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A wave of public information has enabled the report of innumerable examples where transposable elements (TEs) fragments are incorporated as exons into mRNAs, however, the proposition that such exonized TEs encode functional proteins is controversial. In order to characterize the gene expression pattern of TE-derived coding sequences identified in the Coffea arabica genome, we analyzed the macroarray profiling and digital northern of transcripts with distinct molecular functions of sequences containing TE-cassettes or not. The evolutionary changes of the gene expression pattern mediated by exonized TE sequences using probes of tissues and stress conditions derived from C. arabica and C. canephora are presented and discussed.

O presente trabalho teve como objetivos (i) analisar a expressão de genes candidatos (GC), que foram previamente identificados em plantas de Coffea canephora,e agora analisar a expressão em plantas de Coffea arabica cultivadas no campo e submetidas e não submetidas ao estresse hídrico e (ii) avaliar a diversidade genética de alguns GCs por meio da busca dos polimorfismos nucléicos. Os experimentos de expressão gênica foram realizados com os cultivares IAPAR59 (tolerantes à seca) e Rubi (sensível à seca) de C. arabica cultivados com e sem irrigação durante a estação seca. As folhas foram amostradas no ano de 2008 foram utilizadas para analisar a expressão de GCs por PCR quantitativo em tempo real (qPCR). Em paralelo, seqüências gênicas correspondentes aos GC foram clonadas e seqüenciadas a partir do DNA genômico de diferentes espécies, clones e cultivares de cafeeiro. Os polimorfismos foram identificados e permitiram de definir os haplótipos encontrados nos genótipos estudados.

The effects of drought-stress on aerial and root architecture, ecophysiology, anatomy and molecular responses were investigated during 2 years using the Iapar59 (drought-tolerant) and Rubi (drought susceptible) cultivars of Coffea arabica. Plants were grown under three water treatments: I-I a non limited watering treatment (irrigated each year during the dry season), NI-NI a limited water treatment (non-irrigated during the dry seasons) and NI-I, a limited watering in year 1 and non-limited watering in year 2 ("recovery"). Six points of measurements were taken along the experiment. For all conditions, the phenotypic plasticity was followed by analyzing architecture, evolutions of biomass, ?13C and sap flow for example.

Promoters that restrict transgenic expressions in a specific organ or tissue and, at a certain stage of plant development or in response to stress are extremely desirable for the improvement of agronomic species by transgenesis. Coffee is among the five most valuable crops to developing nations, and its production is of great economic interest to Brazil. Commercial production is based mainly on the specie Coffea arabica, which is tetraploid, perennial and has low genetic variability, impairing the genetic improvement by traditional methods. Thus, genetic modification appears as a good way to obtain coffee plants more suitable for different purposes. The aim of this work was to isolate an active promoter from endosperm of coffee seeds. Initially, a contig preferentially expressed in coffee fruits was identified by Electronic Northern, using the database of the Brazilian Coffee Genome Project. The organ specificity of the transcript was confirmed in fruit, particularly in seed endosperm, by RT-PCR, RT-qPCR and Northern blot. Then, the promoter was isolated by the 5' RACE method using the Universal Genome Walker Kit (Clontech). A fragment of 1.2 kb and three truncated versions (407 bp, 785 bp and 1.0 kb) were isolated, starting at the first ATG codon of the protein, in order to evaluate the expression profile of different modules. The sequences were in silico analyzed and all fragments exhibited promoter-specific cis elements. In the present work we constructed expression cassettes in which the four versions of the promoter were cloned in the binary vector pBI121 by replacing the 35S promoter that regulates the ?-glucuronidase (uidA/gus) gene. The constructed binary vectors were used to transform tobacco (Nicotiana tabacum) through Agrobacterium tumefaciens. Histochemical analysis of GUS activity show that the 1.2 kb, 1.0 kb e 785 pb fragments promotes expression in leaves, flowers and fruits, with differences in the activity level, and all three fragments have low activity in roots. Finally, the 407 bp fragment promotes the expression of Gus only in tobacco seeds. Future work will be performed to determine in which seed tissue this promoter is active and which domains are involved in the tissue-specificity. (Texte intégral)

