Coffea arabica var. Laurina (Bourbon pointu, BP) est un mutant naturel de C. arabica ‘Bourbon’ (B). La mutation laurina est récessive, monolocus, Mendélienne et ses effets pléiotropiques. Ces effets ont été étudiés sur le développement des graines et chez les plantules en histologie, morphologie et biochimie. La standardisation de la croissance en taille des graines était primordiale, notamment pour l'étude fine de la composition en polysaccharides pariétaux (PP). Les stades (st) de développement ont été reliés à l'âge des fruits et leur aspect histo-morphologique (st 1-2, 3, 4 : croissance ; st 5 à 7 : maturation). L'étude de la composition en PP a mis en évidence trois phases : φ 1 (st 1-2, 3), φ 2 (st 3, 4, première partie du 5), et φ 3 (deuxième partie du 5, st 6 et 7). La mutation affecte les φ 1 et 2, probablement par le biais du périsperme. Chez la plantule, la mutation affecte la longueur des hypocotyles exposés à la lumière en provoquant le semi-nanisme de BP par rapport à B par un nombre moindre de cellules. Les teneurs en ABA, auxine et cytokinines sont aussi touchées. La composition en PP et en acides chlorogéniques (CQA) n'est affectée ni par la mutation, ni par la lumière pendant la croissance des plantules (sauf pour les CQA ses racines). Un effet-organe est mis en évidence. La mutation réduit la teneur en caféine (CAF) et la différence variétale est présente à la lumière et à l'obscurité. Ces résultats permettent de mieux caractériser les effets pléiotropiques de la mutation laurina. Ils permettent une meilleure 1/ compréhension de l'évolution des PP de la graine au cours de son développement et 2/ connaissance des hormones, CAF, CQA et PP des plantules. / Coffea arabica var Laurina (Bourbon pointu, BP), is a natural mutant of C. arabica ‘Bourbon’ (B). The laurina mutation is recessive, monolocus and Mendelian, with pleiotropic effects. This work highlighted new effects of this mutation, with histological, morphological and biochemical approaches on seeds development and on seedlings. Simultaneous fitting of growth curves of B and BP and their standardization allow comparisons of these varieties and fine study of polysaccharide cell wall (PCW) composition over time. Thus, anatomic specificities, development stages (st) and fruit age were linked (growth: st 1-2 to 4; maturation: st 5 to 7). When taking in account the CWP evolution in time-course, three phases occurred: φ 1 (st 1-2, 3), φ 2 (st 3, 4 and the beginning of 5), and φ 3 (the end of 5, st 6, 7). The two first were affected by the mutation probably through maternal effect (perisperm). In seedlings, the hypocotyls semi-dwarfism in light growth condition is an effect of the mutation and was due to a lower cell number than in B. The phytohormones content was also affected (ABA, auxine, cytokinins). But, neither CWP composition nor chlorogenics acids (CQA) content were affected by the mutation, or growth condition (expected for CQA content in roots). The composition in CWP and CQA was different depending on organs. Finally, caffeine (CAF) content was affected by the mutation in light or darkness conditions. These results represent an important step in 1/ the characterization of the pleiotropic effects of the laurina mutation, 2/ the understanding of the CWP evolution in seed time course, and 3/ new knowledge on seedlings (hormone, CAF, CQA and CWP composition).

