Pang Yee Man Flora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.v / Table of Contents --- p.vii / List of Figures --- p.xii / Symbols and Abbreviations --- p.xv / Scientific Names --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Vitellogenesis --- p.2 / Chapter 1.2 --- Vitellogenin --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Vitellogenin synthesis in the liver --- p.4 / Chapter 1.3 --- Regulation of vitellogenin synthesis --- p.5 / Chapter 1.3.1 --- Estradiol --- p.5 / Chapter 1.3.1.1 --- Mechanism of action --- p.6 / Chapter 1.3.1.2 --- Estradiol-stimulated vitellogenin expression --- p.7 / Chapter 1.3.1.3 --- Memory effects --- p.9 / Chapter 1.3.2 --- Testosterone --- p.10 / Chapter 1.3.3 --- Cortisol --- p.13 / Chapter 1.3.4 --- Progesterone --- p.14 / Chapter 1.3.5 --- Growth Hormone --- p.14 / Chapter 1.3.6 --- Prolactin --- p.15 / Chapter 1.3.7 --- Thyroid hormone --- p.15 / Chapter 1.4 --- Growth factors --- p.16 / Chapter 1.4.1 --- Activin --- p.16 / Chapter 1.4.1.1 --- Structure --- p.16 / Chapter 1.4.1.2 --- Functions --- p.17 / Chapter 1.4.2 --- Epidermal growth factors (EGF) --- p.18 / Chapter 1.4.2.1 --- Structure --- p.18 / Chapter 1.4.2.2 --- Functions --- p.19 / Chapter 1.5 --- Objectives of the present study --- p.20 / Chapter Chapter 2 --- Expression of Goldfish Vitellogenin in vivo and in vitro --- p.25 / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Materials --- p.26 / Chapter 2.2.2 --- Sequencing --- p.27 / Chapter 2.2.3 --- Cell culture --- p.28 / Chapter 2.2.4 --- RNA extraction --- p.29 / Chapter 2.2.5 --- Northern hybridization --- p.31 / Chapter 2.2.6 --- Slot blot hybridization --- p.32 / Chapter 2.2.7 --- Data analysis --- p.33 / Chapter 2.2.8 --- SDS-PAGE analysis --- p.33 / Chapter 2.2.9 --- in situ hybridization --- p.34 / Chapter 2.3 --- Results --- p.37 / Chapter 2.3.1 --- Validation of vitellogenin mRNA detection --- p.37 / Chapter 2.3.2 --- Basal and estradiol-stimulated vitellogenin expression and production invivo --- p.38 / Chapter 2.3.3 --- Localization of vitellogenin expression in the liver --- p.39 / Chapter 2.3.4 --- Expression of vitellogenin in vitro --- p.40 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 3 --- Effects of Steroids on the Expression of Goldfish Vitellogenin in vitro --- p.60 / Chapter 3.1 --- Introduction --- p.60 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Materials --- p.62 / Chapter 3.2.2 --- Animal --- p.62 / Chapter 3.2.3 --- Primary culture of dispersed hepatic cells --- p.62 / Chapter 3.2.4 --- Drug treatment --- p.64 / Chapter 3.2.5 --- Total RNA isolation --- p.64 / Chapter 3.2.6 --- Messenger RNA isolation --- p.65 / Chapter 3.2.7 --- Slot blot analysis --- p.66 / Chapter 3.2.8 --- Data analysis --- p.68 / Chapter 3.2.9 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.68 / Chapter 3.2.10 --- Cloning of aromatase cDNA --- p.69 / Chapter 3.2.11 --- Sequencing --- p.70 / Chapter 3.3 --- Results --- p.71 / Chapter 3.3.1 --- Effect of 17-β estradiol on vitellogenin mRNA expression --- p.71 / Chapter 3.3.2 --- Effect of testosterone on vitellogenin mRNA expression --- p.71 / Chapter 3.3.3 --- Detection of aromatase mRNA expression in the liver by RT-PCR --- p.72 / Chapter 3.3.4 --- Effect of aromatase inhibitors on testosterone-stimulated vitellogenin expression --- p.73 / Chapter 3.4 --- Discussion --- p.81 / Chapter Chapter 4 --- Effects of Epidermal Growth Factor (EGF) and Activin on the Expression of Vitellogenin in the Goldfish Hepatic Cells in vitro --- p.86 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and Methods --- p.88 / Chapter 4.2.1 --- Materials --- p.88 / Chapter 4.2.2 --- Primary culture of dispersed hepatic cells --- p.89 / Chapter 4.2.3 --- Slot blot analysis --- p.91 / Chapter 4.2.4 --- Data analysis --- p.91 / Chapter 4.3 --- Results --- p.92 / Chapter 4.3.1 --- Effect of activin on vitellogenin mRNA expression --- p.92 / Chapter 4.3.2 --- Effect of EGF and TGF-α on vitellogenin mRNA expression --- p.93 / Chapter 4.4 --- Discussion --- p.99 / Chapter Chapter 5 --- General Discussion --- p.104 / Chapter 5.1 --- Overview --- p.104 / Chapter 5.2 --- Contribution of the present study --- p.106 / Chapter 5.2.1 --- Expression of goldfish vitellogenin in vivo and in vitro --- p.106 / Chapter 5.2.2 --- Effects of steroids on the expression of goldfish vitellogenin in vitro --- p.106 / Chapter 5.2.3 --- Effects of EGF and activin on the expression of vitellogenin in the goldfish hepatic cells in vitro --- p.107 / Chapter 5.3 --- Future prospects --- p.108

