Des recherches en sciences judiciaires ont montré récemment une possible corrélation entre le temps d’entreposage d’échantillons de fluides corporels et la dégradation de l’ARN dans ceux-ci. Le moment où une tache a été déposée sur une scène de crime peut être important pour déterminer la pertinence d’un échantillon dans une enquête. Dans ce mémoire, nous rapportons les profils de dégradation de quatre ARN différents mesurés par RT-qPCR, soit l’ARN ribosomique 18S et les ARNm de la β-actine, de la glyceraldehyde-3-phosphate déhydrogénase et de la cyclophiline A, obtenus de taches de sang, de salive et de sperme, entreposés à la température de la pièce ou au congélateur à -80°C sur une période de 6 mois. Nos résultats montrent une faible variation interindividuelle pour le sang et le sperme, mais une différence importante entre les donneurs pour la salive. De plus, le profil de dégradation est semblable pour tous les transcrits, mais diffère entre les fluides. La congélation des échantillons stabilise les ARN avant leur analyse. Finalement, la quantité d’ARN détecté est en relation avec le temps d’entreposage et pourrait être utilisée afin d’estimer l’âge des échantillons lorsque l’impact des conditions d’entreposage sur la dégradation de l’ARN sera mieux connu. / Recent studies in forensic science have shown a possible correlation between the degradation rate of some RNA transcripts and the age of bloodstains. The time of deposition of a stain can be of major importance to determine the relevance of a sample in a forensic investigation. In this thesis, we describe the degradation profiles of the 18S ribosomal RNA and the β-actin, glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A mRNAs, measured by RT-qPCR and obtained from dried blood, semen and saliva stains stored at room temperature or frozen at -80°C up to 6 months. Our results showed low inter-individual variation for blood and semen stains, but a high variation was observed between donors for saliva. Moreover, degradation profile of each transcripts was similar, but differed between fluids. Freezing samples prevented RNA degradation over time. Finally, RNA quantity was in relation with the time of storage and could be used to estimate the time since deposition of a stain when the effects of various storage conditions on RNA degradation profiles will be better documented. / Projet de recherche réalisé en collaboration avec la section Biologie/ADN du Laboratoire de sciences judiciaires et de médecine légale (LSJML) de Montréal.

O feijão é importante fonte carboidratos, vitaminas, minerais e fibras. No Brasil, a produtividade desta leguminosa é baixa e um dos fatores é a ocorrência de doenças como a antracnose causada pelo Colletothrichum lindemuthianum, que gera perdas de até 100% da produção. Plantas possuem diversos mecanismos de defesa contra patógenos e relatos apontam que o silício é capaz não só de promover mudanças morfológicas nas folhas, mas também de ativar os genes de resistência. O presente trabalho foi dividido em três estudos que tinham como objetivo: (1) entender a resposta de três cultivares de feijoeiro ao silício disponível na solução nutritiva; (2) identificar a contribuição do Si na expressão de genes relacionados à infecção pelo fungo através da construção de duas bibliotecas subtrativas por supressão (SSH), visando selecionar genes diferencialmente representados durante a infecção da planta com a raça 65 de C. lindemuthianum (a) e durante a infeção da planta na presença de uma maior dose silicato de potássio (75 ppm) no substrato (b); (3) identificar a resposta de dez transcritos selecionados no Estudo 2 para tentar entender a resposta dos mesmos em diferentes períodos (0; 6; 42; 72 h) após a inoculação, com ou sem suplemento de Si. Como resultados, foi observado que para as três cultivares avaliadas o Si começa a ser absorvido 14 dias após o transplante. Também foi identificado por de microscopia de varredura (MEV) que não há diferença significativa entre o número de tricomas e cada cultivar, mas que para o número de estômatos a cultivar IAC-Harmonia destacou-se das demais. Além disso, quando as três cultivares foram suplementadas com Si, houve a formação de uma cera epicuticular descrita como mecanismo de defesa da planta contra fungos; e que através de EDX (Energy-dispersive X-ray spectroscopy) foi possível constatar que plantas tratadas com Si apresentam maior teor deste elemento nas folhas. Através de inoculações com a raça 65 do patógeno verificou-se o efeito do mineral na redução da severidade da doença nas cultivares IAC-Harmonia e Pérola. No segundo estudo, duas bibliotecas de hibridização subtrativa por supressão (SSH), foram construídas visando selecionar os genes diferencialmente expressos entre plantas inoculadas e não-inoculadas (A) e entre plantas inoculadas e tratadas ou não com 75 ppm de Si (B). Foram geradas 991 sequências únicas, anotadas através do GeneOntolgy. Quinze genes de cada biblioteca foram selecionados para os experimentos