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-10T13:05:43Z No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-10T13:06:06Z (GMT) No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) / Made available in DSpace on 2018-04-10T13:06:06Z (GMT). No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) Previous issue date: 2018-02-20 / Protease inhibitors have a broad biotechnological application, which goes since the development of several drugs to your utilization as a bioinsecticide, antifungal and as an antibacterial agent. However, those are found in small quantities in their natural sources, which unfeasible it utilization in industrial scale. Therefore, the heterologous production ends up as a method that allows the increase of scale production of those proteins. The Inga laurina Trypsin Inhibitor (ILTI) previously characterized showed an inhibitory effect in proteases extracted from the midgut of insects, besides reducing its larval developments by up to 84%, therefore, boing a candidate to be used as a potential bioinsecticide. Thus, the present work aimed at the heterologous production of ILTI in Komagataella phaffii (Pichia pastoris), followed by the optimization of the culture modes in a bioreactor with the objective of maximizing the production of the recombinant protein. For this, the gene that codifies ILTI were cloned in the expression vector pPIC9K, followed by your insertion on the strain GS115 by electroporation. PCR analysis showed that the recombinant vector was integrated into the genome of the yeast, and all the clones obtained had MutS genotype. The expression was performed for 96 hours by adding 0.5% methanol. An analysis of the proteins on the supernatant of the recombinant strain culture, by SDS-PAGE, confirmed the production of a protein with a size close to 20 KDa. Data from MALDI-TOF confirmed that the obtained protein is, in fact, the recombinant ILTI. Furthermore, inhibitory assays showed that the produced protein had activity against trypsin. Thus, culture in bioreactors was performed to optimize the production of this heterologous protein. In order to increase its expression, fed-batch was performed where on the batch phase the biomass production was favored, and the feeding phase was programmed to continuously supply methanol, based on the methanol specific consumption and its specific growth velocity, using methanol as carbon source. During the fermentations, 351.27 UIT were obtained in the extract of crude fermentation broth, and a specific activity of 2.07 UIT/mg protein. Although widely used as a host for the production of heterologous proteins, studies of the production of protease inhibitors in K. phaffii are still very limited. Until the moment, there is no report in the optimization of the production of serine protease inhibitors in K. phaffii, making this study pioneering and essential for the beginning of scaling up the process of this technology. / Os inibidores de protease possuem uma ampla aplica????o biotecnol??gica que vai desde o desenvolvimento de diversos f??rmacos at?? sua utiliza????o como bioinseticidas, antif??ngicos e como agentes antibacterianos. Por??m, estes s??o encontrados em pequenas quantidades nas suas fontes naturais, o que inviabiliza sua utiliza????o em escala industrial. Sendo assim, a produ????o heter??loga acaba sendo um m??todo que permite o aumento da escala de produ????o dessas prote??nas. O inibidor de tripsina de Inga laurina (ILTI) foi caracterizado previamente e mostrou possuir efeito inibit??rio em proteases extra??das do trato digestivo de insetos, al??m de diminuir em at?? 84% seu desenvolvimento larval, sendo, portanto, um candidato a ser utilizado como um poss??vel bioinseticida. Dessa forma, o presente trabalho teve como objetivo a produ????o heter??loga do inibidor ILTI, em Komagataella phaffii (Pichia pastoris), seguido da otimiza????o dos modos de cultivo em biorreator com o objetivo de maximizar a produ????o da prote??na recombinante. Para isso, o gene que codifica o ILTI foi clonado no vetor de express??o pPIC9K seguindo de sua inser????o na cepa GS115 por eletropora????o. An??lises de PCR mostraram que o vetor recombinante foi integrado ao genoma da levedura e que todos os clones obtidos possu??am gen??tipo MutS. A indu????o da express??o foi realizada durante 96 horas por meio da adi????o de metanol ?? 0,5%. A an??lise de prote??nas presentes no sobrenadante da cultura da cepa recombinante, por meio de SDS-PAGE, confirmou a produ????o de uma prote??na com tamanho pr??ximo a 20 KDa. Dados obtidos em MALDI-TOF confirmaram que a prote??na obtida ?? de fato ILTI recombinante. Al??m disso, ensaios inibit??rios mostraram que a prote??na produzida possui atividade contra tripsina. Dessa forma, cultivo em biorreatores foram realizados com a finalidade de otimizar a produ????o dessa prote??na heter??loga. A fim de aumentar sua express??o foram realizadas bateladas alimentadas, onde durante a fase de batelada foi favorecida a produ????o de biomassa, e a fase de alimenta????o programada para fornecer metanol de forma cont??nua, com base nos dados de velocidade espec??fica de consumo de metanol e velocidade de crescimento espec??fica nesta fonte de carbono. Ao longo das fermenta????es realizadas foi obtido 351,27 UIT no extrato bruto do caldo fermentado e uma atividade espec??fica de 2,07 UIT/mg de prote??na. Apesar de ser amplamente utilizada como hospedeira para a produ????o de prote??nas heter??logas, estudos da produ????o de inibidores em K. phaffii ainda s??o muito limitados. At?? o momento n??o existem relatos de otimiza????o da produ????o de inibidores de serinoproteases em K. phaffii, sendo esse estudo pioneiro e essencial para o in??cio do processo de escalonamento dessa tecnologia.

