Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

‘It's the end of the university as we know it (and I feel fine)’: the Generation Y student in higher education discourse

This paper examines discussions of Generation Y within higher education discourse, arguing the sector’s use of the term to describe students is misguided for three reasons. First, portraying students as belonging to Generation Y homogenises people undertaking higher education as young, middle-class and technologically literate. Second, speaking of Generation Y students allows constructivism to be reinvented as a ‘new’ learning and teaching philosophy. Third, the Generation Y university student has become a central figure in concerns about technology’s role in learning and teaching. While the notion of the ‘Generation Y student’ creates the illusion that higher education institutions understand their constituents, ultimately, it is of little value in explaining young adults’ educational experiences.

Six degrees of cohesion in patent citation network

While the phrase “six degrees of separation” is widely used to characterize a variety of humanderived networks, in this study we show that in patent citation network, related patents are connected with an average distance of 6, whereas an average distance for a random pair of nodes in the graph is approximately 15. We use this information to improve the recall level in prior-art retrieval in the setting of blind relevance feedback without any textual knowledge.

ICT for blended learning Queensland University of Technology, Australia

This report focuses on blended learning within the Queensland University of Technology (QUT) which is one of Australia’s largest public universities. Although the university in its current format was established in 1989, it contains several previous institutions that can be traced to the earliest forms of technical and teacher education in Queensland in the 19th century (Kyle et al., 1999). The focal point of the report is the experience of QUT’s Faculty of Education which was formed from the amalgamation of several teacher training colleges servicing pre-school and kindergarten, primary and secondary teacher education. While the broader university currently employs approximately 4,000 staff and has about 40,000 students, QUT’s Faculty of Education employs around 170 staff and has approximately 5,000 enrolled students. The Faculty of Education at QUT is the largest provider of pre-service teacher education in Australia and is one of the largest producers of educational research. A major theme of the Faculty of Education is its focus on education and research that provides teachers, schools and educational authorities with practical solutions to the multifaceted issues facing contemporary education.

The ascendancy of oracy in university courses: Implications for teachers and second language users

This chapter reports on a study of oracy in a first-year university Business course, with particular interest in the oracy demands for second language-using international students. The research is relevant at a time when Higher Education is characterised by the confluence of increased international enrolments, more dialogic teaching and learning, and imperatives for teamwork and collaboration. Data sources for the study included videotaped lectures and tutorials, course documents, student surveys, and an interview with the lecturer. The findings pointed to a complex, oracy-laden environment where interactive talk fulfilled high-stakes functions related to social inclusion, the co-construction of knowledge, and the accomplishment of assessment tasks. The salience of talk posed significant challenges for students negotiating these core functions in their second language. The study highlights the oracy demands in university courses and foregrounds the need for university teachers, curriculum writers and speaking test developers to recognise these demands and explicate them for the benefit of all students.

Developing a Second Life virtual field trip for university students : an action research approach

Background: Integrating 3D virtual world technologies into educational subjects continues to draw the attention of educators and researchers alike. The focus of this study is the use of a virtual world, Second Life, in higher education teaching. In particular, it explores the potential of using a virtual world experience as a learning component situated within a curriculum delivered predominantly through face-to-face teaching methods. Purpose: This paper reports on a research study into the development of a virtual world learning experience designed for marketing students taking a Digital Promotions course. The experience was a field trip into Second Life to allow students to investigate how business branding practices were used for product promotion in this virtual world environment. The paper discusses the issues involved in developing and refining the virtual course component over four semesters. Methods: The study used a pedagogical action research approach, with iterative cycles of development, intervention and evaluation over four semesters. The data analysed were quantitative and qualitative student feedback collected after each field trip as well as lecturer reflections on each cycle. Sample: Small-scale convenience samples of second- and third-year students studying in a Bachelor of Business degree, majoring in marketing, taking the Digital Promotions subject at a metropolitan university in Queensland, Australia participated in the study. The samples included students who had and had not experienced the field trip. The numbers of students taking part in the field trip ranged from 22 to 48 across the four semesters. Findings and Implications: The findings from the four iterations of the action research plan helped identify key considerations for incorporating technologies into learning environments. Feedback and reflections from the students and lecturer suggested that an innovative learning opportunity had been developed. However, pedagogical potential was limited, in part, by technological difficulties and by student perceptions of relevance.

Move it, use it : university-school collaboration for learning to teach physical education in primary schools

Reports of increasing numbers of obese Australian children and adolescents have raised the alarm to be proactive in reducing this so called epidemic. It has evoked a call for greater emphasis on teaching physical education in schools, as a measure for attaining fitness not only with obese students but for all students. This paper emphasises how preservice teachers need to be a key target for implementing physical education (PE) reform in schools, as many primary teachers will be generalists and may not be confident enough to implement PE effectively. Through a review of existing literature, teaching practices essential for the effective promotion and implementation of PE were identified under six broad categories: personal-professional skills development, addressing system requirements, pedagogical practices, managing student behaviour, providing feedback to students, and reflecting on practice. Subsequently, the development of these practices in preservice teachers is considered in the context of a university-school collaboration where preservice teachers taught physical education to primary school students for one day per week over a four week period. These authentic teaching experiences provided the preservice teachers with vital opportunities to put theory into practice and interact with “real-world” students. Self-evaluative data from 38 of these preservice teachers, in the form of a five-part Likert scale survey and extended response survey, demonstrated that they were able to develop the majority of the essential teaching practices identified by literature. In particular, the preservice teachers developed self efficacy, enthusiasm, and motivation for teaching PE, facets which are often found to be lacking in generalist primary teachers and yet are essential if children’s perceptions and habits regarding physical activity are to be changed.

