems to become activated by more than one specific activator. The association of complement with de-endothelialization and expression of several complement activators indicate a possible role of endothelial dysfunction and/or impaired clearance mechanisms. Impaired complement regulation seems to be associated with increased complement activation in IA walls. These results stress the role of chronic inflammation in IA wall pathobiology and the regulatory role of complement within this process. Imaging inflammation would possibly enhance the diagnostics of rupture-prone IAs, and targeting IA treatment to prevent chronic inflammation might improve IA treatment in the future.

Rab8 and Rab8-interacting proteins as players in cell polarization

Rab8 and its interacting proteins as regulators of cell polarization During the development of a multi-cellular organism, progenitor cells have to divide and migrate appropriately as well as organize their differentiation with one another, in order to produce a viable embryo. To divide, differentiate and migrate cells have to undergo polarization, a process where internal and external components such as actin, microtubules and adhesion receptors are reorganized to produce a cell that is asymmetric, with functionally different surfaces. Also in the adult organism there is a continuous need for these processes, as cells need to migrate in response to tissue damage and to fight infection. Improper regulation of cell proliferation and migration can conversely lead to disease such as cancer. GTP-binding proteins function as molecular switches by cycling between a GTP-bound (active) conformation and a GDP-bound (inactive) conformation. The Ras super-family of small GTPases are found in all eukaryotic cells. They can be functionally divided into five subfamilies. The Ras family members mainly regulate gene expression, controlling cell proliferation and differentiation. Ras was in fact the first human oncogene to be characterized, and as much as 30% of all human tumors may be directly or indirectly caused by mutations of Ras molecules The Rho family members mainly regulate cytoskeletal reorganization. Arf proteins are known to regulate vesicle budding and Rab proteins regulate vesicular transport. Ran regulates nuclear transport as well as microtubule organization during mitosis. The focus of the thesis of Katarina Hattula, is on Rab8, a small GTPase of the Rab family. Activated Rab8 has previously been shown to induce the formation of new surface extensions, reorganizing both actin and microtubules, and to have a role in directed membrane transport to cell surfaces. However, the exact membrane route it regulates has remained elusive. In the thesis three novel interactors of Rab8 are presented. Rabin8 is a Rab8-specific GEF that localizes to vesicles where it presumably recruits and activates its target Rab8. Its expression in cells leads to remodelling of actin and the formation of polarized cell surface domains. Optineurin, known to be associated with a leading cause of blindness in humans (open-angle glaucoma), is shown to interact specifically with GTP-bound Rab8. Rab8 binds to an amino-terminal region and interestingly, the Huntingtin protein binds a carboxy-terminal region of optineurin. (Aberrant Huntingtin protein is known to be the cause Huntington s disease in humans.) Co-expression of Huntingtin and optineurin enhanced the recruitment of Huntingtin to Rab8-positive vesicular structures. Furthermore, optineurin promoted cell polarization in a similar way to Rab8. A third novel interactor of Rab8 presented in this thesis is JFC1, a member of the synaptogamin-like protein (Slp) family. JFC1 interacts with Rab8 specifically in its GTP-bound form, co-localizes with endogenous Rab8 on tubular and vesicular structures, and is probably involved in controlling Rab8 membrane dynamics. Rab8 is in this thesis work clearly shown to have a strong effect on cell shape. Blocking Rab8 activity by expression of Rab8 RNAi, or by expressing the dominant negative Rab8 (T22N) mutant leads to loss of cell polarity. Conversely, cells expressing the constitutively active Rab8 (Q67L) mutant exhibit a strongly polarized phenotype. Experiments in live cells show that Rab8 is associated with macropinosomes generated at ruffling areas of the membrane. These macropinosomes fuse with or transform into tubules that move toward the cell centre, from where they are recycled back to the leading edge to participate in protrusion formation. The biogenesis of these tubules is shown to be dependent on both actin and microtubule dynamics. The Rab8-specific membrane route studied contained several markers known to be internalized and recycled (1 integrin, transferrin, transferrin receptor, cholera toxin B subunit (CTxB), and major histocompatibility complex class I protein (MHCI)). Co-expression studies revealed that Rab8 localization overlaps with that of Rab11 and Arf6. Rab8 is furthermore clearly functionally linked to Arf6. The data presented in this thesis strongly suggests a role for Rab8 as a regulator for a recycling compartment, which is involved in providing structural and regulatory components to the leading edge to participate in protrusion formation.

Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Stock assessment report on Queensland east coast red throat emperor (Lethrinus miniatus) fishery

The Red Throat Emperor fishery was assessed using an age-structured model that incorporated all available information on catch, catch per unit effort (CPUE) and age structure and a surplus production model fitted to the catch and CPUE data. The Great Barrier Reef (GBR) was divided into five regions: Townsville, Mackay, Storm Cay, Swain reefs, and Capricorn Bunker. Age structure varied greatly between regions, with fish aged 5-8 years predominating in the Townsville region, 4-7 years in the Mackay, Storm Cay and Swains regions, and 2-3 years in the Capricorn-Bunker region. These differences were explained by different age-dependent vulnerabilities to fishing between the regions. The age-structured model estimated that exploitable biomass fell to about 60% of virgin biomass in the late 1990s, due mainly to years of poor recruitment, but recovered to around 70% by 2004. Further recovery can be expected due to the fishery not meeting its total allowable commercial catch (TACC) of 700 t in recent years. The current TACC of 700 t, combined with a recreational-charter catch of around 450 t, contains little margin for error, especially in view of high year-to-year variability of recruitment of red throat emperor and stresses on the GBR from land clearing, coastal development and climate change. The state of the population needs to be monitored closely. Further data on age structures after 2000 will provide more certainty to this assessment.

Harvest strategy evaluations and co-management for the Moreton Bay Trawl Fishery

Non-Technical Summary Seafood CRC Project 2009/774. Harvest strategy evaluations and co-management for the Moreton Bay Trawl Fishery Principal Investigator: Dr Tony Courtney, Principal Fisheries Biologist Fisheries and Aquaculture, Agri-Science Queensland Department of Agriculture, Fisheries and Forestry Level B1, Ecosciences Precinct, Joe Baker St, Dutton Park, Queensland 4102 Email: tony.courtney@daff.qld.gov.au Project objectives: 1. Review the literature and data (i.e., economic, biological and logbook) relevant to the Moreton Bay trawl fishery. 2. Identify and prioritise management objectives for the Moreton Bay trawl fishery, as identified by the trawl fishers. 3. Undertake an economic analysis of Moreton Bay trawl fishery. 4. Quantify long-term changes to fishing power for the Moreton Bay trawl fishery. 5. Assess priority harvest strategies identified in 2 (above). Present results to, and discuss results with, Moreton Bay Seafood Industry Association (MBSIA), fishers and Fisheries Queensland. Note: Additional, specific objectives for 2 (above) were developed by fishers and the MBSIA after commencement of the project. These are presented in detail in section 5 (below). The project was an initiative of the MBSIA, primarily in response to falling profitability in the Moreton Bay prawn trawl fishery. The analyses were undertaken by a consortium of DAFF, CSIRO and University of Queensland researchers. This report adopted the Australian Standard Fish Names (http://www.fishnames.com.au/). Trends in catch and effort The Moreton Bay otter trawl fishery is a multispecies fishery, with the majority of the catch composed of Greasyback Prawns (Metapenaeus bennettae), Brown Tiger Prawns (Penaeus esculentus), Eastern King Prawns (Melicertus plebejus), squid (Uroteuthis spp., Sepioteuthis spp.), Banana Prawns (Fenneropenaeus merguiensis), Endeavour Prawns (Metapenaeus ensis, Metapenaeus endeavouri) and Moreton Bay bugs (Thenus parindicus). Other commercially important byproduct includes blue swimmer crabs (Portunus armatus), three-spot crabs (Portunus sanguinolentus), cuttlefish (Sepia spp.) and mantis shrimp (Oratosquilla spp.). Logbook catch and effort data show that total annual reported catch of prawns from the Moreton Bay otter trawl fishery has declined to 315 t in 2008 from a maximum of 901 t in 1990. The number of active licensed vessels participating in the fishery has also declined from 207 in 1991 to 57 in 2010. Similarly, fishing effort has fallen from a peak of 13,312 boat-days in 1999 to 3817 boat-days in 2008 – a 71% reduction. The declines in catch and effort are largely attributed to reduced profitability in the fishery due to increased operational costs and depressed prawn prices. The low prawn prices appear to be attributed to Australian aquacultured prawns and imported aquacultured vannamei prawns, displacing the markets for trawl-caught prawns, especially small species such as Greasyback Prawns which traditionally dominated landings in Moreton Bay. In recent years, the relatively high Australian dollar has resulted in reduced exports of Australian wild-caught prawns. This has increased supply on the domestic market which has also suppressed price increases. Since 2002, Brown Tiger Prawns have dominated annual reported landings in the Moreton Bay fishery. While total catch and effort in the bay have declined to historically low levels, the annual catch and catch rates of Brown Tiger Prawns have been at record highs in recent years. This appears to be at least partially attributed to the tiger prawn stock having recovered from excessive effort in previous decades. The total annual value of the Moreton Bay trawl fishery catch, including byproduct, is about $5 million, of which Brown Tiger Prawns account for about $2 million. Eastern King Prawns make up about 10% of the catch and are mainly caught in the bay from October to December as they migrate to offshore waters outside the bay where they contribute to a large mono-specific trawl fishery. Some of the Eastern King Prawns harvested in Moreton Bay may be growth overfished (i.e., caught below the size required to maximise yield or