The association of single nucleotide polymorphisms with endometrial cancer risk and prognosis

Endometrial cancer is one of the most common female diseases in developed nations and is the most commonly diagnosed gynaecological cancer in Australia. The disease is commonly classified by histology: endometrioid or non-endometrioid endometrial cancer. While non-endometrioid endometrial cancers are accepted to be high-grade, aggressive cancers, endometrioid cancers (comprising 80% of all endometrial cancers diagnosed) generally carry a favourable patient prognosis. However, endometrioid endometrial cancer patients endure significant morbidity due to surgery and radiotherapy used for disease treatment, and patients with recurrent disease have a 5-year survival rate of less than 50%. Genetic analysis of women with endometrial cancer could uncover novel markers associated with disease risk and/or prognosis, which could then be used to identify women at high risk and for the use of specialised treatments. Proteases are widely accepted to play an important role in the development and progression of cancer. This PhD project hypothesised that SNPs from two protease gene families, the matrix metalloproteases (MMPs, including their tissue inhibitors, TIMPs) and the tissue kallikrein-related peptidases (KLKs) would be associated with endometrial cancer susceptibility and/or prognosis. In the first part of this study, optimisation of the genotyping techniques was performed. Results from previously published endometrial cancer genetic association studies were attempted to be validated in a large, multicentre replication set (maximum cases n = 2,888, controls n = 4,483, 3 studies). The rs11224561 progesterone receptor SNP (PGR, A/G) was observed to be associated with increased endometrial cancer risk (per A allele OR 1.31, 95% CI 1.12-1.53; p-trend = 0.001), a result which was initially reported among a Chinese sample set. Previously reported associations for the remaining 8 SNPs investigated for this section of the PhD study were not confirmed, thereby reinforcing the importance of validation of genetic association studies. To examine the effect of SNPs from the MMP and KLK families on endometrial cancer risk, we selected the most significantly associated MMP and KLK SNPs from genome-wide association study analysis (GWAS) to be genotyped in the GWAS replication set (cases n = 4,725, controls n = 9,803, 13 studies). The significance of the MMP24 rs932562 SNP was unchanged after incorporation of the stage 2 samples (Stage 1 per allele OR 1.18, p = 0.002; Combined Stage 1 and 2 OR 1.09, p = 0.002). The rs10426 SNP, located 3' to KLK10 was predicted by bioinformatic analysis to effect miRNA binding. This SNP was observed in the GWAS stage 1 result to exhibit a recessive effect on endometrial cancer risk, a result which was not validated in the stage 2 sample set (Stage 1 OR 1.44, p = 0.007; Combined Stage 1 and 2 OR 1.14, p = 0.08). Investigation of the regions imputed surrounding the MMP, TIMP and KLK genes did not reveal any significant targets for further analysis. Analysis of the case data from the endometrial cancer GWAS to identify genetic variation associated with cancer grade did not reveal SNPs from the MMP, TIMP or KLK genes to be statistically significant. However, the representation of SNPs from the MMP, TIMP and KLK families by the GWAS genotyping platform used in this PhD project was examined and observed to be very low, with the genetic variation of four genes (MMP23A, MMP23B, MMP28 and TIMP1) not captured at all by this technique. This suggests that comprehensive candidate gene association studies will be required to assess the role of SNPs from these genes with endometrial cancer risk and prognosis. Meta-analysis of gene expression microarray datasets curated as part of this PhD study identified a number of MMP, TIMP and KLK genes to display differential expression by endometrial cancer status (MMP2, MMP10, MMP11, MMP13, MMP19, MMP25 and KLK1) and histology (MMP2, MMP11, MMP12, MMP26, MMP28, TIMP2, TIMP3, KLK6, KLK7, KLK11 and KLK12). In light of these findings these genes should be prioritised for future targeted genetic association studies. Two SNPs located 43.5 Mb apart on chromosome 15 were observed from the GWAS analysis to be associated with increased endometrial cancer grade, results that were validated in silico in two independent datasets. One of these SNPs, rs8035725 is located in the 5' untranslated region of a MYC promoter binding protein DENND4A (Stage 1 OR 1.15, p = 9.85 x 10P -5 P, combined Stage 1 and in silico validation OR 1.13, p = 5.24 x 10P -6 P). This SNP has previously been reported to alter the expression of PTPLAD1, a gene involved in the synthesis of very long fatty acid chains and in the Rac1 signaling pathway. Meta-analysis of gene expression microarray data found PTPLAD1 to display increased expression in the aggressive non-endometrioid histology compared with endometrioid endometrial cancer, suggesting that the causal SNP underlying the observed genetic association may influence expression of this gene. Neither rs8035725 nor significant SNPs identified by imputation were predicted bioinformatically to affect transcription factor binding sites, indicating that further studies are required to assess their potential effect on other regulatory elements. The other grade- associated SNP, rs6606792, is located upstream of an inferred pseudogene, ELMO2P1 (Stage 1 OR 1.12, p = 5 x 10P -5 P; combined Stage 1 and in silico validation OR 1.09, p = 3.56 x 10P -5 P). Imputation of the ±1 Mb region surrounding this SNP revealed a cluster of significantly associated variants which are predicted to abolish various transcription factor binding sites, and would be expected to decrease gene expression. ELMO2P1 was not included on the microarray platforms collected for this PhD, and so its expression could not be investigated. However, the high sequence homology of ELMO2P1 with ELMO2, a gene important to cell motility, indicates that ELMO2 could be the parent gene for ELMO2P1 and as such, ELMO2P1 could function to regulate the expression of ELMO2. Increased expression of ELMO2 was seen to be associated with increasing endometrial cancer grade, as well as with aggressive endometrial cancer histological subtypes by microarray meta-analysis. Thus, it is hypothesised that SNPs in linkage disequilibrium with rs6606792 decrease the transcription of ELMO2P1, reducing the regulatory effect of ELMO2P1 on ELMO2 expression. Consequently, ELMO2 expression is increased, cell motility is enhanced leading to an aggressive endometrial cancer phenotype. In summary, these findings have identified several areas of research for further study. The results presented in this thesis provide evidence that a SNP in PGR is associated with risk of developing endometrial cancer. This PhD study also reports two independent loci on chromosome 15 to be associated with increased endometrial cancer grade, and furthermore, genes associated with these SNPs to be differentially expressed according in aggressive subtypes and/or by grade. The studies reported in this thesis support the need for comprehensive SNP association studies on prioritised MMP, TIMP and KLK genes in large sample sets. Until these studies are performed, the role of MMP, TIMP and KLK genetic variation remains unclear. Overall, this PhD study has contributed to the understanding of genetic variation involvement in endometrial cancer susceptibility and prognosis. Importantly, the genetic regions highlighted in this study could lead to the identification of novel gene targets to better understand the biology of endometrial cancer and also aid in the development of therapeutics directed at treating this disease.

