We have synthesized dimethyl derivatives of the GFP chromophore (p-HOBDI) and several of its derivatives, and their photochemistry and photophysics were investigated using various steady-state and time-resolved techniques as follows. We first consider the effect of the £]-barrel on the optical properties of the GFP chromophore (p-HOBDI) experimentally by selective variation of the protonation state of chromophores and different solvents. Each of these forms shows a complex solvatochromic behavior and is governed by both polar and acid/base properties of the solvents. In contrast to their solution behavior, some O-alkyl GFP chromophore (alkoxy-BDI) derivatives exhibit large fluorescent enhancement in the solid state. The color of the crystalline BDI is tuned by substituent-mediated crystal packing, showing the potential applications in optoelectronic devices. Using femtosecond polarization-sensitive infrared (IR) spectrosceopy of the C=O stretching mode of the HOBDI, we have then discovered a near complete twisting around the ethylenic bridge between the phenolate and imidazolidinone groups upon electronic excitation. Cis/trans isomerization induced by the rotation around the bridge is thought to be responsible for the behavior of blinking in fluorescent protein; however, the mechanism of the thermal reverse isomerization is more problematic. Thus we synthesized BDI derivatives with decreasing para-donating ability, HO, CH3O, CH3, H, and Cl, and used a Hammett plot for the rate study. With a positive â value, we conceived, for the first time, a novel nucleophilic addition/elimination mechanism. Finally, the GFP chromophore falls into the general category of hydroxyarene photoacids, which exhibit high excited-state acidities but neutral ground states. A hydroxyl substituent at the meta position shows enhanced charge transfer and greater acidity in the excited state. As a result, we have demonstrated that the fast quenching of the excited state by internal conversion to the ground state is much slower in meta- than in para-HOBDI derivatives. This allows studies of this ultrafast intermolecular ESPT that competes with isomerization. The photoinduced dynamics of the meta isomer of GFP chromophore was further investigated using femtosecond transient absorption and fluorescence upconversion spectroscopies.

Made available in DSpace on 2015-05-07T14:49:24Z (GMT). No. of bitstreams: 1 parte2.pdf: 5319403 bytes, checksum: 9db382d5d0f4d7aa4560566932ec9ac8 (MD5) Previous issue date: 2013-01-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In this dissertation, the author discusses environmental education, conservation units and law as instruments that can be used in favor of local sustainable development. The aim is to find common ground between instruments analyzed so that they can be used jointly in favor of improving the quality of life and strengthening the work of public servants and the community involved with environmental issues. This is a case study, starting from the analysis of practices in environmental education encompassed by APA Araripe region. For both, there was an interdisciplinary effort to provide general information about the area the reader, approaching biophysical and social aspects. We tried to carry out a survey and evaluation of the main activities focused on environmental education in the region since 1988, mainly in the municipalities of Bodocó, Crato and Garden. At the same time we sought legal instruments in order to facilitate its understanding by all those involved with the Environmental Education in Protected Areas. So besides a ransom of history of environmental education in the region, it is intended that knowledge of the existence and scope of the laws may make it more effective and efficient practices both as standards. / Na presente dissertação de mestrado, o autor aborda a Educação Ambiental, as Unidades de Conservação e o Direito como instrumentos que podem ser utilizados em favor do desenvolvimento sustentável local. O intuito é encontrar pontos em comum entre instrumentos analisados de modo que possam ser utilizados conjuntamente em favor da melhoria da qualidade de vida e reforçando o trabalho de servidores públicos e da coletividade envolvidos com as questões ambientais. Trata-se de um estudo de caso, partindo do análise das práticas de Educação ambiental na região englobada pela APA Chapada do Araripe. Para tanto, realizou-se um esforço interdisciplinar para fornecer dados gerais sobre a região ao leitor, abordando-se aspectos biofísicos e sociais. Procurou-se realizar levantamento e avaliação das principais atividades voltadas para a Educação ambiental na região desde 1988, principalmente nos municípios de Bodocó, Crato e Jardim. Ao mesmo tempo buscou-se instrumentos jurídicos aplicáveis no intuito de facilitar sua compreensão por todos aqueles que estão envolvidos com a Educação Ambiental em Unidades de Conservação. Assim, além de um resgate de histórico de da Educação Ambiental na Região, pretende-se que o conhecimento da existência e do alcance das leis possa tornar mais efetivas e eficazes tanto as práticas quanto as normas.

