Yes / N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. / University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W.

Corrosion of reinforcing steel in concrete exposed to chloride-laden environments is a well-known and documented phenomenon. The need for cost effective systems for protection against corrosion has become increasingly clear since the first observations of severe corrosion damage to interstate bridges in the 1960's. As one potential solution to the mounting problem of corrosion deterioration of structures, corrosion-inhibiting admixtures have been researched and introduced into service. This report conveys the results of a three-part laboratory study of corrosion inhibiting admixtures in concrete. The commercial corrosion inhibiting admixtures for concrete have been analyzed by three evaluation methods, including: • Conventional concrete corrosion cell prisms under ponding, • Black steel reinforcing bars immersed in simulated concrete pore solutions, • Electrochemical screening tests of special carbon steel specimens in electrochemical corrosion cells containing filtered cement slurry solution. The purposes of the study include: • Determining the influence of a series of commercially available corrosion inhibiting admixtures on general concrete handling, performance and durability properties not related to corrosion. • Determining the effectiveness of corrosion inhibiting admixtures for reduction or prevention of corrosion of reinforcing steel in concrete, relative to untreated systems, under laboratory conditions. • Conducting a short-term pore solution immersion test for inhibitor performance and relating the results to those of the more conventional long-term corrosion monitoring techniques that employ admixtures in reinforced concrete prisms. • Determining whether instantaneous electrochemical techniques can be applied in screening potential inhibitor admixtures. Concrete properties under test included air content, slump, heat of hydration, compressive strength, and electrical indication of chloride permeability. Monitoring of concrete prism specimens included macro-cell corrosion current, mixed-cell corrosion activity as indicated by linear polarization, and ancillary temperature, relative humidity, and chloride concentration documentation. Simulated pore solution specimens were analyzed on the basis of weight loss and surface area corroded as a function of chloride exposure. Electrochemical screening involved polarization resistance of steel in solution. Results include corrosion potential, polarization resistance and corrosion current density. / Master of Science

Uma das marcantes características morfológicas e funcionais dos crustáceos e de outros artrópodes é a presença de um exoesqueleto que cria uma barreira física para o crescimento desses animais. Nos crustáceos, a muda é um evento cíclico, dividido em 5 estágios, e um deles compreende a troca desse exoesqueleto, permitindo o aumento de tamanho. O início, período e a frequência do ciclo de muda dependem da idade e do sexo do animal e de fatores ambientais e fisiológicos. Hormônios como os ecdiesteróides e o hormônio inibidor da muda produzidos e secretados pelos órgãos Y e X, respectivamente, atuam diretamente no ciclo de muda, porém outros hormônios podem regular, de forma positiva ou negativa, este processo. A melatonina é um hormônio encontrado amplamente no reino animal, porém em crustáceos, diferentemente do que ocorre nos vertebrados, a sua síntese e secreção não estão relacionadas com a presença ou ausência de luz, e seu papel na muda tem sido pouco investigado. Os animais foram aclimatados no laboratório à temperatura 22±2 °C e ciclo claro-escuro 12h:12h LD, sendo os experimentos realizados nesta mesma condição. Considerando o acima exposto, os objetivos do presente trabalho foram (1) verificar a produção de melatonina no siri azul Callinectes sapidus, através da investigação da expressão das enzimas AANAT e ASMT no pedúnculo óptico e hepatopâncreas, bem como os níveis hemolinfáticos da indolamina; (2) avaliar se existe um perfil oscilatório diário na expressão gênica dos fatores relacionados com a muda, CasMIH e CasEcR1; (3) verificar se a manipulação com melatonina exógena influencia essa expressão. Para isso, técnicas de imunohistoquímica, citometria de fluxo, ensaio imunoenzimático e PCR quantitativo foram empregadas. Nossos resultados demonstraram uma oscilação dos níveis hemolinfáticos de melatonina em siris em pré-muda, com pico às 8 horas; entretanto, no estágio de intermuda os níveis deste hormônio foram menores e constantes ao longo de 24 horas. Não pudemos comprovar a presença das enzimas da via