The occurrence of Boron (B) toxicity in Australian soils is recognised as a limiting factor for cereal productivity. A number of loci conferring tolerance to B toxicity have been identified in barley and chromosomally mapped. However, a lack of knowledge relating to the physiological and molecular events that occur under B toxicity and the molecular basis for B stress tolerance has been a bottleneck in harnessing available genetic diversity in barley and wheat. The recent advances in functional genomics provided an opportunity to examine B stress in barley in more detail. The aim of this project was to analyse genes differentially expressed under B stress in tolerant and intolerant barley to identify candidate genes involved in B toxicity tolerance. Two experimental approaches, Suppression Subtractive Hybridization (SSH) and microarray were adopted. Firstly, SSH was performed to examine gene expression in roots of selected tolerant and intolerant doubled haploid lines from a Clipper (B intolerant) X Sahara 3771 (B tolerant) mapping population, grown under moderate B stress. The SSH experiment aimed to investigate the early transcriptional response of B tolerant barley lines to B stress in order to identify the basis for B toxicity tolerance in roots. Differential screening of the subtracted library generated from B treated plants identified a total of 111 non-redundant clones up-regulated in bulked tolerant lines. On the other hand 94 clones were differentially expressed under non-treated conditions. Among the clones identified from subtracted library generated from B treated plants, metabolism was the largest functional category, representing 21% of the clones. The largest functional category in the subtracted library generated from non treated plants was cellular transport, representing 19% of the clones. Based on sequence similarity, about 170 transcripts identified in this experiment were assigned to chromosomal segments (bins) on the three homoeologous genomes of bread wheat. In total, 36 clones from the subtracted library generated from B treated plants were analysed as candidates. Nine were genetically mapped within the region of B tolerance QTL on three chromosomes (2H, 4H and 6H). The genes mapped to 4H and 6H QTL have the highest association with these loci in the Clipper X Sahara 3771 doubled haploid mapping population. A 4H B tolerance QTL candidate gene was identified as a B transporter gene with similarity to the Arabidopsis BOR1 gene. Genes identified to be differentially expressed in the tolerant lines from SSH suggest activation of a diverse defence response in the roots of barley plants under B stress. Data from SSH experiment indicate that cell wall-plasma membrane cytoskeleton continuum constitute the first action site against B toxicity and the influence of toxic B on K+ uptake could be the key initiating factor. In the second approach, the Affymetrix 22K Barley1 GeneChip(TM) was used to investigate B stress adaptation processes in barley. Gene expression was profiled in leaves of Sahara 3771 and Clipper plants grown under various B concentrations. The results show that the two genotypes respond differently to B toxicity. The B intolerance of Clipper is expressed through the induction of a high number of probe sets (2310) even at a low B concentration of 100 µM. In contrast, Sahara 3771 responded to a high B concentration (2000 µM) through the induction of only a few hundred (266) probe sets. In Sahara 3771 no change in the expression level of any probe sets was observed at 100 µM B. Altogether 286 probe sets showed differential expression in Sahara 3771 under three levels of B treatment (500, 1000 and 2000 µM). About 30% of these were down-regulated and about 70% were up-regulated in Sahara 3771 in response to B treatment. Most of the probe sets (59%) up-regulated in Sahara 3771 did not respond to B treatment in Clipper. These genes are either salt stress responsive or related to plant defense and thus could play a key role in protecting barley plants from the toxic effects of B. Two differentially expressed probe sets annotated as B transporters were identified between Sahara 3771 and Clipper under control condition. These two B