Ecological economics has arisen over a period of three decades with a strong emphasis on the essential need to recognise the embeddedness of the economy in the biophysical. However, that element of realism is not matched by an equally well informed social theory. Indeed the tendency has been to adopt mainstream economic concepts, theories and models formulated of the basis of a formal mathematical deductivist approach that pays little or no attention to social reality. Similarly mainstream economic methods are employed as pragmatic devices for communication. As a result ecological economics has failed to develop its own consistent and coherent theory and failed to make the link between the social and the economic. In order to reverse this situation the social and political economy must be put to the fore and that is the aim of social ecological economics. This paper provides a brief overview of the arguments for such a development. The prospect is of unifying a range of critical thought on the social and environmental crises with the aim of informing the necessary social ecological transformation of the economy. / Series: SRE - Discussion Papers

Single cell migration in artificial collagen gels as an in vitro model system in the context of cancer are studied. Cell and matrix mechanical properties are determined using atomic force microscopy and an advanced analysis method. Matrix pore-size is studied using a novel approach and analysis method. A novel, minimally invasive approach to determine the amount of displacement of the cell microenvironment due to force generation of single cells during migration in artificial 3D collagen gels is introduced. An automated analysis and user friendly software to analyze high-throughput cell invasion is introduced. These methods are used to study cell migration and mechanical properties of the breast cancer cell lines MDA-MB-231 and MCF-7 and the influence of cell nuclear elasticity is investigated. Using mouse embryonic fibroblasts, the role of focal adhesion kinase (FAK) during cell migration is studied using FAK deficient knock-out cell lines FAK-/- and control FAK+/+ as well as kinase-dead mutants FAKR454/R454 and control FAKWT/WT.:Abstract i Acknowledgements iii 1 Introduction 1 2 Background 5 2.1 Cancer — An ever-changing Disease 5 2.1.1 Carcinogenesis and Neoplasm 6 2.1.2 Hallmarks of Cancer 7 2.1.3 Metastasis— The malignant Progression of Cancer 7 2.1.4 Metastatic Cascade 9 2.2 The Cell— Where it begins 10 2.2.1 Actomyosin Complex 12 2.2.1.1 Actin Monomer 12 2.2.1.2 Polymerization 12 2.2.1.3 Structures 14 2.2.1.4 Actin Cortex 15 2.2.1.5 Filopodia 16 2.2.1.6 Lamellipodium 16 2.2.1.7 Invadopodium 17 2.2.1.8 Stress Fibers 17 2.2.1.9 Actin in Cancer and Metastasis 17 2.2.1.10 Myosin and Actin 18 2.2.2 Focal Adhesions 19 2.2.3 Microtubules 20 2.2.4 Intermediate Filaments 21 2.2.5 Cellular Stiffness 22 2.2.6 Nuclear Deformability 23 2.3 The Extracellular Matrix— Where it happens 24 2.3.1 Components and Structure 25 2.3.2 Collagen as a Model System 26 2.3.2.1 Collagen I Fibril Formation 27 2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28 2.4 Single Cell Migration— Why it spreads 29 3 Materials and Methods 31 3.1 Cell Culture 31 3.1.1 Cancer Cells 31 3.1.2 Mouse fibroblasts 32 3.1.3 Pharmacological treatment 34 3.2 Collagen matrices 34 3.3 Cell Elasticity 36 3.3.1 Atomic Force Microscopy 36 3.3.2 Preparation 