Le caféier Coffea arabica var. Laurina, aussi appelé Bourbon Pointu, est apparu à la Réunion suite à une mutation spontanée de la variété Bourbon. Cette mutation Laurina, monolocus et récessive, a des effets pléiotropiques qui différencie le Bourbon pointu du Bourbon. Au niveau morphologique, elle se caractérise par un nanisme, un port pyramidal et une forme pointue de ses graines. A un niveau moléculaire, la teneur des grains en caféine est fortement réduite. Malgré des caractéristiques agronomiques d'intérêt et d'excellentes qualités organoleptiques, peu d'études sont disponibles sur ce mutant naturel. Ce travail a ainsi pour objectif d'étudier la mutation Laurina et d'en caractériser les effets afin de mieux décrire les cascades de réaction mises en place. Il se base sur la comparaison du Bourbon pointu avec sa variété parente Bourbon. Au niveau morphologique, des mesures de croissance végétative ont permis l'analyse et l'explication de la forme pyramidale du mutant. Au niveau histologique, l'étude de l'apex, centre initiateur des organes de surface, et de différents entre-noeuds de l'axe orthotrope a permis d'expliquer l'origine du nanisme en terme de division et d'élongation cellulaire. Des hypothèses quant à l'action de la mutation Laurina sur certaines hormones ont été émises à partir des résultats d'application de gibbérelline exogène. Ces études macro et microscopiques ont été couplées au niveau moléculaire à la recherche de gènes différentiellement exprimés entre les deux variétés. La comparaison des transcriptomes des deux variétés à un stade précoce post-cotylédonaire avait pour but la recherche de gènes candidats impliqués dans les premières cascades de réactions menant aux effets pléiotropiques observés. Le clonage différentiel basé sur la méthode SSH (Hybridation Suppressive Soustractive) couplé à une étape de tri à haut débit (macro-array) a été appliqué à ces fins. L'ensemble des résultats décrit plus précisément les effets pléiotropiques induits par la mutation Laurina. La description précise des effets pléiotropiques de la mutation ouvre des pistes quant à la caractérisation moléculaire de la mutation à travers une approche gène candidat.

Thesis (MSc) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2008. / Bibliography: p. 120-124. / Introduction -- Environmental factors affecting the emission of biogenic Volatile organic compounds -- Materials and experimental procedures -- Quantification using sold-phase microextraction in a dynamic system: technique development -- The emission profile of Tristaniopsis laurina -- Study of the effect of elevated atmospheric CO₂ levels on the emission of BVOCS from Australian native plants -- Conclusions and future work. / Biogenic Volatile Organic Compounds (BVOCs) emitted by plants can affect the climate and play important roles in the chemistry of the troposphere. As ambient atmospheric carbon dioxide (CO₂) levels are rapidly increasing knowledge of the effect of elevated atmospheric CO₂ on plant BVOC emissions is necessary for the development of global climate models. -- During this study, the effect of elevated atmospheric CO2 mixing ratios on BVOC emissions from Corymbia citriodora (Lemon Scented Gum) and Tristaniopsis laurina (Water Gum) was determined for the first time through the combination of Solid-Phase Microextraction (SPME), Gas Chromatography-Flame Ionisation Detection (GC-FID), Gas Chromatography-Mass Spectrometry (GC-MS) and an environment chamber. For C. citriodora elevated atmospheric CO₂ led to a decrease in the emission rate of α-pinene, β-pinene, eucalyptol, citronellal and β-caryophyllene, however, elevated CO₂ had no effect on the emission rate of citronellol. The emission profile of T. laurina has been determined for the first time. For T. laurina elevated CO₂ led to a decrease in the emission rate of α-pinene but the emission rates of β-pinene, limonene, eucalyptol and citronellol were unaffected. The results obtained in this work confirm that the effect of elevated atmospheric CO₂ on plant BVOC emissions is species-specific. / Mode of access: World Wide Web. / 124 leaves ill. (some col.)