A novel graph-based method for targeted ligand-protein fitting

A thesis submitted to the Faculty of Creative Arts, Technologies & Science, University of Bedfordshire, in partial & fulfilment of the requirements for the degree of Master of Philosophy.The determination of protein binding sites and ligand -protein fitting are key to understanding the functionality of proteins, from revealing which ligand classes can bind or the optimal ligand for a given protein, such as protein/ drug interactions. There is a need for novel generic computational approaches for representation of protein-ligand interactions and the subsequent prediction of hitherto unknown interactions in proteins where the ligand binding sites are experimentally uncharacterised. The TMSite algorithms read in existing PDB structural data and isolate binding sites regions and identifies conserved features in functionally related proteins (proteins that bind the same ligand). The Boundary Cubes method for surface representation was applied to the modified PDB file allowing the creation of graphs for proteins and ligands that could be compared and caused no loss of geometric data. A method is included for describing binding site features of individual ligands conserved in terms of spatial relationships allowed identification of 3D motifs, named fingerprints, which could be searched for in other protein structures. This method combine with a modification of the pocket algorithm allows reduced search areas for graph matching. The methods allow isolation of the binding site from a complexed protein PDB file, identification of conserved features among the binding sites of individual ligand types, and search for these features in sequence data. In terms of spatial conservation create a fingerprint ofthe binding site that can be sought in other proteins of/mown structure, identifYing putative binding sites. The approach offers a novel and generic method for the identification of putative ligand binding sites for proteins for which there is no prior detailed structural characterisation of protein/ ligand interactions. It is unique in being able to convert PDB data into graphs, ready for comparison and thus fitting of ligand to protein with consideration of chemical charge and in the future other chemica! properties

Probabilistic methods in the analysis of protein interaction networks

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Reports to the President

A compilation of annual reports for the 1999-2000 academic year, including a report from the President of the Massachusetts Institute of Technology, as well as reports from the academic and administrative units of the Institute. The reports outline the year's goals, accomplishments, honors and awards, and future plans

UNSTABLE TERRITORIES OF REPRESENTATION: Architectural Experience and the Behaviour of Forms, Spaces and the Collective Dynamic Environment

This thesis applies an interdisciplinary cybernetic and phenomenological analysis to contemporary theories of representation and interpretation of architecture, resulting in a speculative theoretical model of architectural experience as a behavioural system. The methodological model adopted for this research defines the main structure of the thesis where the narrative and the contributing parts of its complexity emerge. The narrative is presented through objectives and hypotheses that shift and slide between architectural representation and its experience based on three key internal components in architecture: the architectural forms and spaces, the active observers that interact with their environment, and finally, the responsive environment. Three interrelated research questions are considered. The first seeks to define the influence of the theoretical instability between complex life processes, emerging technologies and active perception upon architecture. The second questions the way in which the architectural experience is generated. The third asks: Does architecture behave? And if so, is it possible to define its behavioural characteristics related to its representation, experience and the medium of communication in-between? The thesis begins by exploring the effect of developments in digitally interactive, biological, and hybrid technologies on representation in architecture. An account of architectural examples considers the shift in the meaning of representation in architecture from the actual and literal to the more conceptual and experimental, from the individual human body and its relations to the multifaceted ecosystem of collective and connected cultures. The writings of Kester Rattenbury, Neil Leach, and Peter Cook among others contribute to the transformation of the ordinary perceptual experience of architecture, the development of experimental practices in architectural theory, and the dynamism of our perception. The thesis goes on to suggest that instability in architectural representation does not only depend on the internal components of the architectural system but also on the principles and processes of complex systems as well as changes in active perception and our consciousness that act as the external influences on the system. Established theoretical endeavours in biology of D’Arcy Thompson, Alan Turing, and John Holland and philosophies of Merleau-Ponty, Richard Gregory, and Deleuze and Guattari are discussed in this context. Pre-programmed and computational models, illustrative and generative, are presented throughout the thesis. In the final stage of the development of the thesis architecture is analysed as a system. This is not an unprecedented notion, however defining the main elements and components of this system and their interactions and thereafter identifying that the system behaves and defining its behavioural characteristics, adds to the knowledge in the field of theoretical and experimental architecture. This thesis considers the behavioural characteristics of architecture to be derived from the hypothetical links and unstable thresholds of its non-dualistic notions of materiality and immateriality, reality and virtuality, and finally, intentionality and interpretation