Polyploidization constitutes a common mode of evolution in flowering plants. This event provides the raw material for the divergence of function in homeologous genes, leading to phenotypic novelty that can contribute to the success of polyploids in nature or their selection for use in agriculture. Mounting evidence underlined the existence of homeologous expression biases in polyploid genomes; however, strategies to analyze such transcriptome regulation remained scarce. Important factors regarding homeologous expression biases remain to be explored, such as whether this phenomenon influences specific genes, how paralogs are affected by genome doubling, and what is the importance of the variability of homeologous expression bias to genotype differences. This study reports the expressed sequence tag assembly of the allopolyploid Coffea arabica and one of its direct ancestors, Coffea canephora. The assembly was used for the discovery of single nucleotide polymorphisms through the identification of high-quality discrepancies in overlapped expressed sequence tags and for gene expression information indirectly estimated by the transcript redundancy. Sequence diversity profiles were evaluated within C. arabica (Ca) and C. canephora (Cc) and used to deduce the transcript contribution of the Coffea eugenioides (Ce) ancestor. The assignment of the C. arabica haplotypes to the C. canephora (CaCc) or C. eugenioides (CaCe) ancestral genomes allowed us to analyze gene expression contributions of each subgenome in C. arabica. In silico data were validated by the quantitative polymerase chain reaction and allele-specific combination TaqMAMA-based method. The presence of differential expression of C. arabica homeologous genes and its implications in coffee gene expression, ontology, and physiology are discussed.

Drought stress significantly affects coffee yield, productivity and quality. Thus, the goal of this study was to investigate the molecular mechanisms underlying the response to drought stress in coffee plants by different approaches. Candidate gene identification was performed by comparing gene expression and protein profile of different genetic materials (tolerant vs. susceptible) as well as, under different conditions of water supply (irrigated vs. non-irrigated). In this work, the genetic materials studied were Conillon clones of Coffea canephora and two cultivars of C. arabica. The applied water stress (PD=-3,0 MPa) to the conillon plants was achieved under green-house conditions and, in the case of arabica, adult plants cultivated under field conditions were used. Under field conditions, leaves were collected during day and night, and the most pronounced observed water-stress was of PD =-1,7 MPa. After selection of candidate genes by different strategies, the expression was confirmed by qPCR analysis. The natural variation of some selected candidate genes was also performed using a set of different genotypes. The data obtained indicated that several genes displayed decreased expression upon water stress and usually these were encoding-genes of proteins involved in photosynthesis. On the other hand, the applied water stress on coffee plants also induced a set of genes such as RD29, DREBA and NAC, which have already been described in literature as genes involved in plant responses to drought. In addition, this study also revealed the importance of other factors controlling the expression of these genes, such as the circadian clock and the age. (Texte intégral)

The coffee business worldwide shows that coffee is economically important for many countries. For Brazil, the largest producer and exporter of coffee, this commodity is usually a major source of income to small farmers. Drought periods have become increasingly intense over the recent years, affecting the production of these farmers as it reduces productivity and can even cause irreversible crop damage. Nowadays, the elucidation of the genetic factors involved in drought tolerance is amongst the priorities of several research groups. Responses to drought stress certainly involve changes in gene expression and the goal of this work, was the characterization of the gene expression profile on leaves of a sensitive clone of C. canephora (clone 22) and a tolerant clone (clone 14), grown under control and water stress conditions. The technique used in this study was the hybridization of DNA macroarrays, which was efficient to identify genes differentially expressed between the treatments used (irrigated and non irrigated) as well as differences between the genotypes studied (clone tolerant x sensitive). These differentially expressed genes are potential candidates for drought tolerance in coffee and may be subject to further experimental studies. The previously selected genes on the macroarrays experiments were further characterized by real-time quantitative PCR assays (qPCR). In general, most of the results obtained by the qPCR analysis corroborated with the differential expression observed with the macroarrays experiments.

