Taka 'i fonua mahu : being Tongan; second generation Tongan migrants

Taka ‘i fonua mahu is a Tongan proverb, which means: "Going about or living in a fruitful land". This thesis analyses the experiences and impacts on migration on being Tongan, particularly Tongan youth in an adopted fruitful land, South East Queensland. The thesis argues that being Tongan in Tonga, has new meaning in the diaspora because of remittances, job prospects, educational opportunity, adapting to a multicultural society, and social justice. These issues are revealed by comparisons made with the experiences of the first generation Tongan migrants, and second generation Tongan migrants, as well as those in New Zealand and America. It argues that the Church, the family and kainga (extended family) impact on the anga fakatonga (Tongan way) and the essence of community as experienced by the first and second generation Tongan migrants. The framework for this analysis is a study of transnationalism, and being Tongan as it is maintained and changed in the diaspora.

DRD2/ANKK1 Taq1A (rs 1800497 C>T) genotypes are associated with susceptibility to second generation antipsychotic-induced akathisia

Rationale Although the advent of atypical, second-generation antipsychotics (SGAs) has resulted in reduced likelihood of akathisia, this adverse effect remains a problem. Extrapyramidal adverse effects are associated with increased drug occupancy of dopamine 2 receptors (DRD2). The A1 allele of the DRD2/ANKK1,rs1800497, is associated with decreased striatal DRD2 density. Objectives The aim of this study was to identify whether the A1(T) allele of the DRD2/ANKK1 was associated with akathisia (measured with the Barnes Akathisia Rating Scale) in a clinical sample of 234 patients treated with antipsychotics. Results Definite akathisia (a score≥ 2 for the global clinical assessment of akathisia) was significantly less common in subjects prescribed SGAs (16.8 %) than those prescribed FGAs (47.6%), p<0.0001. Overall, 24.1% of A1+ (A1A2/A1A1) patients treated with SGAs had akathisia compared to 10.8% of A1- (A2A2) patients. A1+ (A1A2/A1A1) patients administered SGAs also had higher global clinical assessment of akathisia scores than A1- subjects (p=0.01). SGAs maintained their advantage over FGAs regarding akathisia even in A1+ patients treated with SGAs. Conclusions These results strongly suggest that A1+ variants of the DRD2/ANKK1 Taq1A allele confer risk for akathisia in patients treated with SGAs and may explain inconsistencies across prior studies comparing FGAs and SGAs.

Prenatal Vitamin D deficiency induces an early and more severe experimental autoimmune encephalomyelitis in the second generation

In a previous study, we demonstrated that mouse adult F(1) offspring, exposed to a vitamin d deficiency during pregnancy, developed a less severe and delayed Experimental Autoimmune Encephalomyelitis (EAE), when compared with control offspring. We then wondered whether a similar response was observed in the subsequent generation. To answer this question, we assessed F(2) females whose F(1) parents (males or females) were vitamin d-deprived when developing in the uterus of F(0) females. Unexpectedly, we observed that the vitamin d deficiency affecting the F(0) pregnant mice induced a precocious and more severe EAE in the F(2) generation. This paradoxical finding led us to assess its implications for the epidemiology of Multiple Sclerosis (MS) in humans. Using the REFGENSEP database for MS trios (the patient and his/her parents), we collected the parents' dates of birth and assessed a potential season of birth effect that could potentially be indicative of the vitamin d status of the pregnant grandmothers. A trend for a reduced number of births in the Fall for the parents of MS patients was observed but statistical significance was not reached. Further well powered studies are warranted to validate the latter finding.

Optimisation of bio-oil extraction process from Beauty Leaf (Calophyllum inophyllum) oil seed as a second generation biodiesel source