The consistency of crash involvement recall across time

Introduction Road safety researchers rely heavily on self-report data to explore the aetiology of crash risk. However, researchers consistently acknowledge a range of limitations associated with this methodological approach (e.g., self-report bias), which has been hypothesised to reduce the predictive efficacy of scales. Although well researched in other areas, one important factor often neglected in road safety studies is the fallibility of human memory. Given accurate recall is a key assumption in many studies, the validity and consistency of self-report data warrants investigation. The aim of the current study was to examine the consistency of self-report data of crash history and details of the most recent reported crash on two separate occasions. Materials & Method A repeated measures design was utilised to examine the self-reported crash involvement history of 214 general motorists over a two month period. Results A number of interesting discrepancies were noted in relation to number of lifetime crashes reported by the participants and the descriptions of their most recent crash across the two occasions. Of the 214 participants who reported having been involved in a crash, 35 (22.3%) reported a lower number of lifetime crashes as Time 2, than at Time 1. Of the 88 drivers who reported no change in number of lifetime crashes, 10 (11.4%) described a different most recent crash. Additionally, of the 34 reporting an increase in the number of lifetime crashes, 29 (85.3%) of these described the same crash on both occasions. Assessed as a whole, at least 47.1% of participants made a confirmed mistake at Time 1 or Time 2. Conclusions These results raise some doubt in regard to the accuracy of memory recall across time. Given that self-reported crash involvement is the predominant dependent variable used in the majority of road safety research, this issue warrants further investigation. Replication of the study with a larger sample size that includes multiple recall periods would enhance understanding into the significance of this issue for road safety methodology.