de síntese da melatonina, uma vez que os anticorpos utilizados não apresentaram homologia às proteínas de C. sapidus. Quanto à expressão gênica, uma oscilação diária semelhante nos transcritos dos genes CasMIH e CasEcR1 ocorreu no hepatopâncreas, independente do estágio de muda. No pedúnculo óptico a oscilação dos genes em questão também foi semelhante, mas apenas na pré-muda; na intermuda houve entre eles uma relação de anti-fase. A administração de melatonina exógena (10-7 mol/siri) levou à inibição da expressão dos genes em relação ao controle: no caso de CasMIH foi de 99,7% no pedúnculo óptico e 100% no hepatopâncreas e o CasEcR1 sofreu inibição de 77% no pedúnculo óptico e 99% no hepatopâncreas. A presença de melatonina na hemolinfa é um forte indício de que o animal a sintetiza e pode estar atuando no ciclo de muda, uma vez que a administração deste hormônio inibiu a transcrição dos genes relacionados ao processo. Diante disso, fica mais clara a relevância de entender a flutuação de hormônios que não estão classicamente envolvidos no ciclo de muda, essencial para o crescimento dos crustáceos, mas que podem apresentar a função de regular este processo, como a melatonina. Ademais, a melatonina poderá ser uma boa ferramenta a ser utilizada no cultivo do siri-azul, como agente indutor da redução do período de intermuda levando à uma ecdise precoce / One of the remarkable morphological and functional features of crustaceans and other arthropods is the presence of an exoskeleton that creates a physical barrier for the animal growth. In crustaceans, molting is a cyclic event usually divided into five stages, one of them comprising the exoskeleton exchange what thus allows the increase in size. The onset, period, and frequency of the molt cycle depend on the animal age and sex, as well as on environmental and physiological factors. Hormones such as ecdysteroids and the molt-inhibiting hormone produced and secreted by the Y- and X- organ, respectively, exert direct effects on the molt cycle. Nevertheless, other hormones are known to positively or negatively regulate this process, such as melatonin. Melatonin is a hormone widely found in the animal kingdom, but in crustaceans, differently from what happens in vertebrates, its synthesis and secretion are not regulated by the presence or absence of light. In fact, its role in the molting process has been poorly investigated. The animals were acclimated in the laboratory at 22±2 °C and light-dark cycle 12h:12h LD, and the experiments were performed under the same condition. Considering the above, the objectives of this study were to: 1) verify the production of melatonin in the blue crab Callinectes sapidus, through the evaluation of the expression of key enzymes involved in the synthesis of melatonin, AANAT and ASMT, in the eyestalk and hepatopancreas, as well as melatonin levels in the hemolymph; 2) evaluate whether there exists a daily oscillatory profile in gene expression of the related molt factors, CasMIH and CasEcR1; (3) whether the exogenous melatonin influences the expression of the latter genes. To achieve these goals, immunohistochemistry, flow cytometry, immunoenzymatic assay, and quantitative PCR techniques were used. Our results demonstrated an oscillation of the hemolymphatic levels of melatonin in premolt crabs, peaking at 8 AM; however, in the intermolt stage, the levels of this hormone were smaller and constant along 24 hours. We were not able to show the presence of the enzymes involved in melatonin synthesis, since the antibodies used had no homology with C. sapidus proteins. We also demonstrated a daily oscillatory profile of CasMIH and CasEcR1 transcripts in hepatopancreas independently of the molt stage. In the eyestalk the oscillatory profile of both genes was also similar, but only in the premolt stage; in intermolt, an antiphase relationship between both genes was found. The exogenous administration of melatonin (10-7 mol/crab) inhibited the expression of CasMIH by 99.7 and 100% in eyestalk and hepatopancreas, respectively, whereas CasEcR1 was inhibited by 77% and 99%, in the eyestalk and hepatopancreas, respectively, compared to saline-treated animals. The presence of melatonin in the hemolymph is a reliable indicator that the animal synthesizes the hormone, and thus melatonin may influence the molt cycle since it inhibited the expression of molt-related genes. Therefore, the relevance of understanding the oscillation of hormones that are not classically involved in the molt cycle - essential for crustacean growth - but which can regulate the process, becomes evident. From an economic standpoint, melatonin may be a useful tool in culturing blue crab, which ultimately can shorten the intermolt stage period leading to an early ecdysis

