Comparative and Functional Analysis of Gene Expression in Ophiostoma Species

Ophiostoma floccosum and Ophiostoma piliferum are polymorphic ascomycete fungi found throughout the world. Both species are important economically as they are known to colonise timber and cause discoloration of wood thus reducing its aesthetic value and subsequently price. Albino variants of the two species, in particular O. piliferum, are used as biological control agents to prevent sapstaining and have been used commercially for the past 15 years to reduce pitch/wood extractives in paper manufacturing. Other members of the genus include the plant pathogens O. novo-ulmi and O. clavigerum, known to have a severe effect on forest health and economy around the world. O. floccosum and O. piliferum have been demonstrated in the laboratory to be fermented in large volumes and they are particularly suitable as hosts capable of secreting extracellular recombinant proteins. This research aimed to investigate the transcriptome and molecular functioning of Ophiostoma floccosum and compare this to transcriptomic data available for Ophiostoma piliferum and other Ophiostoma species, O. novo-ulmi, O. clavigerum and O. piceae. This research contributes to the development of O. floccosum and O. piliferum as hosts for protein expression and advances the knowledge of gene expression and molecular functioning in this genus. To gain insight into the molecular functioning of O. floccosum, an expressed sequence tag (EST) collection from yeast-like growth (blastospores) was created during early phase growth. A total of 1207 EST sequences with an average length of 713 bp were identified. Clustering and assembly of the high-quality EST data set resulted in the identification of 598 unique putative transcripts (UPTs). Functional classification of these UPTs, using both homology searching and ab-initio methods, indicated that the majority of protein transcripts produced were involved in metabolism and cell proliferation. Up-regulation of mitochondrial transcripts involved in respiration and the presence of transcripts homologous to enzymes involved in the tri-carboxylic acid cycle indicated that aerobic respiration was likely the preferred method of ATP production in O. floccosum blastospores. However, the putative identification of genes encoding alcohol dehydrogenases within O. floccosum ESTs and the presence of homologues in other Ophiostoma species would suggest that these Ophiostoma species are also likely to be capable of metabolic functioning under anaerobic conditions. To identify homologous genes between Ophiostoma species, the O. floccosum EST data set was compared to 20,783 ESTs from other Ophiostoma species including O. piliferum, O. novo-ulmi, O. clavigerum and O. piceae. All UPTs identified within each of the datasets were aligned resulting in the identification of 347 clusters containing EST sequences from more than one Ophiostoma species. Six were identified that had homologues in all of the datasets excluding O. piceae. Three of the six homologous UPTs were predicted to function in core metabolism with two of the UPTs identified as encoding enzymes used in the glycolysis pathway and one encoding a 60S ribosomal protein. The other three homologous UPTs were thought to have a functional role in protein fate and were putatively identified as being a superoxide dismutase, heat-shock protein and a structural alpha-B chain tubulin gene. Of the 347 clusters, 86 of these contained transcripts identified in the O. floccosum EST datasets, and of these 86, only 10 fragments did not align with any significant homology to other fungal sequences contained in the NCBI non redundant database, indicating that the majority these transcripts are conserved in other fungal species. Predicted genes within the Ophiostoma EST datasets were also investigated to determine codon usage and to identify the presence of genes predicted to encode proteases. Both are important factors in recombinant protein expression. Protease production can severely inhibit the production of recombinant protein in fungal hosts. Based on sequence homology to known proteases, putative proteases were identified in all of the Ophiostoma species investigated with the exception of O. piceae. Homologues for all six peptidase groups were identified including a possible glutamic acid protease and proportionally high numbers of serine and metallo-protease homologues. This research constitutes the first reported findings of putative peptidases in the aspartic, cysteine, glutamic and threonine peptidase families in Ophiostoma species. Key to the over-expression of recombinant proteins is the optimisation of codons in a cloned gene to better utilise available tRNA species within the recombinant host. No codon bias was apparent between up-regulated and lower frequency transcripts in O. floccosum, O. piliferum, O. clavigerum and O. novo-ulmi. Codon usage was found to be consistent between these Ophiostoma species. However, a large difference between the codon usage in mitochondrially encoded genes compared to nuclear encoded genes in O. floccosum was indicated. To optimise the efficiency of a recombinant expression system, we sought to identify promoters in both O. floccosum and O. piliferum that may be applied to a vector system. Using EST data, the most up-regulated UPTs identified from O. floccosum and O. piliferum ESTs were a putative subunit 4 of the NADH-ubiquinone oxidoreductase protein (NADH-UR4) and a possible heat-shock protein (HSP), respectively. A unique hydrolase gene was also identified by molecular probing of O. floccosum genomic DNA. This putative 96 kd protein, called PLIP-Lg, was predicted to be a mitochondrial A1 phospholipase based on both nucleotide and predicted amino acid sequence structure and homology. These gene sequences were investigated using genome walking methods to further elucidate nucleotide sequences in the 5' and 3' directions. In silico investigation of the 5' promoter region of the genes identified a number of predicted transcription factor binding sites, including possible TATA boxes identified previously in the promoter region of an O. floccosum protein. Additionally, RT-PCR methods were used to compare the expression of these transcripts throughout growth in both the mycelial and blastospore forms. All three predicted genes were found to be transcribed throughout growth in both morphological forms and, thus, the use of their promoters in a vector system would not be limited to one morphology. However, the level of expression in blastospores compared to mycelial growth varied by up to 20 fold. Therefore, the morphological form of the fungi did influence the level of expression of these genes and is a factor for consideration for future promoter use. This PhD thesis research provides the first comprehensive investigation into gene expression and the transcriptome of O. floccosum while also providing the first comparative look into similarities between the transcriptomes of several Ophiostoma species. Subsequently, this research adds to the knowledge of metabolic functioning in Ophiostoma species and illustrates the usefulness of EST analysis in determining core molecular functioning within this group. Further to addressing these goals, the research will augment future research into various biotechnological applications for the genus, specifically the development of O. floccosum and O. piliferum as hosts for recombinant protein expression

