Les cyanobactéries sont des organismes photosynthétiques capables de convertir le CO₂ en composés organiques et de produire de l’oxygène en utilisant l’énergie lumineuse. Néanmoins, de fortes intensités lumineuses saturent l'appareil photosynthétique, ce qui conduit à la production d'espèces réactives de l'oxygène, dangereuses pour la cellule. Pour y faire face, la photoactive orange carotenoid protein (OCP) induit une dissipation thermique de l’énergie excédentaire récoltée par le complexe d’antennes, le phycobilisome (PBS), afin de diminuer l’énergie arrivant aux centres photochimiques. L'OCP est composé de deux domaines), le domaine C-terminal (CTD) et le domaine N-terminal (NTD), reliés par un domaine de liason flexible (linker). Pendant la photoactivation, le caroténoïde est transféré vers le NTD, les domaines se séparent et le NTD peut interagir avec le PBS. Trois familles d'OCP coexistent (OCPX, OCP1 et OCP2) dans les cyanobactéries modernes. Outre l'OCP, de nombreuses cyanobactéries contiennent également des homologues des domaines OCP, le CTDH et HCP. Les HCP sont une famille de protéines caroténoïdes présentant différents traits photoprotecteurs. La plupart d'entre eux sont de très bons quenchers d'oxygène singulet, et un subclade est capable d'interagir avec le PBS et d'induire une dissipation de l'énergie thermique comme l'OCP. Le rôle de CTDH était inconnu. La présence de ces homologues parallèlement à l'OCP a conforté l'idée générale que l'OCP a une origine évolutive modulaire et que la CTDH et HCP pourraient interagir pour former un complexe OCP-like ayant des caractéristiques et une fonction similaires à celles de l'OCP. Dans cette thèse, je présente la première caractérisation des protéines CTDH. Les CTDH sont des dimères se liant à une molécule de caroténoïde. Le rôle principal de la CTDH est de transférer son caroténoïde au HCP. De plus, les CTDH sont capables de récupérer les caroténoïdes des membranes contrairement aux HCP. Ces résultats suggèrent fortement que les CTDH sont des transporteurs de caroténoïde qui assurent le chargement en caroténoïde sur les HCP. Ce nouveau mécanisme de translocation des caroténoïdes pourrait être multidirectionnel. La résolution de deux structures tridimensionnelles de l'ApoCTDH d'Anabaena a montré que la queue C-terminale du CTDH (CTT) peut adopter différentes conformations. De plus, l'analyse de mutation a démontré que le CTT joue un rôle essentiel dans la translocation des caroténoïdes. Enfin, je rapporte une caractérisation moléculaire du linker reliant les domaines de différents OCP modernes et son rôle au cours de l'évolution de l'OCP. Tout d’abord, j’ai caractérisé les OCP des trois subclades, y compris l’OCPX non caractérisé. OCPX et OCP2 présentent une désactivation rapide par rapport à OCP1. Alors que OCP1 et OCPX peuvent dimériser, OCP2 est stable en tant que monomère. Enfin, j'ai constaté que le linker est essentiel pour la désactivation de l'OCP et qu'il régule la photoactivation. Dans OCP1 et OCPX, le linker ralentit la photoactivation, tandis que dans OCP2, il augmente le taux de photoactivation. L'analyse bioinformatique complète cette caractérisation et fournit une image claire de l'évolution de l'OCP pour répondre efficacement aux conditions de stress. / Cyanobacteria are photosynthetic organisms capable of CO₂ conversion into organic compounds and production of O2 by using light energy. Nevertheless, high light intensities saturate the photosynthetic apparatus leading to production of reactive oxygen species, which are dangerous for the cell. To cope with this, the photoactive Orange Carotenoid Protein (OCP) induces thermal dissipation of the excess energy harvested by the antenna complex, the phycobilisome (PBS) to decrease the energy arriving at the photochemical centers. The OCP is composed of two domains connected by a flexible linker, the C-terminal domain (CTD) and the N-terminal domain (NTD). During photoactivation, the carotenoid is translocated to the NTD, the domains separate and the NTD is able to interact with the PBS. Three OCP families co-exist (OCPX, OCP1 and OCP2) in modern cyanobacteria. In addition to the OCP, many cyanobacteria also contain homologs of OCP domains, the CTDH and HCP. The HCPs are a family of carotenoid proteins with different photoprotective traits. Most of them are very good singlet oxygen quenchers, and one sub-clade is able to interact with the PBS and to induce thermal energy dissipation like OCP. The role of CTDH was unknown. The presence of these homologs in parallel to the OCP supported the general idea that the OCP has a modular evolutionary origin and that the CTDH and HCP can interact forming an OCP-like complex with similar characteristics and function than the OCP.In this thesis, I present the first characterization of the CTDH proteins. CTDHs are dimers binding a carotenoid molecule. The main role of the CTDH is to transfer its carotenoid to the HCP. In addition, CTDHs are able to uptake carotenoids from membranes but not HCPs. These results strongly suggested that the CTDHs are carotenoid carriers that ensure the proper carotenoid loading into HCPs. This novel carotenoid translocation mechanism could be multidirectional. The resolution of two tridimensional structures of the ApoCTDH from Anabaena showed that the C-terminal tail of the CTDH (CTT) can populate different conformations. Moreover, mutational analysis demonstrated that the CTT has an essential role in carotenoid translocation. Finally, I report a molecular characterization of the flexible linker connecting the domains of different modern OCPs and its role during the evolution of the OCP. First, I characterized OCPs from the three subclades, including the uncharacterized OCPX. OCPX and OCP2 present a fast deactivation compared with OCP1. While OCP1 and OCPX can dimerize, OCP2 is stable as monomer. Finally, I found that the linker is essential for the OCP deactivation and it regulates the photoactivation. In OCP1 and OCPX the linker slows down the photoactivation, while in OCP2 it increases the photoactivation rate. Bioinformatic analysis complements this characterization and provides a clear picture of the evolution of the OCP to respond efficiently to stress conditions.