transporter probe sets did not respond to B treatment but showed opposing expression patterns in the two varieties. One of these probe sets (Contig21126_at) is similar to the B transporter gene isolated from the SSH experiment that maps to the 4H tolerance locus. The map location and expression of this B transporter gene suggest that it could be the borate anion efflux transporter predicted by the proposed efflux model of B tolerance in Sahara 3771 barley. The other B transporter gene (Contig14139_at) showed over expression in Clipper under control condition and could be contributing to high B accumulation in Clipper which needs further investigation. Data from both experiments have indicated that B toxicity triggers oxidative stress and that jasmonate-based signaling plays a key role in B toxicity tolerance. SSH data indicate that Sahara 3771 which evolved in the harsh environment of Africa is more efficient in osmoregulation and ROS scavenging than Clipper. This trait is likely to give Sahara 3771 an edge over Clipper in tolerating toxic the effect of B. In addition to the efflux mechanism, which becomes less efficient with increasing B supply, Sahara 3771 appears to apply a number of other mechanisms for alleviating or withstanding toxic B induced stress to sustain growth. Some of these mechanisms are already known to be used by plants to cope with a number of stresses. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Transcriptomics and gene expression profiling enables the elucidation of the genetic response of an organism to various environmental cues. Transcriptomics enables the deciphering of differences between two closely related organisms to the same environment and in contrast, enables the elucidation of genetic responses of the same organism to different environmental cues. Two major methods are utilized for the study of transcriptomes, high-throughput sequencing and microarray analysis. High-throughput sequencing technologies such as the Illumina platform are relatively new and protocols must be developed for the analyses of transcriptomes (RNA-sequencing). A RNA-seq protocol was developed and refined for the Illumina sequencing platform. This protocol was then utilized for the de novo sequencing of the steelhead salmon transcriptome. Hatchery steelhead exhibit a reduced fitness compared to wild steelhead that has been shown to be genetically based. Consequently, the steelhead transcriptome was assembled, annotated, and used to identify gene expression differences between hatchery and wild fish. We uncovered many differentially expressed genes involved in metabolic processes and growth and development. This work has created a better understanding of the genetic differences between hatchery and wild steelhead salmon. Brachypodium distachyon is a monocot grass important as a model for cereal crops and potential biofuels feedstocks. To better understand the genetic response of this plant to different environmental cues, a comprehensive assessment of the transcriptomic response was conducted under a variety of conditions including diurnal/circadian light/dark/temperature environments and different abiotic stress conditions. Using a whole-genome tiling DNA microarray, we identified that the majority of transcripts in Brachypodium exhibit a daily rhythm in their abundance that is conserved between rice and Brachypodium. We also identified numerous cis-regulatory elements dictating these rhythmic expression patterns. We also identified the genetic response to abiotic stresses such as salinity, drought, cold, heat, and high light. We uncovered a core set of genes which responds to all stresses, indicating a core stress response. A large number of transcription factors were uncovered as potential nodes for regulating the abiotic stress response in Brachypodium. Moreover, promoter elements that drive specific responses to discrete abiotic stresses were uncovered. Altogether, the transcriptome analyses in this work furthers our understandings of how particular organisms respond to environmental cues and better elucidates the relationship between genes and the environment. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Oct. 5, 2011 - April 5, 2012.