Estudio de la interacción leguminosas-hongos fitopatógenos mediante una aproximación de genómica funcional (proteómica y transcriptómica)

The objectives pursued in this PhD thesis are to study the legume responses to phytopathogenic fungi by using a functional genomics approach (transcriptomics and proteomics). The pea (Pisum sativum) and the model legume Medicago truncatula were chosen to study their stress responses to Erysiphe pisi and Mycosphaerella pinodes pathogens. The pea crop constitutes an important source of protein for human consumption, being one of the most grown grain legume in the world, meanwhile M. truncatula is an important forage legume which is being used as a model plant for use in molecular and classical genetic studies. Moreover, within the phytopathogenic fungi, powdery mildew and ascochyta blight are one the most plant pathogenic fungi that seriously constrain crop production worldwide. Within the functional genomics approaches carried out to study the defense mechanisms in legumes to phytopathogenic fungi infection, a differential expression proteomic approach was carried out in order to unveil which resistance mechanisms are involving in the defense response of the pea to E. pisi and M. pinodes infections. The results obtained from the application of this approach in both studies are presented. The changes observed reflect a metabolic adjustment performed by the pea in response to phytopathogen fungi infection. To go deeper into the knowledge of the defence mechanism related to the response to E. pisi infection in legumes, two different transcriptomics approaches were performed using the model legume M. truncatula. Thus, M. truncatula - E. pisi pathosystem was analyzed by using microarray and quantitative real-time PCR platforms, respectively. These results revealed a wide variety of mechanisms and pathways involved in M. truncatula in response to E. pisi infection, including an important number of genes belonging to diverse functional groups, which will help to develop new genetic tools in the breeding program in legumes.Los objetivos planteados en esta tesis doctoral son analizar las respuestas de las leguminosas a la infección de hongos fitopatógenos mediante el uso de una aproximación de genómica funcional (transcriptómica y proteómica). El guisante (Pisum sativum) y la leguminosa modelo Medicago truncatula fueron elegidas para estudiar sus respuestas de estrés a los patógenos Erysiphe pisi y Mycosphaerella pinodes. Dentro de las aproximaciones de genómica funcional llevadas a cabo para estudiar los mecanismos de defensa de las leguminosas a la infección de hongos fitopatógenos, una aproximación de proteómica diferencial fue llevada a cabo con el objetivo de revelar cuáles son los mecanismos de resistencia implicados en las respuestas de defensa del guisante a la infección de E. pisi y M. pinodes. Los resultados obtenidos de la aplicación de esta aproximación en ambos estudios son presentados. Los cambios observados reflejan un ajuste metabólico del guisante en respuesta a la infección de estos hongos fitopatógenos. Para profundizar en el conocimiento de los mecanismos de defensa de las leguminosas en respuesta a la infección de E. pisi, dos aproximaciones transcriptómicas diferentes fueron realizadas usando la leguminosa modelo M. truncatula. En consecuencia, el patosistema M. truncatula - E. pisi fue analizado mediante dos plataformas, microarray y PCR cuantitativa en tiempo real, respectivamente. Estos resultados revelaron una amplia variedad de mecanismos y rutas implicadas en las respuestas de defensa de M. truncatula en respuesta a la infección de E. pisi, incluyendo un importante número de genes pertenecientes a diversos grupos funcionales, los cuales ayudaran en el desarrollo de nuevas herramientas genéticas en los programas de mejora vegetal en leguminosas

Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica EstruturalThe microbial plant cell wall degradation is one of the most important processes in the global turnover of atmospheric carbon dioxide. The work presented in this thesis addressed the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens, essential to the process of cellulose degradation, and aimed to study some of the components involved in their architecture (cohesins and dockerins) and efficiency (Carbohydrate-Binding Modules - CBMs). For this I used a combination of Nuclear Magnetic Resonance (NMR), X-ray crystallography and computer modeling techniques. My objective was to help rationalize the molecular determinants of specificity of CBMs, including the CtCBMs of families 11, 30 and 44, and the mechanisms of molecular recognition between cohesins and dockerins. In Chapter I, I present a general introduction to the theme of degradation of plant cell walls, with special attention to the cellulosome and its components. In Chapter II, I discuss the structural characteristics of the CtCBM11 based on the structures obtained by NMR at 25 and 50 °C and the structure obtained by crystallography. I found that although similar, the structures show some differences, particularly regarding the binding cleft area, which explains the negative results obtained by co-crystallization. In Chapter III and IV I study the molecular determinants of specificity in modules CtCBM11, 30 and 44, based on NMR and computer modeling data. I found that the atoms of the cellooligosaccharides most important for binding are the ones at positions 2 and 6 of the central units of the ligands. Moreover, I characterized the mechanisms responsible for selection and binding of these modules to various substrates. I established that binding occurs by a mechanism for conformational selection, where the topology of the residues of the protein, the conformation of the ligand and the number of glucose units, play a fundamental role. Chapters V and VI reveal the determination of the 3D structure of the cohesin-module X-dockerin complex of C. thermocellum and the cohesin-dockerin complex of B. cellulosolvens, respectively. Both complexes belong to the type II and their analysis allowed obtaining important information about the structural features that define the cohesin-dockerin interaction. The structure belonging to C. thermocellum revealed that the module X is essential for the stability of the complex. Moreover, for the first time the 3D structure of a cohesin-dockerin complex from B. cellulosolvens was determined. In this complex the dockerin is rotated 180º when compared to other complexes. This gives the cellulosome plasticity. In the final chapters, I present the NMR and X-ray crystallography techniques I used throughout the study. Finally, I draw some general conclusions about all the work done.Fundação para a Ciência e Tecnologia - SFRH/BD/35992/2007, and projects PTDC/QUI/68286/2006, PTDC/QUI-BIQ/100359/2008 and PTDC/BIA-PRO/103980/200

Network analysis of large scale object oriented software systems

PhD ThesisThe evolution of software engineering knowledge, technology, tools, and practices has seen progressive adoption of new design paradigms. Currently, the predominant design paradigm is object oriented design. Despite the advocated and demonstrated benefits of object oriented design, there are known limitations of static software analysis techniques for object oriented systems, and there are many current and legacy object oriented software systems that are difficult to maintain using the existing reverse engineering techniques and tools. Consequently, there is renewed interest in dynamic analysis of object oriented systems, and the emergence of large and highly interconnected systems has fuelled research into the development of new scalable techniques and tools to aid program comprehension and software testing. In dynamic analysis, a key research problem is efficient interpretation and analysis of large volumes of precise program execution data to facilitate efficient handling of software engineering tasks. Some of the techniques, employed to improve the efficiency of analysis, are inspired by empirical approaches developed in other fields of science and engineering that face comparable data analysis challenges. This research is focused on application of empirical network analysis measures to dynamic analysis data of object oriented software. The premise of this research is that the methods that contribute significantly to the object collaboration network's structural integrity are also important for delivery of the software system’s function. This thesis makes two key contributions. First, a definition is proposed for the concept of the functional importance of methods of object oriented software. Second, the thesis proposes and validates a conceptual link between object collaboration networks and the properties of a network model with power law connectivity distribution. Results from empirical software engineering experiments on JHotdraw and Google Chrome are presented. The results indicate that five considered standard centrality based network measures can be used to predict functionally important methods with a significant level of accuracy. The search for functional importance of software elements is an essential starting point for program comprehension and software testing activities. The proposed definition and application of network analysis has the potential to improve the efficiency of post release phase software engineering activities by facilitating rapid identification of potentially functionally important methods in object oriented software. These results, with some refinement, could be used to perform change impact prediction and a host of other potentially beneficial applications to improve software engineering techniques