It has been well documented that fruit and vegetable (F/V) intake is linked to lower risk of mortality and chronic disease. Raman resonance spectroscopy is a valid indicator of F/V intake and quantifies that intake by measuring skin carotenoid levels. In this study, 46 children, participated in a 6-week feeding study wherein they were randomly assigned to one of two groups: 1) consuming a high-carotenoid (HC) breakfast/snack food, or 2) consuming a placebo bar, every day. The HC food contained 4.3mg carotenoids per serving and the placebo contained none. Skin carotenoids were measured every two weeks using a BioPhotonic scanner. The treatment group had a mean increase in scanner score of 5,802 Raman intensity units which was significantly higher than the mean increase of the placebo group, 1,771. In this study we found that consumption of 120 gm of a high-carotenoid food significantly increased skin carotenoid levels in children ages 5-18 over a 6 week period. Packaging type as well as storage conditions play a role in preserving carotenoids which are sensitive to light, temperature, and oxygen. Care must be taken when choosing packaging and storage conditions for foods containing carotenoids. Two shelf-life studies, one at room-temperature and another in frozen storage, were conducted on the HC breakfast/snack food to determine the best method of packaging to maintain quality and preserve carotenoids. The food was randomly packed into one of three packaging types for both studies. The packages used in the frozen study were then randomly assigned to one of three freezer storage methods. Room-temperature samples were analyzed on days 3, 7, 10, and 14 and samples in frozen storage were pulled every month for 5 months. Measures of water activity, moisture content, color values (L*a*b*, chroma, and hue), and carotenoid content were analyzed in each sample. At the conclusion of each study, L*, b*, chroma, and hue were significantly affected by packaging type. No significant associations were found in any other measures. From this study we draw the conclusion that cellophane packaging or packaging with a N2 backflush would be the best options for use with this food to best retain its quality.

Fleshy fruits in different species can display large variation in color. A link between fruit color and seed dispersal success is suggested by previous studies showing that high intensity of fruit color increases the visitation rate in seed-dispersing birds. Wild tomato species (Solanum spp.) are excellent model organisms for research on genetic basis of differential fruit color development during the ripening process. Despite polymorphism in fruit color, all tomato species have yellow flowers due to accumulation of carotenoid pigments, which suggests that the carotenoid pathway is intact. Thus, regulatory changes controlling enzymes activity during fruit maturation are likely to have played a role in fruit color evolution. Our transcriptome analysis of wild tomato species revealed that differences in cyc-b and crtr-b2 expression could explain the diversity of fruit color. Additionally, co-expression analysis elucidated regulators of the carotenoid pathway. Combined with Sanger sequencing of carotenoid pathway genes, we identified genes that may underlie differences in fruit color during tomato evolution.