value), although the optimum size-at-capture was not determined in this study. Banana Prawns typically make up about 5% of the catch, but can exceed 20%, particularly following heavy rainfall. Economic analysis of the fishery From the economic survey, cash profits were, on average, positive for both fleet segments in both years of the survey. However, after the opportunity cost of capital and depreciation were taken into account, the residual owner-operator income was relatively low, and substantially lower than the average share of revenue paid to employed skippers. Consequently, owner-operators were earning less than their opportunity cost of their labour, suggesting that the fleets were economically unviable in the longer term. The M2 licensed fleet were, on average, earning similar boat cash profits as the T1/M1 fleet, although after the higher capital costs were accounted for the T1/M1 boats were earning substantially lower returns to owner-operator labour. The mean technical efficiency for the fleet as a whole was estimated to be 0.67. That is, on average, the boats were only catching 67 per cent of what was possible given their level of inputs (hours fished and hull units). Almost one-quarter of observations had efficiency scores above 0.8, suggesting a substantial proportion of the fleet are relatively efficient, but some are also relatively inefficient. Both fleets had similar efficiency distributions, with median technical efficiency score of 0.71 and 0.67 for the M2 and T1/M1 boats respectively. These scores are reasonably consistent with other studies of prawn trawl fleets in Australia, although higher average efficiency scores were found in the NSW prawn trawl fleet. From the inefficiency model, several factors were found to significantly influence vessel efficiency. These included the number of years of experience as skipper, the number of generations that the skipper’s family had been fishing and the number of years schooling. Skippers with more schooling were significantly more efficient than skippers with lower levels of schooling, consistent with other studies. Skippers who had been fishing longer were, in fact, less efficient than newer skippers. However, this was mitigated in the case of skippers whose family had been involved in fishing for several generations, consistent with other studies and suggesting that skill was passed through by families over successive generations. Both the linear and log-linear regression models of total fishing effort against the marginal profit per hour performed reasonably well, explaining between 70 and 84 per cent of the variation in fishing effort. As the models had different dependent variables (one logged and the other not logged) this is not a good basis for model choice. A better comparator is the square root of the mean square error (SMSE) expressed as a percentage of the mean total effort. On this criterion, both models performed very similarly. The linear model suggests that each additional dollar of average profits per hour in the fishery increases total effort by around 26 hours each month. From the log linear model, each percentage increase in profits per hour increases total fishing effort by 0.13 per cent. Both models indicate that economic performance is a key driver of fishing effort in the fishery. The effect of removing the boat-replacement policy is to increase individual vessel profitability, catch and effort, but the overall increase in catch is less than that removed by the boats that must exit the fishery. That is, the smaller fleet (in terms of boat numbers) is more profitable but the overall catch is not expected to be greater than before. This assumes, however, that active boats are removed, and that these were also taking an average level of catch. If inactive boats are removed, then catch of the remaining group as a whole could increase by between 14 and 17 per cent depending on the degree to which costs are reduced with the new boats. This is still substantially lower than historical levels of catch by the fleet. Fishing power analyses An analysis of logbook data from 1988 to 2010, and survey information on fishing gear, was performed to estimate the long-term variation in the fleet’s ability to catch prawns (known as fishing power) and to derive abundance estimates of the three most commercially important prawn species (i.e., Brown Tiger, Eastern King and Greasyback Prawns). Generalised linear models were used to explain the variation in catch as a function of effort (i.e., hours fished per day), vessel and gear characteristics, onboard technologies, population abundance and environmental factors. This analysis estimated that fishing power associated with Brown Tiger and Eastern King Prawns increased over the past 20 years by 10–30% and declined by approximately 10% for greasybacks. The density of tiger prawns was estimated to have almost tripled from around 0.5 kg per hectare in 1988 to 1.5 kg/ha in 2010. The density of Eastern King Prawns was estimated to have fluctuated between 1 and 2 kg per hectare over this time period, without any noticeable overall trend, while Greasyback Prawn densities were estimated to have fluctuated between 2 and 6 kg per hectare, also without any distinctive trend. A model of tiger prawn catches was developed to evaluate the impact of fishing on prawn survival rates in Moreton Bay. The model was fitted to logbook data using the maximum-likelihood method to provide estimates of the natural mortality rate (0.038 and 0.062 per week) and catchability (which can be defined as the proportion of the fished population that is removed by one unit of effort, in this case, estimated to be 2.5 ± 0.4 E-04 per boat-day). This approach provided a method for industry and scientists to develop together a realistic model of the dynamics of the fishery. Several