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.

Specification-driven approach for protocol design of distributed computing systems

An important issue in the design of a distributed computing system (DCS) is the development of a suitable protocol. This paper presents an effort to systematize the protocol design procedure for a DCS. Protocol design and development can be divided into six phases: specification of the DCS, specification of protocol requirements, protocol design, specification and validation of the designed protocol, performance evaluation, and hardware/software implementation. This paper describes techniques for the second and third phases, while the first phase has been considered by the authors in their earlier work. Matrix and set theoretic based approaches are used for specification of a DCS and for specification of the protocol requirements. These two formal specification techniques form the basis of the development of a simple and straightforward procedure for the design of the protocol. The applicability of the above design procedure has been illustrated by considering an example of a computing system encountered on board a spacecraft. A Petri-net based approach has been adopted to model the protocol. The methodology developed in this paper can be used in other DCS applications.

Groove Binding Ligands for the Interaction with Parallel-Stranded ps-Duplex DNA and Triplex DNA

DNA adopts different conformations not only based on novel base pairs, but also with different chain polarities. Besides several duplex structures (A, B, Z, parallel stranded (ps)-DNA, etc.), DNA also forms higher-order structures like triplex, tetraplex, and i-motif. Each of these structures has its own biological significance. The ps-duplexes have been found to be resistant to certain nucleases and endonucleases. Molecules that promote triple-helix formation have significant potential. These investigations have many therapeutic advantages which may be useful in the regulation of the expression of genes responsible for certain diseases by locking either their transcription (antigene) or translation (antisense). Each DNA minor groove binding ligand (MGBL) interacts with DNA through helical minor groove recognition in a sequence-specific manner, and this interferes with several DNA-associated processes. Incidentally, these ligands interact with some non-B-DNA and with higher-order DNA structures including ps-DNA and triplexes. While the design and recognition of minor grooves of duplex DNA by specific MGBLs have been a topic of many reports, limited information is available on the binding behavior of MGBLs with nonduplex DNA. In this review, we summarize various attempts of the interaction of MGBLs with ps-DNA and DNA triplexes.

Use of modal energy distribution in the design of honeycomb sandwich decks

Through the example of a spacecraft equipment deck, which is generally made of honeycomb sandwich construction, it is shown that modal energy distribution can be used as an effective guideline in improving the deck's frequencies to meet the restrictions imposed upon it. The kinetic energy distribution is employed as a basis for redistributing various packages on the deck. Strain energy distribution is used to identify areas which can be stiffened by bonding �doublers� on the face sheets and the doubler thickness is obtained from a sensitivity analysis.

CFD Based Design Optimizations of the Diffuser of a Gas Dynamically Driver Laser Cavity

Based on the an earlier CFD analysis of the performance of the gas-dynamically controlled laser cavity [1]it was found that there is possibility of optimizing the geometry of the diffuser that can bring about reductions in both size and cost of the system by examining the critical dimensional requirements of the diffuser. Consequently,an extensive CFD analysis has been carried out for a range of diffuser configurations by simulating the supersonic flow through the arrangement including the laser cavity driven by a bank of converging – diverging nozzles and the diffuser. The numerical investigations with 3D-RANS code are carried out to capture the flow patterns through diffusers past the cavity that has multiple supersonic jet interactions with shocks leading to complex flow pattern. Varying length of the diffuser plates is made to be the basic parameter of the study. The analysis reveals that the pressure recovery pattern during the flow through the diffuser from the simulation, being critical for the performance of the laser device shows its dependence on the diffuser length is weaker beyond a critical lower limit and this evaluation of this limit would provide a design guideline for a more efficient system configuration.The observation based on the parametric study shows that the pressure recovery transients in the near vicinity of the cavity is not affected for the reduction in the length of the diffuser plates up to its 10% of the initial size, indicating the design in the first configuration that was tested experimentally has a large factor of margin. The flow stability in the laser cavity is found to be unaffected since a strong and stable shock is located at the leading edge of the diffuser plates while the downstream shock and flow patterns are changed, as one would expect. Results of the study for the different lengths of diffusers in the range of 10% to its full length are presented, keeping the experimentally tested configuration used in the earlier study [1] as the reference length. The conclusions drawn from the analysis is found to be of significance since it provides new design considerations based on the understanding of the intricacies of the flow, allowing for a hardware optimization that can lead to substantial size reduction of the device with no loss of performance.