Evaluating the taxa that provide shared pollination services across multiple crops and regions

Many pollinator species visit multiple crops in multiple regions, yet we know little about their pollination service provisioning at local and regional scales. We investigated the floral visitors (n = 13,200), their effectiveness (n = 1718 single visits) and response to landscape composition across three crops avocado, mango and macadamia within a single growing region (1 year), a single crop (3 years) and across different growing regions in multiple years. In total, eight wild visitor groups were shared across all three crops. The network was dominated by three pollinators, two bees (Apis mellifera and Tetragonula spp.) and a fly, Stomorhina discolor. The visitation network for the three crops was relatively generalised but with the addition of pollen deposition data, specialisation increased. Sixteen managed and wild taxa were consistently present across three years in avocado, yet their contribution to annual network structure varied. Node specialisation (d’) analyses indicated many individual orchard sites across each of the networks were significantly more specialised compared to that predicted by null models, suggesting the presence of site-specific factors driving these patterns. Identifying the taxa shared across multiple crops, regions and years will facilitate the development of specific pollinator management strategies to optimize crop pollination services in horticultural systems

The spirit of cleavage : pedagogy, gender, and reform in early nineteenth-century British women's fiction

By the end of the eighteenth century, women's education had become a topic of serious cultural debate. In my dissertation I examine the ways in which six early nineteenth-century noncanonical British women novelists--Eliza Fenwick, Mary Hays, Amelia Opie, Hannah More, Sydney Owenson, and Mary Brunton--attempt to reconstruct culturally dominant gender representations through their discourse on education. More pointedly, I measure the possible efficacy of these reformist efforts in light of the political and cultural forces and conditions which demand their suppression or co-option. My analysis suggests that these writers were accomplished readers of the polemics and politics of their period, creatively appropriating turn-of-the-century intellectual and philosophical debates and constructing an alternative history through their fictions. Far from homogeneous in their responses to the cultural text of their era, these women and their fictions are marked by differences in politics, nationality, class, and religion, yet they all attempt to transform female pedagogical practices and dominant gender constructions through an appeal to balance and reconciliation. For disparate reasons, these novels defy customary, binary constructions of complementary sex-based schooling by revalorizing or rewriting culturally prevalent notions of a properly feminine education in the decorative accomplishments and arguing for women's access to masculine, rational pedagogy in both form and content. Because education plays a pivotal role in the ideological construction of gender, in envisioning a comprehensive alternative mode of female instruction which reconciles the masculine and feminine, these novelists also construct an alternative gender representation for the early nineteenth-century woman, a vision of gender parity which translates into expanded opportunity, cultural agency, and socio-political significance for British women. In challenging dominant notions of rationality, furthermore, these novelists also rewrite conventional terms of cultural cohesion in an attempt to augment communal benefits and individual happiness. Such efforts, however, are qualified by the author's limited concern with reconstructing gender through education for the turn-of-the-century male, as well as by the shift in the underlying logic for these educational and gender reforms from a matter of rights to one of religion, a transition which gradually lends to an appropriation of these disruptive efforts by the dominant order. Nevertheless, through their discourse on education these women breach any illusions of social consensus and stability, thereby creating the fissure, the opening, the "spirit of cleavage" in the cultural fabric that remains to disrupt dominant prescriptions throughout the nineteenth century. By choosing education as their point of intervention, these six writers adopt the position of the intellectual, a primary site of opposition that helps clear a space from which to gain the perspective, resistance, and mobility necessary to begin to envision and effect lasting, far-reaching cultural change