With only 689 genes Mycoplasma pneumoniae (M. pneumoniae) is among the simplest known organisms. Because of this simplicity, mycoplasma represents an attractive organism for systems-wide analyses. Such approaches aiming at the whole quantitative understanding of an entire organism are expected to illustrate the basic principles of life. Strand-specific tiling arrays complemented by transcriptome sequencing, were combined with more than 252 spotted arrays to study M. pneumoniae transcriptional organization. An important presence of alternative transcripts (42%) within operons and a high frequency of antisense RNA (89) were detected. Metabolism was also studied in detail. A manually curated metabolic network allowed the definition of a minimal medium with 19 essential nutrients. This has been complemented with measurements of biomass indicators, metabolites and fluxes. Integration with transcriptional profiling has provided keys in the metabolic regulation. Protein organization and interactions have been addressed systematically by Tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The biochemical analysis revealed 178 protein complexes which have been complemented by structural models, single-article electron microscopy and electron tomography. By integrating the datasets from these different approaches, we show that this small bacterium harbors an unexpected complexity with features such as the frequent occurrence of alternative transcripts and antisense RNA, a small but tightly controlled metabolic network and a high level of proteome organization. / Amb només 689 gens Mycoplasma pneumoniae es troba entre els organismes més simples que es coneixen. Degut a aquesta simplicitat, mycoplasma representa un organisme atractiu per dur a terme estudis a nivell genòmic. S'espera d'aquests treballs que pretenen descriure de manera quantitativa l'organisme sencer que ajudin a entendre els principis bàsics de la vida. Per tal l'estudiar amb profunditat del transcriptoma, s'ha fet ús d'una combinació de dades de "tiling arrays" amb especificitat de cadena, ultraseqüenciació i més de 252 microarrays. Després d'analitzar els resultats s'ha detectat una alta presència de transcrits alternatius (42%) dintre operons i una alt contingut de ARN de tipus "antisense" (89). També s'ha realitzat un estudi detallat del metabolisme. S'ha revisat i completat manualment el mapa metabòlic de M. pneumoniae, fet que ha permès el disseny d'un medi mínim amb l'ús de 19 ingredients essencials. El mapa s'ha completat amb diferents mesures d'indicadors de biomassa, metabòlits i fluxos. També s'ha estudiat la regulació de metabolisme mitjançant microarrays. Per altra banda, s'han mesurat sistemàticament les interaccions proteïna-proteïna mitjançant "Tandem affinity purification-mass spectrometry (TAP-MS)". Aquest anàlisis ha detectat 178 complexes diferents, els quals han estat complementats amb models estructurals, microscòpia electrònica i tomografia electrònica. Mitjançant la integració d'aquestes col·leccions de dades, es pot mostrar que aquest petit bacteri amaga un inesperada complexitat amb característiques com la freqüència de transcrits alternatius i ARN "antisense", una xarxa metabòlica petita però fortament controlada i una alta organització del proteoma.

Artificial nighttime lighting (ANL) is known to adversely affect animals, but little is known what the consequences are to plants. Two genotypes of Populus balsamifera, a common urban tree, were used to investigate how ANL impacts plants. While the two genotypes varied in their physiological sensitivity to ANL, poorer levels of net leaf carbon assimilation compared to control samples suggested that ANL perturbed the perception of time of day for these plants. Gene set analysis on a subset of PopGenExpress microarray samples identified time of day specific processes in P. balsamifera, and a set of candidate ANL-sensitive genes were identified from these. Transcript measurements from the two genotypes revealed that ANL affects plants at the molecular level, for the diel cycling of the putative ANL-sensitive genes was perturbed. Together, these results suggest that ANL affects plants at the physiological and molecular level by perturbing their perception of time of day.

Artificial nighttime lighting (ANL) is known to adversely affect animals, but little is known what the consequences are to plants. Two genotypes of Populus balsamifera, a common urban tree, were used to investigate how ANL impacts plants. While the two genotypes varied in their physiological sensitivity to ANL, poorer levels of net leaf carbon assimilation compared to control samples suggested that ANL perturbed the perception of time of day for these plants. Gene set analysis on a subset of PopGenExpress microarray samples identified time of day specific processes in P. balsamifera, and a set of candidate ANL-sensitive genes were identified from these. Transcript measurements from the two genotypes revealed that ANL affects plants at the molecular level, for the diel cycling of the putative ANL-sensitive genes was perturbed. Together, these results suggest that ANL affects plants at the physiological and molecular level by perturbing their perception of time of day.

