JEREISSATI, Camila Barbosa Pinheiro. Análise proteômica de plastídeos do endosperma de sementes em desenvolvimento de pinhão manso (Jatropha curcas L.). 2015. 127 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Vitor Campos (vitband@gmail.com) on 2016-09-01T22:55:24Z No. of bitstreams: 1 2015_teses_cbpjereissati.pdf: 1568499 bytes, checksum: 2f41598abfda8cf6bcab51817b1b742a (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-02T21:06:57Z (GMT) No. of bitstreams: 1 2015_teses_cbpjereissati.pdf: 1568499 bytes, checksum: 2f41598abfda8cf6bcab51817b1b742a (MD5) / Made available in DSpace on 2016-09-02T21:06:57Z (GMT). No. of bitstreams: 1 2015_teses_cbpjereissati.pdf: 1568499 bytes, checksum: 2f41598abfda8cf6bcab51817b1b742a (MD5) Previous issue date: 2015 / Jatropha curcas L. is a plant native to America and belongs to the Euphorbiaceae family. Currently it is gaining economical interest mainly because it is an oilseed crop with potential to produce biodiesel. However, presence of phorbol esters (a class of diterpenes) that are the major toxic constituents of the seeds, limits a better usage of the plant, by making the use of the residue, obtained after the oil extraction from the seeds, unfeasible for animal feed, due to its pro-carcinogenic activity and inflammatory action. Proteomic analysis of the plastids isolated from developing seeds of Jatropha is important because the synthesis of fatty acid as well as phorbol esters, the two most attractive compounds in the study of Jatropha curcas, occur in plastids. Proteomic analysis of this organelle is crucial to better understand and explore not only the biosynthetic pathway of these two compounds but other metabolic pathways , and addtionaly providing foundation for researchs that aimed to develope genotypes with more suitable characteristics for industrial applications. In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled online to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in three different plastids proteins databases i.e. PPDB, SUBA and PlProt, while homologues for 13 proteins were not found in any of these three databases but were marked as plastidial by at least one of the three prediction programs used (TargetP, ChloroP and PlantMPloc). Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, this study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level. / O pinhão manso (Jatropha curcas L.) é uma planta nativa da América, pertencente à família Euphorbiaceae. Atualmente, ela desperta interesse econômico principalmente por se tratar de uma oleaginosa com potencial para a produção de biodiesel. Entretanto, a presença de ésteres de forbol (uma classe de diterpeno), que são os principais constituintes tóxicos das sementes, limita uma melhor utilização dessa planta, por inviabilizar o uso do resíduo de extração do óleo das sementes na alimentação animal, bem como, por apresentar atividade pró-carcinogênica e ação inflamatória. A análise proteômica de plastídeos, isolados de sementes em desenvolvimento de pinhão manso, é uma importante vertente de estudo, pois tanto a síntese de ácidos graxos como dos ésteres de forbol, os dois compostos mais atrativos no estudo de Jatropha curcas, ocorrem nos plastídeos. O estudo proteômico dessa organela torna-se crucial para melhor compreender e explorar não somente as vias biossintéticas desses dois compostos, como de outras vias metabólicas, além de proporcionar um conjunto de dados que pode ser utilizado em pesquisas voltadas para o desenvolvimento de genótipos com características mais adequadas para aplicações industriais. No presente trabalho, realizou-se uma análise proteômica de plastídeos isolados do endosperma de sementes em desenvolvimento do pinhão manso, que estavam nos estágios iniciais de deposição de lipídios e proteínas de reserva (25-30DAA), confirmados por meio de análises histológica e histoquímica. As proteínas extraídas dos plastídeos foram digeridas com tripsina e os peptídeos foram aplicados no sistema de nano-LC EASYII acoplado online ao espectrômetro de massa nano ESI LTQ-Orbitrap velos, o que resultou na identificação 1103 proteínas, representando 804 grupos de proteínas, dos quais 923 foram consideradas identificações verdadeiras. Isso expandiu consideravelmente o repertório de proteínas do pinhão manso até agora identificas. Dentre as proteínas identificadas, apenas 5 são codificadas pelo genoma plastidial, e nenhuma delas está envolvida na fotossíntese, o que evidencia a natureza não fotossintética dos plastídeos isolados. Homólogos de 824, dentre as 923 proteínas identificadas, estavam presentes nos bancos de dados PPDB, SUBA e PlProt, enquanto homólogos para 13 proteínas não foram encontrados em nenhum dos três bancos de dados plastidiais, mas foram detectados como plastidiais por pelo menos um dos três programas de predição de localização subcelular utilizados (TargetP, ChloroP, PlantMPloc). A classificação funcional mostrou que a maioria das proteínas identificadas pertencia ao metabolismo dos aminoácidos, seguido dos metabolismos dos carboidratos, energético e dos lipídios. As subunidades maiores e menores da Rubisco foram identificadas, e sua presença nos plastídeos foi considerada uma característica adaptativa para contrabalancear a perda de um terço do carbono na forma de CO2 como um resultado da conversão de carboidratos em óleo através da glicólise. Apesar de enzimas envolvidas na biossíntese de diversos precursores dos diterpenóides terem sido identificadas, não foi detectado nenhuma terpeno sintase/ciclase, o que sugere que os plastídeos isolados do endosperma de sementes em desenvolvimento não sintetizam ésteres de forbol, apesar de uma grande quantidade desse composto ser encontrada neste tecido. Como conclusão, o presente trabalho proporciona insights sobre as principais vias biossíntéticas, e sobre características peculiares dos plastideos isolados do endosperma de sementes em desenvolvimento.