37 3.3.3 Data Aquisition 38 3.3.4 Data Analysis 38 3.4 Matrix Stiffness 40 3.4.1 Preparation 40 3.4.2 Data Aquisition 41 3.4.3 Data Analysis 41 3.5 Invasion Assay 42 3.5.1 Preparation 42 3.5.2 Data aquisition 44 3.5.3 Data Analysis 44 3.6 Matrix Topology 48 3.6.1 Preparation 49 3.6.2 Data Acquisition 50 3.6.3 Data Analysis 51 3.6.3.1 Binarization 51 3.6.3.2 Pore-Size 53 3.6.3.3 Fiber Thickness 54 3.7 Fiber Displacement 55 3.7.1 Preparation 56 3.7.2 Data Aquisition 56 3.7.3 Data analysis 57 3.7.3.1 Fiber Displacement 59 3.7.3.2 Cell Segmentation 60 3.7.3.3 Shell Analysis 61 3.8 A toolset to understand Single Cell Migration and what influences it 62 4 Results 65 4.1 Cell Elasticity 65 4.1.1 Example Force-Distance Curves 66 4.1.2 Single Cell Elasticity 67 4.2 Matrix Stiffness 69 4.3 Invasion 71 4.4 Matrix Topology 75 4.5 Influence of Cell Nucleus on Cell Migration 79 4.5.1 Cellular Elasticity 79 4.5.2 Invasion 81 4.6 Fiber Displacement 89 4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93 4.7.1 FAK Knock-Out 93 4.7.2 Kinase-dead FAK Mutant 96 5 Discussion 103 References 107 / Die Einzelzellmigration in künstlichen Kollagennetzwerken als ein in vitro Modellsystem im Kontext von Krebs wurde studiert. Mechanische Eigenschaften von Zellen und der verwendeten Kollagennetzwerke wurden mithilfe der Atomic Force Microscopy (AFM) und weiterentwickelten Analysemethoden bestimmt. Die Porengröße der verwendeten Kollagennetzwerke wurde mit einer neuentwickelten Auswertemethode analysiert. Eine neuartige, minimal-invasive Methode zur Bestimmung der Verformung der Mikroumgebung von Zellen während der Migration verursacht durch Kräftegenerierung der Zelle wird beschrieben. Die Analyse des Invasions-Assays wurde automatisiert und eine nutzerfreundliche Software entwickelt, mit der große Datenmengen ausgewertet werden können. Diese Methoden wurden verwendet, um mechanische Eigenschaften und Migration der humanen Brustkrebszellinien MDA-MB-231 und MCF-7 zu studieren. Die Rolle der focal adhesion kinase (FAK) wurde mithilfe von embryonalen Maus-Fibroblasten studiert. Sowohl eine FAK knock-out Zellinie FAK-/- und Kontrolle FAK+/+, als auch eine kinase-dead Mutante FAKR454/R454 und Kontrolle FAKWT/WT wurden hinsichtlich ihrer Invasion und Verformung der Mikroumgebung analysiert.:Abstract i Acknowledgements iii 1 Introduction 1 2 Background 5 2.1 Cancer — An ever-changing Disease 5 2.1.1 Carcinogenesis and Neoplasm 6 2.1.2 Hallmarks of Cancer 7 2.1.3 Metastasis— The malignant Progression of Cancer 7 2.1.4 Metastatic Cascade 9 2.2 The Cell— Where it begins 10 2.2.1 Actomyosin Complex 12 2.2.1.1 Actin Monomer 12 2.2.1.2 Polymerization 12 2.2.1.3 Structures 14 2.2.1.4 Actin Cortex 15 2.2.1.5 Filopodia 16 2.2.1.6 Lamellipodium 16 2.2.1.7 Invadopodium 17 2.2.1.8 Stress Fibers 17 2.2.1.9 Actin in Cancer and Metastasis 17 2.2.1.10 Myosin and Actin 18 2.2.2 Focal Adhesions 19 2.2.3 Microtubules 20 2.2.4 Intermediate Filaments 21 2.2.5 Cellular Stiffness 22 2.2.6 Nuclear Deformability 23 2.3 The Extracellular Matrix— Where it happens 24 2.3.1 Components and Structure 25 2.3.2 Collagen as a Model System 26 2.3.2.1 Collagen I Fibril Formation 27 2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28 2.4 Single Cell Migration— Why it spreads 29 3 Materials and Methods 31 3.1 Cell Culture 31 3.1.1 Cancer Cells 31 3.1.2 Mouse fibroblasts 32 3.1.3 Pharmacological treatment 34 3.2 Collagen