Depuis les années 2000, les odeurs de fruits cuits, évoquant le pruneau, la figue, ou encore la pêche cuite sont de plus en plus fréquemment retrouvées dans les vins rouges de la région bordelaise. Les marqueurs responsables de ces nuances associés à l’état de maturité des raisins sont inconnus. Dans un premier temps, nous montrons que ces nuances, qui étaient jadis très rarement rencontrées dans les vins d’une appellation que nous avons appelée A, figurent aujourd’hui parmi les arômes considérés comme typiques de ces vins. Cependant, les dégustateurs qui assimilent ce caractère au type de cette appellation tendent à confondre l’arôme de fruits cuits du vin jeune à celui du caractère oxydé des vins prématurément vieillis. Les analyses par CPG-O, CPG-SM et CPG-CPG-SM de moûts et de vins marqués par des nuances de fruits cuits ont permis de valider l’existence de zones odorantes rappelant l’odeur de ces échantillons. L’analyse par GC-MS de nombreux échantillons de moût et de vin montrent que le furanéol (caramel), la γ-nonalactone (coco, pêche cuite), la (Z)-1,5-octadien-3-one (géranium) contribuent à cet arôme. Nous révélons également l’existence d’un composé à odeur de fruits cuits, de type lactone et non identifié à ce jour. L’impact de la (Z)-1,5-octadien-3-one est clarifiée pour la première fois dans les moûts. Nous précisons également l’incidence de la date de récolte des raisins de Merlot et Cabernet-Sauvignon sur l’arôme et la composition fine des moûts et des vins. La vinification de raisins récoltés tardivement voire flétris produit des vins marqués par des nuances de fruits cuits accompagnées de teneurs élevées en furaneol et γ-nonalactone. / In the 2000s, aromas of dried fruits, referring to prune, fig or cooked peach are found more and more often in Bordeaux red wines. The markers responsible for these aromas related to the grape maturity are unknown. Firstly, we show that these shades, which were rarely encountered in wines from an appellation named A in this study, are now part of the distinctive aromas of these wines. However, the tasters that assimilate these characteristics to the distinctive aromas and the type of the appellation tend to get mistaken between the cooked fruit aromas from young wines and the ones from the oxidized character of prematurely aged wines. The GC-O, GC-MS and GC-GC-MS analysis of musts and wines, that had shades of cooked fruits aromas, allowed to confirm the realness of a scented area reminding of the scent of the samples. The analysis by GC-MS of number of must and wine samples show that the furaneol (caramel), the γ-nonalactone (coconut, cooked peach), the (Z)-1,5-octadien-3-one (geranium) take part in this aroma. We also reveal the presence of a lactone type compound having a cooked fruit aroma, but not identified so far. The influence of the (Z)-1,5-octadien-3-one has been identified for the first time in musts. The influence of the harvest date of Merlot and Cabernet-Sauvignon grapes on the aromas and fine composition of musts and wines is also specified. Winemaking processing of grapes that have been late harvested, even with a withered appearance produces wines with shades of cooked fruits aromas and high content of furaneol and γ-nonalactone.