Tsang, Kwok On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 147-165). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.xiv / LIST OF TABLES --- p.XX / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- "Mikania micrantha, a problematic weed around the world" --- p.1 / Chapter 1.1.1 --- Current situation --- p.1 / Chapter 1.1.2 --- Properties of M micrantha --- p.2 / Chapter 1.1.3 --- Control methods of M. micrantha --- p.6 / Chapter 1.1.3.1 --- Manual removal --- p.6 / Chapter 1.1.3.2 --- Chemical control methods --- p.7 / Chapter 1.1.3.3 --- Biological control methods --- p.7 / Chapter 1.2 --- Parasitic plants --- p.10 / Chapter 1.2.1 --- Introduction --- p.10 / Chapter 1.2.2 --- Modes of parasitism --- p.10 / Chapter 1.2.3 --- Biology of Cuscula spp. --- p.13 / Chapter 1.2.3.1 --- Seed germination --- p.15 / Chapter 1.2.3.2 --- I lost detection and parasitism --- p.17 / Chapter 1.2.3.3 --- Reproduction --- p.19 / Chapter 1.2.3.4 --- Impacts on hosts --- p.21 / Chapter 1.3 --- Previous researches on the control of M. micrantha by cuscuta --- p.23 / Chapter 1.4 --- Research significance --- p.25 / Chapter 1.4.1 --- Knowledge gap --- p.25 / Chapter 1.4.2 --- Experimental objectives and significance --- p.26 / Chapter 1.4.3 --- Thesis layout --- p.28 / Chapter CHAPTER 2 --- Germination biology of Cuscuta japonica --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- "Cuscuta seeds collection, treatment and storage" --- p.34 / Chapter 2.2.2 --- Imbibition --- p.35 / Chapter 2.2.3 --- Germination --- p.35 / Chapter 2.2.4 --- Emergence --- p.36 / Chapter 2.2.5 --- Germination dynamics --- p.37 / Chapter 2.2.6 --- Statistical analysis --- p.37 / Chapter 2.3 --- Results --- p.38 / Chapter 2.3.1 --- Imbibition test --- p.38 / Chapter 2.3.2 --- Germination test --- p.40 / Chapter 2.3.3 --- Emergence test --- p.42 / Chapter 2.3.4 --- Germination dynamic --- p.43 / Chapter 2.4 --- Discussion --- p.44 / Chapter 2.4.1 --- Seed dormancy --- p.44 / Chapter 2.4.2 --- Germination requirements --- p.48 / Chapter 2.4.3 --- Emergence ability --- p.51 / Chapter 2.4.4 --- Germination dynamics --- p.52 / Chapter 2.5 --- Conclusions --- p.54 / Chapter CHAPTER 3 --- Life cycle of C. japonica --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.1 --- Site description --- p.57 / Chapter 3.2.2 --- Data collection --- p.62 / Chapter 3.3 --- Results --- p.64 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Life cycle of C. japonica in Dragon's Back and its implication --- p.71 / Chapter 3.4.2 --- Life cycle of (\ japonica in Shan Tong Road and Yau King Lane --- p.74 / Chapter 3.5 --- Conclusions --- p.80 / Chapter CHAPTER 4 --- Effect of infestation by C. japonica and C. campcstris on the growth of M. micrantha --- p.82 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and Methods --- p.84 / Chapter 4.2.1 --- Sites description --- p.84 / Chapter 4.2.2 --- Plant materials --- p.85 / Chapter 4.2.3 --- Infestation --- p.86 / Chapter 4.2.4 --- Harvest of plant materials --- p.87 / Chapter 4.2.5 --- Chlorophyll extraction and concentration determination --- p.87 / Chapter 4.2.6 --- Measurements --- p.88 / Chapter 4.2.7 --- Statistical analysis --- p.89 / Chapter 4.3 --- Results --- p.89 / Chapter 4.3.1 --- "Changes in length of stem, leaf size and number of leaves" --- p.89 / Chapter 4.3.2 --- Changes in biomass of hosts and parasites --- p.94 / Chapter 4.3.3 --- Changes in the chlorophyll concentration --- p.97 / Chapter 4.4 --- Discussion --- p.99 / Chapter 4.2.1 --- Cuscuta as a strong sink to the host --- p.99 / Chapter 4.2.2 --- Growth of cuscuta and comparison of its influence on M micrantha --- p.104 / Chapter 4.5 --- Conclusions --- p.106 / Chapter CHAPTER 5 --- Effect of C. japonica infestation on the activities of anti-oxidative enzymes of M. micrantha --- p.107 / Chapter 5.1 --- Introduction --- p.107 / Chapter 5.2 --- Materials and Methods --- p.110 / Chapter 5.2.1 --- Plant materials --- p.110 / Chapter 5.2.2 --- Infestation --- p.111 / Chapter 5.2.3 --- Harvest of plant materials --- p.111 / Chapter 5.2.4 --- Measurement of enzyme activity --- p.112 / Chapter 5.2.4.1 --- Reagent preparation --- p.112 / Chapter 5.2.4.2 --- Extraction method --- p.112 / Chapter 5.2.4.3 --- Enzyme activity determination --- p.113 / Chapter 5.3 --- Results --- p.115 / Chapter 5.3.1 --- SOD activity --- p.115 / Chapter 5.3.2 --- CAT activity --- p.116 / Chapter 5.3.3 --- POD activity --- p.117 / Chapter 5.4 --- Discussion --- p.1 19 / Chapter 5.4.1 --- Changes in SOD activity --- p.120 / Chapter 5.4.2 --- Changes in