Peperomia pellucida (L.) HBK (Piperaceae) (erva de jaboti) é uma herbácea amplamente encontrada nos trópicos e que possui diversas propriedades biológicas. Seus estudos fitoquímicos haviam demonstrado a presença da pellucidina A, uma rara dinorlignana ciclobutânica, que seria formada por acoplamento oxidativo de 2,4,5- triidroxi-estireno seguido de metilações. Nesse trabalho, foram caracterizados o ácido 2,4,5-trimetoxi-cinâmico, 2,4,5-trimetoxi-estireno, 2,4,5-trimetoxi-benzaldeído, dilapiol, 5,6,7-trimetoxi-flavona, sesamina, além da pellucidina A. Estudos de aspectos dinâmicos envolvidos na formação da pellucidina A incluíram a ontogenia e respostas à diferentes tratamentos como estresse hídrico, predação por herbívoros, ácido jasmônico e luz UV360. O tratamento com ácido jasmônico resultou num significativo incremento do dilapiol enquanto que, o tratamento sob luz UV360 resultou no aumento na produção da pellucidina A sugerindo um mecanismo de cicloadição [2+2] para sua biossíntese. A administração de diferentes precursores in vivo revelou que a L-[2-13C]- fenilalanina (0,75%), ácido [8-13C]-cinâmico (1,32%), ácido [8-13C]-ferúlico (0,51%), ácido 2,4,5-trimetoxi-[8-13C]-cinâmico (7,9%) e o 2,4,5-trimetoxi-estireno (13,3%) foram incorporados à pellucidina A. Ensaios de conversão enzimática indicaram a descarboxilação do ácido 2,4,5-trimetoxi-cinâmico em 2,4,5-trimetoxi-estireno enquanto que o 2,4,5-trimetoxi-estireno foi dimerizado em pellucidina A através da reação de cicloadição [2+2] sensibilizada pela presença da 5,6,7-trimetoxi-flavona (18,45%), tal qual a benzofenona (11,15%). Assim, sugere-se a sequência L-fenilalanina, ácido cinâmico, ácido 2,4,5-trimetoxi-cinâmico, 2,4,5-trimetoxi-estireno e pellucidina A, sendo a última etapa através de mecanismo fotoquímico tendo como sensibilizador a 5,6,7-trimetoxi-flavona. / Peperomia pellucida (L.) HBK (Piperaceae) (erva de jaboti) is an herbaceous plant that is widespread in the tropics and have several biological properties. Previous reports described the presence of pellucidin A, a rare dinorlignan having the unique cyclobutane moiety, that was supposedly formed by oxidative coupling of the precursor 2,4,5-trihydroxy-styrene followed by methylations steps. In this study, a comprehensive phytochemical study resulted in the description of 2,4,5-trimethoxy-cinnamic acid, 5,6,7-trimethoxy-flavone, 2,4,5-trimethoxy-styrene, 2,4,5-trimethoxy-benzaldehyde, dillapiol and sesamin in addition to pellucidin A. Studies of the dynamic aspects involved in the formation of pellucidin A included changes during ontogeny and responses to different treatments such as drought stress, herbivory, jasmonic acid and UV360 light. The treatment with jasmonic acid resulted in a significant increase in dillapiol whereas treatment under UV360 light resulted in an increase in production of pellucidin A, suggesting that a cycloaddition [2+2] mechanism is involved in its formation. The in vivo administration of different precursors to plants of P. pellucida revealed that L-[2-13C]-phenylalanine (0.75%), [8-13C]-cinnamic acid (1.32%), [8-13C]-ferulic acid (0.51%) 2,4,5-trimethoxy-[8-13C]-cinnamic acid (7.9%) and 2,4,5-trimethoxy-[8-13C]-styrene (13.3%) were incorporated into pellucidin A. The enzymatic conversion assays indicated decarboxylation of 2,4,5-trimethoxy-cinnamic acid into 2,4,5-trimethoxy-styrene while the 2,4,5-trimethoxy-styrene was dimerized in the pellucidin A by cycloaddition reaction [2+2] sensitized by 5,6,7-trimethoxy-flavone (18.45%), as well as by benzophenone (11.15%). Thus, we suggest the sequence L-phenylalanine, cinnamic acid, 2,4,5-trimethoxy-cinnamic acid, 2,4,5-trimethoxy-styrene and pellucidin A, the last step being carried out by a photochemical mechanism having 5,6,7- trimethoxy-flavone as a sensitizer.