A neural network based automatic generation control design through reinforcement learning

This paper presents the design and implementation of a learning controller for the Automatic Generation Control (AGC) in power systems based on a reinforcement learning (RL) framework. In contrast to the recent RL scheme for AGC proposed by us, the present method permits handling of power system variables such as Area Control Error (ACE) and deviations from scheduled frequency and tie-line flows as continuous variables. (In the earlier scheme, these variables have to be quantized into finitely many levels). The optimal control law is arrived at in the RL framework by making use of Q-learning strategy. Since the state variables are continuous, we propose the use of Radial Basis Function (RBF) neural networks to compute the Q-values for a given input state. Since, in this application we cannot provide training data appropriate for the standard supervised learning framework, a reinforcement learning algorithm is employed to train the RBF network. We also employ a novel exploration strategy, based on a Learning Automata algorithm,for generating training samples during Q-learning. The proposed scheme, in addition to being simple to implement, inherits all the attractive features of an RL scheme such as model independent design, flexibility in control objective specification, robustness etc. Two implementations of the proposed approach are presented. Through simulation studies the attractiveness of this approach is demonstrated.

A new approach to the design of reinforcement schemes for learning automata

On introduit une nouvelle classe de schémas de renforcement des automates d'apprentissage utilisant les estimations des caractéristiques aléatoires de l'environnement. On montre que les algorithmes convergent en probabilité vers le choix optimal des actions. On présente les résultats de simulation et on suggère des applications à un environnement à plusieurs apprentissages

Effect of Inlet Jet injection angle on a calandria based reactor-An investigation with numerical analysis of heat and mass transfer for an optimum reactor design

Heat and mass transfer studies in a calandria based reactor is quite complex both due to geometry and due to the complex mixing flow. It is challenging to devise optimum operating conditions with efficient but safe working range for such a complex configuration. Numerical study known to be very effective is taken up for investigation. In the present study a 3D RANS code with turbulence model has been used to compute the flow fields and to get the heat transfer characteristics to understand certain design parameters of engineering importance. The angle of injection and of the coolant liquid has a large effect on the heat transfer within the reactor.

Legislativo pós-1988 : reflexões e perspectivas

Reúne uma série de trabalhos sobre o Poder Legislativo brasileiro, com ênfase para a Câmara dos Deputados, no contexto institucional da vigência da Constituição de 1988, a Constituição Cidadã e é organizado em três blocos temáticos: "Organização e funcionamento do Congresso Nacional", " Função comunicativa e interacional do Parlamento" e "Formação do corpo legislativo".

Geolocalização, redes sociais e dispositivos móveis: proposta de sistema gráfico

A geolocalização, os dispositivos móveis cada vez mais sofisticados e eficientes e a disponibilidade instantânea de mapas de todos os lugares do mundo, permitiram o desenvolvimento de novos aplicativos, os chamados serviços baseados em geolocalização; além disso, as redes sociais online têm se consagrado como ferramentas de relações entre pessoas. A combinação destas tecnologias levou ao surgimento de programas como o Foursquare, que alcançou a marca dos 30 milhões de usuários em 2013, quatro anos depois de seu lançamento, o que mostra a importância deste tipo de aplicativo; através do estudo destes aplicativos, observa-se a valorização da vizinhança como fator de afinidade social. Da análise dos elementos de wayfinding, depreende-se que a cidade incorpora marcos com valor emocional. Ao final, é proposto um sistema gráfico que torne visíveis estas referências emocionais.