Blumeria graminis f. sp. hordei is a biotroph pathogen that causes powdery mildew disease in barley. In this study, Pallas01 and Pallas03 barley lines having Mla1, Ml (Al2) and Mla6, Mla14 R-genes were inoculated with Bgh103(64/01) race of the Blumeria graminis f. sp. hordei having avirulence and virulence to Pallas01 and Pallas03, respectively. The proteins were isolated from the three biological replicates of 12, 24, and 48 hpi samples following the method in Rampitsch et al., 2006. These there biological replicates of three time points together with the mock inoculated plant proteins were separated on 2D-PAGE using IPG strips of 4-7 pH values as three technical replicates, resulting 108 gels. The gels were analyzed using PdQuest (Bio Rad) in order to assess up- or down-regulated protein spots by comparing against controls and the samples having resistance or susceptible responses with each other. According to the analysis, 36 proteins were found to be differentiated and among them 18 proteins were found up-regulated and 8 proteins were found down-regulated. The spots were manually v excised and subjected to the nano-LC-ESI-MS/MS analysis (Proteome Factory, Germany). The MASCOT algorithm was used for identification of the possible proteins. The experimental pI and MW values were used for selecting the differentiated proteins from the mass results. The relative abundance of each of the 38 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be carbohydrate metabolism related. The relative distribution of the proteins into four main functional categories was taken into consideration. Statistical tests (Students&amp / #8223 / T-test) were carried among the identified proteins in order to reveal statistically significant proteins throughout the study. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, most of the proteins were found to be located in cytoplasm or chloroplast.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Jatropha curcas L. is a plant native to America and belongs to the Euphorbiaceae family. Currently it is gaining economical interest mainly because it is an oilseed crop with potential to produce biodiesel. However, presence of phorbol esters (a class of diterpenes) that are the major toxic constituents of the seeds, limits a better usage of the plant, by making the use of the residue, obtained after the oil extraction from the seeds, unfeasible for animal feed, due to its pro-carcinogenic activity and inflammatory action. Proteomic analysis of the plastids isolated from developing seeds of Jatropha is important because the synthesis of fatty acid as well as phorbol esters, the two most attractive compounds in the study of Jatropha curcas, occur in plastids. Proteomic analysis of this organelle is crucial to better understand and explore not only the biosynthetic pathway of these two compounds but other metabolic pathways , and addtionaly providing foundation for researchs that aimed to develope genotypes with more suitable characteristics for industrial applications. In this study, we performed a proteomic analysis of plastids isolated from the endosperm of developing Jatropha curcas seeds that were in the initial stage of deposition of protein and lipid reserves. Proteins extracted from the plastids were digested with trypsin, and the peptides were applied to an EASY-nano LC system coupled online to an ESI-LTQ-Orbitrap Velos mass spectrometer, and this led to the identification of 1103 proteins representing 804 protein groups, of which 923 were considered as true identifications, and this considerably expands the repertoire of J. curcas proteins identified so far. Of the identified proteins, only five are encoded in the plastid genome, and none of them are involved in photosynthesis, evidentiating the nonphotosynthetic nature of the isolated plastids. Homologues for 824 out of 923 identified proteins were present in three different plastids proteins databases i.e. PPDB, SUBA and PlProt, while homologues for 13 proteins were not found in any of these three databases but were marked as plastidial by at least one of the three prediction programs used (TargetP, ChloroP and PlantMPloc). Functional classification showed that proteins belonging to amino acids metabolism comprise the main functional class, followed by carbohydrate, energy, and