matrices 34 3.3 Cell Elasticity 36 3.3.1 Atomic Force Microscopy 36 3.3.2 Preparation 37 3.3.3 Data Aquisition 38 3.3.4 Data Analysis 38 3.4 Matrix Stiffness 40 3.4.1 Preparation 40 3.4.2 Data Aquisition 41 3.4.3 Data Analysis 41 3.5 Invasion Assay 42 3.5.1 Preparation 42 3.5.2 Data aquisition 44 3.5.3 Data Analysis 44 3.6 Matrix Topology 48 3.6.1 Preparation 49 3.6.2 Data Acquisition 50 3.6.3 Data Analysis 51 3.6.3.1 Binarization 51 3.6.3.2 Pore-Size 53 3.6.3.3 Fiber Thickness 54 3.7 Fiber Displacement 55 3.7.1 Preparation 56 3.7.2 Data Aquisition 56 3.7.3 Data analysis 57 3.7.3.1 Fiber Displacement 59 3.7.3.2 Cell Segmentation 60 3.7.3.3 Shell Analysis 61 3.8 A toolset to understand Single Cell Migration and what influences it 62 4 Results 65 4.1 Cell Elasticity 65 4.1.1 Example Force-Distance Curves 66 4.1.2 Single Cell Elasticity 67 4.2 Matrix Stiffness 69 4.3 Invasion 71 4.4 Matrix Topology 75 4.5 Influence of Cell Nucleus on Cell Migration 79 4.5.1 Cellular Elasticity 79 4.5.2 Invasion 81 4.6 Fiber Displacement 89 4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93 4.7.1 FAK Knock-Out 93 4.7.2 Kinase-dead FAK Mutant 96 5 Discussion 103 References 107

Malonyl-thioesters are reactive at the thioester carbonyl and the carboxylate moieties, as seen in acyl transfer or hydrolysis and decarboxylation. Enzymes use these reactive centers to perform different enzyme chemistry throughout metabolism. This enzyme chemistry coupled with the inherent reactivity of malonyl-thioesters makes structure-function studies difficult. When malonyl-thioesters are used for structure-function studies, it usually results in a hydrolyzed or decarboxylated product. There are examples, however, where this is overcome, many of which are discussed throughout this thesis. To overcome the inherent reactivity of malonyl-thioesters and enzymes, analogs have been synthesized to perform structure-function studies. Initial studies focused on altering the thioester carbonyl to limit hydrolysis and decarboxylation; however, these studies revealed the importance of retaining the thioester carbonyl to be positioned in the oxyanion hole. My thesis work focused on the synthesis, characterization, and use in structure-function studies of malonyl-thioester analogs that either preserve the thioester carbonyl or alter it to an ester or amide, and alter the carboxylate to a sulfonate or nitro group. After synthesizing the methylmalonyl-CoA analogs, we performed structure-function studies with methylmalonyl-CoA decarboxylase. This case study revealed the potential of these analogs to both inhibit decarboxylase activity and their use in structure-function studies to gain mechanistic insights. This successful study prompted us to continue these structure-function studies in enzymes with different chemistries such as an epimerase or bi-functional acyltransferase/decarboxylase. The widespread use of these methylmalonyl-CoA analogs also motivated us to add more malonyl-thioester analogs to our toolbox. I have preliminary data that these malonyl-thioester analogs inhibit β-keto-acyl-synthase III, an enzyme involved in fatty acid production in <i>E. coli</i>. This inhibition gives us confidence that these analogs will be useful in structure-function studies that will reveal answers to long standing mechanism and protein-protein interaction questions in the polyketide and fatty acid synthase field.