de validação por RT-qPCR. Para a Biblioteca A, 11/15 genes foram positivamente regulados, e em B, 14/15. No terceiro estudo ficou evidenciado que a inoculação com o patógeno alterou positivamente a expressão de sete genes, enquanto que o tratamento com 75 ppm de Si alterou a expressão de oito genes, em pelo menos um dos tempos avaliados / Beans are an important source of carbohydrates, vitamins, minerals and fibers. In Brazil, this legume still has low productivity and one of the factors involved is the occurrence of diseases such as anthracnose, caused by the fungus Colletothrichum lindemuthianum, which causes losses in production of up to 100%. Plants present several defense mechanisms against pathogens and the reports indicate that silicon does not only promote morphological changes in leaves, but also activates resistance genes. This work was divided into three studies aiming: (1) to understand the response of three bean cultivars to a silicon source in a nutrient solution, (2) to identify the contribution of Si in the expression of genes related to the infection by the fungus by constructing two subtractive suppression libraries (SSH), to select genes differentially represented during infection of the plant with race 65 of C. lindemuthianum (a) and during infection of the plant in the presence of higher dose of potassium silicate (75 ppm) in the substrate (b), (3) to identify the response of ten selected transcripts in Study 2 in various periods (0, 6, 42, 72 h) after inoculation, with or without supplemental Si. As a result, it was observed that for all three cultivars Si begins to be absorbed 14 days after transplantation. Was also identified by microscopy (SEM) that there is no significant difference between the number of trichomes among cultivars, but that the number of stomata for the IAC-Harmonia stood out from the rest. Moreover, when the three cultivars were supplemented with Si, thus forming an epicuticular wax described as a defense mechanism against plant fungi, and that by EDX (Energy-dispersive X-ray spectroscopy) it was found that plants treated with Si have higher content of this element in leaves. Through inoculations with race 65 of the pathogen it was verified the effect of the mineral in reducing disease severity in IAC-Pérola and IAC - Harmonia. In the second study, two libraries from suppression subtractive hybridization (SSH) were constructed in order to select the differentially expressed genes between inoculated and non-inoculated (A) and between plants inoculated and treated or not with 75 ppm of Si (B). In total, 991 unique sequences were generated, those recorded by GeneOntolgy. Fifteen genes from each library were selected for the validation experiments by RT-qPCR. For library A, 11/15 genes were positively regulated, and in B, 14/15. In the third study it is showed that inoculation with the pathogen positively altered expression of seven genes, whereas treatment with 75 ppm of Si changed the expression of eight genes, in at least one of the times analyzed

Nous avons développé un nouveau test de prédiction de l’efficacité de thérapies anti-cancéreuses. Ce concept se base sur la détermination d’une signature moléculaire tumorale par RT-qPCR. Cette signature est issue d’un algorithme de normalisation innovant de comparaison des données d’expression relative des cibles de la tumeur avec celles de tissus de référence. Cette normalisation offre à chaque cible de la signature un rang et un score spécifique permettant de hiérarchiser les voies pro-tumorales afin de trouver la ou les cibles dominantes responsables du développement de la tumeur. La signature comprend des cibles donnant des informations générales sur le statut et l’hétérogénéité de la tumeur, sur l’angiogenèse et la lymphangiogenèse, sur le microenvironnement tumoral et enfin sur l’activité migratoire. Une validation préclinique dans les modèles du cancer colorectal, de la prostate et du glioblastome, a montré que le test est prédictif de l’efficacité thérapeutique. / We developed a new tool for prediction of cancer treatment efficacy. Our process is based on the determination of the molecular signature which is intended to provide a clinician’s decision tool helping to select which tumor signaling pathway(s) has/have to be targeted for best therapeutic effect. This signature representing a scoring obtained by RT-qPCR through a sequential normalization process of the expression level of target genes in the tumor compared to cancer type-specific references. These genes were selected because of a good knowledge of related biological functions, a correlation between expression level and aggressiveness of the tumor, the existence of a therapeutic arsenal already in clinical use. This signature is validated in a preclinical model of colorectal cancer and prostate cancer and glioblastoma. The results obtained show that the test we developed allows to identify the most important signaling pathway implicated in the disease and to choose the best drug.