lipid metabolisms. The small and large subunits of Rubisco were identified, and their presence in plastids is considered to be an adaptive feature counterbalancing for the loss of one-third of the carbon as CO2 as a result of the conversion of carbohydrate to oil through glycolysis. While several enzymes involved in the biosynthesis of several precursors of diterpenoids were identified, we were unable to identify any terpene synthase/cyclase, which suggests that the plastids isolated from the endosperm of developing seeds do not synthesize phorbol esters. In conclusion, this study provides insights into the major biosynthetic pathways and certain unique features of the plastids from the endosperm of developing seeds at the whole proteome level. / O pinhÃo manso (Jatropha curcas L.) à uma planta nativa da AmÃrica, pertencente à famÃlia Euphorbiaceae. Atualmente, ela desperta interesse econÃmico principalmente por se tratar de uma oleaginosa com potencial para a produÃÃo de biodiesel. Entretanto, a presenÃa de Ãsteres de forbol (uma classe de diterpeno), que sÃo os principais constituintes tÃxicos das sementes, limita uma melhor utilizaÃÃo dessa planta, por inviabilizar o uso do resÃduo de extraÃÃo do Ãleo das sementes na alimentaÃÃo animal, bem como, por apresentar atividade prÃ-carcinogÃnica e aÃÃo inflamatÃria. A anÃlise proteÃmica de plastÃdeos, isolados de sementes em desenvolvimento de pinhÃo manso, à uma importante vertente de estudo, pois tanto a sÃntese de Ãcidos graxos como dos Ãsteres de forbol, os dois compostos mais atrativos no estudo de Jatropha curcas, ocorrem nos plastÃdeos. O estudo proteÃmico dessa organela torna-se crucial para melhor compreender e explorar nÃo somente as vias biossintÃticas desses dois compostos, como de outras vias metabÃlicas, alÃm de proporcionar um conjunto de dados que pode ser utilizado em pesquisas voltadas para o desenvolvimento de genÃtipos com caracterÃsticas mais adequadas para aplicaÃÃes industriais. No presente trabalho, realizou-se uma anÃlise proteÃmica de plastÃdeos isolados do endosperma de sementes em desenvolvimento do pinhÃo manso, que estavam nos estÃgios iniciais de deposiÃÃo de lipÃdios e proteÃnas de reserva (25-30DAA), confirmados por meio de anÃlises histolÃgica e histoquÃmica. As proteÃnas extraÃdas dos plastÃdeos foram digeridas com tripsina e os peptÃdeos foram aplicados no sistema de nano-LC EASYII acoplado online ao espectrÃmetro de massa nano ESI LTQ-Orbitrap velos, o que resultou na identificaÃÃo 1103 proteÃnas, representando 804 grupos de proteÃnas, dos quais 923 foram consideradas identificaÃÃes verdadeiras. Isso expandiu consideravelmente o repertÃrio de proteÃnas do pinhÃo manso atà agora identificas. Dentre as proteÃnas identificadas, apenas 5 sÃo codificadas pelo genoma plastidial, e nenhuma delas està envolvida na fotossÃntese, o que evidencia a natureza nÃo fotossintÃtica dos plastÃdeos isolados. HomÃlogos de 824, dentre as 923 proteÃnas identificadas, estavam presentes nos bancos de dados PPDB, SUBA e PlProt, enquanto homÃlogos para 13 proteÃnas nÃo foram encontrados em nenhum dos trÃs bancos de dados plastidiais, mas foram detectados como plastidiais por pelo menos um dos trÃs programas de prediÃÃo de localizaÃÃo subcelular utilizados (TargetP, ChloroP, PlantMPloc). A classificaÃÃo funcional mostrou que a maioria das proteÃnas identificadas pertencia ao metabolismo dos aminoÃcidos, seguido dos metabolismos dos carboidratos, energÃtico e dos lipÃdios. As subunidades maiores e menores da Rubisco foram identificadas, e sua presenÃa nos plastÃdeos foi considerada uma caracterÃstica adaptativa para contrabalancear a perda de um terÃo do carbono na forma de CO2 como um resultado da conversÃo de carboidratos em Ãleo atravÃs da glicÃlise. Apesar de enzimas envolvidas na biossÃntese de diversos precursores dos diterpenÃides terem sido identificadas, nÃo foi detectado nenhuma terpeno sintase/ciclase, o que sugere que os plastÃdeos isolados do endosperma de sementes em desenvolvimento nÃo sintetizam Ãsteres de forbol, apesar de uma grande quantidade desse composto ser encontrada neste tecido. Como conclusÃo, o presente trabalho proporciona insights sobre as principais vias biossÃntÃticas, e sobre caracterÃsticas peculiares dos plastideos isolados do endosperma de sementes em desenvolvimento.