Do controle social à gestão participativa: perspectivas (pós soberanas) da participação política no SUS

Este trabalho parte da hipótese de que discutir a participação política no SUS exige a problematização das opções e concepções que orientam sua definição como controle social, operacionalizado por meio de mecanismos de representação de interesses. A intenção de promover uma reflexão sobre a participação política no SUS pé remetida ao cotidiano institucional, como desafio de construção de modos de gestão participativos. A proposta metodológica baseis-se em uma abordagem filosófica, que tem por objetivo delinear os conceitos e dispositivos de gestão propostos no campo da Saúde Coletiva, as inovações teóricas que ofertam ao debate sobre a gestão em saúde, tendo por marcador o tema da política. Denominamos matrizes conceituais os dois planos filosófico que selecionamos para estudo no campo da Saúde Coletiva, a saber, o Planejamento em Saúde e o Modelo Assistencial em Defesa da Vida. O conceito de Política que adotamos se define não em termos de igualdade (formal) que se contrapões às diferenças (sociais), mas como coprodução de realidade que se concretiza nas relações entre Igualdade e Diferença, como acesso e uso dos bens-comuns, em sua capacidade indeterminada e aberta de criação de valor. Pensar a participação nestes termos significa tecer participações como possibilidade de instituir normas, e não apenas com controle da execução e fiscalização das normas existentes. Nessa concepção propõe-se pensar a gestão como coprodução de saúde, a partir da publicização e articulação reticular da dimensão normativa da atividade humana, o que implica questionar a produção concreta (portanto local) das políticas e intervenções públicas. Dentre as principais questões teóricas discutidas, destacamos a articulação de redes de cooperação e a construção de saberes, artifícios técnicos e dispositivos que viabilizem a produção e legitimação do valor-Saúde como bem comum. Perspectiva que explicita as implicações que desejamos incorporar ao conceito de gestão participativa, como possível tecnologia de governo pós-soberana. A participação na saúde é pensada, deste modo, a partir do problema de constituição de uma política pública que consiga permanecer aberta e imprevisível, resguardando, contudo, condições materiais de igualdade. Nesse sentido, uma política pública que rompa com os mecanismos da soberania moderna ao incorporar em seus desenhos institucionais a imprevisibilidade da produção normativa, forjando-se como dispositivo ético (portanto aberto e comum) de produção de valor.

O design de Victor Burton

Victor Burton é um designer que dedicou a maior parte do seu trabalho ao livro. A fascinação por este objeto começou ainda na infância devido ao contato com as obras raras da biblioteca da família, o que aguçou o desejo de se tornar designer exclusivamente para projetar livros. Sua atuação no mercado editorial brasileiro começou no final dos anos 1970, na editora Confraria dos Amigos do Livro. Como o maior interesse de Victor no livro é a relação entre texto e imagem, os livros iconográficos se tornaram seu principal objetivo e são nos livros desta natureza onde melhor conseguimos visualizar seu estilo. Victor desenvolveu uma linguagem gráfica própria que redefiniu o padrão do mercado editorial brasileiro. Numa época em que o projeto gráfico, principalmente a capa do livro, entre tantas cores e atrativos disputam a atenção do consumidor nas prateleiras das livrarias, já não é tão fácil identificar nem a editora nem a autoria do projeto gráfico e da capa. Entretanto, os livros de Victor Burton possuem um estilo que nos permite reconhecer sua assinatura. Desta forma, a questão que norteou este trabalho foi por que conseguimos identificar os livros do designer Victor Burton? Sendo assim, o objetivo deste trabalho foi enumerar e identificar os aspectos gráficos que caracterizam o estilo deste designer nos livros iconográficos. Para isso, nos baseamos no método descritivo desenvolvido por Guilherme Cunha Lima, em O Gráfico Amador. A partir das características levantadas, pudemos identificar os principais elementos que nos permite reconhecer a autoria dos trabalhos desenvolvidos por Victor Burton. O uso desses aspectos gráficos reflete o trabalho meticuloso do designer Victor Burton que consegue criar uma narrativa visual auxiliando a leitura do texto através de uma nova leitura gráfica, sobretudo nos livros iconográficos