aspects need to be developed further to make this model acceptable to industry. Firstly, there is considerable evidence to suggest that temperature influences prawn catchability. This ecological effect should be incorporated before developing meaningful harvest strategies. Secondly, total effort has to be allocated between each species. Such allocation of effort could be included in the model by estimating several catchability coefficients. Nevertheless, the work presented in this report is a stepping stone towards estimating essential fishery parameters and developing representative mathematical models required to evaluate harvest strategies. Developing a method that allowed an effective discussion between industry, management and scientists took longer than anticipated. As a result, harvest strategy evaluations were preliminary and only included the most valuable species in the fishery, Brown Tiger Prawns. Additional analyses and data collection, including information on catch composition from field sampling, migration rates and recruitment, would improve the modelling. Harvest strategy evaluations As the harvest strategy evaluations are preliminary, the following results should not be adopted for management purposes until more thorough evaluations are performed. The effects, of closing the fishery for one calendar month, on the annual catch and value of Brown Tiger Prawns were investigated. Each of the 12 months (i.e., January to December) was evaluated. The results were compared against historical records to determine the magnitude of gain or loss associated with the closure. Uncertainty regarding the trawl selectivity was addressed using two selectivity curves, one with a weight at 50% selection (S50%) of 7 g, based on research data, and a second with S50% of 14 g, put forward by industry. In both cases, it was concluded that any monthly closure after February would not be beneficial to the industry. The magnitude of the benefit of closing the fishery in either January or February was sensitive to which mesh selectivity curve that was assumed, with greater benefit achieved when the smaller selectivity curve (i.e., S50% = 7 g) was assumed. Using the smaller selectivity (S50% = 7 g), the expected increase in catch value was 10–20% which equates to $200,000 to $400,000 annually, while the larger selectivity curve (S50% = 14 g) suggested catch value would be improved by 5–10%, or $100,000 to $200,000. The harvest strategy evaluations showed that greater benefits, in the order of 30–60% increases in the tiger annual catch value, could have been obtained by closing the fishery early in the year when annual effort levels were high (i.e., > 10,000 boat-days). In recent years, as effort levels have declined (i.e., ~4000 boat-days annually), expected benefits from such closures are more modest. In essence, temporal closures offer greater benefit when fishing mortality rates are high. A spatial analysis of Brown Tiger Prawn catch and effort was also undertaken to obtain a better understanding of the prawn population dynamics. This indicated that, to improve profitability of the fishery, fishers could consider closing the fishery in the period from June to October, which is already a period of low profitability. This would protect the Brown Tiger Prawn spawning stock, increase catch rates of all species in the lucrative pre-Christmas period (November–December), and provide fishers with time to do vessel maintenance, arrange markets for the next season’s harvest, and, if they wish, work at other jobs. The analysis found that the instantaneous rate of total mortality (Z) for the March–June period did not vary significantly over the last two decades. As the Brown Tiger Prawn population in Moreton Bay has clearly increased over this time period, an interesting conclusion is that the instantaneous rate of natural mortality (M) must have increased, suggesting that tiger prawn natural mortality may be density-dependent at this time of year. Mortality rates of tiger prawns for June–October were found to have decreased over the last two decades, which has probably had a positive effect on spawning stocks in the October–November spawning period. Abiotic effects on the prawns The influence of air temperature, rainfall, freshwater flow, the southern oscillation index (SOI) and lunar phase on the catch rates of the four main prawn species were investigated. The analyses were based on over 200,000 daily logbook catch records over 23 years (i.e., 1988–2010). Freshwater flow was more influential than rainfall and SOI, and of the various sources of flow, the Brisbane River has the greatest volume and influence on Moreton Bay prawn catches. A number of time-lags were also considered. Flow in the preceding month prior to catch (i.e., 30 days prior, Logflow1_30) and two months prior (31–60 days prior, Logflow31_60) had strong positive effects on Banana Prawn catch rates. Average air temperature in the preceding 4-6 months (Temp121_180) also had a large positive effect on Banana Prawn catch rates. Flow in the month immediately preceding catch (Logflow1_30) had a strong positive influence on Greasyback Prawn catch rates. Air temperature in the preceding two months prior to catch (Temp1_60) had a large positive effect on Brown Tiger Prawn catch rates. No obvious or marked effects were detected for Eastern King Prawns, although interestingly, catch rates declined with increasing air temperature 4–6 months prior to catch. As most Eastern King Prawn catches in Moreton Bay occur in October to December, the results suggest catch rates decline with increasing winter temperatures. In most cases, the prawn catch rates declined with the waxing lunar phase (high luminance/full moon), and increased with the waning moon (low luminance/new moon). The SOI explains little additional variation