Os potenciais evocados auditivos de longa latência fornecem dados objetivos sobre a funcionalidade das estruturas corticais auditivas. O objetivo do presente estudo foi caracterizar a maturação do sistema auditivo central em crianças com audição normal por meio destes potenciais. Foram avaliados 56 indivíduos de ambos os sexos, com audição dentro dos padrões de normalidade, comprovada por audiometria e imitanciometria, sendo 46 crianças, de três a 12 anos, e 10 adultos jovens, os quais foram incluídos a título de comparação com a casuística infantil. O equipamento utilizado disponibilizava dois canais de registro, sendo um destinado à captação dos potenciais evocados auditivos de longa latência e outro, ao registro do artefato gerado pelo movimento ocular, visando ao seu controle. Os potenciais foram registrados com os indivíduos em estado de alerta, assistindo a um vídeo mudo, por meio de eletrodos posicionados em Cz (ativo) e A2 (referência) e os movimentos oculares, por eletrodos em posição supra e infra-orbital esquerda; o eletrodo terra foi colocado em A1. Foram analisados os valores de latência e de amplitude dos componentes P1, N1 e P2, bem como a morfologia destes componentes de acordo com a idade. Para verificar a reprodutibilidade dos potenciais registrados, foi realizado um estudo duplo-cego com a introdução da análise de uma avaliadora experiente em Eletrofisiologia, a qual não mostrou diferenças estatisticamente significantes da análise feita pela autora da pesquisa. Como resultado, constatou-se, quanto à morfologia, melhora na definição dos componentes com o avanço da idade. Foi observada diminuição nos valores de latência dos componentes P1, N1 e P2 com o avanço da idade. Não foi observada variação nos valores de amplitude com o avanço da idade para os componentes N1 e P2, exceto para o componente P1, que teve sua amplitude diminuída. Não foi observada diferença estatisticamente significante entre os sexos. Pôde-se observar que o processo maturacional do sistema auditivo central acontece de maneira gradativa, sendo as maiores modificações observadas ao se comparar crianças e adultos. / The long latency auditory evoked potentials provide objective data on the function of hearing cortical structures. This study aims at characterizing the maturation of the central hearing system, in normally hearing children, through these potentials. Fifty-six subjects from both genders, with hearing within normality patterns, verified by means of audiometry and imitanciometry, were evaluated, being 46 children in the age range 3-12, and 10 young adults, who were included for comparison with the infantile sample. The equipment utilized had two recording channels: one to catch the long latency auditory evoked potentials, and the other, to record the artifact generated by ocular movement, aiming at its control. The potentials were recorded with subjects alert, while watching a mute video, through electrodes positioned in Cz (active) and A2 (reference), and the ocular movements, through electrodes in left supra and infra-orbital position and the ground electrode was placed in A1. Values of latency and of amplitude for components P1, N1 and P2, as well as the morphology of these components, according to age, were analyzed. In order to verify the reproducibility of the recorded potentials, a double blind study was carried out, by introducing the analysis of an experienced Electrophysiology evaluator, whose analysis did not show statistically significant differences, as compared to that performed by the author of the research. Thus, as to morphology, improvement in the definition of components was seen, as age increased. A decrease in latency values of components P1, N1 and P2 was observed, as age advanced. No variation was verified in amplitude values, as age increased, for components N1 and P2, except for component P1, whose amplitude was diminished. No statistically significant difference was seen between genders. It was verified that the maturational process of the central hearing system takes place in a gradual fashion, being the greatest modifications seen, when comparing children and adults.

Macrophages are sentinels present in most tissues of the body, where they recognise and respond to biological dangers. Recognition and uptake of particles is mediated through phagocytic receptors which upon activation induce appropriate responses. These responses need to be tightly regulated in order to destroy pathogens but prevent uncontrolled inflammation. Phagocytosis is an evolutionarily conserved process required for host defence and homeostasis. During phagocytosis, particles are recognised by cell surface receptors that trigger rearrangement of the actin cytoskeleton and internalization of the bound particle into a de novo, membranous organelle known as the phagosome. Regulation of phagocytosis and phagosome maturation can be achieved through changes in transcription/translation and differential recruitment of proteins but also through their non-translational modifications. Here I explored the role of ubiquitylation in the phagosome biogenesis of Interferon-gamma (IFN-ɣ) activated macrophages. Ubiquitylation is a diverse, reversible post-translational modification which is not only involved in protein degradation but also in vesicle trafficking and immune signalling. My data shows that phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ. I applied a targeted AQUA peptide approach by which we quantified ubiquitin chain linkage peptides from phagosome samples by PRM. This data shows that all chain linkages apart from M1/linear chains are present on phagosomes. Furthermore, IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, I characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. I demonstrated that most ubiquitin chains are on the cytoplasmic site of the phagosome enabling an interaction of ubiquitin chains with cytosolic proteins such as Rab7. Rab7 a major regulator of vesicle trafficking could be shown to be ubiquitylated on phagosomes. I further showed that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation and RNF115 is responsible for most of the increase of K63 polyubiquitylation of phagosomal proteins. Knock-down of RNF115 promotes phagosome maturation and induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome and disruption of this pathway induces pro-inflammatory responses in macrophages. In conclusion, this is the first study showing unbiasedly that ubiquitylation plays an important role in vesicle trafficking to the lysosome.