Otimização da co-pirólise de gasóleo pesado com embalagens de PEAD pós-uso utilizando planejamento fatorial

A co-pirólise é uma rota promissora, uma vez que minimiza o impacto ambiental causado pela disposição do plástico de maneira inadequada, evita seu acúmulo em lixões e permite um melhor aproveitamento de um recurso natural não-renovável, o petróleo, matéria prima importante para a geração de energia e obtenção de produtos químicos. O presente trabalho teve como objetivo a definição das condições experimentais mais propícias à obtenção de líquidos pirolíticos com alta fração de óleo diesel, resultantes da co-pirólise de polietileno de alta densidade (PEAD) pós-consumo com gasóleo pesado tilizando-se catalisador de FCC (Fluid Catalytic Cracking). Como instrumento de otimização das condições experimentais, optou-se pela Metodologia Planejamento Fatorial. Foi também estudado o efeito das condições experimentais, como: a temperatura de reação, a relação gasóleo/polietileno e a quantidade de catalisador no meio reacional. As amostras de polietileno, gasóleo e catalisador foram submetidas à co-pirólise em sistema de leito fixo, sob fluxo constante de nitrogênio, variando-se a temperatura entre 450 C a 550 C, a quantidade de PEAD no meio reacional foi de 0,2 a 0,6 g, e a quantidade de catalisador foi de zero a 0,06 g, mantendo-se fixa a quantidade de gasóleo em 2 g. Foram efetuadas as caracterizações física e química do gasóleo, polietileno pós-uso e do catalisador. Como resultado, obteve-se a produção de 87% de fração de óleo diesel em duas condições diferentes: (a) 550 0C de temperatura sem catalisador; (b) 500 0C de temperatura e 25% de catalisador FCC. Em ambos os casos, a quantidade de gasóleo pesado e PEAD foram constantes (2 g Gasóleo; 0,2 g PEAD), assim com o tempo de reação de 15 minutos. A fração de óleo diesel obtida neste estudo alcançou o poder calorífico de 44,0 MJ/Kg que é similar ao óleo diesel comercial. É importante ressaltar que em ambos os casos nenhum resíduo foi produzido, sendo uma rota ambientalmente importante para reciclagem de embalagens plásticas contaminadas com óleo lubrificante originárias de postos de serviço, visando à recuperação de ambos conteúdo energético e orgânico dos resíduos de embalagens plásticas pós-uso

Reformas de um projeto gráfico: uma análise da coleção do Sítio do Picapau Amarelo em três tempos

Este trabalho busca compreender as atuais configurações dos livros infantojuvenis no mercado editorial, com a entrada na chamada era digital e o aumento crescente na produção de e-books por parte das editoras, ao mesmo tempo em que livros impressos ainda continuam sendo produzidos em larga escala e reinventados constantemente. Partindo do conceito de remediação (BOLTER e GRUSIN, 2000), procura-se descobrir por quais mudanças os livros impressos passaram ao longo dos anos, quais são suas novas configurações gráficas face à atual realidade digital, e com quais critérios esses livros estão sendo transformados em e-books. Para tal, foi feito um estudo de caso dos livros infantojuvenis de Monteiro Lobato, importante autor para a literatura brasileira, com uma análise comparativa da coleção do Sítio do Picapau Amarelo, em quatro edições (três impressas e uma digital) e três épocas diferentes (anos 1940, 1980 e 2000). Focando na parte gráfica e estética dos livros (que foi a parte que se alterou) e com a observação dos protocolos de leitura (CHARTIER, 2011) presentes nas diferentes edições, procura-se compreender as transformações pelas quais esses livros passaram ao longo dos anos, em decorrência do contexto sociocultural em que foram produzidos, além de suas atuais configurações no contexto da cultura da convergência (JENKINS, 2009). Após a análise realizada pôde-se perceber que talvez o processo de remediação no mercado editorial ainda não esteja tão presente quanto se pensava inicialmente, apesar de estar caminhando lentamente nessa direção.