Le latex de Carica papaya est un milieu riche en cystéine protéinases. Celles-ci ont été régulièrement utilisées en cosmétique ou pour l’attendrissement de la viande. Mais ces protéines ont aussi un intérêt pharmaceutique. En effet, le latex est bien connu pour posséder une activité antifongique mais aussi une activité anthelminthique. Ces effets sont régulièrement attribués aux cystéine protéinases qui se trouvent en concentration importante dans le latex. Malgré ces concentrations importantes en protéinases, d’autres protéines restent actives dans ce milieu. C’est le cas de la glutamine cyclase, qui a été extraite intacte de ce milieu et cristallisée. Sa structure nous a révélé une architecture particulière en ‘’&61538;-propeller‘’ à cinq pales avec double fermeture. Cette structure lui confère sa très grande stabilité. <p>Les industries pharmaceutiques sont aussi à la recherche de protéines très stables et résistantes aux protéinases endogènes. Nous avons donc entrepris l’étude du protéome de Carica papaya afin de mettre en évidence d’autres protéines minoritaires relativement stables pouvant conférer au latex son activité anthelminthique. Cette analyse a permis la mise en évidence de différentes protéines appartenant à diverses familles des « pathogenesis related protéins » (PR-proteins): une &61538;-1,3 glucosidase, une analogue à la barwin, une thaumatine et deux chitinases.<p>Nous nous sommes particulièrement intéressés à ces deux dernières au cours de cette thèse. Une caractérisation de ces deux protéines a permis de montrer que celles-ci étaient bien deux protéines distinctes, identifiées comme chitinases majeure et mineure selon leur abondance dans le latex. Elles sont relativement stables et résistantes à la protéolyse. Une analyse de la séquence de la chitinase majeure a montré que celle-ci était homologue à la chitinase issue de l’orge et une analyse de sa structure révèle la présence d’une grande concentration en prolines localisées principalement dans les neuf boucles de sa structure. Cela pourrait expliquer sa grande résistance vis à vis des cystéine protéinases.<p>La cristallisation de cette même chitinase en présence de N-acétyl-glucosamine comme additif, a conduit à une structure contenant trois molécules de GlcNac, deux dans le centre actif de notre protéine et une participant au réseau cristallin. Aucune structure de chitinase n’avait encore pu être obtenue en co-cristallisation avec un substrat. A partir des deux GlcNac observés dans le centre actif, nous avons reconstruit un complexe chitinase/(GlcNac)4. L’analyse de ce complexe a permis de mettre en évidence de nouvelles interactions entre (GlcNac)4 et les acides aminés du centre actif ainsi que de confirmer le mécanisme de la famille GH 19.<p> Des tests préliminaires sur nématodes ont finalement confirmé l’activité anthelminthique du latex et montré que la chitinase pouvait aussi être un bon nématocide<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Cheung Sao Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves xxiv-xxxvii). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Abstract in Chinese --- p.viii / List of Figures and Tables --- p.x / List of Abbreviations --- p.xix / Table of Content --- p.xxiii / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Human Liver Cancer --- p.1 / Chapter 1.1.1 --- Incidence of Hepatocellular Carcinoma --- p.1 / Chapter 1.1.2 --- Causes and Symptoms of Hepatocellular Carcinoma --- p.4 / Chapter 1.1.3 --- Treatment Options for Hepatocellular Carcinoma --- p.4 / Chapter 1.1.4 --- Multi-drug Resistance --- p.5 / Chapter 1.1.4.1 --- Mechanisms of Multi-drug Resistance --- p.5 / Chapter 1.2 --- Traditional Chinese Medicine --- p.10 / Chapter 1.2.1 --- Sophora flavescens and Radix Sophorae --- p.10 / Chapter 1.2.2 --- Flavonoid and its Sub-classification --- p.13 / Chapter 