Fong, Yu Hang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 102-107). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong, Ho Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 103-109). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Wan Tai Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 102-111). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgements --- p.vii / General Abbreviations --- p.ix / Abbreviations of Chemicals --- p.xi / Table of Contents --- p.xii / List of Figures --- p.xvi / List of Tables --- p.xvi / Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Soybeans --- p.1 / Chapter 1.1.1 --- Nutrient composition of soybean --- p.1 / Chapter 1.1.2 --- Nitrogen fixation and assimilation in soybean --- p.3 / Chapter 1.1.3 --- The role in nitrogen allocation and controlling the nitrogen sink-source relationship of asparagine --- p.3 / Chapter 1.1.4 --- Characterization of asparagine synthetase --- p.8 / Chapter 1.1.4.1 --- Biochemistry and molecular background of plant asparagine synthetase --- p.8 / Chapter 1.1.4.2 --- Asparagine synthetase in Arabadopsis thaliana --- p.9 / Chapter 1.1.4.3 --- "Asparagine synthesis in soybean, Glycine max" --- p.10 / Chapter 1.1.4.4 --- "Asparagine synthetase in rice, Oryza sativa" --- p.11 / Chapter 1.2 --- Seed protein quality and quantity improvement --- p.13 / Chapter 1.2.1 --- Nutrition composition of rice --- p.13 / Chapter 1.2.2 --- Molecular approaches for improving seed storage protein quality --- p.14 / Chapter 1.2.2.1 --- Protein sequence modification --- p.14 / Chapter 1.2.2.2 --- Synthetic genes --- p.16 / Chapter 1.2.2.3 --- Overexpression of homologous genes --- p.17 / Chapter 1.2.2.4 --- Transfer and expression of heterologous genes --- p.18 / Chapter 1.2.2.5 --- "Manipulation of pathway synthesizing essential amino acids, aspartate family amino acid" --- p.19 / Chapter 1.2.3 --- Research in improving rice seed protein quality and quantity --- p.22 / Chapter 1.3 --- Hypothesis and objective of this study --- p.23 / Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Plant materials --- p.25 / Chapter 2.1.2 --- Bacterial strains and vectors --- p.26 / Chapter 2.1.3 --- Growth conditions for soybean --- p.26 / Chapter 2.1.4 --- Chemicals and reagents --- p.26 / Chapter 2.1.5 --- "Buffer, solution and gel" --- p.26 / Chapter 2.1.6 --- Commercial kits --- p.27 / Chapter 2.1.7 --- Equipments and facilities used --- p.27 / Chapter 2.1.8 --- Primers --- p.27 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Growth condition for plant materials --- p.28 / Chapter 2.2.1.1 --- General conditions for planting soybean --- p.28 / Chapter 2.2.1.2 --- Soybean seedlings for gene expression profile analysis --- p.28 / Chapter 2.2.1.3 --- Mature soybean for gene expression profile analysis --- p.29 / Chapter 2.2.1.4 --- Mature soybean for cloning of AS I and AS2 full length cDNA --- p.30 / Chapter 2.2.1.5 --- Mature soybean seed for amino acid profile analysis --- p.30 / Chapter 2.2.1.6 --- General conditions for planting transgenic rice in CUHK --- p.30 / Chapter 2.2.1.7 --- Transgenic rice seedling for PCR screening --- p.31 / Chapter 2.2.1.8 --- Transgenic rice for functional test and seed for biochemical analysis --- p.31 / Chapter 2.2.2 --- Molecular techniques --- p.32 / Chapter 2.2.2.1 --- Total RNA extraction --- p.32 / Chapter 2.2.2.2 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.2.2.3 --- Northern blot analysis --- p.33 / Chapter 2.2.2.3.1 --- Chemiluminescent detection --- p.33 / Chapter 2.2.2.3.2 --- Film development --- p.34 / Chapter 2.2.2.4 --- Preparation of single-stranded DIG-labeled PCR probes --- p.34 / Chapter 2.2.2.4.1 --- Primer design for the PCR probes of --- p.34 / Chapter 2.2.2.4.2 --- Amplification of AS1 and AS2 internal PCR fragments --- p.34 / Chapter 2.2.2.4.3 --- Quantitation of purified AS1 and AS2 PCR fragments --- p.35 / Chapter 2.2.2.4.4 --- Biased PCR to make single-stranded DNA probes --- p.35 / Chapter 2.2.2.4.5 --- Probe quantitation --- p.36 / Chapter 2.2.2.5 --- Probe specificity test --- p.37 / Chapter 2.2.2.6 --- Cloning of full length cDNA --- p.37 / Chapter 2.2.2.6.1 --- First strand cDNA synthesis from RNA of high protein content soybean leaf --- p.37 / Chapter 2.2.2.6.2 --- PCR for amplification of AS1 and AS2 