Η θερμοκρασία ανάπτυξης αποτελεί παράγοντα μεγάλης σημασίας στην οντογένεση των ιχθύων, αφού ως ποικιλόθερμοι οργανισμοί είναι συνεχώς εκτεθειμένοι στις μεταβολές του περιβάλλοντός τους. Έχει παρατηρηθεί πως η θερμοκρασία ανάπτυξης δύναται να προκαλέσει μετατόπιση του χρονοδιαγράμματος των οντογενετικών γεγονότων και πλαστικότητα σε μορφολογικούς και φυσιολογικούς χαρακτήρες (πχ στο μυοσκελετικό και το καρδιαγγειακό σύστημα). Ωστόσο, μέχρι σήμερα δεν έχει μελετηθεί η επίδραση της θερμοκρασίας ανάπτυξης στο πρότυπο της γονιδιακής έκφρασης του zebrafish και σκοπός της παρούσας εργασίας είναι η μελέτη του ολικού μεταγραφικού προτύπου νυμφών zebrafish. Για το λόγο αυτό σχεδιάστηκαν δύο πειράματα, όπου τρεις θερμοκρασιακές συνθήκες ανάπτυξης (22, 28 και 32oC) εφαρμόστηκαν στην πρώιμη οντογενετική περίοδο: για το διάστημα 0-20 dpf* (1ο πείραμα) και 10-20 dpf (2ο πείραμα). Πραγματοποιήθηκε απομόνωση ολικού RNA από τα άτομα ηλικίας 20 dpf όλων των πληθυσμών και από τα άτομα ηλικίας 10 dpf του πληθυσμού των 28oC του 2ου πειράματος και ακολούθησε υβριδοποίηση σε ολιγονουκλεοτιδικές μικροσυστοιχίες Affymetrix, με 15.509 αντιπροσωπευτικές αλληλουχίες γονιδίων (probe sets). Τα 21 μεταγραφικά προφίλ επεξεργάσθηκαν με τα εξειδικευμένα προγράμματα ανάλυσης μικροσυστοιχιών, dChip και MeV (v.4.5.1). Η κανονικοποίηση και το φιλτράρισμα των δεδομένων των μικροσυστοιχιών απέδωσε μεταγραφικά πρότυπα με 9.488 probe sets. Με τεχνικές πολυπαραμετρικής στατιστικής ανάλυσης (HCL και PCA) πραγματοποιήθηκαν οι συγκρίσεις των μεταγραφικών προτύπων μεταξύ των πειραματικών πληθυσμών για κάθε θερμοκρασία ανάπτυξης και οντογενετικό στάδιο. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων μεταξύ των δύο πειραμάτων, ii) σαφή διαχωρισμό των προτύπων των 28oC και 32oC ως προς αυτά των 22oC και στα δύο πειράματα, και iii) σαφή διαχωρισμό των προτύπων των 28oC διαφορετικού οντογενετικού σταδίου (ηλικίας 10 dpf vs 20 dpf). Θα αναμέναμε τα πρότυπα έκφρασης των 28oC να παρουσιάζουν παρόμοιο πρότυπο, κάτι που δεν παρατηρείται. Αυτό οφείλεται στο πειραματικό σφάλμα που υπεισέρχεται από το διαφορετικό χρόνο πραγματοποίησης των δύο πειραμάτων και τις ρυθμίσεις κατά την υβριδοποίηση. Έτσι πραγματοποιήθηκε η κανονικοποίηση των “28”, που απαλείφει το πειραματικό σφάλμα. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων των 28oC και 32oC ως προς αυτά των 22oC ως αποτέλεσμα της επίδρασης της θερμοκρασίας ανάπτυξης, ii) σαφή διαχωρισμό των προτύπων των 22oC των δύο πειραμάτων ως αποτέλεσμα της επίδρασης της περιόδου εφαρμογής και διάρκειας της θερμοκρασιακής αγωγής και iii) σαφή διαχωρισμό των πρότυπων των 28oC (ηλικίας 10 dpf vs 20 dpf) ως αποτέλεσμα της επίδρασης του οντογενετικού σταδίου. Ακολούθως, πραγματοποιήθηκε ανάλυση σημαντικότητας (SAM) και λειτουργική γονιδιωματική ανάλυση (λογισμικό DAVID) των στατιστικώς σημαντικών γονιδίων που διαφοροποιούν τα πρότυπα. Η ανάλυση των γονιδίων, ως προς την ιστοειδική έκφραση ανέδειξε γονίδια που σχετίζονται με την ανάπτυξη του ματιού, των θωρακικών πτερυγίων και του εγκεφάλου στους πληθυσμούς των 22oC έναντι των 28oC και 32oC. Η παρούσα εργασία αποδεικνύει την επίδραση της θερμοκρασίας ανάπτυξης και της διάρκειας της θερμοκρασιακής αγωγής, καθώς επάγει μηχανισμούς πλαστικότητας στο επίπεδο της γονιδιακής έκφρασης. Τέλος, υποδεικνύεται πως η πρώιμη οντογενετική περίοδος τείνει να είναι περισσότερο θερμοευαίσθητη, καθώς παρατηρείται εντονότερη επίδραση της θερμοκρασίας (στην περίπτωση των 22οC)στην ανάπτυξη του ατόμου. *dpf: days post fertilization / Developmental temperature plays a principal role in the ontogeny of fish. It is known that developmental temperature may shift the initiation time of the ontogenetic stages and induce plasticity in morphological and physiological characters e.g. the musculoskeletal and the cardiovascular system. However, its effect on the gene expression pattern has not previously been attempted for zebrafish. In the present study, zebrafish Affymetrix microarrays of 15,509 probe sets were used to map the transcriptome profile of: a) 20 dpf* old zebrafish