1.2.3 --- Flavonoid and Human Health --- p.15 / Chapter 1.3 --- Cell Death --- p.17 / Chapter 1.3.1 --- Necrosis --- p.17 / Chapter 1.3.2 --- Apoptosis --- p.17 / Chapter 1.3.3 --- Signaling Pathways in Apoptosis --- p.18 / Chapter 1.3.3.1 --- Extrinsic (Death Receptor-mediated) Pathway --- p.20 / Chapter 1.3.3.2 --- Intrinsic (Mitochondrial) Pathway --- p.21 / Chapter 1.3.3.3 --- Cysteine Aspartatic Acid Proteases --- p.21 / Chapter 1.4 --- Research Objective (s) --- p.22 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Cell Lines --- p.23 / Chapter 2.1.1.1 --- HepG2 --- p.24 / Chapter 2.1.1.2 --- RHepG2 --- p.24 / Chapter 2.1.1.3 --- WRL-68 --- p.25 / Chapter 2.1.2 --- Culture Media --- p.26 / Chapter 2.1.2.1 --- Rosewell Park Memorial Institute( RPMl) 1640 Medium --- p.26 / Chapter 2.1.2.2 --- Dulbecco's Modified Eagle's Medium (DMEM) --- p.26 / Chapter 2.1.3 --- Animals --- p.27 / Chapter 2.2 --- Traditional Chinese Medicines and Conventional Anti-cancer Drugs --- p.27 / Chapter 2.3 --- Antibodies --- p.29 / Chapter 2.4 --- Chemicals --- p.30 / Chapter 2.5 --- Reagents and Buffers --- p.34 / Chapter 2.5.1 --- Reagents for Silica Gel Column Chromatography --- p.34 / Chapter 2.5.2 --- Buffers for Common Use --- p.34 / Chapter 2.5.3 --- Reagents for Cell Viability Assay --- p.35 / Chapter 2.5.4 --- Reagents and Buffers for Typical Apoptosis Experiments --- p.35 / Chapter 2.5.4.1 --- Cell Cycle Analysis --- p.35 / Chapter 2.5.4.2 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.35 / Chapter 2.5.4.3 --- DNA Fragmentation Detection --- p.35 / Chapter 2.5.5 --- Reagents and Buffers for Western Blot Study --- p.36 / Chapter 2.5.5.1 --- Whole-cell Protein Extraction --- p.38 / Chapter 2.5.5.2 --- Mitochondrial and Cytosolic Fraction Protein Extraction --- p.38 / Chapter 2.5.6 --- Reagents and Buffers for Mitochondrial Transmembrane Potential Depolarization Measurement --- p.39 / Chapter 2.5.7 --- Reagents and Buffers for in vivo Animal Study --- p.39 / Chapter 2.5.8 --- Reagents and Buffers for Two-Dimensional Gel Electrophoresis --- p.40 / Chapter 2.5.8.1 --- Sample Preparation --- p.40 / Chapter 2.5.8.2 --- First Dimension Gel Electrophoresis - Isoelectric Focusing (IEF) --- p.40 / Chapter 2.5.8.3 --- Second Dimension Gel 日ectrophoresis - SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.40 / Chapter 2.5.8.4 --- Silver Staining --- p.41 / Chapter 2.5.9 --- Reagents for Mass Spectrometry Preparation --- p.42 / Chapter 2.5.9.1 --- Destaining --- p.42 / Chapter 2.5.9.2 --- Trypsin Digestion --- p.42 / Chapter 2.5.9.3 --- Desalting of Peptide Mixture --- p.43 / Chapter 2.5.10 --- Reagents and Buffers for Real-Time PCR --- p.43 / Chapter 2.6 --- Methods --- p.44 / Chapter 2.6.1 --- Isolation of Bioactive Constituents by Silica Gel Column Chromatography --- p.44 / Chapter 2.6.2 --- Cell Viability Assay --- p.45 / Chapter 2.6.3 --- Typical Apoptosis Experiments --- p.45 / Chapter 2.6.3.1 --- Cell Cycle Analysis --- p.46 / Chapter 2.6.3.2 --- Annexin V-FITC/ PI Staining Experiment --- p.47 / Chapter 2.6.3.3 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.48 / Chapter 2.6.3.4 --- DNA Fragmentation Reaction --- p.48 / Chapter 2.6.4 --- Western Blot Study --- p.49 / Chapter 2.6.4.1 --- Whole-cell Protein Extraction --- p.49 / Chapter 2.6.4.2 --- Mitochondrial and Cytosolic Fraction Protein Extraction --- p.50 / Chapter 2.6.5 --- Caspase Activity Determination --- p.54 / Chapter 2.6.6 --- Mitochondrial Transmembrane Potential Depolarization Measurement --- p.55 / Chapter 2.6.7 --- in vivo Animal Study --- p.56 / Chapter 2.6.8 --- Two-Dimensional Gel Electrophoresis --- p.58 / Chapter 2.6.8.1 --- Sample Preparation --- p.58 / Chapter 2.6.8.2 --- First Dimension Electrophoresis - Isoelectric Focusing (IEF) --- p.59 / Chapter 2.6.8.3 --- Second Dimension Electrophoresis - SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.60 / Chapter 2.6.8.4 --- Silver Staining --- p.61 / Chapter 2.6.9 --- Mass Spectrometry Preparation --- p.63 / Chapter 2.6.9.1 --- Destaining and Trypsin Digestion --- p.63 / Chapter 2.6.9.2 --- Peptide Extraction --- p.63 / Chapter 2.6.9.3 --- Desalting of Peptide Mixture --- p.64 / Chapter 2.6.10 --- Real-Time PCR --- p.65 / Chapter 2.6.11 --- Cellular Glutathione Level Detection --- p.69 / Chapter 2.7 --- Statistical Analysis --- p.70 / Chapter Chapter 3 --- RESULTS AND DISCUSSIONS - CYTOTOXICITY OF FLAVONOIDS ISOLATED FROM RADIX SOPHORAE --- p.72 / Chapter 3.1 --- Screening of Cytotoxic Flavonoids from Radix Sophorae --- p.72 / Chapter 3.2 --- Cytotoxicity of Leachianone A on Human Hepatoma Cell Lines --- p.74 / Chapter 3.3 --- Cytotoxicity of Leachianone A on Human Normal Liver Cell Line --- p.77 / Chapter 3.4 --- Cytotoxicity of Sophoraflavone J on Human Hepatoma Cell Line --- p.79 / Chapter 3.5 --- Cytotoxicity of Sophoraflavone J on Human Normal Liver Cell Line --- p.79 / Chapter 3.6 --- Cytotoxicities of Cisplatin and Taxol on Human Hepatoma as well as Normal Liver Cell Lines --- p.81 / Chapter 3.7 --- Conclusion --- p.86 / Chapter Chapter 4 --- "RESULTS AND DISCUSSIONS - MECHANISTIC STUDY OF LEACHIANONE A-INDUCED CELL DEATH IN HEPATOMA CELLS, HepG2 and RHepG2" --- p.88 / Chapter 4.1 --- Promotion of Cell Cycle Arrest --- p.88 / Chapter 4.2 --- Induction of Apoptosis as Evidenced by Phosphatidylserine Externalization and DNA Fragmentation --- p.93 / Chapter 4.2.1 --- Occurrence of Phosphatidylserine Externalization --- p.94 / Chapter 4.2.2 --- DNA Fragmentation Detection --- p.99 / Chapter 4.2.2.1 --- Terminal Deoxynucleotidyl Transferase(TdT)-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.99 / Chapter 4.2.2.2 --- DNA Laddering Pattern in Agarose Gel Electrophoresis --- p.103 / Chapter 4.3 --- Recruitment of Multiple Signaling Pathways in Leachianone A-induced Apoptosis --- p.105 / Chapter 4.3.1 --- "Activation of Caspases-3, -8, and -9" --- p.105 / Chapter 4.3.2 --- Altered Expressions of Bcl-2 Family Proteins --- p.112 / Chapter 4.3.3 --- Loss of Mitochondrial Membrane Potential --- p.115 / Chapter 4.4 --- in vivo Tumor Growth Inhibition in HepG2-bearing Nude Mice --- p.121 / Chapter 4.5 --- Conclusion --- p.127 / Chapter Chapter 5 --- RESULTS AND DISCUSSIONS - MECHANISTIC STUDY OF SOPHORAFLAVONE J-INDUCED CELL DEATH IN HEPATOMA CELLS HepG2 --- p.132 / Chapter 5.1 --- Execution of Cellular Apoptosis --- p.133 / Chapter 5.2 --- Involvement of Multiple Signaling Pathways in Sophoraflavone J-induced Apoptosis --- p.138 / Chapter 5.3 --- Differential Proteomes of Control and Sophoraflavone J-treated HepG2 Cells --- p.148 / Chapter 5.4 --- Conclusion --- p.167 / Chapter Chapter 6 --- OVERALL CONCLUSION AND FUTURE PERSPECTIVES --- p.169 / References --- p.xxiv

Philosophiae Doctor - PhD / The role of phenolic acids in mitigating salt stress tolerance have been well documented. However, there are contradicting reports on the effect of exogenously applied phenolic acids on the growth and development of various plants species. A general trend was observed where phenolic acids were shown to inhibit plant growth and development, with the exception of a few documented cases. One of these such cases is presented in this thesis. This study investigates the role of exogenously applied p-coumaric acid (p-CA) on physio-biochemical and molecular responses of chia seedlings under salt stress. This study is divided into three parts. Part one (Chapter 3) focuses on the impact of exogenous p-coumaric acid on the growth and development of chia seedlings. In this section, chia seedlings were supplemented with exogenous p-CA and the various biochemical and plant growth parameters were measured. The results showed that exogenous p-CA enhanced the growth of chia seedlings. An