CAT and POD activity --- p.122 / Chapter 5.4.3 --- Effects and implications of the changes in the activities of the anti-oxidative enzymes --- p.123 / Chapter 5.5 --- Conclusions --- p.124 / Chapter CHAPTER 6 --- Host range of C. japonica --- p.126 / Chapter 6.1 --- Introduction --- p.126 / Chapter 6.2 --- Materials and methods --- p.130 / Chapter 6.2.1 --- Field study --- p.130 / Chapter 6.2.1.1 --- Site description --- p.130 / Chapter 6.2.2.2 --- Data collection --- p.130 / Chapter 6.2.2 --- Greenhouse study --- p.131 / Chapter 6.2.2.1 --- Site description --- p.131 / Chapter 6.2.2.2 --- Plants selection --- p.131 / Chapter 6.2.2.3 --- Experimental setup --- p.132 / Chapter 6.2.2.4 --- Statistical analysis --- p.133 / Chapter 6.3 --- Results --- p.133 / Chapter 6.3.1 --- Field study --- p.133 / Chapter 6.3.2 --- Greenhouse study --- p.137 / Chapter 6.4 --- Discussion --- p.138 / Chapter 6.4.1 --- Field study --- p.138 / Chapter 6.4.2 --- Greenhouse study --- p.140 / Chapter 6.4.3 --- Implications on application --- p.141 / Chapter 6.5 --- Conclusions --- p.143 / Chapter CHAPTER 7 --- General Summary and Conclusions --- p.144 / REFERENCES --- p.147 / APPENDIX A --- p.166 / APPENDIX B --- p.173 / APPENDIX C --- p.176

Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus. In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro. Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity. In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and without a PGRP. Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 737

Uma das marcantes características morfológicas e funcionais dos crustáceos e de outros artrópodes é a presença de um exoesqueleto que cria uma barreira física para o crescimento desses animais. Nos crustáceos, a muda é um evento cíclico, dividido em 5 estágios, e um deles compreende a troca desse exoesqueleto, permitindo o aumento de tamanho. O início, período e a frequência do ciclo de muda dependem da idade e do sexo do animal e de fatores ambientais e fisiológicos. Hormônios como os ecdiesteróides e o hormônio inibidor da muda produzidos e secretados pelos órgãos Y e X, respectivamente, atuam diretamente no ciclo de muda, porém outros hormônios podem regular, de forma positiva ou negativa, este processo. A melatonina é um hormônio encontrado amplamente no reino animal, porém em crustáceos, diferentemente do que ocorre nos vertebrados, a sua síntese e secreção não estão relacionadas com a presença ou ausência de luz, e seu papel na muda tem sido pouco investigado. Os animais foram aclimatados no laboratório à temperatura 22±2 °C e ciclo claro-escuro 12h:12h LD, sendo os experimentos realizados nesta mesma condição. Considerando o acima exposto, os objetivos do presente trabalho foram (1) verificar a produção de melatonina no siri azul Callinectes sapidus, através da investigação da expressão das enzimas AANAT e ASMT no pedúnculo óptico e hepatopâncreas, bem como os níveis hemolinfáticos da indolamina; (2) avaliar se existe um perfil oscilatório diário na expressão gênica dos fatores relacionados com a muda, CasMIH e CasEcR1; (3) verificar se a manipulação com melatonina exógena influencia essa expressão. Para isso, técnicas de imunohistoquímica, citometria de fluxo, ensaio imunoenzimático e PCR quantitativo foram empregadas. Nossos resultados demonstraram uma oscilação dos níveis hemolinfáticos de melatonina em siris em pré-muda, com pico às 8 horas; entretanto, no estágio de intermuda os níveis deste hormônio foram menores e constantes ao longo de 24 horas. Não pudemos comprovar a presença das enzimas da via de síntese da melatonina, uma vez que os anticorpos utilizados não apresentaram homologia às proteínas de C. sapidus. Quanto à expressão gênica, uma oscilação diária semelhante nos transcritos dos genes CasMIH e CasEcR1 ocorreu no hepatopâncreas, independente do estágio de muda. No pedúnculo óptico a oscilação dos genes em questão também foi semelhante, mas apenas na pré-muda; na intermuda houve entre eles uma relação de anti-fase. A administração de melatonina exógena (10-7 mol/siri) levou à inibição da expressão dos genes em relação ao controle: no caso de CasMIH foi de 99,7% no pedúnculo óptico e 100% no hepatopâncreas e o CasEcR1 sofreu inibição de 77% no pedúnculo óptico e 99% no hepatopâncreas. A presença de melatonina na hemolinfa é um forte indício de que o animal a sintetiza e pode estar atuando no ciclo de muda, uma vez que a administração deste hormônio inibiu a transcrição dos genes relacionados ao processo. Diante disso, fica mais clara a relevância de entender a flutuação de hormônios que não estão classicamente envolvidos no ciclo de muda, essencial para o crescimento dos