full length cDNA --- p.38 / Chapter 2.2.2.6.3 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.38 / Chapter 2.2.2.6.4 --- Ligation of DNA inserts into pBluescript II KS(+) T-vector --- p.39 / Chapter 2.2.2.6.5 --- Preparation of E. coli DH5α CaCl2-mediaed competent cells --- p.39 / Chapter 2.2.2.6.6 --- Transformation of E. coli DH5α competent cell --- p.40 / Chapter 2.2.2.7 --- Screening of recombinant plasmids --- p.40 / Chapter 2.2.2.7.1 --- Isolation of recombinant plasimid DNA from bacterial cells --- p.41 / Chapter 2.2.2.7.2 --- PCR screening on recombinant plasmids --- p.41 / Chapter 2.2.2.7.3 --- DNA gel electrophoresis --- p.41 / Chapter 2.2.2.8 --- Sequencing and homology search --- p.42 / Chapter 2.2.2.9 --- Functional test using transgenic plant --- p.43 / Chapter 2.2.2.9.1 --- Preparation of chimeric gene constructs and recombinant plasmids --- p.43 / Chapter 2.2.2.9.2 --- Agrobacterium mediated transformation into rice calli to regenerate transgenic AS1/ AS2 rice --- p.44 / Chapter 2.2.2.10 --- PCR Screenig of homozygous and heterozygous transgenic plants --- p.44 / Chapter 2.2.2.10.1 --- Isolation of genomic DNA from transgenic plants --- p.45 / Chapter 2.2.2.10.2 --- PCR screening using genomic DNA --- p.46 / Chapter 2.2.2.11 --- Quantitative PCR analysis on transgenic plants --- p.48 / Chapter 2.2.3 --- Biochemical Analysis --- p.49 / Chapter 2.2.3.1 --- Quantitative amino acid analysis in mature soybean seeds --- p.49 / Chapter 2.2.3.2 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.49 / Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Amino acid analysis on mature soybean seeds --- p.50 / Chapter 3.2 --- Expression pattern analysis of AS genes by Northern Blot analysis --- p.54 / Chapter 3.2.1 --- Making of single strand digoxigenin (DIG)-labeled probe --- p.54 / Chapter 3.2.2 --- Probe specificity --- p.57 / Chapter 3.2.3 --- AS expression level under light/dark treatments by Northern Blot analysis --- p.58 / Chapter 3.2.4 --- AS expression level in young seedlings by Northern Blot analysis --- p.62 / Chapter 3.2.5 --- AS expression level in podding soybean by Northern Blot analysis --- p.64 / Chapter 3.3 --- Cloning of AS genes from high protein content soybeans --- p.66 / Chapter 3.3.1 --- "PCR amplification of AS1 and AS2 full length cDNA from the first-strand cDNA of high portein content cultivar soybean, YuDoul2" --- p.66 / Chapter 3.3.2 --- Nucleotide sequences analysis of AS1 and AS2 full-length cDNA clones --- p.68 / Chapter 3.4 --- Construction of AS1 and AS2 transgenic rice --- p.75 / Chapter 3.4.1 --- Construction of AS1 and AS2 constructs --- p.75 / Chapter 3.4.2 --- Transformation of chimeric gene constructs into Agrobacterium tumefaciens --- p.75 / Chapter 3.4.3 --- Agrobacterium mediated transformation into Oryza sativa calli to regenerate transgenic rice --- p.76 / Chapter 3.4.4 --- PCR screening of transgene from transgenic AS1 and AS2 rice --- p.76 / Chapter 3.4.5 --- Quantitative PCR analysis of the transgene expression --- p.81 / Chapter 3.4.6 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.83 / Chapter 4 --- Discussion --- p.89 / Chapter 4.1 --- The role of asparagine and asparagine synthetase in nitrogen assimilation and sink-source relationship in soybean --- p.89 / Chapter 4.2 --- Comparative study of AS between different high seed protein content crops --- p.92 / Chapter 4.3 --- The attempt to find out the reason for the strong AS1 expression detected in high protein soybean cultivars --- p.92 / Chapter 4.4 --- Other factors affecting seed protein contents --- p.93 / Chapter 4.5 --- Rice seed quality improvement by nitrogen assimilation enhancement --- p.94 / Chapter 4.6 --- Comparative study of amino acid profile and seed total protein in other transgenic rice --- p.95 / Chapter 4.7 --- Possible reason of higher seed protein content in AS2 transgenic rice --- p.96 / Chapter 4.8 --- Selectable marker --- p.97 / Chapter 5 --- Conclusion and Prespectives --- p.99 / Chapter 6 --- References --- p.102 / Chapter 7 --- Appendix --- p.112 / Appendix I: Major chemicals and reagents used in this research --- p.112 / "Appendix II: Major buffer, solution and gel used in this research" --- p.114 / Appendix III: Commercial kits used in this research --- p.117 / Appendix IV: Major equipments and facilities used in this research --- p.118 / Appendix V: Primer list --- p.119