The role of immunoglobulins and their transporters in urogenital Chlamydial infections

Chlamydia trachomatis infections of the male and female reproductive tracts are the world's leading sexually transmitted bacterial disease, and can lead to damaging pathology, scarring and infertility. The resolution of chlamydial infection requires the development of adaptive immune responses to infection, and includes cell-mediated and humoral immunity. Whilst cluster of differentiation (CD)4+ T cells are known to be essential in clearance of infection [1], they are also associated with immune cell infiltration, autoimmunity and infertility in the testes [2-3]. Conversely, antibodies are less associated with inflammation, are readily transported into the reproductive tracts, and can offer lumenal neutralization of chlamydiae prior to infection. Antibodies, or immunoglobulins (Ig), play a supportive role in the resolution of chlamydial infections, and this thesis sought to define the function of IgA and IgG, against a variety of chlamydial antigens expressed during the intracellular and extracellular stages of the chlamydial developmental cycle. Transport of IgA and IgG into the mucosal lumen is facilitated by receptor-mediated transcytosis yet the expression profile (under normal conditions and during urogenital chlamydial infection) of the polymeric immunoglobulin receptor (pIgR) and the neonatal Fc receptor (FcRn) remains unknown. The expression profile of pIgR and FcRn in the murine male reproductive tract was found to be polarized to the lower and upper reproductive tract tissues respectively. This demonstrates that the two receptors have a tissue tropism, which must be considered when targeting pathogens that colonize different sites. In contrast, the expression of pIgR and FcRn in the female mouse was found to be distributed in both the upper and lower reproductive tracts. When urogenitally infected with Chlamydia muridarum, both male and female reproductive tracts up-regulated expression of pIgR and down-regulated expression of FcRn. Unsurprisingly, the up-regulation of pIgR increased the concentration of IgA in the lumen. However, down-regulation of FcRn, prevented IgG uptake and led to an increase or pooling of IgG in lumenal secretions. As previous studies have identified the importance of pIgR-mediated delivery of IgA, as well as the potential of IgA to bind and neutralize intracellular pathogens, IgA against a variety of chlamydial antigens was investigated. The protection afforded by IgA against the extracellular antigen major outer membrane protein (MOMP), was found to be dependent on pIgR expression in vitro and in vivo. It was also found that in the absence of pIgR, no protection was afforded to mice previously immunized with MOMP. The protection afforded from polyclonal IgA against the intracellular chlamydial antigens; inclusion membrane protein A (IncA), inclusion membrane proteins (IncMem) and secreted chlamydial protease-like activity factor (CPAF) were produced and investigated in vitro. Antigen-specific intracellular IgA was found to bind to the respective antigen within the infected cell, but did not significantly reduce inclusion formation (p > 0.05). This suggests that whilst IgA specific for the selected antigens was transported by pIgR to the chlamydial inclusion, it was unable to prevent growth. Similarly, immunization of male mice with intracellular chlamydial antigens (IncA or IncMem), followed by depletion CD4+ T cells, and subsequent urogenital C. muridarum challenge, provided minimal pIgR-mediated protection. Wild type male mice immunized with IncA showed a 57 % reduction (p < 0.05), and mice deficient in pIgR showed a 35 % reduction (p < 0.05) in reproductive tract chlamydial burden compared to control antigen, and in the absence of CD4+ T cells. This suggests that pIgR and secretory IgA (SIgA) were playing a protective role (21 % pIgR-mediated) in unison with another antigen-specific immune mechanism (36 %). Interestingly, IgA generated during a primary respiratory C. muridarum infection did not provide a significant amount of protection to secondary urogenital C. muridarum challenge. Together, these data suggest that IgA specific for an extracellular antigen (MOMP) can play a strong protective role in chlamydial infections, and that IgA targeting intracellular antigens is also effective but dependent on pIgR expression in tissues. However, whilst not investigated here, IgA targeting and blocking other intracellular chlamydial antigens, that are more essential for replication or type III secretion, may be more efficacious in subunit vaccines. Recently, studies have demonstrated that IgG can neutralize influenza virus by trafficking IgG-bound virus to lysosomes [4]. We sought to determine if this process could also traffic chlamydial antigens for degradation by lysosomes, despite Chlamydia spp. actively inhibiting fusion with the host endocytic pathway. As observed in pIgR-mediated delivery of anti-IncA IgA, FcRn similarly transported IgG specific for IncA which bound the inclusion membrane. Interestingly, FcRn-mediated delivery of anti-IncA IgG significantly decreased inclusion formation by 36 % (p < 0.01), and induced aberrant inclusion morphology. This suggests that unlike IgA, IgG can facilitate additional host cellular responses which affect the intracellular niche of chlamydial growth. Fluorescence microscopy revealed that IgG also bound the inclusion, but unlike influenza studies, did not induce the recruitment of lysosomes. Notably, anti-IncA IgG recruited sequestosomes to the inclusion membrane, markers of the ubiquitin/proteasome pathway and major histocompatibility complex (MHC) class I loading. To determine if the protection against C. muridarum infection afforded by IncA IgG in vitro translated in vivo, wild type mice and mice deficient in functional FcRn and MHC-I, were immunized, depleted of CD4+, and urogenitally infected with C. muridarum. Unlike in pIgR-deficient mice, the protection afforded from IncA immunization was completely abrogated in mice lacking functional FcRn and MHC-I/CD8+. Thus, both anti-IncA IgA and IgG can bind the inclusion in a pIgR and FcRn-mediated manner, respectively. However, only IgG mediates a higher reduction in chlamydial infection in vitro and in vivo suggesting more than steric blocking of IncA had occurred. Unlike anti-MOMP IgA, which reduced chlamydial infection of epithelial cells and male mouse tissues, IgG was found to enhance infectivity in vitro, and in vivo. Opsonization of EBs with MOMP-IgG enhanced inclusion formation of epithelial cells in a MOMP-IgG dose-dependent and FcRn-dependent manner. When MOMP-IgG opsonized EBs were inoculated into the vagina of female mice, a small but non-significant (p > 0.05) enhancement of cervicovaginal C. muridarum shedding was observed three days post infection in mice with functional FcRn. Interestingly, infection with opsonized EBs reduced the intensity of the peak of infection (day six) but protracted the duration of infection by 60 % in wild type mice only. Infection with EBs opsonized in IgG also significantly increased (p < 0.05) hydrosalpinx formation in the oviducts and induced lymphocyte infiltration uterine horns. As MOMP is an immunodominant antigen, and is widely used in vaccines, the ability of IgG specific to extracellular chlamydial antigens to enhance infection and induce pathology needs to be considered. Together, these data suggest that immunoglobulins play a dichotomous role in chlamydial infections, and are dependent on antigen specificity, FcRn and pIgR expression. FcRn was found to be highly expressed in upper male reproductive tract, whilst pIgR was dominantly expressed in the lower reproductive tract. Conversely, female mice expressed FcRn and pIgR in both the lower and upper reproductive tracts. In response to a normal chlamydial infection, pIgR is up-regulated increasing secretory IgA release, but FcRn is down-regulated preventing IgG uptake. Similarly to other studies [5-6], we demonstrate that IgA and IgG generated during primary chlamydial infections plays a minor role in recall immunity, and that antigen-specific subunit vaccines can offer more protection. We also show that both IgA and IgG can be used to target intracellular chlamydial antigens, but that IgG is more effective. Finally, IgA against the extracellular antigen MOMP can afford protection, whist IgG plays a deleterious role by increasing infectivity and inducing damaging immunopathology. Further investigations with additional antigens or combination subunit vaccines will enhance our understanding the protection afforded by antibodies against intracellular and extracellular pathogenic antigens, and help improve the development of an efficacious chlamydial vaccine.