crustáceos, mas que podem apresentar a função de regular este processo, como a melatonina. Ademais, a melatonina poderá ser uma boa ferramenta a ser utilizada no cultivo do siri-azul, como agente indutor da redução do período de intermuda levando à uma ecdise precoce / One of the remarkable morphological and functional features of crustaceans and other arthropods is the presence of an exoskeleton that creates a physical barrier for the animal growth. In crustaceans, molting is a cyclic event usually divided into five stages, one of them comprising the exoskeleton exchange what thus allows the increase in size. The onset, period, and frequency of the molt cycle depend on the animal age and sex, as well as on environmental and physiological factors. Hormones such as ecdysteroids and the molt-inhibiting hormone produced and secreted by the Y- and X- organ, respectively, exert direct effects on the molt cycle. Nevertheless, other hormones are known to positively or negatively regulate this process, such as melatonin. Melatonin is a hormone widely found in the animal kingdom, but in crustaceans, differently from what happens in vertebrates, its synthesis and secretion are not regulated by the presence or absence of light. In fact, its role in the molting process has been poorly investigated. The animals were acclimated in the laboratory at 22±2 °C and light-dark cycle 12h:12h LD, and the experiments were performed under the same condition. Considering the above, the objectives of this study were to: 1) verify the production of melatonin in the blue crab Callinectes sapidus, through the evaluation of the expression of key enzymes involved in the synthesis of melatonin, AANAT and ASMT, in the eyestalk and hepatopancreas, as well as melatonin levels in the hemolymph; 2) evaluate whether there exists a daily oscillatory profile in gene expression of the related molt factors, CasMIH and CasEcR1; (3) whether the exogenous melatonin influences the expression of the latter genes. To achieve these goals, immunohistochemistry, flow cytometry, immunoenzymatic assay, and quantitative PCR techniques were used. Our results demonstrated an oscillation of the hemolymphatic levels of melatonin in premolt crabs, peaking at 8 AM; however, in the intermolt stage, the levels of this hormone were smaller and constant along 24 hours. We were not able to show the presence of the enzymes involved in melatonin synthesis, since the antibodies used had no homology with C. sapidus proteins. We also demonstrated a daily oscillatory profile of CasMIH and CasEcR1 transcripts in hepatopancreas independently of the molt stage. In the eyestalk the oscillatory profile of both genes was also similar, but only in the premolt stage; in intermolt, an antiphase relationship between both genes was found. The exogenous administration of melatonin (10-7 mol/crab) inhibited the expression of CasMIH by 99.7 and 100% in eyestalk and hepatopancreas, respectively, whereas CasEcR1 was inhibited by 77% and 99%, in the eyestalk and hepatopancreas, respectively, compared to saline-treated animals. The presence of melatonin in the hemolymph is a reliable indicator that the animal synthesizes the hormone, and thus melatonin may influence the molt cycle since it inhibited the expression of molt-related genes. Therefore, the relevance of understanding the oscillation of hormones that are not classically involved in the molt cycle - essential for crustacean growth - but which can regulate the process, becomes evident. From an economic standpoint, melatonin may be a useful tool in culturing blue crab, which ultimately can shorten the intermolt stage period leading to an early ecdysis

This thesis concerns a melanoma-derived growth regulatory factor that inhibited proliferation of several malignant human cell lines, and, in particular, a line designated UCT-BR-1, which was derived from a human breast cancer metastasis. The work is presented in four chapters. Chapter 1 provides a review of the relevant literature at the time of writing; Chapters 2 and 3 describe the experimental work that was done; and in Chapter 4 I discuss the implications of my results for current and future work in growth factors. Experimental results are presented as Charts (which may be Figures or Tables) and the methods and experimental protocols that I used are described in the Chart legends and not in the main text of the thesis. The Appendix contains details of the tissue culture techniques and descriptions of the cell lines that were used. Sources of the various laboratory materials as well as the methods that were employed for the more routine procedures are also described in the appendix.