La rose est l'une des plantes ornementales les plus populaires, dont les composés volatils sont non seulement impliqués dans les interactions des fleurs avec l’environnement au sens large, mais aussi largement utilisés dans l’industrie des arômes et parfums. Le chapitre 1 décrit l'histoire de la culture de la rose, les usages de son parfum, les connaissances actuelles sur la biosynthèse des composés de ce parfum, ainsi que les voies de biosynthèse des composés volatils qui ont été récemment élucidées chez différentes plantes. Les chapitres expérimentaux 2 et 3 analysent les fonctions de deux gènes exprimés dans les pétales de rose. Ils codent pour des protéines Nudix hydrolase 1 (NUDX1). Le gène NUDX1-1 (nommé RhNUDX1 dans la publication) a été découvert en comparant les transcriptomes de deux cultivars de rose, Rosa x hybrida cv. 'Papa Meilland' (PM) très parfumé et R. x hybrida cv. 'Rouge Meilland' (RM), dépourvu de parfum. Le gène RhNUDX1-1 n'est exprimé que chez PM et son expression est corrélée avec la production de monoterpènes dans les pétales, en particulier de géraniol. Lors de l'étude d'une descendance issue du croisement de R. chinensis cv. ‘Old Blush’ (OB) et de R. x wichurana (Rw), le gène orthologue RcNUDX1-1a, présentant la même fonction, a été caractérisé chez OB. Un gène paralogue, RwNUDX1-2, a été découvert chez Rw et il a été démontré que son expression présentait une corrélation avec la production sesquiterpènes, en particulier de E,E-farnesol. Une série d'analyses in vitro et in vivo ainsi qu'une analyse de corrélation ont permis de vérifier la fonction de RhNUDX1-1, qui hydrolyse le géranyl diphosphate (GPP) en géranyl monophosphate (GP). Une phosphatase non identifiée pourrait catalyser la transformation du GP en géraniol. Des expériences de fusion avec la Green Fluorescent Protein (GFP), suivies de transformation transitoire de feuilles de tabac, ont révélé que RhNUDX1-1 était localisée dans le cytoplasme. Les mêmes approches (analyses QTL, essais enzymatiques et expression transitoire) ont également été appliquées à RwNUDX1-2, démontrant sa fonction dans la production de E,E-farnesol. La cartographie de RwNUDX1-2 et la localisation subcellulaire de la protéine sont encore à l'étude. De plus, la cristallographie des protéines et la modélisation ont été employées pour étudier le mécanisme de l'interaction NUDX1-substrat et les acides aminés potentiellement importants pour la reconnaissance du substrat. Collectivement, ces données révèlent une voie alternative pour la biosynthèse des terpènes, en particulier le géraniol et E,E-farnesol, via l'hydrolyse des prényl diphosphates par les enzymes NUDX1. Nos résultats montrent que la production de composés volatils dans les pétales est fortement corrélée avec l’expression des gènes des voies de biosynthèse. Par conséquent, la régulation transcriptionnelle de RcNUDX1-1a et RwNUDX1-2 joue probablement un rôle important dans la production de parfum. Les promoteurs de RcNUDX1-1a, RcNUDX1-1b, et RwNUDX1-2 et deux facteurs de transcription (FT), RcbHLH79 (OB TF) et RwbHLH79 (Rw TF) ont ainsi été isolés et testés (Chapitre 4). Les FT candidats ont été choisis lors d’une analyse RNA-Seq (Chapitre 5). En utilisant des tests d'expression transitoire avec le gène rapporteur GUS (β-glucuronidase) dans les pétales de rose, il a été montré que les trois promoteurs pouvaient entraîner l'expression de GUS. Les deux FT ont ensuite été introduits dans des feuilles de tabac avec les promoteurs testés, pour voir s'ils étaient capables d'activer ces promoteurs. Aucune transactivation significative n'a été détectée, même si Rw TF semblait pouvoir activer une construction témoin (promoteur du gène de la tomate TPS5. Les transcriptomes de quatre cultivars de rose, dont deux produisent du géraniol mais pas de E,E-farnesol et deux autres produisent du E,E-farnesol mais pas de géraniol, ont été analysés (Chapitre 5) et ont abouti à une liste de FT putatifs pour une étude plus approfondie / Roses are one of the most popular ornamental plants, whose volatiles are not only involved in environmental interactions but also widely used for industries. Chapter 1 describes the cultivation history of roses, usages of rose