No description available.

Allopolyploids are formed by interspecific hybridization and whole genome duplication, with the resulting organism contains multiple distinct subgenomes in one nucleus. Subgenomic interactions result in massive genetic and epigenetic reconstruction, contributing to variable phenotypic traits noted in newly formed allopolyploids. To better understand these mechanisms in the context of molecular biology, evolution, and plant breeding, plant biologists study the model organism Brassica napus (farmed as canola or oilseed rape). With white-flowering and yellow-flowering progenitors, flower color phenotype of B. napus presents an opportunity to examine subgenomic interactions. CAROTENOID CLEAVAGE DIOXYGENASE 4 (CCD4) is known to play a major role in determining flower color phenotype of carotenoid-synthesizing angiosperms. Here, we investigate the genetic and epigenetic role of CCD4 orthologs and their role in flower color phenotype of B. napus.

No description available.

No description available.

De fortes illuminations peuvent être dommageables voire même létales pour les organismes photosynthétiques. Une des stratégies utilisées pour se protéger de tels effets délétères consiste à augmenter la dissipation thermique de l’énergie absorbée en excès au niveau des antennes. Chez les cyanobactéries une protéine photo-active, l’Orange Carotenoid Protein (OCP), contrôle ce processus. Une fois photo-activée l’OCP interagit avec le coeur des phycobilisomes (PBs, les antennes collectrices majoritaires chez les cyanobactéries) et déclenche le mécanisme, entrainant à la fois une baisse de l’énergie parvenant aux photosystèmes et une diminution de la fluorescence des PBs. L’énergie absorbée en excès est dissipée sous forme de chaleur. Pour que les PBs regagnent leur pleine capacité de transfert, une autre protéine nommée Fluorescence Recovery Protein (FRP) est requise. La FRP accélère la désactivation de l’OCP. Dans ce manuscrit, je vais présenter ma contribution à la compréhension du mécanisme de photo-protection lié à l’OCP.J’ai continué la caractérisation de la FRP chez Synechocystis PCC 6803, organisme modèle utilisé dans nos études. J’ai montré que la FRP de Synechocystis est plus courte que ce qui est indiqué dans Cyanobase, commençant en fait à la méthionine 26. Mes résultats ont aussi révélé que la photo-protection n’a lieu que lorsque le ratio OCP/FRP est élevé.Le plus grand aboutissement de ma thèse a été la reconstitution in vitro du mécanisme de photo-protection lié à l’OCP en utilisant de l’OCP, de la FRP et des PBs isolés. J’ai montré que la lumière est requise uniquement pour la photo-activation de l’OCP et que l’attachement de l’OCP au PB ne demande aucune illumination. Ce n’est qu’une fois photo-activée que l’OCP peut interagir avec le PB et entrainer la diminution de fluorescence (quenching). En se basant sur les résultats obtenus in vitro nous avons proposé un modèle moléculaire pour le mécanisme de photo-protection lié à l’OCP. Le système de reconstitution in vitro a été utilisé pour évaluer l’importance d’un pont salin conservé (Arg155-Glu244) entre les deux domaines de l’OCP et a révélé que celui-ci stabilise la forme inactive de l’OCP. La photo-activation entraine rupture du pont salin, l’Arg155 étant ensuite impliquée dans l’interaction entre OCP et PB. Le site d’attachement de l’OCP au coeur du PB a aussi été étudié en utilisant le système in vitro. Nos résultats ont montré que les émetteurs terminaux du PB ne sont pas requis et que le site primaire de quenching est un trimère d’allophycocyanine émettant à 660nm. Enfin nous avons étudié les propriétés des états excités du caroténoïde dans l’OCP photo-activée, montrant qu’un de ces états a un caractère de transfert de charge très prononcé et peut avoir un rôle principal dans la dissipation de l’énergie. Nos résultats suggèrent fortement que non seulement l’OCP induit dissipation de l’énergie absorbée sous forme de chaleur mais aussi que l’OCP agit directement comme dissipateur d’énergie. / Strong light can cause damage and be lethal for photosynthetic organisms. An increase of thermal dissipation of excess absorbed energy at the level of photosynthetic antenna is one of the processes protecting against deleterious effects of light. In cyanobacteria, a soluble photoactive carotenoid binding protein, Orange Carotenoid Protein (OCP) mediates this process. The photoactivated OCP by interacting with the core of phycobilisome (PB; the major photosynthetic antenna of cyanobacteria) triggers the photoprotective mechanism, which decreases the energy arriving at the reaction centres and PSII fluorescence. The excess energy is dissipated as harmless heat. To regain full PB capacity in low light intensities, theFluorescence Recovery Protein (FRP) is required. FRP accelerates the deactivation of OCP.In this work, I present my input in the understanding of the mechanism underlying the OCPrelated photoprotection. I further characterized the FRP of Synechocystis PCC6803, the model organism in our studies. I established that the Synechocystis FRP is shorter than what it was proposed in Cyanobase and it begins at Met26. Our results also revealed the great importance of a high OCP to FRP ratio for existence of photoprotection. The most remarkable achievement of this thesis is the in vitro reconstitution of the OCPrelated mechanism using isolated OCP, PB and FRP. I demonstrated that light is only needed for OCP photoactivation but OCP binding to PB is light independent. Only the photoactivated OCP is able to bind the PB and quench all its fluorescence. Based on our in vitro experiments we proposed a molecular model of OCP-related photoprotection. The in vitro reconstituted system was applied to examine the importance of a conserved salt bridge (Arg155-Glu244) between the two domains of OCP and showed that this salt bridge stabilises the inactive form of OCP. During photoactivation this salt bridge is broken and Arg155 is involved in the interaction between the OCP and the PB. The site of OCP binding in the core of a PB wasalso investigated with the in vitro reconstituted system. Our results demonstrated that the terminal energy emitters of the PB are not needed and that the first site of fluorescence quenching is an APC trimer emitting at 660 nm. Finally, we characterised the properties of excited states of the carotenoid in the photoactivated OCP showing that one of these states presents a very pronounced charge transfer character that likely has a principal role in energy dissipation. Our results strongly suggested that the OCP not only induces thermal energy dissipation but also acts as the energy dissipator.