O melhoramento da qualidade dos grãos de café com o objetivo de agregar maior valor e aumentar a produtividade é um desafio de longa data. Entretanto, com o desenvolvimento da biologia molecular e da bioquímica permitiu-se um novo nível de melhoramento a partir do conhecimento das moléculas e das vias bioquímicas com influência direta na qualidade e no aroma da bebida. O desenvolvimento do fruto de café é caracterizado por várias mudanças nos tecidos e alterações bioquímicas, ilustrando a importância do seu estudo para melhor compreender as características finais dos grãos de café. O objetivo deste trabalho é desenvolver uma ferramenta eficaz de identifcação do conteúdo protéico expresso nos diferentes estágios de desenvolvimento de frutos da espécie Coffea arabica com possível influência no aroma e sabor da bebida de café. A metodologia utilizada neste trabalho baseia-se na estratégia proteômica do tipo "bottom-up", com digestão protéica e análise por cromatografia líquida de fase reversa e espectrometria de massa em três estágios de maturação de grãos de café variedade arábica: grãos verdes (151 dias após a floração), maduros (210 dias após a floração) e secos (grãos maduros deixados ao ambiente por 13 dias), coletados no ano de 2008. Os dados demonstram que doze proteínas puderam ser identificadas através de buscas de similiaridade feitas em um banco de dados da Embrapa. Embora a metodotologia proposta neste trabalho tenha apresentado uma riqueza de peptídios para análise em cromatografia líquida e MALDI-TOF quando comparada a outras, faz-se necessário a busca de metedologias alternativas, pois a abundância relativa da proteína 11 S constitui um empecilho para a identificação de um maior número de moléculas.

Aiming at the establishment of a breeding program based on marker-assisted selection for drought tolerance on coffee, the present work had as a goal, the identification of candidate genes for this trait by using an integrated approach (gene expression and proteomics). The data was obtained by comparing gene expression and protein profile of different genetic materials (Tolerant vs. Susceptible) as well as, under different conditions of water supply (Irrigated vs. Non-irrigated). In this work, the genetic materials studied were the conillon clones 22 and 14 (Coffea canephora) and the cultivars Rubi and Iapar 59 (Coffea arabica). The applied water stress (?PD=-3,0 MPa) to the conillon plants was achieved under green-house conditions and, in the case of Arabica, adult plants cultivated under field conditions were studied. In the later case, leaves were collected during day and night, and the most pronounced observed water-stress was of ?PD=-1,7 MPa. After selection of candidate genes by different strategies, the expression was confirmed by qPCR analysis. The data obtained indicated that several genes displayed decreased expression upon water stress and usually these were encoding-genes of proteins involved in photosynthesis. On the other hand, the applied water stress on coffee plants also induced a set of genes such as RD29, DREBA and NAC, which have already been described in literature as genes involved in plant responses to drought. In the case of a Dehydrin encoding-gene, the data obtained in this work confirmed previously observed differential expression on C. canephora, on C. arabica, also. However, this study revealed the importance of other factors controlling the expression of these genes, such as the circadian clock, the age as well as the profile of the plants and, these observations make the interpretation of differential expression data obtained from field trials even more complex.