The molecular bases of training adaptation

Skeletal muscle is a malleable tissue capable of altering the type and amount of protein in response to disruptions to cellular homeostasis. The process of exercise-induced adaptation in skeletal muscle involves a multitude of signalling mechanisms initiating replication of specific DNA genetic sequences, enabling subsequent translation of the genetic message and ultimately generating a series of amino acids that form new proteins. The functional consequences of these adaptations are determined by training volume, intensity and frequency, and the half-life of the protein. Moreover, many features of the training adaptation are specific to the type of stimulus, such as the mode of exercise. Prolonged endurance training elicits a variety of metabolic and morphological changes, including mitochondrial biogenesis, fast-to-slow fibre-type transformation and substrate metabolism. In contrast, heavy resistance exercise stimulates synthesis of contractile proteins responsible for muscle hypertrophy and increases in maximal contractile force output. Concomitant with the vastly different functional outcomes induced by these diverse exercise modes, the genetic and molecular mechanisms of adaptation are distinct. With recent advances in technology, it is now possible to study the effects of various training interventions on a variety of signalling proteins and early-response genes in skeletal muscle. Although it cannot presently be claimed that such scientific endeavours have influenced the training practices of elite athletes, these new and exciting technologies have provided insight into how current training techniques result in specific muscular adaptations, and may ultimately provide clues for future and novel training methodologies. Greater knowledge of the mechanisms and interaction of exercise-induced adaptive pathways in skeletal muscle is important for our understanding of the aetiology of disease, maintenance of metabolic and functional capacity with aging, and training for athletic performance. This article highlights the effects of exercise on molecular and genetic mechanisms of training adaptation in skeletal muscle.