larvae at three developmental temperatures, i.e. 22oC, 28oC and 32oC (1st experiment) and b) 20 dpf old zebrafish larvae, which were all grown at 28oC for the first 10 days and subsequently divided into three groups, which were grown at 22oC, 28oC and 32oC, respectively; the profile of 10 dpf old larvae was also measured (2nd experiment). We have isolated total RNA from the above populations and then, hybridization of RNA samples has been done on oligonucleotide Affymetrix microarrays of 15,509 probe sets. All 21 profiles were normalized and filtered (dChip software), and multivariate statistical analysis techniques were used on the normalized 9,488 probe set expression profiles (TM4 MeV software). Hierarchical Clustering (HCL) and Principal Component Analysis (PCA) on expression profiles indicated: a) clear separation of the two experiments based on their transcriptomic patterns, b) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC in both experiments and c) clear separation of the 28oC profiles based on the developmental stage. We would expect expression profiles of 28oC to be clustered together, though this was not observed because of experimental parameters during the hybridization, as the two experiments were carried out independently on different dates. So the normalization of “28” profiles took place, in order to eliminate the experimental noise. HCL and PCA, then, indicated: a) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC, as the effect of developmental temperature, b) clear separation of 22oC profiles of the two experiments, based on the effect of the period and duration of thermal conditions and c) clear separation of the 28oC profiles based on the developmental stage. Then, Significant Analysis of Microarrays (SAM) and Functional Genomic Classification Analysis (DAVID software) of statistically significant genes was carried out. Analysis of genes based on tissue-specific expression indicated characteristic genes for the development of the eye, pectoral fins and brain in 22oC profiles versus 28oC and 32oC profiles. The present study has proved that thermal effect is determinative among the early ontogenetic stage, especially in the case of longer cold thermal period, and developmental temperature may induce plastic response of gene expression, that could affect the fate of fish. *dpf: days post fertilization

Asthma is a respiratory syndrome associated with airflow limitation, bronchial hyperresponsiveness and inflammation of the airways in the lungs. Despite the ongoing research efforts, the outstanding heterogeneity displayed by the multiple forms in which this condition presents often hampers the attempts to determine and classify the phenotypic and endotypic biological structures at play, even when considering a limited assembly of asthmatic subjects. To increase our understanding of the molecular mechanisms and functional pathways that govern asthma from a systems medicine perspective, a computational workflow focused on the identification of personalized transcriptomic modules from the U-BIOPRED study cohorts, by the use of the novel MODifieR integrated R package, was designed and applied. A feature selection of candidate asthma biomarkers was implemented, accompanied by the detection of differentially expressed genes across sample categories, the production of patient-specific gene modules and the subsequent construction of a set of core disease modules of asthma, which were validated with genomic data and analyzed for pathway and disease enrichment. The results indicate that the approach utilized is able to reveal the presence of components and signaling routes known to be crucially involved in asthma pathogenesis, while simultaneously uncovering candidate genes closely linked to the latter. The present project establishes a valuable pipeline for the module-driven study of asthma and other related conditions, which can provide new potential targets for therapeutic intervention and contribute to the development of individualized treatment strategies.

