Made available in DSpace on 2014-08-20T13:59:15Z (GMT). No. of bitstreams: 1 tese_marcia_vaz_ribeiro.pdf: 713659 bytes, checksum: 2dc33e63ce136dffc6ae1dfd420cfab3 (MD5) Previous issue date: 2011-10-31 / The medicinal Alternanthera (Amaranthaceae) species, such as A. philoxeroides, present a great variety of bioactive compounds, among which are the betacyanins, nitrogen pigments that belong to the betalains class. These compounds are widely used as additives for food and drugs, due to their antioxidant action and lack of toxicity, which have already been proven. Techniques have been developed in order to improve productivity and performance of this pigment, one of those is the use of in vitro elicitors or in vivo stressing agents. Both have an important role in the transduction process of signals that regulate the defense genes in plants, acting as stimulators of production or degradation of several primary and secondary metabolites. This work aimed to assess, in A. philoxeroides plants, the growth and production characteristics of betacyanin in in vitro plants; the levels of photosynthetic pigments, betacyanins, lipid peroxidation and antioxidant enzymes activity in in vivo plants under salt stress, and also to quantify the level of betacyanin and the 5GT-DBs gene expression in the biosynthetic route of this compound in in vitro plants submitted to elicitation by NaCl and by tyrosine. For this, three trials were conducted. In the first one, A. philoxeroides explants were inoculated in MS medium with increasing NaCl concentrations (0, 50, 100, 150, 200 and 250 mM) for 35 days. In the second one, plants from in vitro cultures were acclimatized in greenhouses and irrigated with a sodium chloride solution (0, 200 and 400 mM) for 30 days. The third trial had two essays, one composed of in vitro A. philoxeroides plants in a liquid MS medium in vermiculite substrate for 35 days. After this period, a NaCl solution (400 mM) was added to the medium and the shoots were collected after 0, 12, 24, 36 and 48 hours of exposure. In the second one, nodal segments were inoculated in MS medium with and without tyrosine (0 e 75 µM), and its aerial parts were collected after 35 days. In the growth analysis, reduction of the averages was observed for the following variables: height, number of shoots, number of sprouts and root number and length; for the plants that have grown in the sodium chloride medium. The highest concentrations of betacyanins were found in the stalks of plants from MS medium, with 50 mM of NaCl. After 30 days of in vivo cultivation, the levels of chlorophyll a, chlorophyll b and carotenoids decreased as the salt concentration increased, while the reason of chlorophyll a/b in plants submitted to a higher salt concentration presented a difference in comparison to the control. Higher levels of betacyanin were observed on stalks, when compared to the leaves, in the highest salt concentrations. On the leaves, there was a significant increase of lipid peroxidation and superoxide dismutase activity. On the roots, there was an increase of enzymes catalase and ascorbate peroxidase. Regarding the analysis of differential expression (qRT-PCR), it was possible to observe that from 12 to 24 hours of salt stress, the 5-GT gene expression firstly increased, then there was a decrease in 36 hours and a new increase in 48 hours. The 5-GT gene also showed increased expression as a response to tyrosine. It was possible to conclude that A. philoxeroides elicited in vitro with sodium chloride present a decrease of the assessed morphological parameters, but in low concentrations betacyanin synthesis is stimulated. Salt stress causes greater degradation in the photosynthetic pigments, increment of betacyanin synthesis in stalks and damage to the cell membranes of the leaves. The increase of antioxidant enzymes activity stimulated the protective system against oxidative stress on in vivo A. philoxeroides plants. It is suggested that in this species, the enzyme bethanidine 5-Oglucosyltransferase reaches its highest expression in 48 hours of exposure to salt elicitation and also when grown in a medium containing tyrosine. / As espécies medicinais do gênero Alternanthera (Amaranthaceae) como A. philoxeroides apresentam uma variedade de compostos bioativos, entre eles as betacianinas, que são pigmentos nitrogenados pertencentes à classe das betalaínas. Esses compostos são amplamente utilizados como aditivos de produtos alimentícios e medicamentos devido à sua comprovada ação antioxidante e ausência de toxicidade. Técnicas têm sido desenvolvidas para aprimorar a produtividade e o rendimento deste pigmento, sendo uma delas o uso de elicitores in vitro ou agentes estressantes in vivo. Ambos apresentam um