The resourceful adolescent project : building psychological resilience in adolescents and their parents

"The model of adolescent depression underlying the Resourceful Adolescent Project (RAP) suggests that adolescent depression and suicide result from a combination of some or all of three interrelating factors: (a) deficits in adolescent psychological wellbeing and coping resources; (b) escalating or chronic family conflict; and (c) depressive adolescent thoughts, feelings and behaviour. This overview of the activities of RAP outlines the need for the project, the benefits of early intervention in preventing adolescent depression, and the empirical and theoretical bases of the programs used in the project. The programs for adolescents and for their parents, their efficacy and the future directions of RAP are also discussed." -- from Libraries Australia

Implementing a systematic process for rapidly embedding sustainability within chemical engineering education: a case study of James Cook University, Australia

Sustainability has emerged as a primary context for engineering education in the 21st Century, particularly the sub-discipline of chemical engineering. However, there is confusion over how to go about integrating sustainability knowledge and skills systemically within bachelor degrees. This paper addresses this challenge, using a case study of an Australian chemical engineering degree to highlight important practical considerations for embedding sustainability at the core of the curriculum. The paper begins with context for considering a systematic process for rapid curriculum renewal. The authors then summarise a 2-year federally funded project, which comprised piloting a model for rapid curriculum renewal led by the chemical engineering staff. Model elements contributing to the renewal of this engineering degree and described in this paper include: industry outreach; staff professional development; attribute identification and alignment; program mapping; and curriculum and teaching resource development. Personal reflections on the progress and process of rapid curriculum renewal in sustainability by the authors and participating engineering staff will be presented as a means to discuss and identify methodological improvements, as well as highlight barriers to project implementation. It is hoped that this paper will provide an example of a formalised methodology on which program reform and curriculum renewal for sustainability can be built upon in other higher education institutions.

On becoming an undergraduate : transition to university

This paper investigates the first year experience of undergraduates with a view to discovering some of the factors which determine a successful negotiation of the transitional phase. The paper begins with a theoretical framework of transition based on the three models of Van Gennep (1960), Viney (1980) and Tinto (1987) and applied to the educational transition from school to University. A new model of transition is presented which looks at the relationship between social and academic adjustment of students to university over time.

Queensland Government Agency Libraries Review: Options Paper

The Queensland Government Agency Libraries Review was undertaken during the period January-June 2010. The research project was sponsored jointly by the Director-General, Department of the Premier and Cabinet, and the Director-General, Department of Public Works. The objective of the project was to examine future options for the Queensland Government library and research services that provide clients with efficient and cost-effective access to the information they need to conduct government business, to develop a picture of the services provided, the information resources managed, the client base and staffing. The review also considered the environmental factors impacting on contemporary government libraries to determine possible strategies that would ensure a strong and sustainable future for the services. The review process culminated in identifying potential options for future service delivery. The preferred option involves a proposal for the centralizing responsibility for the coordination of a network of Queensland Government Libraries and Research Centres (QGLR). The establishment of a network of research centres, with a central point for coordination of common strategies, systems and processes will enable library and research services to flow around individual agencies, and provide clearer avenues for multi-disciplinary work that characterises contemporary government policy and government services. The key elements of the proposal include the development of a single library and research portal for all government officers, which can be tailored to meet their particular research needs; increasing the visibility and accessibility of information and knowledge resources to those officers regardless of their agency affiliation; introducing better, more cost effective purchasing and licensing arrangements; and the potential to have a voice of influence in the strategic agenda for the government’s use of information and research.

Experiences of a walking intervention in a University community : Qualitative perspectives

Background: This study explored the experiences of university employees that participated in a walking intervention that encouraged individuals to walk more throughout their workday. The 10-week program was comprised of 5 phases (i.e. baseline, anticipating barriers, short planned walks, longer planned walks and maintenance) and utilized a pedometer diary and an online website for logging steps. The pedometer diary included “action plans” for addressing barriers and planning walking and the online dashboard provided graphical outputs that allowed participants to visualize whether they were reaching or exceeding their step targets. Methods: A subsample of 12 academic and administrative employees from the study completed open ended questionnaires at the end of the study. The questions focused on capturing the major themes of benefits/mediators and problems/moderators of the program and were assessed using phenomenological approaches. Results: Participants found a raised consciousness of physical inactivity throughout the work day. They also found it useful to have a graphical display of physical activity patterns, but found time constraints and lack of managerial support to be the primary barriers/moderators of the program. Those most likely to withdraw from the program experienced technical difficulties with objective monitors and the online website. Conclusions: Findings highlight the value in being involved in a group forum and provide insights into the challenges of supporting such programs within the workplace.

Digital Storytelling within a Service-Learning Partnership: Technology as Product and Process for University Students and Culturally and Linguistically Diverse High School Youth

This chapter describes a university/high school partnership focused on digital storytelling. It also explains the multi-stage process used to establish this successful partnership and project. The authors discuss the central role that technology played in developing this university/high school partnership, a collaboration that extended the impact of a digital storytelling project to reach high school students, university students, educators, high school administrators, and the local community. Valuing a reflective process that can lead to the creation of a powerful final product, the authors describe the impact of digital storytelling on multiple stakeholders, including the 13 university students and 33 culturally and linguistically diverse high school youth who participated during the fall of 2009. In addition, the chapter includes reflections from university and high school student participants expressed during focus groups conducted throughout the project. While most participants had a positive experience with the project, complications with the technology component often caused frustrations and additional challenges. Goals for sharing this project are to critically evaluate digital storytelling, describe lessons learned, and recommend good practices for others working within a similar context or with parallel goals.