Reports to the President

A compilation of annual reports for the 1981-1982 academic year, including a report from the President of the Massachusetts Institute of Technology, as well as reports from the academic and administrative units of the Institute. The reports outline the year's goals, accomplishments, honors and awards, and future plans

Development of novel gelatin-binding proteins for targeting therapeutics to cartilage lesions in the osteoarthritic joint

Osteoarthritis (OA) is the most common chronic joint disease appearing to be increasing in prevalence amongst a population that is now on average living significantly longer. Age is a well-established risk factor for OA. Type II (TII ) gelatin, derived from TII collagen is found abundantly in damaged regions of the OA joint, making it an ideal target for binding to target a therapeutic. Retention and integration of mesenchymal stem cells (MSCs) within the damaged regions of the OA joint could facilitate not only joint repair, a prolonged improvement in pain and mobility for OA patients, but also could be defined as the first disease modifying OA therapeutic. The collagen binding domain (CBD) of Matrix-metalloproteinase-2 (MMP-2) binds to TII gelatin and has previously been used by the group as a starting point for designing mutants with increased affinity for TII gelatin. 222 is a chimeric protein previously developed by the Hollander group and proven to bind with an affinity fourteen times greater than CBD to TII gelatin. However, it was concluded that 222 would be challenging to exploit therapeutically because of poor solubility and variable efficacy, therefore the aim of this thesis was to further enhance binding efficacy and/or to improve solubility of 222. Subsequent designed mutant proteins here were intended to be used to coat mesenchymal stem cells (MSCs), to promote potent adherence to TII gelatin in the OA joint. In this work a combination of in silico and in vitro experiments were conducted with the aim of developing proteins that bind with a greater affinity to TII gelatin than 222. Firstly, binding residues were assessed in silico for surface exposure and stability, to impact upon binding and be recoverable in vitro. Four mutants were selected to take to in vitro experiments. Asn (N) 11, 69 and 127 (equivalents in the three modules of 222) were, identified as the most important binding residues. As their mutation to Ala (A) caused the greatest decrease in binding affinity, when assessed in vitro using a TII gelatin binding assay. All three important residues were then mutated to every alternate in silico. Docking predicted 222W, with Trp (W) (substituted at position 11, 69 and 127) as the only such mutant predicted in silico to have stronger binding affinity than 222. Use of a Maltose-Binding Protein (MBP) tag was successful in aiding soluble expression of this mutant 222W. However, this mutation seemed also to alter the characteristics of the protein, preventing 3C protease cleavage, meaning protein of interest (POI) alone could not be isolated. Alongside, the CamSol webserver was utilised to design six mutants with increasing solubility. The CamSol tool predicted mutant protein CamSol6 (CS6) to be the most soluble with three substitutions Val (V) 4 to Glu (E); Phe (F) 6 to E; Tyr (Y) 9 to E and an insertion of EEE between Gly (G) 97 and Y98. CS6 was expressed, purified, and characterised for solubility and binding to TII gelatin. An amorphous precipitation assay with polyethylene glycol (PEG) and ammonium sulfate was used to give a measure of apparent solubility. CS6 was more soluble than both CBD and 222, however binding to TII gelatin was reduced compared to 222. The work presented in this thesis has identified key residues important for the binding of 222 to TII gelatin, as well as those important for enhancing solubility. To be used as a therapeutic a protein must be shown to be stable, soluble, demonstrate minimal heterogeneity, minimal contamination as well as being suitable to scale, with consistent and reproducible expression, purification and physiochemistry. Further work is warranted to achieve these properties and develop a strongly binding and more soluble mutant of 222, with a balance of both characteristics for optimal therapeutic potential