Preliminary bond-slip model for CFRP sheets bonded to steel plates

Carbon fiber reinforced polymer (CFRP) sheets have established a strong position as an effective method for innovative structural rehabilitation. However, the use of externally bonded CFRP in the repair and rehabilitation of steel structures is a relatively new technique that has the potential to improve the way structures are repaired. An important step toward understanding bond behaviour is to have an estimation of local bond stress versus slip relationship. The current study aims to establish the bond-slip model for CFRP sheets bonded to steel plate. To obtain the shear stress versus slippage relationship, a series of double strap tension type bond tests were conducted. This paper reports on the findings of the experimental studies. The strain and stress distributions measured in the specimens for two different bond lengths. The results show a preliminary bi-linear bond-slip model may be adopted for CFRP sheet bonded with steel plate.

The essential parameters of a resource-based carrying capacity assessment model : an Australian case study

Carrying capacity assessments model a population’s potential self-sufficiency. A crucial first step in the development of such modelling is to examine the basic resource-based parameters defining the population’s production and consumption habits. These parameters include basic human needs such as food, water, shelter and energy together with climatic, environmental and behavioural characteristics. Each of these parameters imparts land-usage requirements in different ways and varied degrees so their incorporation into carrying capacity modelling also differs. Given that the availability and values of production parameters may differ between locations, no two carrying capacity models are likely to be exactly alike. However, the essential parameters themselves can remain consistent so one example, the Carrying Capacity Dashboard, is offered as a case study to highlight one way in which these parameters are utilised. While examples exist of findings made from carrying capacity assessment modelling, to date, guidelines for replication of such studies in other regions and scales have largely been overlooked. This paper addresses such shortcomings by describing a process for the inclusion and calibration of the most important resource-based parameters in a way that could be repeated elsewhere.

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites

Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.

Replicating satellite RNA induces sequence-specific DNA methylation and truncated transcripts in plants

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding β-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ∼22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants

A series of improved vectors have been constructed that are suitable for use in Agrobacterium tumefaciens-mediated monocot transformation. These binary vectors have several useful features, including the selectable marker genes bar (phosphinothricin resistance) or hph (hygromycin resistance) driven by either the cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin promoter, a high-copy-number replication origin that allows reliable mini-prep DNA isolation from Escherichia coli, and a polylinker sequence into which target genes can be easily inserted. A significant improvement has been made to the hph gene by the introduction of an intron into its coding region. The presence of the intron abolishes hph expression in A. tumefaciens, rendering the bacterium susceptible to the selective agent hygromycin B. The use of such an intron-hph vector thus enables the antibiotic in plant culture media to function as both a selective agent for transformed tissue and as a contraselective agent for A. tumefaciens growth, thus minimising the overgrowth of A. tumefaciens on plant tissues during transformation. Furthermore, the intron appears to be correctly spliced in plant cells and significantly enhances hph expression in transformed rice tissue. In our experiments, the use of the intron-hph vector increased the frequency of rice transformation and has enabled the production of transgenic barley.

The Yd2 resistance to barley yellow dwarf virus is effective in barley plants but not in their leaf protoplasts

The Yd2 gene for “resistance” to barley yellow dwarf virus (BYDV) has been widely used in barley (Hordeum vulgare). We have tested Australian isolates of BYDV of varying severity against barley genotypes with and without the Yd2 gene and report here a positive relationship between symptoms and virus levels determined by ELISA. Cultivar Shannon is the result of backcrossing the resistant line CI 3208 to cultivar Proctor, a susceptible line. It appears to be intermediate in reaction to BYDV between Proctor and CI 3208, although it carries the major gene, Yd2. Unlike the whole plant studies, no significant differences were observed with regard to the ability of protoplasts derived from these various genotypes to support BYDV replication. It is therefore demonstrated for the first time that the Yd2 gene is not among the small number of resistance genes which are effective against virus replication in isolated protoplasts.