Active matter represents a unique class of non-equilibrium systems, including examples ranging from cellular structures to large-scale biological tissues. These systems exhibit intriguing spatiotemporal dynamics, driven by the constituent particles’ continuous energy expenditure. Such active-matter systems, featuring complex hydrodynamics, are described by sophisticated mathematical models, typically using partial differential equations (PDEs). PDEs modeling hydrodynamics, such as the Navier-Stokes equations, are analytically intractable, and notoriously challenging to study computationally. The challenges include the need for consistent numerical methods along with their efficient and scalable high-performance computer implementation to solve the PDEs numerically. However, when considering new theoretical PDE models, such as active hydrodynamics, conventional approaches often fall short due to the specialization made in the numerical methods to study certain specific models. The inherent complexity and nonlinearity of active-matter PDEs add to the challenge. Hence, the computational study of such active-matter PDE models requires rapidly evolving high-performance computer software that can easily implement new numerical methods to solve these equations in biologically realistic three-dimensional domains. This presents a rich, yet underexplored territory demanding scalable computational frameworks that apply to a large class of PDEs. In this thesis, we introduce a computational framework that effectively allows for using multiple numerical methods through a context-aware template expression system akin to an embedded domain-specific language. This framework primarily aims at solving lengthy PDEs associated with active hydrodynamics in complex domains, while experimenting with new numerical methods. Existing PDE-solving codes often lack this flexibility, as they are closely tied to a PDE and domain geometry that rely on a specific numerical method. We overcome these limitations by using an object-oriented implementation design, and show experiments with adaptive and numerically consistent particle-based approach called Discretization-Corrected Particle Strength Exchange (DC-PSE). DC-PSE allows for the higher-order discretization of differential operators on arbitrary particle distributions leading to the possibility of solving active hydrodynamic PDEs in complex domains. However, the curse of dimensionality makes it difficult to numerically solve three-dimensional equations on single-core architectures and warrants the use of parallel and distributed computers. We design a novel template-expression system and implement it in the scalable scientific computing library OpenFPM. Our methodology offers an expression-based embedded language, enabling PDE codes to be written in a form that closely mirrors mathematical notation. Leveraging OpenFPM, this approach also ensures parallel scalability. To further enhance our framework's versatility, we employ a \textit{separation-of-concerns} abstraction, segregating the model equations from numerics, and domain geometry. This allows for the rapid rewriting of codes for agile numerical experiments across different model equations in various geometries. Supplementing this framework, we develop a distributed algebra system compatible with OpenFPM and Boost Odeint. This algebra system opens avenues for a multitude of explicit adaptive time-integration schemes, which can be selected by modifying a single line of code while maintaining parallel scalability. Motivated by symmetry-preserving theories of active hydrodynamics, and as a first benchmark of our template-expression system, we present a high-order numerically convergent scheme to study active polar fluids in arbitrary three-dimensional domains. We derive analytical solutions in simple Cartesian geometries and use them to show the numerical convergence of our algorithm. Further, we showcase the scalability of the computer code written using our expression system on distributed computing systems. To cater to the need for solving PDEs on curved surfaces, we present a novel meshfree numerical scheme, the Surface DC-PSE method. Upon implementation in our scalable framework, we benchmark Surface DC-PSE for both explicit and implicit Laplace-Beltrami operators and show applications to computing mean and Gauss curvature. Finally, we apply our computational framework to exploring the three-dimensional active hydrodynamics of biological flowing matter, a prominent model system to study the active dynamics of cytoskeletal networks, celluar migration, and tissue mechanics. Our software framework effectively tackles the challenges associated to numerically solving such non-equilibrium spatiotemporal PDEs. We perform linear perturbation analysis of the three-dimensional Ericksen-Leslie model and find an analytical expression for the critical active potential or, equivalently, a critical length of the system above which a spontaneous flow transition occurs. This spontaneous flow transition is a first realization of a three-dimensional active Fr\'eedericksz transition. With our expression system, we successfully simulate 3D active fluids, finding phases of spontaneous flow transitions, traveling waves, and spatiotemporal chaos with increasing active stress. We numerically find a topological phase transition similar to the Berezinskii–Kosterlitz–Thouless transition (BKT transition) of the two-dimensional XY model that occurs in active polar fluids after the spontaneous flow transition. We then proceed to non-Cartesian geometries and show the application of our software framework to solve the active polar fluid equations in spherical domains. We find spontaneous flows in agreement with recent experimental observations. We further showcase the framework to solve the equations in 3D annular domains and a `peanut' geometry that resembles a dividing cell. Our simulations further recapitulate the actin flows observed in \textit egg extracts within spherical shell geometries, showcasing our framework's versatility in handling complex geometrical modifications of model equations. Looking ahead, we hope our framework will serve as a foundation for further advancements in computational morphogenesis, fostering collaboration and using the present techniques in biophysical modeling.