L’esca est une maladie du bois de la vigne complexe et mal connue, contre laquelle aucun moyen de lutte efficace n’existe à ce jour. Ce travail s’est concentré sur les interactions précoces entre Vitis vinifera L. et les champignons associés au « young esca » P. chlamydospora et P. aleophilum dans deux types de tissus lignifiés : l’entre-nœud et le nœud (modèle plaie de taille). La colonisation 6 et 12 semaines après traitement des souches transformées P. aleophilum::gfp7 et P. chlamydospora::gfp1 a été observée. Les deux espèces coloniseraient différents tissus dans les premières semaines suivant l’infection. Les fibres du xylème constitueraient un tissu essentiel lors de l’interaction précoce entre P. aleophilum et la vigne, tandis que P. chlamydospora::gfp1 a seulement colonisé les vaisseaux du xylème après 12 semaines. Le bois de la vigne présenterait des réponses spécifiques à la présence de P. aleophilum 6 semaines après traitement, puis générales à la blessure 12 semaines après traitement, en microscopie. L’hypothèse de la spécificité de la réponse induite dans le bois de la vigne par ces deux espèces a été confirmée en étudiant l’expression de 11 gènes associés à la défense 10 h, 24 h, 48 h et 120 hpi. La réponse précoce du bois de la vigne serait spécifique suivant l’identité des pathogènes. Les tissus de l’entre-nœud ont été induits différemment par la blessure par rapport aux tissus dans la région nodale. Un modèle pour le criblage d’agents de biocontrôle ou d’éliciteurs contre l’esca en quelques mois en conditions de laboratoire a permis le développement d’un moyen de lutte durable et novateur, l’eau ozonée. L’eau ozonée présente des propriétés sporicides remarquables contre P. aleophilum in vitro. In planta l’application d’eau ozonée sur une blessure infectée en modèle plaie de taille a réduit de moitié la quantité de mycélium capable de se développer dans le bois 9 semaines après inoculation. / Esca is a complex pathosystem, poorly understood, affecting grapevine’s trunk. Currently there is not efficient tools to control esca disease. This study investigated early events between grapevine and fungi associated to young esca P. aleophilum and P. chlamydospora in two different woody tissues: at the internode and at the nodal (pruning wound model) levels. The colonization of transformed strains P. aleophilum::gfp7 and P. chlamydospora::gfp1 was observed 6 and 12 weeks post inoculation. Fungal species colonized different tissues during the first weeks of infection. Xylem fibres could be the key site of P. aleophilum-grapevine early interactions, whereas P. chlamydospora::gfp1 only colonized xylem vessels 12 weeks post inoculation. Grapevine wood may respond specifically to P. aleophilum after 6 weeks. This response seemed to be related to healing in microscopy, and thus aspecific 12 weeks post inoculation. The specificity of induced response in the wood of Vitis vinifera L. was confirmed by studying gene expressions of 11 defense-related genes 10 h, 24 h, 48 h and 120 hpi. The early response of woody tissues could be specific to pathogens identity in grapevine. Wounding damages induced defense-related genes differently according the tissues (internode vs node). A model for screening biological control agents or elicitors in laboratory conditions, within months, has been used to develop a sustainable and original control tool: ozonated water. Ozonated water presented remarkable sporicidal properties on P. aleophilum in vitro. Infected pruning wound treated with ozonated water reduced the P. aleophilum inoculum able to develop in the injured wood by a factor two 9 weeks post inoculation.