The Beauty Leaf tree (Calophyllum inophyllum) is a potential source of non-edible vegetable oil for producing future generation biodiesel because of its ability to grow in a wide range of climate conditions, easy cultivation, high fruit production rate, and the high oil content in the seed. This plant naturally occurs in the coastal areas of Queensland and the Northern Territory in Australia, and is also widespread in south-east Asia, India and Sri Lanka. Although Beauty Leaf is traditionally used as a source of timber and orientation plant, its potential as a source of second generation biodiesel is yet to be exploited. In this study, the extraction process from the Beauty Leaf oil seed has been optimised in terms of seed preparation, moisture content and oil extraction methods. The two methods that have been considered to extract oil from the seed kernel are mechanical oil extraction using an electric powered screw press, and chemical oil extraction using n-hexane as an oil solvent. The study found that seed preparation has a significant impact on oil yields, especially in the screw press extraction method. Kernels prepared to 15% moisture content provided the highest oil yields for both extraction methods. Mechanical extraction using the screw press can produce oil from correctly prepared product at a low cost, however overall this method is ineffective with relatively low oil yields. Chemical extraction was found to be a very effective method for oil extraction for its consistence performance and high oil yield, but cost of production was relatively higher due to the high cost of solvent. However, a solvent recycle system can be implemented to reduce the production cost of Beauty Leaf biodiesel. The findings of this study are expected to serve as the basis from which industrial scale biodiesel production from Beauty Leaf can be made.

Experimental and statistical investigation of Australian native plants for second-generation biodiesel production

This work explores the potential of Australian native plants as a source of second-generation biodiesel for internal combustion engines application. Biodiesels were evaluated from a number of non-edible oil seeds which are grow naturally in Queensland, Australia. The quality of the produced biodiesels has been investigated by several experimental and numerical methods. The research methodology and numerical model developed in this study can be used for a broad range of biodiesel feedstocks and for the future development of renewable native biodiesel in Australia.

Physio-chemical assessment of beauty leaf (Calophyllum inophyllum) as second-generation biodiesel feedstock

Recently, second-generation (non-vegetable oil) feedstocks for biodiesel production are receiving significant attention due to the cost and social effects connected with utilising food products for the production of energy products. The Beauty leaf tree (Calophyllum inophyllum) is a potential source of non-edible oil for producing second-generation biodiesel because of its suitability for production in an extensive variety of atmospheric condition, easy cultivation, high fruit production rate, and the high oil content in the seed. In this study, oil was extracted from Beauty leaf tree seeds through three different oil extraction methods. The important physical and chemical properties of these extracted Beauty leaf oils were experimentally analysed and compared with other commercially available vegetable oils. Biodiesel was produced using a two-stage esterification process combining of an acid catalysed pre-esterification process and an alkali catalysed transesterification process. Fatty acid methyl ester (FAME) profiles and important physicochemical properties were experimentally measured and estimated using equations based on the FAME analysis. The quality of Beauty leaf biodiesels was assessed and compared with commercially available biodiesels through multivariate data analysis using PROMETHEE-GAIA software. The results show that mechanical extraction using a screw press produces oil at a low cost, however, results in low oil yields compared with chemical oil extraction. High pressure and temperature in the extraction process increase oil extraction performance. On the contrary, this process increases the free fatty acid content in the oil. A clear difference was found in the physical properties of Beauty leaf oils, which eventually affected the oil to biodiesel conversion process. However, Beauty leaf oils methyl esters (biodiesel) were very consistent physicochemical properties and able to meet almost all indicators of biodiesel standards. Overall this study found that Beauty leaf is a suitable feedstock for producing second-generation biodiesel in commercial scale. Therefore, the findings of this study are expected to serve as the basis for further development of Beauty leaf as a feedstock for industrial scale second-generation biodiesel production.

Transition pedagogy : a third generation approach to FYE : a case study of policy and practice for the higher education sector

Current research and practice related to the first year experience (FYE) of commencing higher education students are still mainly piecemeal rather than institution-wide with institutions struggling to achieve cross-institutional integration, coordination and coherence of FYE policy and practice. Drawing on a decade of FYE-related research including an ALTC Senior Fellowship and evidence at a large Australian metropolitan university, this paper explores how one institution has addressed that issue by tracing the evolution and maturation of strategies that ultimately conceptualize FYE as “everybody's business.” It is argued that, when first generation co-curricular and second generation curricular approaches are integrated and implemented through an intentionally designed curriculum by seamless partnerships of academic and professional staff in a whole-of-institution transformation, we have a third generation approach labelled here as transition pedagogy. It is suggested that transition pedagogy provides the optimal vehicle for dealing with the increasingly diverse commencing student cohorts by facilitating a sense of engagement, support and belonging. What is presented here is an example of transition pedagogy in action.

