Design, synthesis and thermal behaviour of a series of well-defined clickable and triggerable sulfonate polymers

In the printing industry, the exploitation of triggerable materials that can have their surface properties altered on application of a post-deposition external stimulus has been crucial for the production of robust layers and patterns. To this end, herein, a series of clickable poly(R-alkyl p-styrene sulfonate) homopolymers, with systematically varied thermally-labile protecting groups, has been synthesised via reversible addition-fragmentation chain transfer (RAFT) polymerisation. The polymer range has been designed to offer varied post-deposition thermal treatment to switch them from hydrophobic to hydrophilic. Suitable RAFT conditions have been identified to produce well-defined homopolymers (Đ, Mw/Mn < 1.11 in all cases) at high monomer conversions (>80% for all but one monomer) with controllable molar mass. Poly(p-styrene sulfonate) with an isobutyl protecting group has been shown to be the most readily thermolysed polymer that remains stable at room temperature, and was thus investigated further by incorporation into a diblock copolymer, P3HT-b-PiBSS, by click chemistry. The strategy for preparation of thermal modifiable block copolymers exploiting R-protected p-styrene sulfonates and azide-alkyne click chemistry presented herein allows the design of new, roll-to-roll processable materials for potential application in the printing industry, particularly organic electronics.

Multimode Analysis of Nanoscale Biomolecular Interactions

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions

Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells.

Interactions between Late Quaternary volcanic and sedimentary processes in the Naples Bay, Southern Tyrrhenian sea

The interactions between Late Quaternary volcanic and sedimentary processes in the Naples Bay, Southern Tyrrhenian sea, are here discussed through the results of the marine geological survey at the scale 1:25.000. The example of the geological map n. 465 “Isola di Procida”, herein presented, has put in evidence the stratigraphy of marine Quaternary deposits and related volcanic seismic units. The volcanic deposits cropping out in the Procida island have been explained as the result of eruptions of local eruptive centres. The geological survey carried out onshore indicates the occurrence of several pyroclastic units linked to the eruptive activity of the Ischian and Phlegrean volcanic complexes, interstratified with the products erupted by local volcanic centres. The occurrence in the pyroclastic sequences of Ischia and Procida islands of several marker horizons and their stratigraphic correlations have allowed to reconstruct the volcanological evolution of the two islands and the interactions with sedimentary processes at the scale of the whole Tyrrhenian margin. Four geological maps at the scale 1:25.000 covering the whole Naples Bay have been reconstructed based on the interpretation of marine geological and geophysical data. The stratigraphic relationships between the seismic units and the eruptive deposits have testified the activity of several monogenetic volcanic centers, whose products are interstratified with marine and continental deposits of the Late Quaternary depositional sequence.

Glutathione Transferases as Detoxification Agents: Structural and Functional Studies