A cana-de-açúcar é uma gramínea C4 usada por séculos como a principal fonte de açúcar e mais recentemente para obtenção de etanol. Devido a sua grande importância no cenário econômico mundial, estudos em cana-de-açúcar são cada vez mais importantes no sentido de prover informações que possam levar ao aumento de produtividade para suprir tanto a demanda interna quanto externa. No entanto, a quantidade de dados moleculares e biotecnológicos disponíveis está muito aquém do necessário, e investimentos na obtenção de novos conhecimentos serão necessários se quisermos evoluir neste campo, assim como manter o nosso país como líder na produção de etanol. Neste trabalho, conduzimos experimentos em campo para comparar variedades contrastantes para a tolerância ao déficit hídrico e realizamos diagnósticos fisiológicos e moleculares para diferenciar as variedades. O estresse hídrico levou à diminuição do crescimento e desenvolvimento de todas as três variedades analisadas. A variedade RB855536 foi identificada como a menos produtiva das três, visto que em condições de déficit hídrico ela diminui mais seu crescimento, sofre mais efeitos do estresse oxidativo, acumula mais osmólitos e tem o ciclo de Calvin menos ativo. A variedade RB867515 teve um melhor desempenho, não acumulou osmólitos, não teve aumento na concentração de prolina, teve sinais menores de estresse oxidativo e um melhor funcionamento do ciclo de Calvin. Além disto, nós observamos a indução de transcritos na via de resposta do ABA e proteínas relacionadas com a fotossíntese, transporte de água e dobramento protéico. A variedade RB92579 teve um padrão similar ao encontrado para a RB867515, mas teve uma maior fotossíntese, um menor estresse oxidativo e uma menor perda de pigmentos. Nossos dados avançaram na identificação de genes envolvidos na resposta ao déficit hídrico em cana-de-açúcar assim como permitiram distinguir as variedades utilizadas no estudo. A partir de uma prospecção inicial de dados de transcriptoma previamente obtidos pelo grupo, foram selecionados fatores de transcrição associados a características agronômicas de interesse. Produzimos anticorpos para 5 TFs de cana-de-açúcar e padronizamos a metodologia de ChIP-Seq utilizando a plataforma de sequenciamento Roche 454. Uma análise dos dados de ChIP-Seq usando anticorpos para a RNA Polimerase II nos permitiu detectar contaminações com DNA humano, provavelmente pela utilização de gDNA como controle das reamplificações, assim como a detecção de mapeamento de muitas regiões repetitivas, o que pode ser normal, podendo indicar locais de ligação da Polimerase II ou então background. O mapeamento de praticamente todos os dados de sorgo em cana evidenciou a similaridade entre estes organismos, mas a maior quantidade de mapeamento em cana evidencia uma maior complexidade de seu genoma. Os resultados foram importantes por permitir o estabelecimento de uma metodologia de mapeamento de regiões regulatórias no genoma da cana-de-açúcar e significa um importante passo no estabelecimento de redes regulatórias associadas a características agronômicas de interesse / Sugarcane is a C4 grass used for centuries as the main source of sugar and more recently for ethanol production. We have seen an increasing interest in recent years to understand sugarcane to improve yield and supply the increasing world demand. However, the amount of molecular data available is far from ideal, and investments in acquiring new knowledge will be necessary to progress in this field if we want to maintain our country as a pioneer in ethanol production. In this work, we conducted field experiments to compare varieties contrasting to drought tolerance and performed physiological and molecular analysis. The stress condition led to reduced growth and development of all three varieties. The variety RB855536 was identified as the least productive, suffered more effects of oxidative stress, has accumulated more osmolytes and has the Calvin cycle less active. The variety RB867515 had a better performance, did not accumulate osmolytes, had no increase in the concentration of proline, had