On the feasibility of using servo-mechanisms in wireless multimedia sensor network deployments

This paper considers the use of servo-mechanisms as part of a tightly integrated homogeneous Wireless Multi- media Sensor Network (WMSN). We describe the design of our second generation WMSN node platform, which has increased image resolution, in-built audio sensors, PIR sensors, and servo- mechanisms. These devices have a wide disparity in their energy consumption and in the information quality they return. As a result, we propose a framework that establishes a hierarchy of devices (sensors and actuators) within the node and uses frequent sampling of cheaper devices to trigger the activation of more energy-hungry devices. Within this framework, we consider the suitability of servos for WMSNs by examining the functional characteristics and by measuring the energy consumption of 2 analog and 2 digital servos, in order to determine their impact on overall node energy cost. We also implement a simple version of our hierarchical sampling framework to evaluate the energy consumption of servos relative to other node components. The evaluation results show that: (1) the energy consumption of servos is small relative to audio/image signal processing energy cost in WMSN nodes; (2) digital servos do not necessarily consume as much energy as is currently believed; and (3) the energy cost per degree panning is lower for larger panning angles.

Resorbable composite scaffolds for bone tissue engineering

Bone loss associated with trauma osteo-degenerative diseases and tumors has tremendous socioeconomic impact related to personal and occupation disability and health care costs. Bone grafting is often critical to surgical therapies. Autogenous bone is presently the preferred grafting material; however, this holds several disadvantages such as donor site morbidity. In the present climate of increasing life expectancy with an ensuing increase in bone-related injuries, orthopaedic surgery is undergoing a paradigm shift from bone-grafting to bone engineering, where a scaffold is implanted to provide adequate load bearing and enhance tissue regeneration. Our group at Queensland University of Technology (QUT) have developed, characterised and tested polycaprolactone/ tricalcium phosphate (PCL/TCP) composite scaffolds for low load-bearing bone defects. These scaffolds are being further developed for application in higher load bearing sites. Our approach emphasizes the importance of the biomaterials’ structural design, the scaffold architecture and structural and nutritional requirements for cell culture. These first-generation scaffolds made from medical grade PCL (mPCL) have been studied for more than 5 years within a clinical setting 1. This paper describes the application of second-generation scaffolds in small and large animal bone defect models and the ensuing bone regeneration as shown by histology and µCT.

The ghrelin receptor isoforms (GHS-R1a and GHS-R1b) and GPR39 : an investigation into receptor dimerisation