in prawn catch rates (~ <2%), although its influence was higher for Banana Prawns. Extrapolating findings of the analyses to long-term climate change effects should be interpreted with caution. That said, the results are consistent with likely increases in abundance in the region for the two tropical species, Banana Prawns and Brown Tiger Prawns, as coastal temperatures rise. Conversely, declines in abundance could be expected for the two temperate species, Greasyback and Eastern King Prawns. Corporate management structures An examination of alternative governance systems was requested by the industry at one of the early meetings, particularly systems that may give them greater autonomy in decision making as well as help improve the marketing of their product. Consequently, a review of alternative management systems was undertaken, with a particular focus on the potential for self-management of small fisheries (small in terms of number of participants) and corporate management. The review looks at systems that have been implemented or proposed for other small fisheries internationally, with a particular focus on self-management as well as the potential benefits and challenges for corporate management. This review also highlighted particular opportunities for the Moreton Bay prawn fishery. Corporate management differs from other co-management and even self-management arrangements in that ‘ownership’ of the fishery is devolved to a company in which fishers and government are shareholders. The company manages the fishery as well as coordinates marketing to ensure that the best prices are received and that the catch taken meets the demands of the market. Coordinated harvesting will also result in increased profits, which are returned to fishers in the form of dividends. Corporate management offers many of the potential benefits of an individual quota system without formally implementing such a system. A corporate management model offers an advantage over a self-management model in that it can coordinate both marketing and management to take advantage of this unique geographical advantage. For such a system to be successful, the fishery needs to be relatively small and self- contained. Small in this sense is in terms of number of operators. The Moreton Bay prawn fishery satisfies these key conditions for a successful self-management and potentially corporate management system. The fishery is small both in terms of number of participants and geography. Unlike other fisheries that have progressed down the self-management route, the key market for the product from the Moreton Bay fishery is right at its doorstep. Corporate management also presents a number of challenges. First, it will require changes in the way fishers operate. In particular, the decision on when to fish and what to catch will be taken away from the individual and decided by the collective. Problems will develop if individuals do not join the corporation but continue to fish and market their own product separately. While this may seem an attractive option to fishers who believe they can do better independently, this is likely to be just a short- term advantage with an overall long-run cost to themselves as well as the rest of the industry. There are also a number of other areas that need further consideration, particularly in relation to the allocation of shares, including who should be allocated shares (e.g. just boat owners or also some employed skippers). Similarly, how harvesting activity is to be allocated by the corporation to the fishers. These are largely issues that cannot be answered without substantial consultation with those likely to be affected, and these groups cannot give these issues serious consideration until the point at which they are likely to become a reality. Given the current structure and complexity of the fishery, it is unlikely that such a management structure will be feasible in the short term. However, the fishery is a prime candidate for such a model, and development of such a management structure in the future should be considered as an option for the longer term.

Parasites as indicators of movement and population connectivity of a non-diadromous, tropical estuarine teleost: king threadfin Polydactylus macrochir

Temporal and spatial patterns in parasite assemblages were examined to evaluate the degree of movement and connectivity of post-recruitment life-history stages of a large, non-diadromous tropical estuarine teleost, king threadfin Polydactylus macrochir, collected from 18 locations across northern Australia. Ten parasites types (juvenile stages of two nematodes and seven cestodes, and adults of an acanthocephalan) were deemed to be suitable for use as biological tags, in that they were considered to have a long residence time in the fish, were relatively easy to find and were morphologically very different to each other which aided discrimination. Univariate and discriminant function analysis of these parasites revealed little difference in temporal replicates collected from five locations, suggesting that the parasite communities were stable over the timeframes explored. Univariate, discriminant function, and BrayCurtis similarity analyses indicated significant spatial heterogeneity, with BrayCurtis classification accuracies ranging from 55 to 100% for locations in north-western and northern Australia, 24 to 88% in the Gulf of Carpentaria, and 39 to 88% on the east coast of Queensland. Few differences were observed among locations separated by <200 km. The observed patterns of parasite infection are in agreement with concurrent studies of movement and connectivity of P. macrochir in that they indicate a complex population structure across northern Australia. These results should be considered when reviewing the management arrangements for this species.