The detection of cellular events is central to understanding biomoleculer processes as well as aid in therapeutic intervention strategies. One of the most fascinating biomoleculer events during the life cycle of a cell is proteolytic cleavage of proteins by enzymes known as proteases. Proteases are ubiquitous and participate in essential functions such as fertilization, embryo development, cell cycle regulation, immune response, tissue remodeling and programmed cell death. As proteases are involved in fundamental cellular processes any dysregulation of protease activity is usually associated with a diseased state. Thus methods for detection of protease activity are desirable as it may facilitate the identification of many pathological conditions which are associated with the aberrant expression and activity of proteases.Towards the goal of a general and modular strategy we have utilized split protein reassembly and coiled coils to develop genetically encoded sensors for detection of proteases. We established our first generation protease design utilizing split firefly luciferase and anti-parallel coiled coils and detected Tobacco Etch Virus (TEV) as a model protease. Two further iterations of the coiled-coil design led to the development of second and third generation of protease sensors which showed substantial improvement in the sensor response and was applied towards detection of therapeutically relevant proteases such as caspase-3, prostate specific antigen (PSA), ß-secretase and calpain-1.We applied our methodolgy to develop protease biosensors for the detection of a family of cysteine protease known as caspases. Caspases are involved in programmed cell death and their misregulation is implicated in cancer as well as neurodegenerative disorders. The panel of caspase biosensors was utilized to investigate caspase cleavage specificity as well as caspase activation in mammalian cytosolic extracts and live mammalian cells. Perhaps more importantly, we discovered cross talk between members of the caspase family which perform different biological functions.Finally, we detail our progress towards mimicking a naturally occurring multicomponent complex formed during programmed cell death, known as the apoptosome which leads to the activation of caspases. We have successfully utilized principles of self assembly and multivalency to assemble multi component complexes which exhibit proteolytic activity similar to the natural apoptosome.

A proof of concept study into the working of network media capture and visualization through the use of Packet Capture in real-time. An application was developed that is able to capture tcp network packets; identify and display images in raw HTTP network traffic through the use of search, sort, error detection, timeout failsafe algorithms in real time. The application was designed for network administrators to visualize raw network media content together with its relevant network source \& address identifiers. Different approaches were tried and tested such as using Perl with GTK+ and Visual Studio C\# .Net. Furthermore two different types of image identification methods were used: raw magic string identification in pure tcp network traffic and HTTP Mime type identification. The latter being more accurate and faster. C# was seen as vastly superior in both speed of prototyping and final performance evaluation. The study presents a novel new way of monitoring networks on the basis of their media content through deep packet inspection

DNA diagnostics are important technologies in molecular and cellular biology. By allowing identification of specific sequences, DNA-based diagnostics potentially provide more accurate and rapid results than protein- or antigen-based diagnostics, primarily because phenotypic changes come much later than changes in genotype. Despite this advantage, there are fewer diagnostic or imaging systems that target DNA than those targeting proteins, antibodies, or antigens.Each type of DNA-based diagnostic has its own, unique set of limitations; however, most can be attributed to issues related to sequence restriction, signal detection, specificity, or some combination thereof. For example, while PCR-based methods allow amplification and assessment of specific DNA sequences, they lack the ability to report information of specific cells, or cell types, within the heterogeneous pool of cells typically found in a tumor biopsy. In addition, none of the currently available DNA detection methods has the potential to be utilized in living cells, a disadvantage which limits the potential applications.The work presented here describes the design and development of a new methodology for the detection of specific double-stranded DNA sequences. This detection method is based on the concept that two inactive fragments of a reporter protein, each coupled to engineered zinc finger DNA-binding motifs, are able to reassemble and form an active complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), can achieve single base-pair specificity, and has the potential to be utilized in living cells.In this dissertation, we discuss the efforts from constructing to refining the system, as well as the future applications of SEER in diagnostics and therapeutics. Chapter I will provide an introduction to DNA detection methods, on which the principles of the SEER system are based. Chapter II describes the design and construction of an enzymatic SEER system, SEER-LAC, using beta-lactamase as the enzyme. In Chapter III, we outline the in vitro characterization of the SEER-LAC system, followed by its optimization in Chapter IV. Chapter V illustrates the efforts to develop SEER system for mammalian cell culture applications. In the final chapter, the future developments and applications of SEER are discussed.