increase in chlorophyll, proline and superoxide oxide contents were also observed in the p-CA treatment relative to the control. We suggested that the increase in chia seedling growth could possibly be via the activation of reactive oxygen species-signalling pathway involving O2− under the control of proline accumulation (Chapter 3). Given the allopathy, nature of p-coumaric acid it is noteworthy that the response observed in this study may be species dependent, as contrasting responses have been reported in other plant species. Part two (Chapter 4) of this study investigates the influence of piperonylic acid (an inhibitor of endogenous p-coumaric acid) on the growth and development of chia seedlings. In trying to illustrate whether p-CA does play a regulatory role in enhancing pseudocereal plant growth, we treated chia seedlings with the irreversible inhibitor of C4H enzyme, to inhibit the biosynthesis of endogenous p-CA. In this section, chia seedlings were treated with piperonylic acid and changes in plant growth, ROS-induced oxidative damage, p-CA content and antioxidant capacity was monitored. Inhibition of endogenous p-CA restricted chia seedling growth by enhancing ROS-induced oxidative damage as seen for increased levels of superoxide, hydrogen peroxide and the extent of lipid peroxidation. Although an increase in antioxidant activity was observed in response to piperonylic acid, this increase was not sufficient to scavenge the ROS molecules to prevent oxidative damage and ultimate cellular death manifested as reduced plant growth. The results presented in this section support our hypothesis that p-CA play an important regulatory role in enhancing chia seedling growth and development as shown in Chapter 3. Part three (Chapter 5) seeks to identify and functionally characterise p-coumaric acid induced putative protein biomarkers under salt stress conditions in chia seedlings. Previous studies have shown that p-CA reversing the negative effect caused by NaCl-induced salt stress. While these studies were able to demonstrate the involvement of p-CA in promoting plant growth under salt stress conditions, they focussed primarily on the physiological aspect, which lacks in-depth biochemical and molecular analysis (ionomic and proteomic data) which could help in detecting the genes/proteins involved in salt stress tolerance mechanisms. A comparative ionomics and proteomic study was conducted, with the aim of elucidating the pivotal roles of essential macro elements and/or key protein markers involved in p-CA induced salt stress tolerance in chia seedlings. With the exception of Na, all the other macro elements were decreased in the salt treatment. Contrary to what was observed for the salt treatment most of the macro elements were increased in the p-CA treatment. However, the addition of exogenous p-CA to salt stressed seedlings showed an increase in essential macro elements such as Mg and Ca which have been shown to play a key role in plant growth and development. In the proteomic analysis we identified 907 proteins associated with shoots across all treatments. Interestingly, only eight proteins were conserved amongst all treatments. A total of 79 proteins were unique to the p-CA, 26 to the combination treatment (NaCl + p-CA) and only two proteins were unique to the salt stress treatment. The unique proteins identified in each of the treatments were functionally characterised to various subcellular compartments and biological processes. Most of the positively identified proteins were localised to the chloroplast and plays key roles in photosynthesis, transportation, stress responses and signal transduction pathways. Moreover, the protein biomarkers identified in this study (especially in the p-CA treatment) are putative candidates for genetic improvement of salt stress tolerance in plants.