Diversos estudos têm demonstrado que os fatores ambientais, tais como a sombra, a topografia, as condições meso-climáticas e temperaturas, afetam a composição química dos grãos de café e, conseqüentemente, a qualidade final do produto. Essas modificações dos fatores ambientais e, principalmente, o aumento dos períodos de déficit hídrico, as mudanças de zonas de produção, as novas práticas culturais (por exemplo, a irrigação) e as necessidades crescentes de uso eficiente da água na agricultura, são fatores que influenciam a fisiologia da planta e, conseqüentemente, as condições de maturação do grão de café. Teores de alguns compostos bioquímicos do fruto de café (cafeína, lipídeos totais, sacarose, ácidos clorogênicos e trigonelina) foram avaliados pela técnica de espectroscopia no infravermelho próximo (NIRS) nos grãos produzidos a partir de plantas adultas de Coffea arabica cv. Rubi, cultivadas em condição de campo, sob diferentes regimes hídricos. Os frutos utilizados neste trabalho foram coletados no mês de maio 2007, sendo que a florada aconteceu no mês de setembro 2006, no campos experimental da Embrapa Cerrados, Brasília-DF. Os resultados dessas análises mostraram que a falta de irrigação durante o período de desenvolvimento dos grãos de café, influenciou a composição química destes. Os grãos obtidos nessas condições (sem irrigação) apresentaram teores mais baixos de cafeína e ácidos clorogênicos (ACG) quando comparados aos grãos oriundos das plantas com irrigação contínua. Em contraste, os teores de sacarose e lipídeos foram superiores nos grãos de plantas cultivadas na condição de sequeiro, quando comparados aos grãos das plantas com irrigação contínua. Os resultados demonstraram que o estresse hídrico também influencia no metabolismo dos grãos de café, tendo um papel importante nos teores de compostos metabólicos que afetam a qualidade final da bebida. Análises estatísticas estão sendo realizadas, para verificar a existência de correlações entre os teores dos compostos bioquímicos, a adubação e os regimes hídricos estudados. Além disto, análises sensoriais serão realizadas para verificar se as modificações observadas têm conseqüências na qualidade de bebida com os grãos da safra 2007-2008.

Os programas de melhoramento genético do cafeeiro consistiram em identificar e utilizar a variabilidade natural existente no gênero Coffea criando novos híbridos adaptados às exigências de maior produtividade e qualidade da bebida. A potencial elevação das temperaturas, em função do aquecimento global, torna-se um desafio real no cumprimento de tais exigências demandando o desenvolvimento de novas variedades tolerantes à seca. O aperfeiçoamento das ferramentas moleculares geradas pelos recentes avanços na genômica do café possibilita o estudo dos mecanismos gênicos e a identificação dos genes responsíveis ao défict hídrico. O objetivo desse estudo foi analisar, em folhas de plantas de Coffea arabica, a expressão de genes candidatos (GC) identificados por análises in silico dos dados do projeto Genoma café, hibridizações de macroarranjos e observações do perfil protéico em géis de eletroforese 2D de folhas de plantas submetidas ao estresse hídrico. Os genes candidatos foram identificados em estudos com clones tolerantes e sensíveis de Coffea canephora variedade Conilon realizado em condições controladas (casa de vegetação). Neste experimento foram utilizadas folhas de plantas de Coffea arabica variedades Iapar 59 (tolerante) e Rubi (sensível), submetidas a diferentes regimes hídricos, em condições de campo. O potencial hídrico das plantas foi acompanhado durante as coletas e mensurado com uma bomba de Sholander. O material biológico utilizado para análise foi referente a três pontos de coletas que representam: potencial hídrico basal, menor potencial hídrico de antemanhã e o potencial de recuperação após o retorno da irrigação. A expressão relativa foi analisada por PCR quantitativo em tempo real (qPCR). Os resultados indicam que foi possível identificar genes diferencialmente expressos, envolvidos na resposta ao déficit hídrico, nos cultivares de Coffea arabica testados, em condições de campo.

Amounts of some biochemical compounds of coffee fruits (caffeine, total lipids, sucrose, chlorogenic acids and trigonellin) were evaluated by the near-infra red spectroscopy (NIRS) in the beans produced from adult plants to Coffea arabica cv. Rubi, cultivated in field condition, under different hydric regimes. Results of these analyses showed that the lack of irrigation during the period of coffee bean development influenced the biochemical composition of these beans. When compared with beans from plants with continuous irrigation, those of plants grown without irrigation presented lower amount of caffeine and chlorogenic acids (CGA) but higher contents of sucrose and total lipids. The results presented here showed that water stress influenced coffee bean metabolism, particularly the contents of biochemical compounds known to affect the coffee cup quality.