Prostate cancer is the second most common cause of cancer-related deaths in Western males. Current diagnostic, prognostic and treatment approaches are not ideal and advanced metastatic prostate cancer is incurable. There is an urgent need for improved adjunctive therapies and markers for this disease. GPCRs are likely to play a significant role in the initiation and progression of prostate cancer. Over the last decade, it has emerged that G protein coupled receptors (GPCRs) are likely to function as homodimers and heterodimers. Heterodimerisation between GPCRs can result in the formation of novel pharmacological receptors with altered functional outcomes, and a number of GPCR heterodimers have been implicated in the pathogenesis of human disease. Importantly, novel GPCR heterodimers represent potential new targets for the development of more specific therapeutic drugs. Ghrelin is a 28 amino acid peptide hormone which has a unique n-octanoic acid post-translational modification. Ghrelin has a number of important physiological roles, including roles in appetite regulation and the stimulation of growth hormone release. The ghrelin receptor is the growth hormone secretagogue receptor type 1a, GHS-R1a, a seven transmembrane domain GPCR, and GHS-R1b is a C-terminally truncated isoform of the ghrelin receptor, consisting of five transmembrane domains. Growing evidence suggests that ghrelin and the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, may have a role in the progression of a number of cancers, including prostate cancer. Previous studies by our research group have shown that the truncated ghrelin receptor isoform, GHS-R1b, is not expressed in normal prostate, however, it is expressed in prostate cancer. The altered expression of this truncated isoform may reflect a difference between a normal and cancerous state. A number of mutant GPCRs have been shown to regulate the function of their corresponding wild-type receptors. Therefore, we investigated the potential role of interactions between GHS-R1a and GHS-R1b, which are co-expressed in prostate cancer and aimed to investigate the function of this potentially new pharmacological receptor. In 2005, obestatin, a 23 amino acid C-terminally amidated peptide derived from preproghrelin was identified and was described as opposing the stimulating effects of ghrelin on appetite and food intake. GPR39, an orphan GPCR which is closely related to the ghrelin receptor, was identified as the endogenous receptor for obestatin. Recently, however, the ability of obestatin to oppose the effects of ghrelin on appetite and food intake has been questioned, and furthermore, it appears that GPR39 may in fact not be the obestatin receptor. The role of GPR39 in the prostate is of interest, however, as it is a zinc receptor. Zinc has a unique role in the biology of the prostate, where it is normally accumulated at high levels, and zinc accumulation is altered in the development of prostate malignancy. Ghrelin and zinc have important roles in prostate cancer and dimerisation of their receptors may have novel roles in malignant prostate cells. The aim of the current study, therefore, was to demonstrate the formation of GHS-R1a/GHS-R1b and GHS-R1a/GPR39 heterodimers and to investigate potential functions of these heterodimers in prostate cancer cell lines. To demonstrate dimerisation we first employed a classical co-immunoprecipitation technique. Using cells co-overexpressing FLAG- and Myc- tagged GHS-R1a, GHS-R1b and GPR39, we were able to co-immunoprecipitate these receptors. Significantly, however, the receptors formed high molecular weight aggregates. A number of questions have been raised over the propensity of GPCRs to aggregate during co-immunoprecipitation as a result of their hydrophobic nature and this may be misinterpreted as receptor dimerisation. As we observed significant receptor aggregation in this study, we used additional methods to confirm the specificity of these putative GPCR interactions. We used two different resonance energy transfer (RET) methods; bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), to investigate interactions between the ghrelin receptor isoforms and GPR39. RET is the transfer of energy from a donor fluorophore to an acceptor fluorophore when they are in close proximity, and RET methods are, therefore, applicable to the observation of specific protein-protein interactions. Extensive studies using the second generation bioluminescence resonance energy transfer (BRET2) technology were performed, however, a number of technical limitations were observed. The substrate used during BRET2 studies, coelenterazine 400a, has a low quantum yield and rapid signal decay. This study highlighted the requirement for the expression of donor and acceptor tagged receptors at high levels so that a BRET ratio can be determined. After performing a number of BRET2 experimental controls, our BRET2 data did not fit the predicted results for a specific interaction between these receptors. The interactions that we observed may in fact represent ‘bystander BRET’ resulting from high levels of expression, forcing the donor and acceptor into close proximity. Our FRET studies employed two different FRET techniques, acceptor photobleaching FRET and sensitised emission FRET measured by flow cytometry. We were unable to observe any significant FRET, or FRET values that were likely to result from specific receptor dimerisation between GHS-R1a, GHS-R1b and GPR39. While we were unable to conclusively demonstrate direct dimerisation between GHS-R1a, GHS-R1b and GPR39 using several methods, our findings do not exclude the possibility that these receptors interact. We aimed to investigate if co-expression of combinations of these receptors had functional effects in prostate cancers cells. It has previously been demonstrated that ghrelin stimulates cell proliferation in prostate cancer cell lines, through ERK1/2 activation, and GPR39 can stimulate ERK1/2 signalling in response to zinc treatments. Additionally, both GHS-R1a and GPR39 display a high level of constitutive signalling and these constitutively active receptors can attenuate apoptosis when overexpressed individually in some cell types. We, therefore, investigated ERK1/2 and AKT signalling and cell survival in prostate cancer the potential modulation of these functions by dimerisation between GHS-R1a, GHS-R1b and GPR39. Expression of these receptors in the PC-3 prostate cancer cell line, either alone or in combination, did not alter constitutive ERK1/2 or AKT signalling, basal apoptosis or tunicamycin-stimulated apoptosis, compared to controls. In summary, the potential interactions between the ghrelin receptor isoforms, GHS-R1a and GHS-R1b, and the related zinc receptor, GPR39, and the potential for functional outcomes in prostate cancer were investigated using a number of independent methods. We did not definitively demonstrate the formation of these dimers using a number of state of the art methods to directly demonstrate receptor-receptor interactions. We investigated a number of potential functions of GPR39 and GHS-R1a in the prostate and did not observe altered function in response to co-expression of these receptors. The technical questions raised by this study highlight the requirement for the application of extensive controls when using current methods for the demonstration of GPCR dimerisation. Similar findings in this field reflect the current controversy surrounding the investigation of GPCR dimerisation. Although GHS-R1a/GHS-R1b or GHS-R1a/GPR39 heterodimerisation was not clearly demonstrated, this study provides a basis for future investigations of these receptors in prostate cancer. Additionally, the results presented in this study and growing evidence in the literature highlight the requirement for an extensive understanding of the experimental method and the performance of a range of controls to avoid the spurious interpretation of data gained from artificial expression systems. The future development of more robust techniques for investigating GPCR dimerisation is clearly required and will enable us to elucidate whether GHS-R1a, GHS-R1b and GPR39 form physiologically relevant dimers.