<p>Better methods and tools are needed in the fight against child pornography. This thesis presents a method for file type categorisation of unknown data fragments, a method for reassembly of JPEG fragments, and the requirements put on an artificial JPEG header for viewing reassembled images. To enable empirical evaluation of the methods a number of tools based on the methods have been implemented.</p><p>The file type categorisation method identifies JPEG fragments with a detection rate of 100% and a false positives rate of 0.1%. The method uses three algorithms, Byte Frequency Distribution (BFD), Rate of Change (RoC), and 2-grams. The algorithms are designed for different situations, depending on the requirements at hand.</p><p>The reconnection method correctly reconnects 97% of a Restart (RST) marker enabled JPEG image, fragmented into 4 KiB large pieces. When dealing with fragments from several images at once, the method is able to correctly connect 70% of the fragments at the first iteration.</p><p>Two parameters in a JPEG header are crucial to the quality of the image; the size of the image and the sampling factor (actually factors) of the image. The size can be found using brute force and the sampling factors only take on three different values. Hence it is possible to use an artificial JPEG header to view full of parts of an image. The only requirement is that the fragments contain RST markers.</p><p>The results of the evaluations of the methods show that it is possible to find, reassemble, and view JPEG image fragments with high certainty.</p>

This study investigates how seven Chinese sentence-final particles (SFP le, ne1, ma, ne2, ba1, ba2 and a; hereafter SFP) and their features are represented in English speakers’ L2 Chinese. In this research, SFPs are analysed as heads instantiating different positions in the CP domain (Paul 2009, 2014, 2015), which are head-final, and in particular, they are considered to carry semantic, syntactic and discourse features. As there is no SFP in English, the features on Chinese SFPs are realised by a variety of syntactic means. Through a proficiency test and six experimental tasks, data from 76 participants (including 18 Chinese native speakers, 20 low-intermediate learners, 20 high-intermediate learners and 18 advanced learners) were collected. Results show that English-speaking L2 learners can easily establish the basic syntactic structure of Chinese SFPs and successfully acquire the features attached to SFPs ma, ba1 and a. However, they have significant difficulty in acquiring the features attached to SFPs le, ne1, ne2 and ba2. In general, syntactic features on Chinese SFPs are intact in L2 grammars, whereas semantic features (i.e. syntax-semantics interfaces) are very vulnerable. In addition, it is found that not all discourse features (syntax-discourse interfaces) are problematic. Findings indicate that both L1 grammar (i.e. L1 transfer) and L2 input (frequency, saliency and complexity) play important roles in affecting learners’ acquisition of the features attached to Chinese SFPs. In particular, learners seem to transfer all feature sets from their L1 English. Semantic features that have been transferred from their L1 English but that are neither confirmed nor disconfirmed by the Chinese input have become dormant in the L2 Chinese, which complements the Dormant Feature Hypothesis (Yuan 2014). Furthermore, the homomorphous SFPs which exhibit a ‘one-to-many’ form-meaning connection are believed to complicate learners’ recognition and acquisition of relevant features on SFPs. It is also demonstrated that the mapping of a feature across CP domains (i.e. from a two-CP structure to a one-CP structure) can be problematic and difficult. The discourse feature needs to be reassembled in L2 grammars, which advances the arguments of the Feature Reassembly Hypothesis (Lardiere 2008, 2009a,b).

Better methods and tools are needed in the fight against child pornography. This thesis presents a method for file type categorisation of unknown data fragments, a method for reassembly of JPEG fragments, and the requirements put on an artificial JPEG header for viewing reassembled images. To enable empirical evaluation of the methods a number of tools based on the methods have been implemented. The file type categorisation method identifies JPEG fragments with a detection rate of 100% and a false positives rate of 0.1%. The method uses three algorithms, Byte Frequency Distribution (BFD), Rate of Change (RoC), and 2-grams. The algorithms are designed for different situations, depending on the requirements at hand. The reconnection method correctly reconnects 97% of a Restart (RST) marker enabled JPEG image, fragmented into 4 KiB large pieces. When dealing with fragments from several images at once, the method is able to correctly connect 70% of the fragments at the first iteration. Two parameters in a JPEG header are crucial to the quality of the image; the size of the image and the sampling factor (actually factors) of the image. The size can be found using brute force and the sampling factors only take on three different values. Hence it is possible to use an artificial JPEG header to view full of parts of an image. The only requirement is that the fragments contain RST markers. The results of the evaluations of the methods show that it is possible to find, reassemble, and view JPEG image fragments with high certainty.