Talaromyces versatilis est un champignon filamenteux d’intérêt industriel grâce à sa capacité deproduction d’enzymes hydrolytiques. La Société Adisseo commercialise un cocktail enzymatiqueproduit par fermentation à partir de T. versatilis, sous le nom de Rovabio™. Ce cocktail est utilisé entant qu'additif alimentaire en nutrition animale, car la grande variété d'enzymes hydrolytiques qu’ilcontient peut dégrader les polysaccharides présents dans l’enveloppe des céréales, améliorant ainsila digestibilité la valeur nutritionnelle des matières premières agricoles. Malgré les efforts consentispour mieux connaître la biologie de T. versatilis, très peu est connu sur ce champignon.L’étudeprésentée ici vise à décrire les réseaux de régulation qui contrôlent l’expression des gènes codantpour ces enzymes hydrolytiques, en utilisant des approches génomiques et transcriptomiques.Avoir accès à une annotation correcte de la séquence génomique et posséder les outilsnécessaires pour l'ingénierie génétique sont essentiels pour réaliser des études de génomiquefonctionnelle. Donc, le premier volet de cette thèse a été l’analyse de la séquence génomique et lacuration manuelle de l'annotation, ce qui nous a conduits à évaluer le vaste potentiel génétique de T.versatilis pour la production et la sécrétion d'enzymes hydrolytiques impliquées dans la dégradationde la lignocellulose. Deuxièmement, un système de délétion des gènes initialement conçu pourAspergillus niger a été adapté à T. versatilis. Cette méthode permet le recyclage du marqueur desélection et est efficace dans des souches dont le système NHEJ est actif (Delmas, et al., 2014, AEM).Au cours de ce travail, deux mutants de délétion de T. versatilis ont été obtenus: ΔxlnR et ΔclrA.La première approche mise en place pour avoir une meilleure compréhension des réseaux derégulation via une vue globale du transcriptome, fut l’utilisation de la technique de RNAseq sur troiséchantillons issus de la souche sauvage de T. versatilis exposée au glucose, à la paille de blé et auglucose et paille de blé simultanément comme sources de carbone, respectivement. Les données ontmontré une augmentation massive des niveaux d’expression de nombreux gènes, en particulier ceuxcodant pour des enzymes hydrolytiques, lorsque le mycélium est exposé à la lignocellulose. Enfin, la dernière partie du projet s’est appuyée sur la la RT-qPCR, technique appropriée pourétudier un nombre limité de gènes dans une grande variété de conditions. Toutefois la normalisationdes données est une étape essentielle du flux de travail qui peut conduire à une interprétationbiologique incorrecte de la régulation des gènes. Le travail effectué sur les données de RNAseq nousa amené à reconsidérer la nature des gènes de référence classiquement utilisés, puisque la plupartd'entre eux présentaient des changements d'expression considérables en présence de lignocellulose.En conséquence, un nouvel ensemble de gènes de référence putatifs a été identifié et la stabilité deleur expression validée par RT-qPCR chez T. versatilis cultivé dans plus de 30 conditions différentes.Des jeux de données de RNAseq de 18 champignons filamenteux phylogénétiquement éloignés ontpar ailleurs été collectés, afin de démontrer que la sélection des gènes candidats pour lanormalisation des données de RT-qPCR chez T. versatilis peut être étendue à d'autres champignons(Llanos et al., 2014, BMC Genomics). Ces aspects méthodologiques validés, nous avons enfin réaliséune étude plus détaillée de la transcription d'un groupe de gènes d'intérêt par RT-qPCR, dans unegrande variété de conditions et 2 souches différentes, la souche sauvage et la mutante ΔxlnR.L'analyse de ces données a permis d'identifier des gènes aux profils d'expression similaires, quirépondent de la même façon aux substrats inducteurs et qui partagent probablement les mêmesmécanismes de régulation. / Talaromyces versatilis is an industrially important enzymes producing filamentous fungus.Adisseo Company commercializes the enzymatic cocktail, produced from T. versatilis fermentation,with the name of Rovabio™. This cocktail is applied as an animal feed additive as it contains a widevariety of hydrolytic enzymes that can degrade the polysaccharides present in the seed-coat and thusimproves the digestibility and increases the nutritional value of the agricultural raw materials.Although efforts have been done to study different aspects of the biology of T. versatilis, very little isknown about this fungus. This study aimed to describe the regulatory networks of genes encodingplant cell wall-degrading enzymes from this biotechnologically important fungus using genomic andtranscriptomic approaches.Having a correct annotation of the genomic sequence together with efficient tools for genomeengineering are essential