La vigne est une plante cultivée dans le monde entier dont l’importance économique est principalement liée à la production de vin. La baie de raisin est également l’un des principaux modèles d’étude pour les fruits non-climatériques notamment pour l’étude des mécanismes contrôlant le mûrissement des baies. Le développement de la baie de raisin est caractérisé par deux phases de croissance séparées par une phase de latence se produisant au moment de la véraison. La baie de raisin est composée de trois tissus principaux: la peau, la pulpe et les graines. La peau et la pulpe présentent une structure et une composition en métabolites distinctes et contribuent de manière différente à la qualité du vin, la pulpe fournissant essentiellement le sucre, les acides aminés et organiques alors que la peau est riche en anthocyanes. A l'heure actuelle, les mécanismes moléculaires impliqués dans le contrôle de la maturation des baies de raisin sont encore mal compris. Si l'ABA, le sucre et différents facteurs de transcription jouent un rôle important dans le contrôle de cette phase de développement, les mécanismes épigénétiques, en particulier la méthylation de l’ADN, apparaissent aussi comme des régulateurs importants du développement et du mûrissement des fruits charnus. Dans ce contexte, Le projet de thèse présenté vise à analyser le rôle de la méthylation de l’ADN (1) dans la maturation des baies de raisin et (2) dans la synthèse des anthocyanes en utilisant comme système modèle des cellules de baie de raisin cultivées in vitro.La culture in vitro de baies de raisin en présence d’inhibiteurs de la méthylation de l'ADN, aboutit à une inhibition de la maturation, suggérant que la méthylation de l’ADN joue un rôle crucial pour cette étape du développement chez la vigne. La pellicule et la chair de baies de raisin récoltées à divers stades de développement ont ensuite été analysées séparément pour déterminer les variations des transcriptomes, de l’abondance de différents métabolites, et de la méthylation de l'ADN. Les principaux résultats indiquent des variations des métabolites et du transcriptome, avec des spécificités liés au tissu analysé. En outre, l'analyse des variations de méthylation de l'ADN à deux stades de développement dans chacun de ces deux tissus révèle l’existence de variations de méthylation spécifiques à chaque tissu, tandis que les variations communes aux deux tissus restent limitées. Ces résultats suggèrent un contrôle de la méthylation de l’ADN spécifique à chaque tissu lors de la maturation de la baie. Cependant les régions différentiellement méthylées identifiées dans chaque tissu, ne sont pas associées à des gènes exprimés différentiellement au cours de la maturation des baies, ce qui pose la question du rôle de la méthylation de l’ADN dans le contrôle de l’expression génique dans les baies.Pour analyser le rôle de la méthylation de l’ADN dans le contrôle de la synthèse des anthocyanes, nous avons utilisé des suspensions de cellules de raisin du génotype Gamay Teinturier (GT), connues pour accumuler des anthocyanes lorsqu’elles sont cultivées à la lumière. L’utilisation de la zébularine, un inhibiteur de la méthylation d’ADN, permet de stimuler l’accumulation d'anthocyanes dans les cellules GT en présence de lumière, et de l’induire à l’obscurité. Les traitements à la zébularine provoquent en outre une limitation de la croissance cellulaire, une modification de l’accumulation des sucres solubles et acides organiques ainsi qu’une reprogrammation importante du transcriptome. Ces résultats suggèrent un effet général de la zébularine sur les cellules GT plutôt qu’un effet spécifique sur l’accumulation d’anthocyanes.Dans l'ensemble, les résultats indiquent que la méthylation de l'ADN est importante pour le contrôle de la maturation des fruits de la vigne, bien que les mécanismes qui sous-tendent les variations de la méthylation et leurs rôles dans les différents tissus de la baie de raisin restent à préciser. / Grapevine is a worldwide cultivated fruit crop with high economic importance mainly because of its usage for vine production. Grape berry is also one of the main models for non-climacteric fruits to study the mechanisms controlling the ripening process. Grape berry development is characterized by two phases of rapid size increase separated by a lag phase at the time of ripening induction. Grape berries are composed of three main tissues, the peel, the pulp and the seeds. Peel and pulp present distinct structure and metabolite composition and contribute in a different way to wine quality, the pulp providing sugar, amino and organic acids whereas the peel is important for anthocyanins and other phenolic compound abundance. At the present time, the molecular mechanisms involved in the control of grape berry ripening are still poorly understood. Recent results indicate that both ABA and sugar may be important signals together with various transcription factors. In addition, epigenetic mechanisms are now emerging as important regulators of fleshy fruit development, DNA methylation being critically important for tomato, sweet range and strawberry ripening.The present project aims at analyzing the potential role of DNA methylation in the control grape berry ripening. It also investigates the potential role of DNA methylation in the synthesis of anthocyanins, a compound of primary importance in peel of red grape berries, using in vitro grown fruit cells. To address these questions, grape berries cultivated in vitro were treated with DNA methylation inhibitors. Treatments resulted in delayed and reduced grape berry ripening, therefore sustaining the idea that DNA methylation plays critical roles at this developmental step. Grape berries harvested at various developmental stages were then dissected and each tissue was separately analyzed for transcriptomic, metabolic and DNA methylation variations. Main results indicate significant and distinct metabolic and transcriptomic variations consistent with each tissue following specific modifications during ripening. In addition, analysis of DNA methylation variations at two developmental stages in each tissue indicates both common and tissue specific changes in DNA methylation patterns during fruit ripening. A very small proportion of DMRs is found similarly in the pup and the peel, but most are tissue specific, also consistent with tissue specific control at this developmental phase. Of note, among the different DMRs identified in each tissue, only a few were associated with differentially expressed genes (DEG) during ripening, whereas most were not, questioning the general role of DNA methylation in the control of gene expression at this developmental transition in grape.As Anthocyanins are the most abundant polyphenolic compounds in the skin of red grape berries, we used grape cell suspensions of the Gamay Teinturier genotype, that are known to accumulate anthocyanins when grown in light conditions, to analyze the potential role of DNA methylation in their synthesis. GT cells cultivated in light conditions were treated with the DNA methyltransferase inhibitor zebularine, they accumulate higher quantities of anthocyanins. Of note, GT cells grown in the absence of light do not accumulate anthocyanins. However, zebularine was sufficient to induce anthocyanin accumulation in the absence of light. Zebularine treatments had significant additional effects on grape cells including, cell growth limitation, and modification of soluble sugar, organic acid or stilbene accumulation, together with important transcriptomic reprogramming, consistent with a general effect on cells rather than a specific effect on anthocyanin accumulation.Taken together, results are consistent with DNA methylation being important in the control of grape fruit ripening, although the precise mechanisms underlying methylation variations and roles in grape berries remain to be deciphered.