No description available.

À partir de l’œuvre du cinéaste et artiste hongrois Péter Forgács, la thèse aborde plus généralement la question du remontage de films amateurs dans le documentaire contemporain et examine les enjeux esthétiques, historiques et politiques du remploi des films de famille. Les concepts de déterritorialisation et de reterritorialisation développés par Gilles Deleuze et Félix Guattari nous permettent d’interroger la manière dont la pratique « archéologique » du remontage ouvre les films de famille à de multiples resignifications. Comment traversent-ils des territoires esthétiques, géographiques, institutionnels et politiques, de mémoire, d’histoire et de pensée ? Comment, à travers le temps, les histoires que contiennent ces rouleaux des pellicules substandard sont-elles traversées par l’Histoire ? Comment ces films peuvent-ils, rétrospectivement, subvertir la construction officielle de l’histoire et de la mémoire et renégocier la perception du temps historique ? Quelle métahistoire construisent-ils ?L’analyse de la déterritorialisation et de la reterritorialisation des films de famille par le remploi, nous permet d’interroger la manière dont la pratique du remontage crée un nouveau territoire cinématographique, où la mémoire et l’histoire du passé comme de l’avenir peuvent être creusées dans et par l’image. Ici apparaissent les enjeux poét(h)iques et politiques du remploi. / Through the work of Hungarian artist and filmmaker Péter Forgács, the thesis addresses the question of the reuse (remontage) of amateur films in contemporary documentary and examines the aesthetic, historical and political implications of the appropriation of home movies. The concepts of deterritorialization and reterritorialization developed by Gilles Deleuze and Félix Guattari allow us to question how the “archeological” practice of reassembly exposes home movies to multiple significations. How can they traverse aesthetic, geographical, institutional and political territories as well as territories of memory, history and thought? How are the stories contained in these rolls of substandard film traversed by History? How can these films subvert the official construction of history and memory and renegotiate the perception of historical time? What metahistory do they establish?The analysis of deterritorialization and reterritorialization of home movies allows us to question how the practice of reassembly creates a new cinematographic territory, in which memory and history of the past, as of the future, can be mined in and by the image. Thus remontage implicates poet(h)ical and political issues.