fragrance, knowledge on the biosynthesis of rose scent compounds, as well as non-canonical biosynthesis pathways of other plant volatiles. Experimental chapters (Chapter 2 and 3) analyse the functions of two genes expressed in rose petals, both encoding Nudix hydrolase 1 (NUDX1) protein. NUDX1-1 gene (named RhNUDX1) was first discovered by comparing the transcriptomes of two rose cultivars, the scented Rosa x hybrida cv. ‘Papa Meilland’ (PM) and the unscented R. x hybrida cv. ‘Rouge Meilland’ (RM). RhNUDX1-1 was only expressed in scented PM and its expression exhibited a positive correlation with the monoterpenoid production in petals, especially geraniol. When studying a rose progeny of R. chinensis cv. ‘Old Blush’ (OB) and R. x wichurana (Rw), an orthologous gene RcNUDX1-1a was found in OB, whose expression also had positive correlation with geraniol emission. A paralogous gene in Rw, RwNUDX1-2, was discovered and it was shown that its expression displayed a correlation with the sesquiterpenoid production, especially E,E-farnesol. A series of in vitro and in vivo assays as well as correlation analyses verified the function of RhNUDX1-1, which hydrolysed geranyl diphosphate (GPP) to geranyl monophosphate (GP). The transformation of GP into geraniol is supposed to be processed by an, as yet, unidentified phosphatase. The prediction of the localisation together with green fluorescent protein (GFP) fusion experiments revealed that RhNUDX1-1 was located in the cytosol. A series of approaches (QTL analyses, enzymatic assays and transient expression studies) were also applied to RwNUDX1-2, demonstrating its function in the production of E,E-farnesol. Mapping of RwNUDX1-2 and subcellular localization of the protein are still under investigation. Furthermore, protein crystallography and protein modelling illustrated the NUDX1-substrate interaction and proposed several residues that may be important for substrate recognition, although further experimental and computational data are required to gain more insight into the enzymatic mechanism. Collectively, these data revealed an alternative pathway for the biosynthesis of terpenoids, especially geraniol and E,E-farnesol, in rose, via the hydrolysis of prenyl diphosphates by NUDX1 enzymes. Transcriptional regulation of RcNUDX1-1a or RwNUDX1-2 probably plays an important role in the scent production by rose petals. Therefore, three promoters, pOB1a (promoter of RcNUDX1-1a), pOB1b (promoter of RcNUDX1-1b, not expressed in rose petals), pRw (promoter of RwNUDX1-2) were cloned and tested (Chapter 4). In addition, two transcription factors (TFs), RcbHLH79 (OB TF) and RwbHLH79 (Rw TF) candidates were chosen via RNA-Seq analysis as their expression correlated with expression of RcNUDX1-1a or RwNUDX1-2, respectively (Chapter 5). Using transient expression assays with a reporter gene, β-glucuronidase (GUS) in rose petals, it was shown that all three promoters could drive the expression of GUS, suggesting that all of them are active. However, quantification of promoter activities is still needed. OB TF and Rw TF were introduced into Nicotiana benthamiana leaves together with the promoters driving GUS , to determine if they were able to activate these promoters. However, no significant transactivation was detected in any promoter-TF combination. The expression of the TF in the progeny was also analysed but, due to the similarity of the sequences of family members, no conclusive data were obtained. Transcriptomes of the petals four roses, two of which produce geraniol but not E,E-farnesol and two that produce E,E-farnesol but not geraniol, were analysed (Chapter 5) and this resulted in a list of putative scent related genes and transcription factors for further study

by Ho Wai Chan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. [254]-271). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