importante papel no processo de transdução de sinais que regulam os genes de defesa nas plantas, agindo como estimuladores para a produção e ou degradação de diversos metabólitos, tanto primários quanto secundários. Assim, o objetivo do presente trabalho foi avaliar em plantas de A. philoxeroides, as características de crescimento e produção de betacianina em plantas cultivadas in vitro; os teores dos pigmentos fotossintéticos, betacianinas, peroxidação lipídica e atividade de enzimas antioxidantes em plantas cultivadas in vivo, sob estresse salino, além de, quantificar o teor de betacianina e a expressão do gene 5GT-DBs envolvido na rota biossintética, deste composto, em plantas in vitro submetidas à elicitação por NaCl e pelo aminoácido tirosina. Para isso, foram conduzidos três experimentos. No primeiro, explantes de A. philoxeroides foram inoculados em meio MS, com concentrações crescentes de NaCl (0, 50, 100, 150, 200 e 250 mM), durante 35 dias. No segundo, plantas provenientes da cultura in vitro foram aclimatizadas em casa de vegetação e irrigadas com solução de cloreto de sódio (0, 200 e 400 mM), por 30 dias. O terceiro experimento contou com dois ensaios, sendo o primeiro composto de plantas de A. philoxeroides cultivadas in vitro, em meio MS líquido, no substrato vermiculita, durante 35 dias. Após esse período, foi adicionada ao meio, solução de NaCl (400 mM) e coletada a parte aérea das plantas após 0, 12, 24, 36 e 48 horas de exposição ao sal, Já o segundo, segmentos nodais foram inoculados em meio MS, na presença e ausência de tirosina (0 e 75 µM), tendo sua parte aérea coletada após 35 dias de cultivo. Nas análises de crescimento observou-se redução das médias para as variáveis altura, número de gemas, número de brotos e no número e comprimento de raiz, nas plantas crescidas nos meios contendo cloreto de sódio. As maiores concentrações de betacianinas foram encontradas nos caules de plantas cultivadas em meio MS com 50 mM de NaCl. Após 30 dias de cultivo in vivo os teores de clorofilas a, clorofila b, e carotenóides decresceram à medida que aumentou a concentração de sal, enquanto a razão clorofila a/b das plantas submetidas à maior concentração de sal apresentou

Depuis plusieurs années il est devenu essentiel de comprendre le déterminisme sexuel des espèces à fort intérêt économique afin d’optimiser leur production au sein d’écloseries émergentes.L’objectif principal de cette thèse était de mettre en évidence les mécanismes impliqués dans la détermination et la différenciation sexuelle, et notamment du sexe femelle, chez l’huître perlière P. margaritifera, espèce hermaphrodite protandre et espèce clé de la perliculture, la seconde ressource économique pour la Polynésie française. Pour atteindre cet objectif, deux approches ont été menées : une approche transcriptomique visant à étudier les mécanismes moléculaires du déterminisme et de la différenciation sexuelle, et une approche expérimentale visant à comprendre le phénomène de la sexualisation par des forçages environnementaux et hormonaux en s’intéressant plus particulièrement au déterminisme et à la différenciation sexuelle femelle.Dans l’approche transcriptomique, le transcriptome de la gonade de P. margaritifera a été séquencé à partir de plusieurs échantillons gonadiques d’huîtres de sexe mâle et femelle à différents stades de développement. Après le séquençage Illumina et l'assemblage du transcriptome, 70 147 contigs ont été obtenus. L’analyse fonctionnelle de ces 70 147contigs, a permis d’identifier des gènes d’intérêt et ainsi de constituer un catalogue de 87 ARNm codant pour 67 protéines impliquées dans la détermination, la différenciation sexuelle et/ou la gamétogenèse. Ensuite une analyse stricte des données de quantification RNAseq a révélé 1 937 contigs exprimés de manière différentielle entre les catégories histologiques des gonades. À partir de l’analyse de leurs profils d’expression au sein de chaque échantillon, un nouveau modèle de la reproduction de P. margaritifera, basé sur une double approche analytique, eg. histo-moléculaire, a été proposé. Ce modèle révèle notamment que le déterminisme sexuel de P. margaritifera chez l’adulte se produirait durant une phase de régression de la gonade. Considérant ainsi les nouveaux stades définis par ce modèle, 9 gènes biomarqueurs de la voie sexuelle femelle ont pu être identifiés révélant un modèle prédictif de la voie sexuelle basé sur 3 rapports d’expressions de gènes impliquant 2 gènes inconnus pmarg-c43476 et pmarg-c54338 et 2 gènes connus pmarg-foxl2 et pmarg-fem1-like. Ce deuxième modèle suggère fortement l'implication de pmarg-foxl2 et pmarg-fem1-like dans le déterminisme du sexe de P. margaritifera. Dans l’approche expérimentale, deux expérimentations