for downstream functional genomics works and characterization of theregulatory networks. Therefore, the first task carried out an analysis of the genomic sequence and amanual curation of the annotation, which led us to assess the vast genetic potential of T. versatilis forthe production and secretion of hydrolytic enzymes involved in the degradation of lignocellulosicmaterials. Secondly, I adapted a gene deletion system initially designed for Aspergillus niger. Thismethod allows recycling of the selection marker and is efficient in a non-homologous end-joining(NHEJ)-proficient strain (Delmas, Llanos et al., 2014, AEM). During this work, two deletion mutants ofT. versatilis were obtained: ΔxlnR and ΔclrA.Towards better understanding of the regulatory network, I first contributed to an RNAseq-basedtranscriptomic study that was performed on the wild type strain of T. versatilis exposed to glucoseand wheat straw as carbon sources. The data showed a massive increase in transcript levels ofnumerous genes, in particular those encoding hydrolytic enzymes, when the mycelium wasincubated with lignocellulose.If RT-qPCR is indeed a suitable technique to study a limited number of genes in a large variety ofconditions, data normalisation is a critical step of the workflow that can lead to incorrect biologicalinterpretation of gene regulation. The work done on the RNA-seq data led me to reconsider the useof the classical reference genes, since most of them exhibited expression changes in the presence oflignocellulosic substrate. I therefore identified a new set of putative reference genes and validatedtheir expression stability by RT-qPCR in T. versatilis cultivated under more than 30 differentconditions. Then, I collected about a hundred RNA-seq datasets from 18 phylogenetically distantfilamentous fungi, to demonstrate that the use of the suitable candidates for RT-qPCR datanormalisation in T. versatilis can be extended to other fungi (Llanos et al., 2014 BMC genomics (minorrevisions)). Thereafter, I performed a more detailed RT-qPCR based transcriptional study of a groupof genes of interest, in a wide variety of conditions and in 2 strains, the wild-type and the ΔxlnRmutant. The analysis of expression data of the genes of interest allowed to identify genes with similarexpression patterns, which probably share the same regulatory mechanisms and also the substratesthat act as inducers for their expression

Des recherches en sciences judiciaires ont montré récemment une possible corrélation entre le temps d’entreposage d’échantillons de fluides corporels et la dégradation de l’ARN dans ceux-ci. Le moment où une tache a été déposée sur une scène de crime peut être important pour déterminer la pertinence d’un échantillon dans une enquête. Dans ce mémoire, nous rapportons les profils de dégradation de quatre ARN différents mesurés par RT-qPCR, soit l’ARN ribosomique 18S et les ARNm de la β-actine, de la glyceraldehyde-3-phosphate déhydrogénase et de la cyclophiline A, obtenus de taches de sang, de salive et de sperme, entreposés à la température de la pièce ou au congélateur à -80°C sur une période de 6 mois. Nos résultats montrent une faible variation interindividuelle pour le sang et le sperme, mais une différence importante entre les donneurs pour la salive. De plus, le profil de dégradation est semblable pour tous les transcrits, mais diffère entre les fluides. La congélation des échantillons stabilise les ARN avant leur analyse. Finalement, la quantité d’ARN détecté est en relation avec le temps d’entreposage et pourrait être utilisée afin d’estimer l’âge des échantillons lorsque l’impact des conditions d’entreposage sur la dégradation de l’ARN sera mieux connu. / Recent studies in forensic science have shown a possible correlation between the degradation rate of some RNA transcripts and the age of bloodstains. The time of deposition of a stain can be of major importance to determine the relevance of a sample in a forensic investigation. In this thesis, we describe the degradation profiles of the 18S ribosomal RNA and the β-actin, glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A mRNAs, measured by RT-qPCR and obtained from dried blood, semen and saliva stains stored at room temperature or frozen at -80°C up to 6 months. Our results showed low inter-individual variation for blood and semen stains, but a high variation was observed between donors for saliva. Moreover, degradation profile of each transcripts was similar, but differed between fluids. Freezing samples prevented RNA degradation over time. Finally, RNA quantity was in relation with the time of storage and could be used to estimate the time since deposition of a stain when the effects of various storage conditions on RNA degradation profiles will be better documented.