Les gibbérellines (GA) sont une classe de phytohormones modulant différents aspects du développement des plantes. La biosynthèse des GA est catalysée par l’activité de différentes classes d’enzymes permettant la formation des formes bioactives. Si les mutants de biosynthèse sont nains, un excès de l’hormone provoque croissance excessive et stérilité. Ainsi, les plantes ont développé des mécanismes efficaces leur permettant de maintenir une concentration optimale de GA bioactives. Un niveau supplémentaire de régulation peut être constitué par une séparation spatiale de la biosynthèse dans différents types cellulaires et organes. A l’aide d’approches variées, nous démontrons qu’une forme intermédiaire est mobile sur de longues distances. Ce transport s’effectue à travers les vaisseaux vasculaires de la plante, et pourrait impliquer des transporteurs. Ensembles, nos résultats révèlent la nature et les propriétés biologiques du transport de GA sur de longues distances chez Arabidopsis. / Gibberellins (GA) are a class of diterpenoid hormones regulating major aspects of plant growth. GA biosynthesis from GGDP is catalyzed by the activity of different classes of enzymes leading to the formation of the active forms of GA. Thus GA biosynthesis mutants are dwarfs and late flowering, while GA overdose causes excessive growth and sterility. Therefore plants have evolved efficient mechanisms to maintain optimal levels of bioactive GA. However, an additional level of regulation may reside in the separation of the GA biosynthetic pathway into distinct cell types and organs. Through micro-grafting, genetic and biochemical approaches, we demonstrate that a GA intermediate is mobile over long distances in Arabidopsis. Moreover, this transport occurs through vascular tissues of the plant, and may involve specific transporters. Altogether, our results reveal the nature and the biological properties of GA long distances transport in Arabidopsis.

Ribonucleoproteins (RNPs) are RNA-protein complexes that carry out a variety of key cellular functions and are essential for the regulation of gene expression. Small nuclear RNPs (snRNPs) are a class of RNPs that regulate gene expression at the level of RNA processing in the nucleus. These RNPs are subject to complex and highly regulated biogenesis pathways in order to ensure sufficient snRNP levels are present within the cell. snRNPs are required for viability of all eukaryotic cells and the importance of proper snRNP function in vivo is further highlighted by the fact that the fatal motor neuron disease spinal muscular atrophy (SMA) is caused by a genetic deficiency in the ubiquitously expressed survival motor neuron (SMN) protein, an essential component of the snRNP biogenesis machinery. The most well characterized targets of SMN for RNP assembly are the spliceosomal snRNPs, which are critical factors that carry out pre-mRNA splicing. However, SMN is not believed to be solely dedicated to spliceosomal snRNP biogenesis but rather is thought to be a general RNP assembly machine. Yet, no other RNP targets of the SMN complex had previously been characterized in a conclusive manner. Understanding the cellular targets of SMN-mediated RNP assembly is critical for elucidating basic mechanisms of RNA regulation. Furthermore, despite increased understanding of the molecular function of SMN in spliceosomal snRNP biogenesis and the cellular basis of SMA in animal models, the molecular mechanisms through which loss of SMN function leads to motor neuron disease remain poorly defined. Thus, identifying additional RNP pathways that are dependent on SMN is key to uncover the molecular mechanisms of SMA and may also help in the design of novel therapeutic approaches to this devastating childhood disorder that is currently untreatable. In an effort to expand on the established RNP targets of SMN for assembly, in this dissertation I explore the hypothesis that SMN is required for the biogenesis and function of U7 snRNP and that disruption of this pathway induced by SMN deficiency contributes to motor neuron pathology in SMA. While structurally analogous to spliceosomal snRNPs, U7 snRNP functions not in splicing but rather in the unique 3’-end processing mechanism of replication-dependent histone mRNAs. Here, I first provide detailed molecular characterization of the in vivo functional requirement of SMN for U7 snRNP biogenesis as well as histone mRNA 3’-end processing and proper histone gene expression. I go on to demonstrate that in a mouse model of SMA U7 snRNP biogenesis and function are severely impaired by SMN deficiency and these defects occur in disease-relevant SMA motor neurons. I then describe the development of a novel molecular strategy