The Protective Effects of CD39 Overexpression in Multiple Low-Dose Streptozotocin–Induced Diabetes in Mice

Islet allograft survival limits the long-term success of islet transplantation as a potential curative therapy for type 1 diabetes. A number of factors compromise islet survival, including recurrent diabetes. We investigated whether CD39, an ectonucleotidase that promotes the generation of extracellular adenosine, would mitigate diabetes in the T cell–mediated multiple low-dose streptozotocin (MLDS) model. Mice null for CD39 (CD39KO), wild-type mice (WT), and mice overexpressing CD39 (CD39TG) were subjected to MLDS. Adoptive transfer experiments were performed to delineate the efficacy of tissue-restricted overexpression of CD39. The role of adenosine signaling was examined using mutant mice and pharmacological inhibition. The susceptibility to MLDS-induced diabetes was influenced by the level of expression of CD39. CD39KO mice developed diabetes more rapidly and with higher frequency than WT mice. In contrast, CD39TG mice were protected. CD39 overexpression conferred protection through the activation of adenosine 2A receptor and adenosine 2B receptor. Adoptive transfer experiments indicated that tissue-restricted overexpression of CD39 conferred robust protection, suggesting that this may be a useful strategy to protect islet grafts from T cell–mediated injury

Implementing complete smokefree policies in mental health inpatient settings Results from a before and after mixed-methods evaluation : results from a before and after mixed-methods evaluation

Abstract Background Tobacco smoking is extremely prevalent in people with severe mental illness (SMI) and has been recognised as the main contributor to widening health inequalities in this population. Historically, smoking has been deeply entrenched in the culture of mental health settings in the UK, and until recently, smokefree policies tended to be only partially implemented. However, recent national guidance and the government’s tobacco control plan now call for the implementation of complete smokefree policies. Many mental health Trusts across the UK are currently in the process of implementing the new guidance, but little is known about the impact of and experience with policy implementation. Methods This paper reports findings from a mixed-methods evaluation of policy implementation across 12 wards in a large mental health Trust in England. Quantitative data were collected and compared before and after implementation of NICE guidance PH48 and referred to 1) identification and treatment of tobacco dependence, 2) smoking-related incident reporting, and 3) prescribing of psychotropic medication. A qualitative exploration of the experience of inpatients was also carried out. Descriptive statistical analyses were performed, and the feasibility of collecting relevant and complete data for each quantitative component was assessed. Qualitative data were analysed using thematic framework analysis. Results Following implementation of the complete smokefree policy, increases in the numbers of patients offered smoking cessation advice (72% compared to 38%) were identified. While incident reports demonstrated a decrease in challenging behaviour during the post-PH48 period (6% compared to 23%), incidents relating to the concealment of smoking materials increased (10% compared to 2%). Patients reported encouraging changes in smoking behaviour and motivation to maintain change after discharge. However, implementation issues challenging full policy implementation, including covert facilitation of smoking by staff, were reported, and difficulties in collecting relevant and complete data for comprehensive evaluation purposes identified. Conclusions Overall, the implementation of complete smokefree policies in mental health settings may currently be undermined by partial support. Strategies to enhance support and the establishment of suitable data collection pathways to monitor progress are required

Instructional leadership in centralised systems: evidence from Greek high-performing secondary schools

This paper examines the enactment of instructional leadership (IL) in high-performing secondary schools (HPSS), and the relationship between leadership and learning in raising student outcomes and encouraging teachers’ professional learning in the highly centralised context of Greece. It reports part of a comparative research study focused on whether, and to what extent, IL has been embraced by Greek school leaders. The study is exploratory, using a qualitative multiple case design to examine two HPSS in Athens. The research design involved a qualitative approach using several different methods, including semi-structured interviews with school principals, deputy heads, subject teachers and subject advisers, plus observation of leadership practice and meetings and scrutiny of relevant policy documents. The findings show that IL is conceptualised as an informal collaborative leadership practice, interwoven with the official multi-dimension role of Greek principals and their ‘semi-IL’ role. In the absence of official IL ‘actors’, teachers’ leadership has been expanding