Norovirus som orsakar vinterkräksjukan är en av de vanligaste vintersjukdomarna i Sverige. Sjukdomstiden varar generellt i en till tre dagar med symptomen kräkning och/eller diarré. Till den totala sjukdomsbilden världen över gällande akut gastroenterit, bidrar norovirus med 18 %. Trots att sjukdomen är mycket vanlig är kunskapen om norovirusets förfarande till stor del okänd.Syftet med studien var att göra en tidsserieanalys, även så kallad One-Step Growth analys, av koncentrationen minus-RNA i celler som infekterats med olika koncentrationer av murint norovirus (MNV). För att detektera minus-RNA användes RT-qPCR med SYBR Green. Målet var att se om startkoncentrationerna av virus vid någon tidpunkt korrelerar med mängden minus-RNA i cellerna. Efter 4 och 8 timmar fanns ett exponentiellt samband mellan den initiala viruskoncentrationen och minus-RNA-uttrycket i cellerna. Koncentrationen minus-RNA i de infekterade cellerna ökade mellan de undersökta tiderna 4, 8 och 24 timmar. Vidare visade resultaten att det krävs 4 timmar för att minus-RNA skulle vara kvantifierbar vid en högre infektionskoncentration av viruspartiklar, medan det krävs 24 timmar för den lägre infektionskoncentrationen av viruspartiklar. / Norovirus causes winter vomiting disease and is one of the commonest cause of winter illness in Sweden. The disease period generally lasts one to three days with symptoms like vomiting and/or diarrhea. To the disease burden of acute gastroenteritis worldwide, norovirus contributes with 18 %. Even though the illness is very common, the knowledge about norovirus is poor and largely unknown.The purpose of the study was to do a time series analysis, a so-called One-Step Growth analysis, of the minus-RNA concentration in cells infected with different concentrations of murine norovirus (MNV). For the detection of minus-RNA RT-qPCR was used with SYBR Green. The goal was to correlate start concentration of virus at any time with the amount of minus-RNA in the cells. At 4 and 8 hours there was an exponential connection by the initial virus concentration and minus-RNA development in the cells. The concentration of minus-RNA in the infected cells increased between 4, 8 and 24 hours. Further, the results can be interpreted as requiring 4 hours for the higher concentrations to become quantifiable, while requiring 24 hours for the lower concentrations to become quantifiable.

Breeding for abiotic stress tolerance is one of main topics in plant breeding. Oilseed rape breeding programs were for a long time focused on morphological and physiological parameters. In this thesis few experiments focused on identification of genes involved in abiotic stress reaction were performed using RT-qPCR (quantitative reverse transcription PCR). Simultaneously SPR (surface plasmon resonance) method were used as modern optical method facilitating very low native protein concentration even in presence of other substances. This method facilitates quantification of concrete proteins by binding them to specific antigen and in oilseed rape research it was not used by now. ERD10 protein was identified by SPR as protein involved in cold stress reaction (or acclimation). The results show ERD10 accumulation in standard conditions affects dynamics of its accumulation change during cold stress. In case we are searching for genotypes great in acclimation ability even during short and warm autumn SPR method should be suitable method for fast, easy and relatively cheap screening of large number of genotypes in breeding collections. Also genes LTI78, RCI2A, NRP1 and two genes for hypothetical proteins were analysed. Their relative expression during cold stress was markedly increased too. Very little is known about these genes and proteins nowadays therefor it will be interesting topic of our oncoming experiment. Relative expression of genes picked according to MALDI-TOF/TOF analysis results was also tested in microspore embryo regenerants stressed by simulated drought. Genes for lactoylglutathione lyase I, phospholipase D 1 and peroxiredoxin antioxidase were tested. In tolerant cultivar was markedly decreased gene expression of peroxiredoxin antioxidase in standard conditions and early stress. These gene will be subject for next research as potential marker for more tolerant genotypes selection.

