Development of Novel Activity-Based Probes for the Detection of Serine Proteases

Cystic Fibrosis (CF) is a genetic disease featuring a chronic cycle of inflammation and infection in the airways of sufferers. Mutations lead to altered ion transport, which in turn causes dehydrated airways and reduced mucociliary clearance which predisposes the patient to infection, resulting in a severe immune response and tissue destruction (1). Airway dehydration is primarily caused by the hyperabsorption of sodium by the epithelial sodium channel (ENaC) (2). ENaC is activated by the action of a number of predominantly trypsin-like Channel Activating Proteases (CAPs) including prostasin, matriptase and furin (3). Additional proteases known to activate ENaC include human airway trypsin (3), plasmin, neutrophil elastase and chymotrypsin (4).

Activity profiling is a valuable technique which involves the use of small inhibitory molecules called Activity-Based Probes (ABPs) which can be used to covalently label the active site of proteases and provide a range of information regarding its structure, catalytic mechanism, location and function within biological systems. The development of novel ABPs for CAPs, would enhance understanding of the role of these proteases in CF airways disease and in particular their role in ENaC activation and airway dehydration. This project investigates the application of a range of novel broad-spectrum ABPs targeting the various subclasses of serine proteases, to include those proteases involved in ENaC activation. Additionally, the application of more selective ABPs in detecting specific serine proteases is investigated.

Compounds were synthesised by Solid-Phase Peptide Synthesis (SPPS) using a standard Fmoc/tBu strategy. Kinetic evaluation of synthesised ABPs against various serine proteases was determined by fluorogenic steady-state enzyme assays. Furthermore, application of ABPs and confirmation of irreversible nature of the compounds was carried out through SDS-PAGE and electroblotting techniques.

Synthesised compounds showed potent irreversible inhibition of serine proteases within their respective targeting class (NAP855 vs Trypsin k3/Ki = 2.60 x 106 M-1 min-1, NFP849 vs Chymotrypsin k3/Ki = 1.28 x 106 M-1 min-1 and NVP800 vs Neutrophil Elastase k3/Ki = 6.41 x 104 M-1 min-1). Furthermore ABPs showed little to no cross-reactivity between classes and so display selectivity between classes. The irreversible nature of compounds was further demonstrated through labelling of proteases, followed by separation and detection via SDS-PAGE and electroblotting techniques. Targeted labelling of active proteases only, was demonstrated by failure of ABPs to detect previously inactivated proteases. Extension of the substrate recognition site within probes resulted in an increased potency and selectivity in the detection of the target proteases. Successful detection of neutrophil elastase from CF sputum samples by NVP800, demonstrated the application of compounds within biological samples and their potential use in identifying further proteases involved in ENaC activation and airway dehydration in CF patients.

Concepção e implementação do time-driven activity based costing numa PME industrial: evidência de um estudo empírico

Dissertação para obtenção do Grau de Mestre em Contabilidade e Finanças Orientador: Mestre Paulino Manuel Leite da Silva

Examining current facilitative practices and projections of activity-based physical education degree programs regarding the participation of individuals with disabilties /

Individuals with disabiliiies are increasingly accessing post secondary education opportunities to further develop their educational and career goals. This study examines the current facilitative practices of Canadian university activity-based physical education degree programs on the participation of individuals with disabilities. A critical orientation and descriptive/interpretative approach allows insight into unique stories and experiences of physical education practitioners and special needs professionals as they attempt to provide equitable educational experiences within a least restrictive environment. Leading practitioners are used to triangulate and strengthen the validity of the data while providing direction and advocacy for future development and inclusion of individuals with disabilities. The study concludes with seven recommendations, each providing university activity-based physical education degree programs with viable opportunities for helping create equitable opportunities for individuals with disabilities.

Power and production in site-based management: the responses of women secondary principals to structural reform in South Australian education

After describing and analysing the purpose and shape of structural reform in Australia, the author uses the data gained from interviews with 16 South Australian women principals, to examine its effects on the work of principals. She uses her analysis to issue some warnings about the current moves to Local School Management in South Australia

Evaluation of user perceptions of an application of activity-based costing

This study examined the extent to which preparers and end-users of Activity-Based Costing information are satisfied with this technique and the factors forming the basis of that belief.

Physical activity based professional development for teachers : the importance of whole school involvement

Objective: This study sought to investigate teachers’ perceptions of a physical activity-related professional development intervention.
Design: Interview-based qualitative approach founded on the interpretive paradigm.
Setting: Purposive selection of one high-rated independent, and one low-rated public primary school from Auckland, New Zealand.
Method: A qualitative approach was used, incorporating a questionnaire used for purposive sampling and a total of eighteen semi-structured interviews with six teachers from two primary schools.
Results: Participants highly valued the inclusion of all teachers in onsite professional development and felt supported by a strong, positive organizational culture.
Conclusion: Participant teachers believed physical activity played an important role within the school setting, and recognized their role in the provision of opportunities for their students. However, they did not necessarily feel confident and competent to facilitate opportunities. A specific professional development intervention was introduced as one form of support to address this need. Teachers valued first-hand experience of professional development and reported particular enjoyment if they participated alongside their colleagues.

Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.