to restore U7 snRNP activity in a setting of SMN deficiency in order to investigate the functional consequences of U7 dysfunction in SMA. Finally, I apply this U7 restoration strategy to a mouse model of SMA using AAV9-mediated gene delivery and establish that disrupted U7 activity contributes to select aspects of motor neuron dysfunction in SMA mice. Collectively, my dissertation work provides a significant expansion in our understanding of RNP pathways controlled by SMN and, for the first time, establishes the contribution of an SMN-dependent RNA pathway to SMA pathology in a mouse model of the disease that best recapitulates the human condition both genetically and phenotypically. The continuation of this work in the future not only may lead to a detailed molecular understanding of the mechanisms of SMA but possibly also to the development of novel therapeutic approaches for this deadly disease that are complementary to SMN upregulation.

Osteoarthritis, a degenerative joint disease that affects nearly 30 million Americans, is characterized by lesions of articular cartilage that often lead to severe pain and loss of joint function. The current economic burden of osteoarthritis is estimated to be approximately $190 billion, and with the prevalence of arthritis expected to rise due to the aging population, the associated costs are forecasted to increase. Debilitating osteoarthritis is managed clinically by the surgical implantation of a cartilage graft or cartilage cells to replace the damaged tissue; however, current repair methods often result in poor long-term outcomes due to inadequate integration of the graft with host cartilage and bone. Thus, there is a significant clinical need for approaches that enable functional connection of grafting devices to the host tissue. To address this challenge, the strategy described in this thesis is a versatile, cup-shaped fibrous scaffold system designed to promote the simultaneous integration of the cartilage graft with both the host cartilage and subchondral bone. This thesis is guided by the hypotheses that 1) graft integration with native cartilage can be strengthened by inducing chondrocyte migration to the graft-cartilage junction through chemotactic factor release from the walls of the cup, and 2) graft integration with host bone and the formation of calcified cartilage can be facilitated by pre-incorporation of calcium phosphate nanoparticles in the base of the cup. To test these hypotheses, a microfiber-based integration cup was designed with degradable, polymer-based walls that release insulin-like growth factor-1, which is well-established for inducing chondrocyte migration, and a base consisting of polymer with calcium deficient apatite nanoparticles. In the first aim of this thesis, the dose of insulin-like growth factor-1 in the cup walls was optimized to enhance the migration of cells from surrounding cartilage into the scaffold, and this design was tested in vitro to ensure that the scaffold supports chondrocyte viability, growth, and biosynthesis of a cartilage-like matrix. In the second aim of this thesis, the composition and dose of calcium phosphate in the base of the cup was optimized to support chondrocyte growth and the production of calcified cartilage-like tissue. Subsequently, in the third aim, the independently developed walls and base were joined into a scaffold that was tested in vitro and in vivo, using a simulated full thickness defect model, to examine its potential for clinical translation. Results from these studies demonstrate that the cup system can be implemented with autologous tissue and cell-based grafting strategies as well as with tissue engineered hydrogel grafts to promote integration with host tissue. Moreover, these investigations have yielded new insights into both chemical and structural parameters that direct chondrocyte migration and calcified cartilage formation. In summary, this thesis describes the design and optimization of a novel, multi-functional device for improving integration of cartilage grafts with host tissues. The impact of the studies in this thesis extends beyond cartilage integration, as the interface scaffold design criteria elucidated here are readily applicable to the formation of interfaces between other grafts and host tissues.