Coffee is an important commodity worldwide with about 125 million people depending on this crop for their livelihoods. Drought stress significantly affects coffee yield with losses estimates in Brazil, varying from 10 to 15 % in recent years. Therefore, the development of molecular tools for rapid generation of drought-tolerant coffee varieties is amongst the priorities of the Brazilian Research Program on Coffee Genomics. The principal aim of this study was to investigate the molecular mechanisms underlying the response to drought stress in coffee plants by an integrated approach. As part of the Brazilian Coffee EST project, two EST libraries from leaves of drought-stressed plants of Coffea arabica cv. Rubi (drought sensitive) and C. canephora clone 14 (drought tolerant), were generated. The plant material of C. arabica and C. canephora were obtained from field and pot trials, respectively. More than 15,000 clones were sequenced and, after trimming and clustering, resulted in 10,924 reads grouped on 6,141 unigenes (1,779 contigs and 4,362 singlets). The approaches used to identify candidate genes (ESTs) underlying stress responses in coffee consisted of an in silico analysis of ESTs generated from drought-stressed and control libraries, physiological characterization, transcription profiling of drought-stressed and control tissues and protein profiling by 2-DE coupled with tryptic peptide identification by MALDI-TOF-MS/MS. These integrated analysis resulted in the identification of several candidate drought-responsive genes. In addition, a mapping population from crosses of C. canephora clones contrasting for this trait is also under way for future association studies and QTL mapping. (Texte intégral)

Made available in DSpace on 2016-10-10T03:52:27Z (GMT). No. of bitstreams: 5 Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.pdf: 1400705 bytes, checksum: 25ed945714edfec0adf9dfaceaa84ed4 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 23851 bytes, checksum: 294cb7010cc40c47642971e073de3dba (MD5) license_rdf: 23892 bytes, checksum: afd5dad10b1d1e6dc10c8c5d25222c7a (MD5) license.txt: 1887 bytes, checksum: 445d1980f282ec865917de35a4c622f6 (MD5) Previous issue date: 2008 A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28–29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14–15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog. Sim Publicado

The majority of transgenic organisms reported in the literature have been made using constitutive promoters. However, there are economic, environmental and biosecurity related restrictions involving indiscriminate (constitutive) expression of heterologous genes. The usage of tissue-specific and induced promoters can resolve those issues by limiting the expression of a transgene to the necessary tissues and conditions. The promoters currently used at Embrapa are transnational properties, burdening the research and causing technological dependence. Therefore, the objective of this work was to find and characterize tissue and organ specific promoter in Coffea spp. We have used the Coffee genome database in silico tools to find genes preferentially expressed in root, leaf and fruit. In this way we found 72 organ-specific candidates: 18 apparently preferentially expressed on leaves, 14 on roots and 40 on fruits. Some of those candidates where tested in vitro using RT-PCR, semi-quantitative PCR, northern blotting and qPCR assays. All four leaf-specific candidates tested (GCFo1, GCFo2, GCFo3 and GCFo4) and at least one of the two fruit-specific candidates tested (GCFr1 e GCFr2) where confirmed to be preferentially expressed on their respective organs. Temporal and spatial expression assays showed that GCFr2 has its expression peak at the endosperm, 180 days after flowering. The highest expressed genes of leaf (GCFo3 and GCFo4) and fruit (GCFr2) were used as probes to isolate its respective promoter through a BAC libraries screening or using the Genome Walker Universal Kit (Clontech). Results concerning gene expression and the molecular characterization of these genes will be presented.

The principal aim of this study was to investigate the molecular mechanisms underlying the response to drought stress in coffee plants by different approaches. In order to indentify candidate genes involved in controlling drought tolerance in coffee plants, different strategies were followed in our laboratories. The first used the nucleic data generated by the Brazilian Coffee EST project to identify candidate genes (CG) by in silico analysis (electronic Northern-blot). Differential expression of these CG was verified in leaves and roots from drought-tolerant and susceptible clones of C. canephora var. Conilon by Northern-blot and quantitative PCR experiments. The second was based on the screening of macroarray membranes spotted with coffee ESTs which were hybridized separately with leaf cDNA probes of the same clones. Finally, 2D gel electrophoresis was also performed to selected proteins presenting differential accumulation in leaves of the same clones. These proteins were analyzed by MALDI-TOF-MS/MS leading to the identification of a new set of CG. Results concerning the identification of CG by these different approaches are presented and discussed.