O relógio biológico de plantas é uma rede regulatória de grande relevância para a adaptação dos organismos ao ambiente. Essa rede é composta por diversas vias de controle transcricional e pós-transcricional que se retroalimentam e geram ritmos biológicos. O controle exercido pelo relógio pode ser observado nos mais diversos aspectos da fisiologia e desenvolvimento de plantas. No presente projeto de pesquisa foi investigada a relação entre o relógio biológico e a sinalização por auxinas, uma classe de fitohormônios, em cana-de-açúcar. Foram utilizadas técnicas de expressão gênica, como RT-qPCR, para definição de um protocolo robusto de avaliação de respostas transcricionais a auxinas em plântulas de cana-de-açúcar geradas por organogênese direta. Após 1h da aplicação de 80 µM auxina sintética ácido 1-naftalenoacético, foi possível observar controle transcricional evidente exercido pela aplicação de auxina sobre alguns genes. Também foi observado variação na resposta obtida, dependendo do horário do ciclo circadiano em que o estímulo era oferecido. Esse fenômeno de controle temporal sobre a resposta a um estímulo é chamado gating, sendo de grande relevância para a atuação do relógio biológico de plantas. A partir dessas observações foram realizadas análises de expressão gênica em larga escala, usando oligoarranjos, para compreensão mais aprofundada da conexão entre o relógio biológico e a sinalização por auxinas em cana-de-açúcar. Entre os genes diferencialmente expressos após estímulo com auxina, foi verificado grande presença de genes relacionados a respostas contraestresse biótico. Além disso, as respostas observadas devem estar sobre o controle do relógio biológico de cana-de-açúcar. Diversos genes relacionados a combate a infecções, como quitinases e taumatinas, tiveram sua expressão alterada após aplicação de auxinas, sendo possível observar diferenças no padrão de expressão dos genes dependendo do horário em que auxina era aplicada. Dessa forma, o relógio biológico de cana-de-açúcar, a partir da sinalização por auxinas, deve exercer controle sobre as respostas a estresses bióticos nesse organismo. Os dados obtidos são inéditos e podem contribuir para o aumento da produtividade de cana-de-açúcar assim como para o desenvolvimento de novas ferramentas biotecnológicas focadas nesse cultivar, o qual apresenta grande relevância econômica / The circadian clock is a regulatory network with great relevance to fitness of plants. This network creates biological rhythms, influencing plants metabolism and their interaction with the environment. The clock is composed of interlocking feedback transcriptional and post-transcriptional pathways. In the presente study, we investigated the interconnection between circadian clock and signaling through auxins, a group of phytohormones with great impact to plant biology. Using RT-qPCR, it was established a protocol to measure transcriptional responses after synthetic auxin 1-naphtalenacetic acid (NAA) treatment. The biological material used was leaves of sugarcane plantlets generated by direct organogenesis. After 1h treatment with 80 µM NAA, we observed obvious transcriptional responses in sugarcane plantlets. It was also possible to detect alterations of transcriptional responses according to the moment when the stimulus was offered. This temporal control is called gating and is of great relevance to plant circadian clocks. We then performed transcriptomic analysis, using oligoarrays, to get a deeper understanding of the results obtained. Indeed, it was verified that auxin stimulus is connected to biotic stress transcriptional responses and that these responses are clock-controlled. Transcripts coding for proteins like chitinases and thaumatins, which are related to biotic stress responses, were differentially expressed after auxin treatment. Also, the response of most genes was daytime-dependent. We conclude that sugarcane circadian clock, through auxin signaling, might exert control under biotic stressresponses in sugarcane. The data obtained are novelty and may contribute to increase sugarcane productivity and/or to development of new biotechnological tools dedicated to this cultivar.