séparées ont été réalisées pour mettre en évidence l’effet i) de plusieurs combinaisons de température et de niveau trophique, et ii) de l’œstradiol-17β administré par injection directe dans la gonade ; sur le sexe, la gamétogenèse et l’expression des neuf gènes biomarqueurs de la voie sexuelle femelle identifiés précédemment. Les résultats ont montré que la condition combinant la température de 28°C et la concentration en algues de 40 000 cellules mL-1 était la plus favorable non seulement à la maturation des gonades mâles et femelles mais aussi au maintien du sexe femelle. Ce serait dans cette condition environnementale qu’il serait possible d’induire un changement de sexe de mâle vers femelle. Dans la seconde expérimentation, il a été clairement démontré que la reproduction de P. margaritifera pouvait être régulée par les hormones œstrogènes. Les résultats montrent un effet négatif de l’œstradiol sur le développement et la différenciation mâle. Enfin les résultats du modèle prédictif de la voie sexuelle de P. margaritifera, suggèrent une programmation génétique du sexe femelle qui toutefois resterait soumise aux conditions environnementales validant ainsi l’hypothèse d’un mode de détermination mixte du sexe chez P. margaritifera. / For several years it has become essential to understand sex determination of species with high economic interest to maximize their production in emerging hatcheries.The main objective of this thesis was to identify the mechanisms involved in sex determination and sex differentiation, and particularly in female sex, in the pearl oyster P. margaritifera, a protandrous hermaphrodite species and the key species of the pearl farming, the second economic resource for French Polynesia. To achieve this goal, two approaches were undertaken: a transcriptomic approach to investigate the molecular mechanisms of sex determinism and sex differentiation, and an experimental approach to understand the phenomenon of sexualization by environmental and hormonal forcing focusing especially on female sex determinism and female sex differentiation.In the transcriptomic approach, the gonad transcriptome of P. margaritifera was sequenced from several samples of male and female oyster gonads at different stages of development. After Illumina sequencing and assembly of the transcriptome, 70,147 contigs were obtained. Functional analysis of these 70,147 contigs identified genes of interest and allowed the constitution of a catalog of 87 mRNAs encoding 67 proteins involved in sex determination, sex differentiation and/or gametogenesis. Then a strict analysis of RNAseq quantification data revealed 1,937 contigs differentially expressed between the histological categories of gonad. From the analysis of their expression profiles in each sample, a new model of reproduction of P. margaritifera, based on dual analytical approach, i.e. histo-molecular, has been proposed. This model shows that sex determination of adult P. margaritifera pearl oysters occur during a regression phase of the gonad. And considering the new stages defined on this model, 9 biomarkers genes of the female sexual pathway have been identified revealing a 3-gene-pair expression ratio based model, which makes it possible to predict the sexual pathway in this hermaphrodite species. This predictive model involves two unknown genes pmarg-c43476 and pmarg-c54338 and 2 known genes pmarg-foxl2 and pmarg-fem1-like, and strongly suggests the involvement of pmarg-foxl2 and pmarg-fem1-like in sex determinism in P. margaritifera.In the experimental approach, two separated experiments were conducted to demonstrate the effect of i) various combinations of temperature and trophic level, and ii) 17β-estradiol administered by direct injection into the gonad; on sex, gametogenesis and expression of the nine biomarkers genes of the female sexual pathway previously identified. The results showed that the condition combining a temperature of 28 °C and a concentration of 40 000 cells of algae mL-1 was the most favorable not only for the maturation of the male and female gonads but also for the maintenance of the female sex. It would be in this environmental condition that it would be possible to induce a sex change from male to female. In the second experiment, it was clearly demonstrated that the reproduction of P. margaritifera could be regulated by estrogen hormones. The results show a negative effect of estradiol on male development and differentiation. Finally the results of the predictive model of the sexual pathway of P. margaritifera, suggest a genetic programming of the female sex, which however remain subject to environmental conditions, thus validating the hypothesis of a mixed sex determinism mode in P. margaritifera.