In the context of climate changes, adaptation of perennial plantations to water constraint becomes a major concern of wood and fruit productivity. Adaptation depends on the level of genetic diversity of breeding and natural populations as well as their plasticity. This project plans to describe adaptive mechanisms under water constraint of three perennial plants of the temperate and tropical regions (Pinus, Eucalyptus and Coffea) by combining analysis of plant architecture, physiology, anatomy and molecular responses to drought stress. The part of the project dealing with coffee will be realized in the fields of the Embrapa-Cerrado center localized near Brasilia because this region is always subjected to a long and regular dry season during the winter. Field trials will be performed with IAPAR59 (drought tolerant) and Rubi (drought sensible) cultivars of Coffea arabica in three water treatments: T1 a non limited watering treatment (irrigated during the dry season), T2 a limited watering treatment (non irrigated during the dry season) and T3 limited watering in year 1 and non limited watering in year 2 (recovering). Six points of measurements will be realized along the two years of the experiment (2008-2009). For all condition, molecular plasticity will be investigated for leaf, stem and root by analysing gene expression by quantitative PCR (qPCR) but also using microarrays using meristematic cells. The phenotypic plasticity will be also followed mainly by measuring leaf area, leaf thickness and stomata number. At the anatomical level, the structure of xylem vessels, the parenchyma's thickness will be assessed. These data will be correlated with ecophysiological measurements like biomass estimates (for leaf, stem and root), hydraulic conductivity, stomatal conductance, and water-use efficiency ([delta]C13). We will also characterize the belowground and aerial architecture of coffee plants. Together, the results should allow (1) the identification of dynamic changes at molecular, morphological and ecophysiological levels, (2) the analysis of their correlations, and (3) GxE interaction identification for the three three perennial plants. Finally, results obtained will be compared to see if common mechanisms could be observed between the three models. A multidisciplinary network of scientists and technicians in molecular biology, genetics, ecophysiology, developmental biology is involved in this project from different research organisms and universities. (Texte intégral)

In a context of climate change, adaptation of perennial plantations to water constraints becomes a major concern for wood and fruit productivity. Adaptation depends on the level of genetic diversity in breeding and natural populations, as well as their plasticity. This project plans to describe adaptive mechanisms under water constraints for three perennial plants of temperate and tropical regions, including Pinus, Eucalyptus and Coffea, through a combined analysis of plant architecture, physiology, anatomy and molecular responses to drought stress.

In a context of climate change, adaptation of perennial plantations to water constraints becomes a major concern for wood and fruit productivity. Adaptation depends on the level of genetic diversity in breeding and natural populations, as well as their plasticity. This project plans to describe adaptive mechanisms under water constraints for three perennial plants of temperate and tropical regions, including Pinus, Eucalyptus and Coffea, through a combined analysis of plant architecture, physiology, anatomy and molecular responses to drought stress.

Candidate genes involved in coffee drought tolerance were selected by in silico analysis (Electronic Northern) of SH2 and SH3 cDNA libraries of the Coffee Genome database. Eighteen "contigs" with in silico differential expression were identified and characterized by Northern-blot experiments using total RNA extracted from leaves of drought tolerant and susceptible clones of Coffea canephora var. Conillon grown in greenhouse under controlled conditions. Physiological analyses showed that the clone 22 (drought susceptible) presented faster decrease of leaf water potential than resistant ones that was attributed to its higher stomatal conductance. Below -3.0MPa, this clone also showed lower photosynthetic activity correlated with elevated oxidative stress. For most of the candidate genes selected by "Electronic Northern" approach, differential gene expression was confirmed by conventional Northern-blot analyses. Interestingly, some of them also presented differential expression patterns between the four clones tested (drought susceptible vs. drought tolerant). Research perspectives are discussed based on these results.