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in μg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 μg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 μCi.mL-1, compared to a mean of 4.79 μCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 μg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 μg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 μg.mL-1, and a dexamethasone treated concentration of 22.84 μg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 μM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 μM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.

Thesis (Ed. D.)--Graduate School of Education, Oral Roberts University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 143-170).

During vertebrate oocyte maturation, the chromosomes progress to and arrest at metaphase of meiosis II in preparation for fertilization. This process includes emission of the first polar body. The second polar body is emitted after fertilization. A number of proteins are accumulated during oocyte maturation. Inhibition of this de novo translation does not appear to affect the progression of meiosis during oocyte maturation. The role of these pools of proteins has yet to be elucidated. Curiously, several of the upregulated proteins are key players in mitosis, including INCENP, a subunit of the chromosome passenger complex implicated in chromosome segregation and cytokinesis. During early stages of development in Xenopus laevis, the embryo cycles through mitosis, also known as embryo cleavage, every 30min with little to no time for transcription/translation. Our goal is to determine if the de novo translation of these mitotic proteins during oocyte maturation has a role in early embryogenesis. We used morpholino oligonucleotides antisense to INCENP mRNA (INCENPmorpho) to inhibit de novo translation during oocyte maturation. Using confocal imaging and the host transfer technique, these injected oocytes were matured, fertilized and assessed for developmental competency. INCENPmorpho and a control morpholino (ctrlmorpho) had no discernable effect on 1st or 2nd polar body emission. Whereas ctrlmorpho embryos developed normally, INCENPmorpho embryos did not cleave. Thus, de novo translation of INCENP during oocyte maturation is necessary for embryogenesis. Specifically, accumulation of INCENP and other mitotic proteins during oocyte maturation may be a common strategy in this species to prepare for the rapid and synchronous mitoses during early embryogenesis.

