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.

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.

A sistematização da gestão baseada em atividades (ABM) - Activity Based Management - para aplicação na área hospitalar : caso Centro de Terapia Intensiva de um hospital universitário público

Esta pesquisa tem por base a aplicação da Gestão Baseada em Atividades ABM - Activity Based Management -, em um Centro de Terapia Intensiva (CTI) de um hospital universitário público. Tem corno objetivo aperfeiçoar a prática gerencial do CTI, estabelecendo relações de causa e efeito no uso dos recursos (humanos, tecnológicos, materiais), permitindo efetuar mudanças para sua melhor utilização, maximizando beneficios a toda organização. O enfoque metodológico foi a pesquisa qualitativa e quantitativa, sendo que o Centro de Terapia Intensiva foi o foco de análise na implementação do ABM. As etapas envolvidas na condução deste trabalho foram a exploratória e a dos seminários, orientadas pela técnica de Thiollent (2000). O trabalho propõe urna sistemática de aplicação do ABM em CTI. A partir de sua aplicação no caso específico - CTI de um hospital público de Porto Alegre - discute os principais benefícios e limitações, bem corno as informações gerenciais proporcionadas. De acordo com análise do conteúdo e os resultados obtidos, pôde-se alicerçar o conhecimento teórico e prático do modelo de gestão proposto, estabelecendo urna nova dimensão na gestão de recursos na área hospitalar, com mais eficiência, assegurando a captura de todo potencial de melhoria identificado.

Activity Based Cost (ABC) : qual a importância deste conceito para o executivo de uma operação?

Trata da importância da informação de custos efetivamente gerenciais para as equipes operacionais das empresas que muito poderiam contribuir para a redução de custos e aumento do volume de vendas se soubessem com maior discernimento a composição dos custos dos produtos com os quais trabalham, sua variabilidade em função da variação do volume de vendas e produção, e conseqüentemente a relação destes custos com as demais variações de níveis de atividade de outros produtos da empresa. Além das ações estratégicas que poderiam ser tomadas, a equipe comercial poderia discutir a efetiva agregação de valor de alguns Itens de seu custo para o cliente e, eventualmente, sugerir a alteração de seu produto visando proporcionar melhor aproveitamento da relação custo - benefício à empresa e ao cliente

A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Nationwide impact and vehicle to grid application of electric vehicles mobility using an activity based model

This paper describes the impact of electric mobility on the transmission grid in Flanders region (Belgium), using a micro-simulation activity based models. These models are used to provide temporal and spatial estimation of energy and power demanded by electric vehicles (EVs) in different mobility zones. The increment in the load demand due to electric mobility is added to the background load demand in these mobility areas and the effects over the transmission substations are analyzed. From this information, the total storage capacity per zone is evaluated and some strategies for EV aggregator are proposed, allowing the aggregator to fulfill bids on the electricity markets.

Activity-based protein profiling: The serine hydrolases

With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.

Costing police custody operations

Presents a simulation study of the costing of police custody operations at a UK police force. The custody operation incorporates the arrest, booking-in, interview, detention and court appearance activities. The Activity Based Costing (ABC) approach is used as a framework to show how costs are generated by the three “drivers” of cost, activity and resource. These relate to the design efficiency of the process, the timing and mix of demand on the process and the cost of resources used to undertake the process respectively. The use of discrete-event simulation allows the incorporation of dynamic (time-dependent) and stochastic (variability) elements in the cost analysis. This enables both the amount and timing of the use of capacity and the generation of cost to be established. The concept of committed and flexible resources directs management decisions to the redeployment of unused capacity or alternatively the identification of additional capacity requirements.

The application of a novel, cell permeable activity-based probe for the detection of cysteine cathepsins.

Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS using in-gel fluorescent scanning, obviating the necessity for downstream biotin-streptavidin conjugation and detection procedures.

Delays in activity based neural networks

In this paper we study the effect of two distinct discrete delays on the dynamics of a Wilson-Cowan neural network. This activity based model describes the dynamics of synaptically interacting excitatory and inhibitory neuronal populations. We discuss the interpretation of the delays in the language of neurobiology and show how they can contribute to the generation of network rhythms. First we focus on the use of linear stability theory to show how to destabilise a fixed point, leading to the onset of oscillatory behaviour. Next we show for the choice of a Heaviside nonlinearity for the firing rate that such emergent oscillations can be either synchronous or anti-synchronous depending on whether inhibition or excitation dominates the network architecture. To probe the behaviour of smooth (sigmoidal) nonlinear firing rates we use a mixture of numerical bifurcation analysis and direct simulations, and uncover parameter windows that support chaotic behaviour. Finally we comment on the role of delays in the generation of bursting oscillations, and discuss natural extensions of the work in this paper.

A framework for life cycle cost estimation of a product family at the early stage of product development

A cost estimation method is required to estimate the life cycle cost of a product family at the early stage of product development in order to evaluate the product family design. There are difficulties with existing cost estimation techniques in estimating the life cycle cost for a product family at the early stage of product development. This paper proposes a framework that combines a knowledge based system and an activity based costing techniques in estimating the life cycle cost of a product family at the early stage of product development. The inputs of the framework are the product family structure and its sub function. The output of the framework is the life cycle cost of a product family that consists of all costs at each product family level and the costs of each product life cycle stage. The proposed framework provides a life cycle cost estimation tool for a product family at the early stage of product development using high level information as its input. The framework makes it possible to estimate the life cycle cost of various product family that use any types of product structure. It provides detailed information related to the activity and resource costs of both parts and products that can assist the designer in analyzing the cost of the product family design. In addition, it can reduce the required amount of information and time to construct the cost estimation system.

Custeio Baseado em Atividades (ABC) Aplicado ao Setor Público: estudo de caso no Colégio Militar do Rio de Janeiro (CMRJ)

O objetivo desta pesquisa foi verificar se o método de Custeio Baseado em Atividades (ABC) é adequado para ser aplicado na mensuração dos custos dos serviços educacionais do Setor Público. O tema se torna relevante devido à necessidade de as Organizações Públicas, que prestam serviços educacionais de permitir, através de seus sistemas de custos, o acompanhamento e a avaliação da gestão orçamentária, financeira e patrimonial. Entretanto, o Sistema Integrado de Administração Financeira do Governo Federal (SIAFI) não evidencia quanto custa cada atividade em nenhuma de suas contas. A análise dos resultados aponta ser adequada a aplicação do método de custeio ABC (Custeio Baseado em Atividades) para evidenciação dos custos das atividades e na gestão dos recursos do Setor Público e, em especial, nas organizações que prestam serviços educacionais, onde praticamente todos os seus custos são indiretos e aparentemente fixos, este fato, segundo Kaplan (1998), tornam estas organizações candidatas ideais ao custeio baseado em atividade (ABC). A metodologia utilizada teve como principal finalidade desenvolver, esclarecer e modificar idéias a fim de fornecer hipótese pesquisável para estudos posteriores conforme ensinam Tripodi, Fellin e Meyer (1981, p.64), por fim, esta pesquisa se caracteriza como qualitativa descritiva e teve como fonte de pesquisa a revisão bibliográfica e análise documental e como estratégia metodológica, utilizou-se de um estudo de caso no Colégio Militar do Rio de Janeiro (CMRJ).