minor signs of oxidative stress and a better functioning of the Calvin cycle. In addition, we observed an induction of transcripts in the ABA response pathway and proteins related to photosynthesis, water transport and protein folding. The variety RB92579 had a pattern similar to that found in RB867515, but presented increased photosynthesis, lower oxidative stress and a lower loss of pigment. We succeded in identifying genes involved in the response to water stress in sugarcane as well as in distinguishing the varieties used in the study. We selected transcription factors associated with agronomical traits from the transcriptome data previously obtained by the group. We produced antibodies for 5 sugarcane TFs and standardized the methodology of ChIP-Seq using the 454 sequencing platform. Data analysis of ChIP-Seq using RNA Pol II antibody allowed us to detect contamination with the human genome, probably due to the use of gDNA in the reamplification control, as well as to map the detection repetitive regions, which may be binding sites of Pol II or background. The data reveals that sorghum and the sugarcane genome are similar despite the complexity of sugarcane. The results were important since they allow the beggining of studies to map regulatory regions in the sugarcane genome, as well as the uncovery of regulatory networks associated with agronomic traits of interest

Mais de 20 milhões de hectares em todo o mundo são atualmente destinados a plantações de Eucalyptus, sendo que o Brasil possui a segunda maior área. No ano de 2007 a rede internacional EUCAGEN, liderada pelo Brasil, África do Sul e Estados Unidos, surgiu com o objetivo de colaboração para a pesquisa genômica do eucalipto. A árvore escolhida para o sequenciamento (Brasuz) foi fornecida pelo Brasil e em 2011 as primeiras sequências foram disponibilizadas. Em todas as fases de seu desenvolvimento, o eucalipto está sob o constante ataque de patógenos, destacando-se a ferrugem, causada pelo Basideomiceto Puccinia psidii Winter como a mais importante doença em regiões tropicais. A doença vem se espalhando rapidamente pelo mundo e recentemente foi relatada na Austrália, centro de origem do eucalipto. Com o objetivo de estudar o mecanismo molecular da interação plantapatógeno entre Eucalyptus grandis e Puccinia psidii, estabeleceu-se um patossistema modelo composto por um isolado monopustular do fungo e plantas resistente e susceptível provenientes de uma progênie de meios irmãos da planta Brasuz. O desenvolvimento do patógeno nos genótipos selecionados foi analisado por meio de microscopia de luz e de epifluorescência, e permitiu o monitoramento da dinâmica do desenvolvimento do fungo nos dois genótipos, com a identificação de todas as etapas de desenvolvimento do patógeno no genótipo susceptível bem como o estágio em que o genótipo resistente bloqueia o seu desenvolvimento. Com base nesses resultados determinou-se seis intervalos de interesse para a realização da análise da expressão gênica por meio da técnica de RNA-Seq. As análises revelaram grandes diferenças no perfil transcricional dos dois genótipos em resposta à presença do patógeno, permitindo a identificação de genes conhecidamente envolvidos em mecanismos de defesa de plantas como Receptores LRR-Quinase, fatores de transcrição WRKY, MYBS e GRAS, Proteínas R TIR-NBS-LRR Proteína Induzida por Injúria e proteínas envolvidas em processos de degradação proteica como F-Box. A comparação dos resultados provenientes das análises histológicas e moleculares permitiram a elaboração de um modelo para explicar os principais processos envolvidos no mecanismo de resistência de Eucalyptus grandis à Puccinia psidii. / More than 20 million hectares are destined to Eucalyptus plantations worldwide and Brazil has the second largest planted area. The International Eucalyptus Genome Network, EUCAGEN, was created in 2007 in order to perform genomic research on Eucalyptus. Brazil provided the biological material from the model tree (Brasuz) to have the complete genome sequenced and the first sequences were released to the scientific community in 