Associations of common breast cancer susceptibility alleles with risk of breast cancer subtypes in BRCA1 and BRCA2 mutation carriers

Introduction: More than 70 common alleles are known to be involved in breast cancer (BC) susceptibility, and several exhibit significant heterogeneity in their associations with different BC subtypes. Although there are differences in the association patterns between BRCA1 and BRCA2 mutation carriers and the general population for several loci, no study has comprehensively evaluated the associations of all known BC susceptibility alleles with risk of BC subtypes in BRCA1 and BRCA2 carriers. Methods: We used data from 15,252 BRCA1 and 8,211 BRCA2 carriers to analyze the associations between approximately 200,000 genetic variants on the iCOGS array and risk of BC subtypes defined by estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and triple-negative- (TN) status; morphologic subtypes; histological grade; and nodal involvement. Results: The estimated BC hazard ratios (HRs) for the 74 known BC alleles in BRCA1 carriers exhibited moderate correlations with the corresponding odds ratios from the general population. However, their associations with ER-positive BC in BRCA1 carriers were more consistent with the ER-positive as

Identification of a BRCA2-Specific modifier locus at 6p24 related to breast cancer risk

Common genetic variants contribute to the observed variation in breast cancer risk for BRCA2 mutation carriers; those known to date have all been found through population-based genome-wide association studies (GWAS). To comprehensively identify breast cancer risk modifying loci for BRCA2 mutation carriers, we conducted a deep replication of an ongoing GWAS discovery study. Using the ranked P-values of the breast cancer associations with the imputed genotype of 1.4 M SNPs, 19,029 SNPs were selected and designed for inclusion on a custom Illumina array that included a total of 211,155 SNPs as part of a multi-consortial project. DNA samples from 3,881 breast cancer affected and 4,330 unaffected BRCA2 mutation carriers from 47 studies belonging to the Consortium of Investigators of Modifiers of BRCA1/2 were genotyped and available for analysis. We replicated previously reported breast cancer susceptibility alleles in these BRCA2 mutation carriers and for several regions (including FGFR2, MAP3K1, CDKN2A/B, and PTHLH) identified SNPs that have stronger evidence of association than those previously published. We also identified a novel susceptibility allele at 6p24 that was inversely associated with risk in BRCA2 mutation carriers (rs9348512; per allele HR = 0.85, 95% CI 0.80-0.90, P = 3.9×10−8). This SNP was not associated with breast cancer risk either in the general population or in BRCA1 mutation carriers. The locus lies within a region containing TFAP2A, which encodes a transcriptional activation protein that interacts with several tumor suppressor genes. This report identifies the first breast cancer risk locus specific to a BRCA2 mutation background. This comprehensive update of novel and previously reported breast cancer susceptibility loci contributes to the establishment of a panel of SNPs that modify breast cancer risk in BRCA2 mutation carriers. This panel may have clinical utility for women with BRCA2 mutations weighing options for medical prevention of breast cancer

Characterization of Puberty in an Australian Population-Based Cohort Study

Purpose: Current knowledge of the characteristics of puberty beyond age at menarche and thelarche is limited, particularly within population-based cohorts. Secular trends and concerns of the health effects of early puberty reinforce the value of contemporary studies characterizing the timing, tempo, duration, and synchronicity of puberty. // Methods: The Childhood to Adolescence Transition Study is a unique Australian cohort of individuals followed annually from late childhood to late adolescence, with up to eight assessments of pubertal stage from 9 to 19 years of age (N = 1,183; 636 females). At each assessment, females reported their Tanner Stage of breast and pubic hair development, while males reported on genital/pubic hair development. Nonlinear mixed-effects models characterized pubertal trajectories and were used to derive each individual’s estimates of timing, tempo, and synchronicity. Parametric survival models were used to estimate the overall duration of puberty. // Results: Timing of mid-puberty (Tanner Stage 3) ranged from 12.5 to 13.5 years, with females developing approximately 6 months before males. Pubertal tempo (at mid-puberty) was similar across sex (between half and one Tanner Stage per year), but the overall duration of puberty was slightly shorter in males. Most females exhibited asynchronous changes of breast and pubic hair development. // Discussion: Estimates of pubertal timing and tempo are consistent with reports of cohorts from two or more decades ago, suggesting stabilization of certain pubertal characteristics in predominantly White populations. However, our understanding of the duration of puberty and individual differences in pubertal characteristics (e.g., synchronicity of physical changes) remains limited