This thesis investigates the mechanisms behind natural silk spinning by integrating rheology, spectroscopy and small angle scattering to better understand this process and to guide our efforts towards mimicking Nature’s ways of producing high performance fibres. As a result of natural selection, arthropods such as spiders and moths have evolved the ability to excrete silk proteins in a highly controlled manner. Spun from liquid feedstocks, silk fibres are used ex vivo to build structures with mechanical properties currently unmatched by industrial filaments. As yet, relatively little attention has been directed to the investigation of spinning under biologically relevant conditions. To better understand how and why silk is spun, this thesis bridges the gap between liquid silk flow properties and structure development. To directly connect the two, I have developed and deployed novel experimental platforms that combine infrared spectroscopy and small angle scattering with rheology. This approach has clarified long-standing ambiguities on the structural root of silk’s apparently complex flow properties. Small angle scattering revealed the length scales involved in the flow induced solidification under a range of spinning conditions. Mo reover, infrared spectroscopy offered a unique perspective into silk’s formation process immediately after excretion. In a similar manner to the post-extrusion tuning of the properties of partly solidified spider silk filaments, this thesis has revealed that silkworm silk fibres are far from completely formed once excreted. One might describe the filaments of mulberry silkworm as seeded molten polymers that form its hydrogen bonding network and crystallises slowly on site. Consequently, it enlightens that post-spinning conditions are equally paramount for silkworm silk, giving an explanation for the relatively poorer mechanical properties. The comparison of silks from a range of species, allowed this hypothesis to be extended to wild silkworm silk. My insights into spinning had the fortuitous repercussion of facilitating silk fibre solubilisation leading to the development of better artificial silk feedstocks flowing like native silks. With these findings, I believe we are now in an improved position to conceive artificial fibres with properties rivalling those of Nature.

Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri is considered to be the most serious disease occurring in farmed striped catfish (Pangasianodon hypophthalmus) in Vietnam. This disease has had an increasing impact over the last ten years and has been reported to cause 50-90% mortality of stocks during a single outbreak. Data obtained from natural outbreaks of E. ictaluri in striped catfish showed the role of environmental factors in the establishment and progression of this disease. At present, factors affecting the virulence and transmission of E. ictaluri in striped catfish are poorly understood. The central hypothesis of this thesis focuses on the complex picture of the environmental factors and infectivity of E. ictaluri in striped catfish. In this study, 80 isolates of E. ictaluri recovered from natural clinical disease outbreaks occurring in striped catfish farms between 2002 and 2011 located in 4 distinct geographical areas within Vietnam were characterised using a variety of methods. The biochemical profiles showed that E. ictaluri isolates from striped catfish in Vietnam have similar phenotypic characteristics to other E. ictaluri isolates from other infected fish species. These data showed high levels of phenotypic homogeneity between the E. ictaluri isolates investigated. The status of isolates recovered from natural infections over time and from geographically distinct farms was evaluated using pulsed-field gel electrophoresis (PFGE), plasmid profile identification and antibiotic sensitivity tests. The PFGE results showed 6 main groups with a similarity of 82% and the corresponding genotypes of the prevalent isolates illustrated annual differences. Three plasmid groups were identified distributed among the isolates investigated, in which high molecular weight plasmids of approximately 35 and 140 kb were found in two of the groups. Plasmid profiles of the present study did not show any trend of geographical region or year of isolation. The 140 kb plasmid has been considered as a multi-antibiotic resistance plasmid which confers resistance to tetracycline, trimethoprim and sulphonamides. All Vietnamese isolates showed a high level of resistance to Oxolinic acid, Sulfadimethoxine/Ormetoprim (Romet), Oxytetracycline and Amoxicillin. A reproducible bacterial immersion challenge model was developed and the LD60 estimated prior to performing subsequent experimental challenge studies. Fish were exposed to 107 cfu ml-1 of E. ictaluri by immersion for up to 30 seconds, resulting in a cumulative percentage mortality of 63%. Edwardsiella ictaluri was recovered and identified from all the dead and moribund fish during these experiments and affected fish showed similar clinical signs and pathology to those reported from natural E. ictaluri infections. The present study resulted in a successful experimental immersion challenge model for E. ictaluri infection in healthy striped catfish. Cohabitation challenges were also developed and produced 15-40% mortality, typical clinical signs and pathology, and successful recovery of the challenge organism demonstrating horizontal transmission of E. ictaluri in striped catfish. Experimental studies were then conducted to investigate the association between pH or salinity of water and susceptibility to E. ictaluri infection in striped catfish. The first experiments were performed in in vitro conditions in which E. ictaluri isolates were cultured in a variety of pH and salt concentrations. In vivo experiments were then designed where striped catfish were exposed to 107 cfu ml-1 of E. ictaluri for 30 seconds and then held at 4 different water pHs (5.5, 6.5, 7.5 and 8.5) or NaCl concentrations (0, 0.5, 1 and 1.5%). The results of in vitro experiments showed that a pH value between 5.5 to 6.5 and salt concentration between 0-0.5% were optimal for the growth of E. ictaluri. The in vivo experiments demonstrated that the cumulative mortality of striped catfish in water at pH 5 and pH 6 was significantly higher than that of fish maintained in more alkaline water (p<0.05). By contrast, the cumulative mortality of the striped catfish maintained in 0.5% salt concentration was significantly lower than those kept in 0%, 1% and 1.5% salt concentration (p<0.05). Clinical signs, lesions and histopathological changes in the affected fish were consistent with those reported in natural infections. This study highlighted the use of pH 8.5 and salinity of 0.5% NaCl as a means of decreasing the susceptibility of striped catfish to E. ictaluri. In conclusion, this study used a variety of methods in order to enhance the understanding of the biochemical, biophysical characteristics, plasmid profile and antibiotic resistance as well as the relatedness of E. ictaluri isolates recovered from farmed striped catfish in Vietnam. This study provided two reliable and reproducible bacterial challenge models (immersion and cohabitation) and emphasised the link between pH and salinity with the infectivity and pathogenicity of E. ictaluri in striped catfish.