2011. During Eucalyptus development, it is constantly exposed to pathogen attack with one of the most threatening diseases being eucalyptus rust caused by the neotropical rust fungus Puccinia psidii Winter which is rapidly spreading around the world and was recently described in Australia. In order to try and understand the molecular plant-microbe interaction between E. grandis and P. psidii we have to create a model system by isolating the pathogen from a single pustle and select resistant and susceptible plants from half-sib population generated using Brasuz as the pollen receptor. We performed light and epifluorescence microscopy analyses, identified all of the stages of the fungal development and recognized the moment which the resistant genotype blocks pathogen development. Based on these results we selected six time points to carry out transcriptomic analysis. Using RNA-Seq analysis we were able to verify large differences in transcriptional profile between resistant and susceptible plants and identify genes known involved in plant defense response such as LRR recptor Kinase, transcription factors (WRKY, MYBS and GRAS), TIR-NBS-LRR Proteins, Woundinduced protein and proteins involved in protein degradation (F-Box). Comparing microscopy and transcriptomic results allowed us to propose a model to explain the molecular mechanism of resistance of Eucalyptus grandis to Puccinia psidii.

Global change poses new threats for life in the oceans forcing marine organisms to respond through molecular acclimatory and adaptive strategies. Although bivalve molluscs are particularly tolerant and resilient to environmental stress, they must now face the challenge of more frequent and severe Harmful Algal Blooms (HABs) episodes. These massive outbreaks of microalgae produce toxins that accumulate in the tissues of these filter-feeder organisms, causing changes in their gene expression profiles, which in turn modify their phenotype in order to maintain homeostasis. Such modifications in gene expression are modulated by epigenetic mechanisms elicited by specific environmental stimuli, laying the foundations for long-term adaptations. The present work aims to examine the links between environmental stress in bivalve molluscs (with especial emphasis on Harmful Algal Blooms) and specific epigenetic marks triggering responses through modifications in gene expression patterns. Overall, a better understanding of the molecular strategies underlying the conspicuous stress tolerance observed in bivalve molluscs will provide a framework for developing a new generation of biomonitoring strategies. In addition, this strategy will represent a valuable contribution to our knowledge in acclimatization, adaptation and survival. With that goal in mind, the present work has generated transcriptomic data using RNA-Seq and microarray technologies, facilitating the characterization and investigation of the epigenetic mechanisms used by the Mediterranean mussel Mytilus galloprovincialis during responses to HAB exposure. That information was made publicly available through a specialized online resource (the Chromevaloa Database, chromevaloa.com) assessing the response of chromatin-associated transcripts to Okadaic Acid. Specific epigenetic marks have been assessed under lab-controlled exposure experiments simulating the natural development of the HAB Florida Red Tide (FRT). Results demonstrate a role for the phosphorylation of histone H2A.X and DNA methylation in the response to FRT in the Eastern oyster Crassostrea virginica. Lastly, the study of co-expression networks based on RNA-Seq data series from the Pacific oyster Crassostrea gigas reveals dynamic transcriptomic patterns that vary with time, stressor and tissue. However, consistent functional profiles support the existence of a core response to general conditions of environmental stress. Such response involves metabolic and transport processes, response to oxidative stress and protein repair or disposal, as well as the activation of immune mechanisms supporting a tightly intertwined neuroendocrine-immune regulatory system in bivalves.