The rapid evolutionary rate of HIV-1 has lead to the emergence of multi-drug resistant variants, emphasizing the need for novel inhibitory methods. One such method could be based upon inhibiting viral gene expression through disruption of HIV-1 RNA processing. A means of accomplishing this goal is through use of modified U1snRNA variants that target highly conserved regions of HIV-1 at its terminal exon and prevent 3’ end formation. The modification consists of a 10-nucleotide substitution at the 5’ end complementary to the conserved HIV-1 regions. When modified U1snRNA targeted to HIV-1 were cotransfected with replication deficient HIV-1 proviruses, western blot indicated a specific and significant reduction in the level of viral protein production (p24 and gp120), while U1 constructs that lacked complementary sequences had no effect on HIV-1 protein expression. To further investigate targeted U1snRNA inhibitory effects on HIV-1, efforts are currently underway to determine if this approach has the ability to suppress protein expression in a gene therapeutic model. To date, suppression of viral protein production has reached 50% when tested with a moderately inhibitory U1snRNA. If shown to be effective, such an inhibition would be increased with the use of combinatory modified U1snRNA constructs producing a synergistic effect.

During S phase of the cell cycle, newly synthesized histones are acetylated in the cytoplasm in patterns specific to DNA replication. Once incorporated into nucleosomes, these histones are rapidly deacetylated by enzymes known as histone deacetylases. Though common in all organisms, the significance of this molecular mechanism is not fully understood. Homologous to HDAC6 in humans and HDA1 in budding yeast, Tetrahymena histone deacetylase 2 (Thd2) has been identified as the only known histone deacetylase that performs this task in the unicellular eukaryote Tetrahymena thermophila. Localizing to the transcriptionally inactive germline nucleus, the micronucleus, Thd2 has been found to deacetylate histones H3 and H4 at K9 and/or K14. In order to gain further insight into the role of deposition-related deacetylation in chromatin maturation, the micronuclear morphology and modification status of H3K27, a known marker for heterochromatin in several eukaryotes, were examined in both vegetative and synchronized complete Δthd2 mutant cells. Immunofluorescence microscopy, DAPI staining and a western blot analysis revealed abnormal phenotypes and the conservation of H3K27 methylation in the absence of Thd2. These findings further indicate a role for Thd2 in the maintenance of chromatin structure and suggest the possibility of another mechanism required for deacetylation at H2K27. Essentially, this demonstrates the importance of deposition-related histone deacetylation in chromatin maturation after DNA replication and further maintenance of chromatin domains.

The rapid evolutionary rate of HIV-1 has lead to the emergence of multi-drug resistant variants, emphasizing the need for novel inhibitory methods. One such method could be based upon inhibiting viral gene expression through disruption of HIV-1 RNA processing. A means of accomplishing this goal is through use of modified U1snRNA variants that target highly conserved regions of HIV-1 at its terminal exon and prevent 3’ end formation. The modification consists of a 10-nucleotide substitution at the 5’ end complementary to the conserved HIV-1 regions. When modified U1snRNA targeted to HIV-1 were cotransfected with replication deficient HIV-1 proviruses, western blot indicated a specific and significant reduction in the level of viral protein production (p24 and gp120), while U1 constructs that lacked complementary sequences had no effect on HIV-1 protein expression. To further investigate targeted U1snRNA inhibitory effects on HIV-1, efforts are currently underway to determine if this approach has the ability to suppress protein expression in a gene therapeutic model. To date, suppression of viral protein production has reached 50% when tested with a moderately inhibitory U1snRNA. If shown to be effective, such an inhibition would be increased with the use of combinatory modified U1snRNA constructs producing a synergistic effect.