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that link the extracellular matrix to the actin cytoskeleton. Uniquely, these adhesion receptors mediate inside-out signal transduction, whereby extracellular adhesion is activated from within the cell by talin, a large cytoskeletal protein that binds to the cytoplasmic tail of the β integrin subunit via its PTB-like F3 domain. Features of the interface between talin1 and small β3 fragments only have been described previously. Through NMR studies of full-length integrin β tails, we have found that β tails differ widely in their interactions with different talin isoforms. The muscle-specific β1D/talin2 complex exhibited particularly high affinity, leading to the X-ray crystal structure of the β1D tail/talin2 F2-F3 complex. Further NMR and biological experiments demonstrated that integrin activation is induced by a concerted series of interactions between the talin F3 domain and the β tail and between the talin F2 domain and the cell membrane. Additional studies revealed the structural determinants of tight talin2/β1D binding and the basis of more general differences between β1 and β3 talin binding. NMR studies were also performed on tyrosine-phosphorylated integrin tails binding to the PTB domains of talin1 and Dok1, an inhibitor of integrin activation; these revealed that phosphorylation can inhibit integrin activation by increasing the affinity of the β tail for talin competitors. Key residues governing this switch were identified, and proteins were engineered with reversed affinities, offering potentially useful biological tools. Taken together, these results reveal the remarkable complexity of structural features that enable talin and its competitors to mediate this important form of transmembrane signalling.

The outcome of chemical reactions proceeding via radical pair (RP) intermediates can be influenced by the magnitude and direction of applied magnetic fields, even for interaction strengths far smaller than the thermal energy. Sensitivity to Earth-strength magnetic fields has been suggested as a biophysical mechanism of animal magnetoreception and this thesis is concerned with simulations of the effects of such weak magnetic fields on RP reaction yields. State-space restriction techniques previously used in the simulation of NMR spectra are here applied to RPs. Methods for improving the efficiency of Liouville-space spin dynamics calculations are presented along with a procedure to form operators directly into a reduced state-space. These are implemented in the spin dynamics software Spinach. Entanglement is shown to be a crucial ingredient for the observation of a low field effect on RP reaction yields in some cases. It is also observed that many chemically plausible initial states possess an inherent directionality which may be a useful source of anisotropy in RP reactions. The nature of the radical species involved in magnetoreception is investigated theoretically. It has been shown that European Robins are disorientated by weak radio-frequency (RF) fields at the frequency corresponding to the Zeeman splitting of a free electron. The potential role of superoxide and dioxygen is investigated and the anisotropic reaction yield in the presence of a RF-field, without a static field, is calculated. Magnetic field effect data for Escherichia coli photolyase and Arabidopsis thaliana cryptochrome 1, both expected to be magnetically sensitive, are satisfactorily modelled only when singlet-triplet dephasing is included. With a view to increasing the reaction yield anisotropy of a RP magnetoreceptor, a brief study of the amplification of the magnetic field experienced by a RP from nearby magnetite particles is presented. Finally in a digression from RPs, Spinach is used to determine the states expected to be immune from relaxation and therefore long-lived in NMR experiments on multi-spin systems.