To make the Coffee cDNA-Macroarrays, a non-reduntant group of clones (Unigene) was initially generated by bioinformatics analysis of the Brazilian Coffee Genome Database. The re-arraying routine was performed with the Q-Bot system (Genetix) and resulted in a non-redundant group of 33,000 clones organized in 86 plates (384). Plasmid DNA from all 33 thousand clones was extracted and representative samples were analyzed by gel electrophoresis. To check the accuracy of the re-arraying routine, 4 samples per plate were sequenced and compared by BlastN analysis against the Coffee Database. The results of this analysis indicated 8% mistakes during the re-arraying routine. Plates containing mistakes were re-arrayed and novel samples sequenced. The next step for the Macroarray construction is the printing of the membranes and the objective of this work was to establish the best protocol for this routine. The type of DNA (plasmid vs. PCR product) and the concentration were the variables evaluated. The results obtained with the hybridization experiments indicated that in general the printing routine with the Q-Bot system was very uniform and accurate among the replicates. In addition, the data obtained indicated that the radioactive signals obtained with the PCR product-spots were stronger than the ones obtained with plasmid DNA due to the different molar ratio and probably also, due to the better denaturing step of a linear DNA fragment.

Orientador: João Carlos Moreschi Coorientador: Umberto Klock Dissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias Agrárias, Programa de Pós-Graduaçao em Engenharia Florestal. Defesa: Curitiba, 2006 Inclui bibliografia e anexos Área de concentraçao: Tecnologia e utilizaçao de produtos florestais O setor produtivo de polpa celulósica e papel vêm contribuindo de forma expressiva para o desenvolvimento sócio-econômico do Brasil. Diversas características podem ser mencionadas para explicar este fato, dentre elas o desenvolvimento de modernas tecnologias que geram florestas com qualidade e produtos de alto valor agregado. Estes pontos possibilitaram às empresas alcançar padrões internacionais de qualidade, produtividade e de proteção ao meio ambiente, nas operações florestais e nos processos industriais empregados. Desta forma, a busca por informações precisas a serem utilizadas nestas operações serão um diferencial para um planejamento seguro. Estabeleceu-se como objetivo geral deste estudo avaliar a influência da idade e classe de produtividade sobre a qualidade da madeira e as propriedades da celulose e do papel em plantios comerciais de Pinus taeda L. As árvores amostradas neste estudo foram provenientes de áreas de reflorestamento da Fazenda Monte Alegre pertencentes à empresa Klabin S.A., localizada no município de Telêmaco Borba, Estado do Paraná. A amostragem foi realizada em plantios comerciais de mesmo grau de melhoramento genético, com 8, 14 e 20 anos de idade, abrangendo três classes de produtividade. As características químicas (holocelulose, lignina, extrativos totais e material inorgânico), anatômicas (dimensões dos traqueóides e porcentagem de lenho tardio) e físicas (densidade básica e umidade) da madeira foram determinados. O processo Kraft de obtenção de celulose (rendimentos, rejeito, número kappa, viscosidade, sólidos e álcali residual do licor negro) e as propriedades do papel (gramatura, umidade, espessura, densidade aparente, volume aparente, comprimento de auto-ruptura, índice de tração, índice de arrebentamento, índice de rasgo e índice de rigidez) foram avaliados. Os valores observados foram submetidos a análise de variância fatorial (ANOVA). Com base nestas avaliações, variações significativas na qualidade da madeira, no processo de obtenção de celulose e nas características do papel em função da variação da idade e classe de produtividade foram observadas. Desta forma, pode-se concluir que a idade e a classe de produtividade condicionaram e exerceram grande influência sobre a qualidade da madeira e as propriedades da celulose e do papel The pulp and paper productive sector comes contributing in an expressive way to the socioeconomic development of Brazil. Many characteristics can be mentioned to explain this fact, amongst them the modern technologies development that generate since forests with quality until products of high aggregate value. These points facilitated to the companies to reach products with international quality patterns, high productivity and environment protection, in the forest operations and the used industrial processes. In this way, the search for precise information will be a differential for a safe planning. The goal of this study was to evaluate the productivity class influence on the wood quality and on pulp and paper properties from plantations of Pinus taeda L. The trees used in this study were sampled in reforestation areas of Klabin S.A - Monte Alegre Farm - located in the municipal district of Telêmaco Borba, State of Paraná. The sampling was accomplished in commercial plantations with same degree of genetic improvement, with ages of 8, 14 and 20 years old, embracing three productivity classes. The wood chemical characteristics (holocellulose, lignin, extractives and inorganic material), anatomical (fibers dimensions and latewood percentage) and physical (specific gravity and moisture content) of wood were determined. The e Kraft process for pulping (yield, reject content, kappa number, viscosity, solids and residual alkali in the black liquor) and the paper properties (grammage, humidity, thickness, apparent density, bulk, breaking length, tensile index, burst index, tear index and stiffness index) were evaluated. The obtained values were submitted the factorial analysis of variance (ANOVA). With base in these evaluations, significant variations in the quality of the wood, the process of cellulose attainment and in the characteristics of the paper in function of the variation of the age and classroom of productivity had been observed. This way, it can be concluded that the age and the productivity class conditioned and exercised great influence on the wood quality and the properties of the pulp and of the paper.