No description available.

No description available.

La importancia de comprender los procesos biológicos ha estimulado el desarrollo de métodos para la detección de interacciones proteína-proteína. Esta tesis presenta PIANA (Protein Interactions And Network Analysis), un programa informático para la integración y el análisis de redes de interacción proteicas. Además, describimos un método que identifica motivos de interacción basándose en que las proteínas con parejas de interacción comunes tienden a interaccionar con esas parejas a través del mismo motivo de interacción. Encontramos que las proteínas altamente conectadas (i.e., hubs) con múltiples motivos tienen mayor probabilidad de ser esenciales para la viabilidad de la célula que los hubs con uno o dos motivos. Finalmente, presentamos un método que predice genes relacionados con cáncer mediante la integración de redes de interacción proteicas, datos de expresión diferenciada y propiedades estructurales, funcionales y evolutivas. El valor de predicción positiva es 71% con sensitividad del 1%, superando a otros métodos usados independientemente. / The importance of understanding cellular processes prompted the development of experimental approaches that detect protein-protein interactions. Here, we describe a software platform called PIANA (Protein Interactions And Network Analysis) that integrates interaction data from multiple sources and automates the analysis of protein interaction networks. Moreover, we describe a method that delineates interacting motifs by relying on the observation that proteins with common interaction partners tend to interact with these partners through the same interacting motif. We find that highly connected proteins (i.e., hubs) with multiple interacting motifs are more likely to be essential for cellular viability than hubs with one or two interacting motifs. Furthermore, we present a method that predicts cancer genes by integrating protein interaction networks, differential expression studies and structural, functional and evolutionary properties. For a sensitivity of 1%, the positive predictive value is 71%, which outperforms the use of any of the methods independently.

No description available.

Analyses of the evolution and function of voltage-gated sodium channel proteins (Navs) have largely been limited to mutations from individual people with diagnosed neuromuscular disease. This project investigates the carboxyl-terminus of the Nav paralog (locus scn4aa 3’) that is preferentially expressed in electric organs of Neotropical weakly-electric fishes (Order Gymnotiformes). As a model system, I used the genus Gymnotus, a diverse clade of fishes that produce species-specific electric organ discharges (EODs). I clarified evolutionary relationships among Gymnotus species using mitochondrial (cytochrome b, and 16S ribosome) and nuclear (rag2, and scn4aa) gene sequences (3739 nucleotide positions from 28 Gymnotus species). I analyzed the molecular evolution of scn4aa 3’, and detected evidence for positive selection at eight amino acid sites in seven Gymnotus lineages. These eight amino acid sites are located in motifs that may be important for modulation of EOD frequencies.