Orientador: Adriana Zerlotti Mercadante / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-09-11T21:13:51Z (GMT). No. of bitstreams: 1 Zepka_LeilaQueiroz_D.pdf: 2815017 bytes, checksum: 8abad3ae0da0bc1b940e100078096285 (MD5) Previous issue date: 2009 / Resumo: contém no texto completo. / Abstract: contém no texto completo. / Doutorado / Doutor em Ciência de Alimentos

O objetivo desta pesquisa foi estudar a formação de partículas de licopeno em pó de colágeno hidrolisado (CH) comercial usando CO2 supercrítico (CO2-SC). O estudo consistiu em três etapas: a medida de solubilidade do CH em CO2-SC por método estático para conhecer a afinidade do CH pelo CO2, a formação de partículas de misturas físicas compostas de diferentes proporções de CH/licopeno (4:1, 6:1, 8:1 e 10:1) através de processamento em uma autoclave com CO2-SC a 140 bar e 50 °C em agitação de 1.250 rpm por 45 min para definir a proporção de CH/licopeno, e o estudo do efeito de outras condições de CO2-SC (150 bar e 50 °C, 150 bar e 60 °C, 250 bar e 50 °C, 250 bar e 60 °C) na formação de partículas de licopeno em CH para identificar a melhor condição de CO2-SC. As partículas resultantes foram caracterizadas com relação à sua morfologia por microscopia eletrônica de varredura, distribuição de tamanho por difração a laser, comportamento térmico por calorimetria diferencial de varredura, estrutura química por espectroscopia de infravermelho de transformada de Fourier, liberação de carga, e estrutura física interna das partículas por microscopia de laser confocal para conhecer o mecanismo de formação das partículas pelo processamento com CO2-SC. Os resultados indicaram que o CH é pouco solúvel em CO2-SC, motivo pelo qual não há potencial para processos de micronização supercrítica relacionados à solubilidade do biomaterial carreador em CO2-SC. No estudo de proporções de CH/licopeno foi observado que o processo com CO2-SC a 140 bar e 50 °C permite a obtenção de partículas de licopeno em CH na forma de pó de coloração vermelha com intensidade dependente da quantidade de licopeno inicial, sem mudanças importantes na estrutura física porosa do CH, e um aumento de tamanho das partículas. Recomenda-se a proporção 10:1 de CH/licopeno, pois nela se evidenciou menor aglomeração de partículas e formação de fissuras na superfície das partículas que facilitam a incorporação do licopeno em CH seguindo um mecanismo de impregnação supercrítica propiciado pela sorção de CO2 e arraste simultâneo de licopeno ao interior da estrutura física das partículas de CH. Na exploração de outras condições de CO2-SC, na temperatura de 60 °C (150/250 bar) obteve-se uma massa de aparência viscoelástica sem utilidade para a formação de partículas. Entanto, na temperatura de 50 °C foi observado que as partículas de licopeno em CH formadas com CO2-SC a 150 bar, quando comparadas com as formadas com CO2-SC a 250 bar, evidenciaram formação de fissuras que resultaram na melhor dispersão e maior carga de licopeno na estrutura interna do CH. Além disso, as análises do comportamento térmico e o espectro de infravermelho destas partículas evidenciaram a formação de interações eletrostáticas entre o licopeno e o CH favorecidas pelo processamento com CO2-SC. Assim, conclui-se que o processamento com CO2-SC a 140/150 bar e 50 °C poderia ser utilizada para o design de partículas de licopeno impregnadas em CH, gerando um ingrediente com possível ampla atividade funcional pela atividade biológica do CH em tecidos conjuntivos e pela atividade antioxidante do licopeno. / The objective of this research was to study the formation of lycopene particles in commercial powder of hydrolyzed collagen (HC) using supercritical CO2 (SC-CO2). The study consisted of three steps: the solubility measurement of HC in SC-CO2 by static method to know the affinity of HC by CO2, the formation of particles of physical mixtures composed of different ratios of HC/lycopene (4:1, 6:1, 8:1 and 10:1) by autoclaving with SC-CO2 at 140 bar and 50 °C under stirring at 1,250 rpm for 45 min to define the HC/lycopene ratio, and the study of the effect of other conditions of SC-CO2 (150 bar and 50 °C, 150 bar and 60 °C, 250 bar and 50 °C, 250 bar and 60 °C) in the particle formation of lycopene in HC to identify the best SC-CO2 condition. The resulting particles were characterized with respect to their morphology by scanning electron microscopy, size distribution by laser diffraction, thermal behavior by differential scanning calorimetry, chemical structure by Fourier transform infrared spectroscopy, charge release, and physical structure of the particles by confocal laser microscopy to know the mechanism of particle formation by SC-CO2 processing. The results indicated that HC is poorly soluble in SC-CO2 and therefore has no potential for supercritical micronization processes related to the solubility of the biomaterial in SC-CO2. In the study of ratios of HC/lycopene it was observed that the process with SC-CO2 at 140 bar and 50 °C allowed to obtain lycopene particles in HC with powder appearance of red color with intensity depending on the amount of initial lycopene, without important changes in the porous physical structure of HC, and an increase in particle size. It is recommended a 10:1 ratio of HC/lycopene, since this ratio showed less particle agglomeration, and formation of fissures on the surface of the particles facilitating the incorporation of lycopene into HC following a mechanism of supercritical impregnation propitiated by CO2 sorption and simultaneous drag of lycopene to the interior of the physical structure of the HC particles. In the exploration of other conditions of SC-CO2, at 60 °C (150/250 bar) a mass of viscoelastic appearance was obtained without utility for the formation of particles. However, at 50 °C was observed that the lycopene particles in HC formed with SC-CO2 at 150 bar, when compared with the particles formed with SC-CO2 at 250 bar evidenced the formation of fissures, which had positive influence in the dispersion and charge of lycopene in the internal structure of HC. In addition, analyzes of the thermal behavior and the infrared spectrum of these particles evidenced the formation of electrostatic interactions between lycopene and HC favored by SC-CO2 processing. Thus, it can be concluded that SC-CO2 processing at 140/150 bar and 50 °C could be used for the design of lycopene particles impregnated in HC, producing an ingredient with possible broad functional activity due to the biological activity of HC in connective tissues and the antioxidant activity of lycopene.