No description available.

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the ever-growing world population. Among the many challenges faced by food producers, crop losses and damage caused by fungal plant pathogens is a major problem. The study of fungal pathogens and the interaction between plants and fungi is therefore essential, and has been carried out for many years. Much has been learned in this time, but the full mechanisms of the various modes of fungal attack and plant defence have still not been elucidated. Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first enzymes to be secreted at the start of fungal attack, and many of them are considered to be essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of fungi, including Botrytis cinerea. An important defence mechanism of plants involves the inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act on fungal ePGs. Many different PGIPs from a number of diverse plant species have been described to date. They are known to have differential inhibition capabilities that often result from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains. Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP activity and a strong resistance response against Botrytis, leading to the characterisation of these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic analysis of these lines it was found that they display differential expression of cell wall metabolism genes and biochemical characteristics that might indicate possible cell wall strengthening compared to wild-type tobacco under uninfecting conditions. The V. vinifera cultivars are all very susceptible to fungal attack, whereas other grapevine species, specifically the North American Vitis species, are known for their strong resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have previously been isolated from these more resistant species. The amino acid sequences of the active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera PGIPs separated into 14 subgroups based on their active domain sequences. For this study, one PGIP from each group was selected for functional analysis in tobacco. The selected PGIP-encoding genes were transformed into tobacco by means of Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco populations consisting of three to twelve individually transformed lines of nine different nonvinifera PGIPs were obtained. A subset of the resultant transgenic lines was infected with B. cinerea in two independent whole plant infections over 11-14 days in order to investigate the disease resistance afforded by the various PGIPs towards this fungus. A line from the previously characterised VvPGIP1 population was included as reference; all the infections were contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs displayed very strong disease reduction in comparison to the WT control: after initial primary lesion formation, the spread of fungal infection was contained and halted in these lines, while wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions, indicating very strong resistance phenotypes. The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1 line and the WT control were also used to further evaluate the previous observation that overexpression might lead to changes in expression of cell wall genes. Analysis of the expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic lines tested. This confirmed previous results and have confirmed in all grapevine PGIP overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell wall. From previous work it is known that downregulation of this gene leads to strengthening of the wall. The results obtained in this study showed that the PGIP-specific resistance phenotype seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance towards B. cinerea provides an interesting angle for further investigation into the structural differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen interactions. / AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie. Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme (SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas. Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage (Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1 plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van infeksie. Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies, spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak. Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op baie sterk weerstandsfenotipes. Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van ‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die plantselwand. Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen interaksies.

The aim of this research is to investigate the extent to which Open Source Software (OSS) adoption behaviour can empirically be shown to be governed by a set of self-reported (driving and inhibiting) salient beliefs of key informants in a sample of organisations. Traditional IS adoption/usage theory, methodology and practice are drawn on. These are then augmented with theoretical constructs derived from IT governance and organisational diagnostics to propose an artefact that aids the understanding of organisational OSS adoption behaviour, stimulates debate and aids operational management interventions. For this research, a combination of quantitative methods (via Fisher's Exact Test) and complimentary qualitative method (via Content Analysis) were used using self-selection sampling techniques. In addition, a combination of data and methods were used to establish a set of mixed-methods results (or meta-inferences). From a dataset of 32 completed questionnaires in the pilot study, and 45 in the main study, a relatively parsimonious set of statistically significant driving and inhibiting factors were successfully established (ranging from 95% to 99.5% confidence levels) for a variety for organisational OSS adoption behaviours (i.e. by year, by software category and by stage of adoption). In addition, in terms of mixed-methods, combined quantitative and qualitative data yielded a number of factors limited to a relatively small number of organisational OSS adoption behaviour. The findings of this research are that a relatively small set of driving and inhibiting salient beliefs (e.g. Security, Perpetuity, Unsustainable Business Model, Second Best Perception, Colleagues in IT Dept., Ease of Implementation and Organisation is an Active User) have proven very accurate in predicting certain organisational OSS adoption behaviour (e.g. self-reported Intention to Adopt OSS in 2014) via Binomial Logistic Regression Analysis.