Analyses of the evolution and function of voltage-gated sodium channel proteins (Navs) have largely been limited to mutations from individual people with diagnosed neuromuscular disease. This project investigates the carboxyl-terminus of the Nav paralog (locus scn4aa 3’) that is preferentially expressed in electric organs of Neotropical weakly-electric fishes (Order Gymnotiformes). As a model system, I used the genus Gymnotus, a diverse clade of fishes that produce species-specific electric organ discharges (EODs). I clarified evolutionary relationships among Gymnotus species using mitochondrial (cytochrome b, and 16S ribosome) and nuclear (rag2, and scn4aa) gene sequences (3739 nucleotide positions from 28 Gymnotus species). I analyzed the molecular evolution of scn4aa 3’, and detected evidence for positive selection at eight amino acid sites in seven Gymnotus lineages. These eight amino acid sites are located in motifs that may be important for modulation of EOD frequencies.

Autoimmuna sjukdomar är en samling komplexa, kroniska, inflammatoriska sjukdomstillstånd som kännetecknas av dysreglering av immunsystemet, vilket resulterar i inflammation och skada av vävnader, celler och organ. Dessa sjukdomar har en betydande inverkan på individens livskvalitet och bidrar ofta till ökad dödsrisk där komorbiditeter föreligger. Emellertid medför den varierande symptombilden för olika autoimmuna sjukdomar betydande utmaningar för att uppnå noggrann diagnos, prognos och utvärdering av behandling. Det finns därför ett påtagligt behov av att upptäcka nya biomarkörer.  I denna studie utfördes en omfattande analys av 944 plasmaprover med hjälp av OlinkR Explore-plattformen, vilket genererade data för 1463 unika proteiner. Baserat på uttrycksdata identifierades proteiner förknippade med de sex utvalda autoimmuna sjukdomarna multipel skleros, myosit, reumatoid artrit, systemisk skleros, Sjögrens sjukdom och systemisk lupus erythematosus samt några av deras definierade subgrupper. Dessa potentiella biomarkörer kommer eventuellt att underlätta tidig diagnos, sjukdomsdifferentiering och prognos. Flertalet av dessa proteiner har ännu aldrig kopplats till de här specifika sjukdomarna i litteraturen, särskilt inte från plasmaprover, vilket ger spännande nya perspektiv för biomarkörsutveckling. Det är dock av största vikt att genomföra robusta valideringsstudier i oberoende kohorter.  Sammanfattningsvis belyser våra resultat den potentiella brukbarheten hos dessa proteomiska plasmabiomarkörer för att förbättra tidig sjukdomsdetektering, karakterisering av subgrupper och sjukdomsdifferentiering att stimulera. Förhoppningsvis kan dessa resultat stimulera till vidare forskning inom området för biomarkörer och potentiella framsteg inom individbaserad medicin. / Autoimmune diseases are complex, chronic, inflammatory conditions characterized by dysregulation of the immune system, resulting in inflammation and damage to various tissues, cells and organs. These diseases significantly impact individuals’ quality of life and often contribute to increased mortality risk in the presence of comorbidities. However, due to the diverse array of symptoms associated with different autoimmune diseases, accurate diagnosis, prognosis, and treatment evaluation pose significant challenges. Thus, there is a pressing need for the discovery of novel biomarkers.  In this study, a comprehensive analysis of 944 plasma samples using the OlinkR Explore platform was conducted, generating data on 1463 unique proteins. Based on the expression data, associated proteins were identified for six selected autoimmune diseases, namely multiple sclerosis, myositis, rheumatoid arthritis, systemic sclerosis, Sjögren’s syndrome, and systemic lupus erythematosus, as well as some of their defined subgroups. These are prospective biomarkers and have the potential to aid in early diagnosis, therapeutic intervention, subgroup identification, disease differentiation, and disease prognosis. Notably, some of these proteins have not been previously associated with the specific diseases in the existing literature, especially not in plasma samples, thereby offering intriguing new perspectives for biomarker development. However, it is of great importance to conduct robust validation studies in independent cohorts to confirm the outcomes of this study.  In summary, our findings highlight the potential utility of these proteomic plasma biomarkers in improving the early detection, subgroup characterization, and disease differentiation of autoimmune diseases. The identification of these proteins will hopefully stimulate further investigation in the field of biomarker research and potential advancements in personalized medicine.