Implementing dance in a Queensland primary school : a descriptive case study

This research documents and analyses the modes of implementation of the Dance component of The Arts Essential Learnings in a Queensland school. The research identifies what makes good practice in dance education and the multiple modes of implementation that support this practice. The primary purpose of this research is to describe the factors that influence dance education, as it is delivered, in a Queensland primary school to inform the development of further support for primary teachers and to improve the quality of dance education in Prep -7 schooling. The literature review investigates dance education both in Queensland, Australia and internationally, identifying current issues related to the delivery of dance in a primary school environment including barriers to implementation of dance, authentic learning and integrated approaches to learning. Based on Engestrom.s reformulation of Vygotsky.s theory of socially mediated learning, the implementation of dance education curriculum was explored through descriptive case study method. The case study was conducted in a regional Queensland school identified as delivering the dance curriculum in a variety of ways. The research project provided opportunities to observe, document and analyse how teachers deal with pedagogical dilemmas and solve logistical problems associated with teaching the dance component of the Arts curriculum in this school. Teachers. practices were contextualised through investigation of the whole school context of dance curriculum development. The findings revealed a range of teaching approaches that influenced teachers. interpretation and children.s experience of the dance curriculum. The features of a supportive whole school and cultural environment for dance were identified. These have been captured in a reworked version of Engestrom.s Second Generation Activity Theory that can be applied to the implementation of dance education in primary schools.

Incremental entrepreneurship : best practice professionalization across generations

This chapter will introduce Australia’s Dennis family – a case of ‘incremental entrepreneurship’ in the business transition from the first to the second generation. Following the second generation’s formal involvement and ownership in the business, Dennis Family Corporation (DFC) undertook a major professionalization process to formalize the family business and ensure its continued success. The members of the second generation have successfully sustained the entrepreneurial spirit of their family business (albeit in a different style), adding value to the firm in an ‘incremental’ manner. Throughout the chapter there will be a strong emphasis on the family element of DFC and the roles that each family member has played. Bert Dennis, as the founder and incumbent leader of the firm, has witnessed major changes to the business he built from the ground up. His children, in particular his son Grant Dennis as the primary next generation issue champion, have seen the changes from another perspective – ensuring the business remains within the family into the second generation and beyond. The professionalization process was sparked by a commitment from the second generation to continue to ‘make a real go’ of the family business rather than simply liquidating and distributing the assets. The dedication of all the family members to this objective has ensured the success of this process, and ultimately, the longevity of the firm. Although DFC has become more ‘professional’, it has not lost its entrepreneurial character; rather, it has improved the ways in which entrepreneurialism is fostered and pursued in the company. In essence, this case outlines how the implementation of appropriate governance and management practices has allowed the Dennis family to overcome the challenges and maximize the opportunities associated with owning and operating a multigenerational family fi rm. From a theoretical perspective, this case uses the concepts of entrepreneurial orientation (EO) (Lumpkin and Dess 1996) and the resource- based view (RBV) (Habbershon and Williams 1999; Barney 1991; Wernerfelt 1984) to demonstrate how the fi rm has leveraged its familiness to foster an enduring spirit of entrepreneurship and to maintain a sustained competitive advantage.

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.