Glutathione transferases (GSTs) are a diverse family of enzymes that catalyze the glutathione-dependent detoxification of toxic compounds. GSTs are responsible for the conjugation of the tripeptide glutathione (GSH) to a wide range of electrophilic substrates. These include industrial pollutants, drugs, genotoxic carcinogen metabolites, antibiotics, insecticides and herbicides. In light of applications in biomedicine and biotechnology as cellular detoxification agents, detailed structural and functional studies of GSTs are required. Plant tau class GSTs play crucial catalytic and non-catalytic roles in cellular xenobiotic detoxification process in agronomically important crops. The abundant existence of GSTs in Glycine max and their ability to provide resistance to abiotic and biotic stresses such as herbicide tolerance is of great interest in agriculture because they provide effective and suitable tools for selective weed control. Structural and catalytic studies on tau class GST isoenzymes from Glycine max (GmGSTU10-10, GmGSTU chimeric clone 14 (Sh14), and GmGSTU2-2) were performed. Crystal structures of GmGSTU10-10 in complex with glutathione sulfenic acid (GSOH) and Sh14 in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH) were determined by molecular replacement at 1.6 Å and 1.75 Å, respectively. Major structural variations that affect substrate recognition and catalytic mechanism were revealed in the upper part of helix H4 and helix H9 of GmGSTU10-10. Structural analysis of Sh14 showed that the Trp114Cys point mutation is responsible for the enhanced catalytic activity of the enzyme. Furthermore, two salt bridges that trigger an allosteric effect between the H-sites were identified at the dimer interface between Glu66 and Lys104. The 3D structure of GmGSTU2-2 was predicted using homology modeling. Structural and phylogenetic analysis suggested GmGSTU2-2 shares residues that are crucial for the catalytic activity of other tau class GSTs–Phe10, Trp11, Ser13, Arg20, Tyr30, Leu37, Lys40, Lys53, Ile54, Glu66 and Ser67. This indicates that the catalytic and ligand binding site in GmGSTU2-2 are well-conserved. Nevertheless, at the ligandin binding site a significant variation was observed. Tyr32 is replaced by Ser32 in GmGSTU2-2 and thismay affect the ligand recognition and binding properties of GmGSTU2-2. Moreover, docking studies revealed important amino acid residues in the hydrophobic binding site that can affect the substrate specificity of the enzyme. Phe10, Pro12, Phe15, Leu37, Phe107, Trp114, Trp163, Phe208, Ile212, and Phe216 could form the hydrophobic ligand binding site and bind fluorodifen. Additionally, side chains of Arg111 and Lys215 could stabilize the binding through hydrogen bonds with the –NO2 groups of fluorodifen. GST gene family from the pathogenic soil bacterium Agrobacterium tumefaciens C58 was characterized and eight GST-like proteins in A. tumefaciens (AtuGSTs) were identified. Phylogenetic analysis revealed that four members of AtuGSTs belong to a previously recognized bacterial beta GST class and one member to theta class. Nevertheless, three AtuGSTs do not belong to any previously known GST classes. The 3D structures of AtuGSTs were predicted using homology modeling. Comparative structural and sequence analysis of the AtuGSTs showed local sequence and structural characteristics between different GST isoenzymes and classes. Interactions at the G-site are conserved, however, significant variations were seen at the active site and the H5b helix at the C-terminal domain. H5b contributes to the formation of the hydrophobic ligand binding site and is responsible for recognition of the electrophilic moiety of the xenobiotic. It is noted that the position of H5b varies among models, thus providing different specificities. Moreover, AtuGSTs appear to form functional dimers through diverse modes. AtuGST1, AtuGST3, AtuGST4 and AtuGST8 use hydrophobic ‘lock–and–key’-like motifs whereas the dimer interface of AtuGST2, AtuGST5, AtuGST6 and AtuGST7 is dominated by polar interactions. These results suggested that AtuGSTs could be involved in a broad range of biological functions including stress tolerance and detoxification of toxic compounds.

The relationship of loneliness and social anxiety with children's and adolescents' online communication

Children and adolescents are now using online communication to form and/or maintain relationships with strangers and/or friends. Relationships in real life are important for children and adolescents in identity formation and general development. However, social relationships can be difficult for those who experience feelings of loneliness and social anxiety. The current study aimed to replicate and extend research conducted by Valkenburg and Peter (2007b), by investigating differences in online communication patterns between children and adolescents with and without selfreported loneliness and social anxiety. Six hundred and twenty-six students aged 10-16 years completed a questionnaire survey about the amount of time they engaged in online communication, the topics they discussed, who they communicated with, and their purposes of online communication. Following Valkenburg and Peter (2007b), loneliness was measured with a shortened version of the UCLA Loneliness Scale (Version 3) developed by Russell (1996), whereas social anxiety was assessed with a sub-scale of the Social Anxiety Scale for Adolescents (La Greca & Lopez, 1998). The sample was divided into four groups of children and adolescents: 220 were “non-socially anxious and non-lonely”, 139 were “socially anxious but not lonely”, 107 were “lonely but not socially anxious”, and 159 were “lonely and socially anxious”. A one-way ANOVA and chi-square tests were conducted to evaluate the aforementioned differences between these groups. The results indicated that children and adolescents who reported being lonely used online communication differently from those who did not report being lonely. Essentially, the former communicated online more frequently about personal things and intimate topics, but also to compensate for their weak social skills and to meet new people. Further analyses on gender differences within lonely children and adolescents revealed that boys and girls communicated online more frequently with different partners. It was concluded that for these vulnerable individuals online communication may fulfil needs of self-disclosure, identity exploration, and social interactions. However, future longitudinal studies combining a quantitative with a qualitative approach would better address the relationship between Internet use and psychosocial well-being. The findings also suggested the need for further exploration of how such troubled children and adolescents can use the Internet beneficially.