OBJETIVO: Determinar la capacidad de la quimioterapia neoadyuvante para lograr citorreducción óptima en cáncer epitelial de ovario avanzado y relacionarla con la sobrevida global y libre de enfermedad. MÉTODO: 22 pacientes con cáncer epitelial avanzado de ovario fueron elegidas en base a los criterios de Vergote: enfermedad voluminosa, implantes múltiples, enfermedad metastásica parenquimatosa; se administró 3 ó 4 ciclos de quimioterapia con carboplatinotaxanos, y posterior cirugía. RESULTADOS: Todas las pacientes fueron catalogadas como estadio III-C o IV según la clasificación de la FIGO. El tipo histológico predominante fue el adenocarcinoma sin especificación con el 45,5 %. El 63,6 % eran tumores poco diferenciados. Todas las pacientes respondieron a la quimioterapia neoadyuvante, incluso en 5 pacientes se reportó respuesta patológica total. Se logró cirugía cito- reductora óptima en 81,8 % de los pacientes. Para un seguimiento promedio de 18 meses: 20 pacientes están vivas (90,9 %), 14 libres de enfermedad. En 4 se produjo recidiva, 4 presentaron persistencia y fallecieron 2 de ellas. La morbilidad perioperatoria fue mínima. La sobrevida libre de enfermedad registró una media de 11,45 meses. El único factor relacionado con sobrevida libre de enfermedad fue la capacidad de realizar cirugía óptima (P= 0,006). CONCLUSIONES: La cirugía citorreductora primaria sigue siendo la primera opción en cáncer de ovario epitelial avanzado, la quimioterapia neoadyuvante seguida de cirugía de intervalo es una opción válida en un subgrupo de pacientes con factores de riesgo adversos. SUMMARY OBJECTIVE: To determine the feasibility of optimal cytoreduction after neoadjuvant chemotherapy in epithelia advanced ovarian cancer and relation it with the global survival and disease free survival. METHOD: 22 patients with advanced epithelial cancer of ovary selected according Vergote´s indications: bulky disease, multiply implants, and parenchyma disease metastasis, it administrated 3 or 4 cycles of chemotherapy with carboplatinumtaxans and after interval surgery. The statistical analysis was based on porcentual relations, variable analysis and Pearson variable correlation test. RESULTS: All the patients were catalogued as stage III-C or IV by FIGO classification. Adenocarcinoma without specification was the most important histological subtype with the (45.5 %). 63.6 % were poor differentiation tumors. All the patients responded to neoadyuvant chemotherapy, even there was a complete pathological response in five. Optimal cytoreduction surgery was achieved in 81.8 % of the patients. The median in follow up was 18 months, 20 patients are lived (90.9 %) and 14 without disease. In 4 patients had persistence of the disease, 2 of them died because of these, in 4 patients recurrent disease was described, Disease free survival registers a media of 11.45 months. The unique factor relationated with disease free survival was the capacity to realize optimal cytoreductive surgery (P= 0.006). CONCLUSIONS: The primary cytoreductive surgery is still the gold standard in treatment of advanced epithelian ovarian carcinoma, neoadjuvant chemotherapy and interval debulky surgery is a valid option in a subgroup of patients with adverse prognostic factors.