Análises baseadas em traços funcionais descrevem as espécies em relação às suas características biológicas e assim complementam as informações taxonômicas. Ainda que diversos trabalhos sejam realizados visando descobrir impactos ambientais após a criação de usinas hidrelétricas, poucos são voltados para investigar padrões de estabilização da estrutura da ictiofauna em termos taxonômicos e funcionais; a resposta funcional em longo prazo, em particular, é totalmente desconhecida. Neste estudo buscamos investigar padrões de reestruturação da assembléia de peixes em termos taxonômicos e funcionais nas zonas de transição, fluvial e lacustre, ao longo de dez anos, englobando momentos anteriores e posteriores à construção da UHE Peixe Angical, rio Tocantins. Nossos resultados revelaram indícios de estabilização taxonômica e funcional dentro da primeira década do reservatório, nas diferentes zonas. Tanto a riqueza taxonômica quanto a riqueza funcional apresentaram declínio progressivo após a instalação da barragem, atingindo níveis mais estáveis após seis anos. Conforme esperado, o aumento na abundância registrado no primeiro ano do reservatório não se manteve ao longo do tempo e foi possível notar um decréscimo acentuado em todos os pontos amostrais. Houve evidente alteração na composição taxonômica e funcional ao longo dos anos, com menor taxa de mudança a partir do 6º ano. Espécies registradas na fase Rio com maiores abundâncias praticamente deixaram de ser registradas após a criação da barragem, enquanto outras espécies com características que lhes permitiram resistir aos filtros ambientais do reservatório se destacaram em número de indivíduos. Espacialmente, foi possível notar diferentes padrões de composição tanto para a estrutura taxonômica quanto para a estrutura funcional da assembléia. Além disso, nossos resultados apontam que a perda de diversidade funcional se dá por fatores estocásticos; e que, apesar de haverem perdas de perfis funcionais através da perda de espécies, ainda não foi possível constatar uma convergência funcional. Portanto, dez anos de represamento foi suficiente para observarmos fortes padrões de alteração sobre a ictiofauna afetada pelo represamento da UHE Peixe Angical, além da eminente estabilização dos atributos taxonômicos e funcionais nas diferentes zonas do reservatório ao longo do tempo. / Functional traits describe species in respect to functional characteristics and therefore complement taxonomic information. Although several studies investigate environmental impacts caused by hydropower dams, few have focused on long-term patterns of functional and taxonomic fish diversity; changes in functional structure, in particular, are largely unknown. In this study, we investigated long-term changes (10 years) in taxonomic and functional diversity across environmental gradients created by the construction of Peixe Angical hydropower dam, Tocantins River, Amazon Basin. Our results revealed trends of functional and taxonomic stabilization 10 years after river regulation, in the different zones of the reservoir. Both the functional and taxonomic diversity showed progressive decrease over time, reaching more stable levels after 6 years. As expected, the increase in fish abundance registered soon after damming was followed by a marked decrease in all sites. We observed strong alteration in functional and taxonomic composition over the years, with more stable values after the sixth year. The most abundant species in the river period virtually disappeared after river damming, replaced by species with adaptations to cope with the environmental filters created by the impoundment. In addition, our results showed that the loss of functional diversity occurred stochastically; despite the loss of functional profiles with the loss of species, it was not possible to determine functional convergence. In conclusion, Peixe Angical hydropower dam induced strong changes in the taxonomic and functional structure of fish assemblages, with trends of stabilization ten years after river impoundment, in the different zones of the reservoir.

This thesis examines the problem of the automated re-assembly of jigsaw puzzles. The objectives of this research are as follows: to provide a clear statement of the jigsaw puzzle re-assembly problem; to find out which solution technique is best suited to this problem; to determine the level of sensitivity of the proposed solution technique when solving different variations of this problem; and to explore solution methods for solving incomplete jigsaw puzzles (puzzles with missing pieces). The jigsaw puzzle re-assembly problem has been investigated only intermittently in the research literature. This work presents an extensive examination of the suitability and efficiency of the standard solution techniques that can be applied to this problem. A detailed comparison between different solution methods including Genetic Algorithms, Simulated Annealing, Tabu Search and Constraint Satisfaction Programming, shows that a constraint-based approach is the most efficient method of generating solutions to the jigsaw puzzle problem. The proposed re-assembly algorithm is successful. Consequently, it can be used in development of automated solution generators for other problems in the same domain, thus creating new theoretical and applied directions in this field of research. One potential theoretical line of research concerns jigsaw puzzles that do not have a complete set of puzzle pieces. These incomplete puzzles represent a difficult aspect of this problem that is outlined but can not be resolved in the current research. The computational experiments conducted in this thesis demonstrate that the proposed algorithm being optimised to re-assemble the jigsaw puzzles is not efficient when applied to the puzzles with missing pieces. Further work was undertaken to modify the proposed algorithm to enable efficient re-assembly of incomplete jigsaw puzzles. Consequently, an original heuristic strategy, termed Empty Slot Prediction, was developed to support the proposed algorithm, and proved successful when applied to certain sub-classes of this problem. The results obtained indicate that no one algorithm can be used to solve the multitude of possible scenarios involved in the re-assembly of incomplete jigsaw puzzles. Other variations of the jigsaw puzzle problem that still remain unsolved are presented as avenues for future research. The solution of this problem involves a number of procedures with significant applications in other computer-related areas such as pattern recognition, feature and shape description, boundary-matching, and heuristic modelling. It also has more practical applications in robotic vision and reconstruction of broken artefacts in archaeology.