Venös tromboembolism (VTE) inkluderar både djup ventrombos (DVT) och lungemboli (PE) och är en vanlig och komplex kardiovaskulär sjukdom med allvarliga kortsiktiga och långsiktiga komplikationer. I dagens kliniska praxis skulle diagnoseringen av VTE gynnas av en plasmaproteinpanel som antingen kan utesluta fall av akut VTE på egen hand eller komplettera den nuvarande biomarkören D-dimer, som i sig är begränsad av låg specificitet. På grund av den höga återfallsfrekvensen och de allvarliga post-syndromen skulle en plasmaproteinpanel som kan bedöma risken för återkommande VTE underlätta för kliniker i efterbehandlingsbeslut. Mot denna bakgrund syftade denna studie till att föreslå två separata plasmaproteinpaneler, en för att utesluta akuta VTE-patienter och en annan för att bedöma risken för återkommande VTE. Med 1463 unika plasmaproteiner screenades plasmaproteomet hos 194 individer från två undergrupper av venös tromboembolism-biomarkörstudien (VEBIOS), närmare bestämt VEBIOS ER och VEBIOS Coag, med hjälp av proximity extension assay (PEA). Både genuttryck (DE) -analys och maskininlärning (ML) -algoritmer användes för att identifiera signifikanta respektive viktiga proteiner. För akut VTE identifierades 10 signifikanta proteiner genom DE, samt en panel bestående av fem proteiner tillsammans med D-dimer hade tillsammans en area under kurvan (AUC) på 0,97 genom ML. För återkommande VTE identifierades inga signifikanta proteiner och den bästa proteinpanelen hade en AUC på 0,62. Vissa av dessa proteiner har tidigare rapporterats vara associerade med VTE och vissa inte, vilket resulterar i ortogonal validering eller påvisande av en potentiell ny biomarkör. Sammanfattningsvis hittades flera intressanta plasmaproteiner som potentiellt skulle kunna användas för att utesluta fall av akut VTE. GP1BA och S100A12 var särskilt intressanta då de var återkommande som högt ansenliga enligt DE och ML. Resultaten från denna studie kommer förhoppningsvis bidra till forskningen gällande förbättrad diagnos av VTE-patienter genom användning av plasmaproteinmarkörer och argumentationen för ytterligare undersökningar för dessa identifierade plasmaproteiner. / Venous thromboembolism (VTE) is a common and complex cardiovascular disorder with serious short- and long-term complications, comprising both deep vein thrombosis (DVT) and pulmonary embolism (PE). In current clinical practice, diagnosis of acute VTE would greatly benefit from a plasma protein panel that can exclude cases of VTE on its own or complement the current biomarker, D-dimer, which is limited by low specificity. Because of the high recurrence rate and serious post-syndromes, a protein panel that can assess the risk of VTE recurrence would help clinicians in post-treatment decision-making. Hence, this study sought out to propose two separate plasma biomarker panels, one to exclude acute VTE patients and another to risk-assess VTE recurrence.  To accomplish this, 1463 unique plasma proteins were used to investigate the plasma proteome of 194 individuals from two subgroups of the venous thromboembolism biomarker study (VEBIOS), specifically VEBIOS ER and VEBIOS Coag, using proximity extension assay (PEA). Both differential expression (DE) analysis and machine learning (ML) algorithms were used to find significant and important proteins respectively. For acute VTE, 10 significant proteins were identified through DE, and a panel of five proteins together with D-dimer had together an area under the curve (AUC) of 0.97 through ML. For VTE recurrency, no significant proteins were identified, and the best protein panel had an AUC of 0.62. Some of these proteins have previously been reported as associated to VTE and some not, resulting in some orthogonal validation or novelty. In summary, several interesting plasma proteins were found that could potentially be used to exclude cases of VTE in an acute setting. GP1BA and S100A12 were particularly interesting as they performed well in both DE and ML. The results in this study will hopefully aid the research of improving diagnosis of VTE patients using plasma biomarkers, strengthening the claim and further investigations for these identified plasma proteins.