Small RNA sequencing of potato leafroll virus-infected plants reveals an additional subgenomic RNA encoding a sequence-specific RNA-binding protein

Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3' proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5' transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ~500. nt from the 3' terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5' transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5' of PLRV genomic RNA. © 2013.

Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes

Efficient transformation of barley cv. Schooner was achieved using Agrobacterium delivery, hygromycin or bialaphos selection and embryogenic callus. Using this system, transgenic plants were generated that contained either the green fluorescent protein gene, or transgenes derived from barley yellow dwarf (BYDV) and cereal yellow dwarf (CYDV) viruses. Many of these plants contained 1-3 transgene copies that were inherited in a simple Mendelian manner. Some plants containing BYDV and/or CYDV derived transgenes showed reduced virus symptoms and rates of viral replication when challenged with the appropriate virus. The ability to transform Schooner is a significant advance for the Australian barley industry, as this elite malting variety is, and has for the last 15 years been, the most widely grown barley variety in eastern Australia.

Separating shadows

This practice-led research is in the field of dance creation and performance. The dancer, unlike many artists from different fields, has to contend with the added complexity of being a creative tool. Grove states, ‘..they are the phenomenon of self-creation twice over, being both the artist and the work of art’ (2005, 44). A dancer’s success relies on the quality of their instrument and their ability to reliably employ it in the creative act (Jowitt 2001a). The traditional rehearsal studio is the creative meeting place for dance artists. A liminal space with specific spatial, temporal and personal considerations: a space fraught with uncertainty and vulnerability, ripe with potential. If harnessed, the creative potential inherent in vulnerability, experienced as increased sensitivity and openness (Press 2002), can facilitate new ways of investigating and doing. This research investigated ways in which dancers prepare themselves during the act of creation and rehearsal within the studio, for the public presentation of a site-specific dance work. Navigating the complexities inherent in performing site-specific dance work is best achieved through the development of a connection with the site during the creation of the work. This is achieved through the provision of rehearsals within the site and ensures seamless replication of rehearsal qualities from studio to non-traditional site. This research received funding and support from the Australia Council for the Arts, Arts Queensland, Brisbane Festival, Kelvin Grove Urban Village, Besen Family Foundation, Korean Culture Arts Foundation and the Australia Korea Foundation. It was presented in the Brisbane Festival, 2006. http://www.accentedbody.com/projects/accented-body

The Child Behavior Checklist and Youth Self-Report in adolescents with epilepsy : testing measurement invariance of the Attention and Thought Problems subscales

The objective of this study was to test for the measurement invariance of the Attention and Thought Problems subscales of the Child Behavior Checklist (CBCL) and Youth Self-Report (YSR) in a population-based sample of adolescents with and without epilepsy. Data were obtained from the 14-year follow-up of the Mater University Study of Pregnancy in which 33 adolescents with epilepsy and 1068 healthy controls were included for analysis. Confirmatory factor analysis was used to test for measurement invariance between adolescents with and without epilepsy. Structural equation modeling was used to test for group differences in attention and thought problems as measured with the CBCL and YSR. Measurement invariance was demonstrated for the original CBCL Attention Problems and YSR Thought Problems. After the removal of ambiguous items (“confused” and “daydreams”),measurement invariance was established for the YSR Attention Problems. The original and reduced CBCL Thought Problems were noninvariant. Adolescents with epilepsy had significantly more symptoms of behavioral problems on the CBCL Attention Problems, β = 0.51, p = 0.002, compared with healthy controls. In contrast, no significant differences were found for the YSR Attention and Thought Problems, β = −0.11, p = 0.417 and β = −0.20, p = 0.116, respectively. In this population-based sample of adolescents with epilepsy, the CBCL Attention Problems and YSR Thought Problems appear to be valid measures of behavioral problems, whereas the YSR Attention Problems was valid only after the removal of ambiguous items. Replication of these findings in clinical samples of adolescents with epilepsy that overcome the limitations of the current study is warranted.