Students’ conceptual knowledge about workplace pedagogies and applications to learning in the workplace

The development of effective workplace pedagogies is integral to work-integrated and work-based learning. The workplace pedagogies that facilitate and support learning include: activities in which individuals engage such as daily work practices, questioning; observing, and listening; interactions with more experienced workers through coaching and modelling; and referencing documented procedures. Each of these dimensions is significant in enhancing processes of workplace learning. Learning can be optimised when these pedagogies are appropriately embedded in the context of and process of participating in normal work activities. Yet learners need to understand the nature of these pedagogies, and how and for what purposes to use these to achieve a range of learning outcomes. This is because it is the worker-learners who play the key roles in the process and outcomes of learning through work. A pilot study was conducted on students’ conceptions of how each of these dimensions of workplace pedagogy help their learning, by providing examples of learning from these sources; and stating their preferences for learning in the workplace. A sample of seventeen students, enrolled in the second year of a Diploma in Nursing course at a Technical and Further Education institution, participated in a survey intended to capture these conceptions and the importance attached to each of them. The findings indicate that these students have basic understanding of how each of seven workplace pedagogic practices can contribute to their learning. They reported relying mostly on daily practices, observing and listening to others, modelling, coaching, and other workers. Their selection of these contributions emphasise significant opportunities for guided learning by others, yet suggest fewer student-initiated interactions, less intensity in interactions, and likelihood that learning is more passive. The data also suggests that these students rely mostly on using academic learning skill, and limited workplace learning skills. It is proposed, therefore, that the knowledge and understandings about workplace learning and pedagogies might be best embedded in the full curriculum and not become add-on shortly before students go on work placement. This approach will allow students to appreciate the significance and use of workplace pedagogies for learning.

Crosstalk between developmental and tumour-specific signalling pathways : kallikrein-related serine peptidases and nodal in prostrate cancer

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated gene expression, altered cellular differentiation, and the nexus between these two concepts. The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream genes. Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other kallikreins are potential adjunct biomarkers. As secreted proteases, kallikreins act through enzyme cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgenresponsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling. The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β-catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β-catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14. Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is reexpressed in prostate cancer and may have a functional role in prostate cancer progression. In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

Sapporo World Window : urban interaction through public and private screens

This paper introduces Sapporo World Window, a screen-based application that is currently under development for the new underway passage at the centre of Sapporo City. There are ten large public screens installed in the space, displaying user-generated videos about various aspects of the city and a real-time map that visualises users’ interaction with the city. The application aims to engage the general public by functioning as a unique ‘point of connection’ for socio-cultural and technological interactions, making the space a lively social place where people can have meaningful experiences of interacting with people and places of Sapporo through mobile phones (keitai) and the public screens in the space. This paper first outlines the contextual background and key concept for the application’s design. Then the paper discusses the user interaction processes, technical specifications, and interface design, followed by the conclusions and outlook.

Processing load and the use of concrete representations and strategies for solving linear equations

This paper is a report of students' responses to instruction which was based on the use of concrete representations to solve linear equations. The sample consisted of 21 Grade 8 students from a middle-class suburban state secondary school with a reputation for high academic standards and innovative mathematics teaching. The students were interviewed before and after instruction. Interviews and classroom interactions were observed and videotaped. A qualitative analysis of the responses revealed that students did not use the materials in solving problems. The increased processing load caused by concrete representations is hypothesised as a reason.

Scripting versus emergence : issues for game developers and players in game environment design

This paper defines and discusses two contrasting approaches to designing game environments. The first, referred to as scripting, requires developers to anticipate, hand-craft and script specific game objects, events and player interactions. The second, known as emergence, involves defining general, global rules that interact to give rise to emergent gameplay. Each of these approaches is defined, discussed and analyzed with respect to the considerations and affects for game developers and game players. Subsequently, various techniques for implementing these design approaches are identified and discussed. It is concluded that scripting and emergence are two extremes of the same continuum, neither of which are ideal for game development. Rather, there needs to be a compromise in which the boundaries of action (such as story and game objectives) can be hardcoded and non-scripted behavior (such as interactions and strategies) are able to emerge within these boundaries.

Engaging parents in STEM: Coteaching and cogenerative dialoguing in a Queensland high school

Diminished student interest in science, technology, engineering and mathematics (STEM) is recognised by educators, researchers and public policy makers as a concerning global trend. Inviting stakeholders like scientists and industry specialists to discuss their work is one means schools use to facilitate student engagement in the sciences. However, these visits generally comprise one-off sessions with minimal relevance to students’ particular and ongoing learning needs. This case study investigated coteaching and cogenerative dialoguing with parents in teaching a Year-8 multidisciplinary unit with science and technology foci. Two parents cotaught alongside the resident teacher and researcher over eight months. This paper concentrates on one parent, a medical scientist by profession. Data sources included video and audio recordings of cogenerative dialogues and classroom interactions, student work samples and journal entries. Data were interrogated using the sociological constructs of fields and capitals and the dialectic of structure|agency. The findings reveal how (a) the parent’s science and technology knowledge was tailored to the students’ needs initially and continually and (b) student-generated data indicated enhanced engagement in science and technology. The research speaks to schools and governments about enhancing STEM education by furthering collaborative relationships with relevant stakeholders.