A alimentação diferencial durante o desenvolvimento das abelhas Apis mellifera desencadeia respostas endógenas em vias de sinalização e sistema endócrino, que promovem o desenvolvimento de fenótipos alternativos nas castas femininas. Rainhas e operárias diferem em sua fisiologia, morfologia, longevidade, função na colônia, comportamento, e principalmente, na ativação do sistema reprodutivo. Em relação aos ovários, resultados prévios baseados em ensaios de microarranjos revelaram um conjunto de genes como diferencialmente expressos (DEGs) em larvas de rainhas e operárias. Esse trabalho teve como objetivo analisar os padrões de expressão desses DEGs em mais detalhe em ovários larvais de rainhas e operárias. Com esse objetivo, foram selecionados 18 DEGs para validação por RTqPCR. Estas análises foram realizadas em ovários dissecados de rainhas e operárias em quatro estágios larvais que representam fases críticas no desenvolvimento ovariano (L4, L5F1, L5F2 e L5F3). Dentre os 18 DEGs candidatos, 11 foram confirmados como de fato diferencialmente expressos. Entre esses, quatro genes que codificam enzimas: short chain dehydrogenase reductase (GB54419), 15-hydroxyprostaglandin dehydrogenase (GB18737), SCPEP1-like gene (GB11273) e glycerol-3-phosphate dehydrogenase (GB50902), exibiram um pico de expressão em L5F1 em ovários de operárias. Dentre os dois genes relacionados à estocagem ou transporte de proteínas, o gene apolipoprotein III (GB20117) encontrou-se mais expresso em operárias enquanto que hexamerin 70b (GB10869) se mostrou superexpresso em ovários de rainhas. Os genes relacionados à tradução de mRNA e vias de sinalização: elongation factor 1? (GB52028), heat shock protein 60 (GB18969), heat shock protein 90 (GB40976) e mitogen-activated protein kinase 3 (GB41845) estavam significativamente superexpressos em ovários de rainhas. O gene OCLP-1 (GB19297), que possui uma função hipotética como inibidor de cistina foi encontrado como superexpresso em ovários de operárias. A fim de avaliar a modulação desses genes por hormônio juvenil, foi realizado o tratamento in vivo de larvas de operárias com aplicação tópica do hormônio. Após seis horas de tratamento, os ovários foram dissecados e as amostras analisadas por RT-qPCR. Dos onze DEGs testados para resposta a HJ, seis se mostraram significativamente modulados pelo hormônio Dois genes, short chain dehydrogenase reductase e heat shock protein 90, que também respondem a ecdisona, são up-regulados por HJ, enquanto os genes OCLP-1, hexamerin 70b, 15-hydroxyprostaglandin dehydrogenase e apolipoprotein III eram downregulados. A expressão diferencial desses genes que codificam enzimas, proteínas de transporte/estocagem e fatores de vias de sinalização, indica que estes genes são importantes no desenvolvimento casta-especifíco dos ovários, uma vez que sua expressão está fortemente modulada durante os estágios em que ocorre a morte celular programada em ovários de operárias. / Differential feeding during larval development of the honey bee (Apis mellifera) triggers endogenous responses in signaling pathways and the endocrine system which promote the development of alternative phenotypes in the female castes. Queens and workers differ in physiology, morphology, longevity, function in the colony, behavior, and, especially so, the activation of the reproductive system. Concerning the ovaries, previous results based on microarray assays revealed a set of differentially expressed genes (DEGs) in queen and worker larvae.This project now aimed to further analyze the expression patterns of DEGs in the ovaries of larval workers and queens. From the microarray assays we selected a set of 18 DEGs for validation by qPCR. These analyses were performed on ovaries dissected from queens and workers of four larval stages representing critical phases of ovary development (L4, L5F1, L5F2, L5F3). Among the 18 DEG candidates, 11 were confirmed as differentially expressed. Four genes that code for enzymes: a short chain dehydrogenase reductase (GB54419), a 15-hydroxyprostaglandin dehydrogenase (GB18737), an SCPEP1-like gene (GB11273) and glycerol-3-phosphate dehydrogenase (GB50902) exhibited an expression peak at L5F1 in worker ovaries. Among the two genes encoding storage or transport proteins, apolipoprotein III (GB20117) was more expressed in workers and hexamerin 70b (GB10869) was overexpressed in queen ovaries. Among the genes related to mRNA translation and signaling pathways: elongation fator 1? (GB52028), heat shock protein 60 (GB18969), heat shock protein 90 (GB40976) and a mitogen-activated protein kinase 3 (GB41845) were found significantly overexpressed in queen ovaries. The gene OCLP-1 (GB19297), which has a hypothetical function as an inhibitor cystine knot peptide, was found higher expressed in worker ovaries. So as to evaluate the modulation of these genes by juvenile hormone, an in vivo treatment of workers larvae was performed with cuticular application of the hormone. After six hours of treatment, the ovaries were dissected and the samples analyzed by RTqPCR. Of the eleven DEGs tested for response to JH, six were significantly modulated by the hormone. Two genes, short chain dehydrogenase reductase and heat shock protein 90, which also respond to ecdysone, were up-regulated by JH, while OCLP-1 hexamerin 70b, 15- hydroxyprostaglandin dehydrogenase and apolipoprotein III were down-regulated.The differential expression of these genes encoding enzymes, storage/transport and signaling pathway proteins indicates that they are important in caste-specific ovary development, as their expression is strongly modulated during stages when programmed cell death takes place.