A diversidade da comunidade endofítica de fungos associada à cana-de-açúcar transgênica tolerante a imazapyr e suas linhas de cultivo não transgênicas foi avaliada por isolamento e ARDRA (Amplified rDNA Restriction Analysis). Cultivares transgênicos e não-transgênicos, e seu manejo (aplicação do herbicida ou remoção manual de daninhas), foram considerados para verificar o possível efeito indireto da cana-deaçúcar geneticamente modificada (GM) sobre a comunidade de fungos endofíticos. O total de quatorze haplótipos de ARDRA foram observados na comunidade endofítica de cana-de-açúcar. O seqüenciamento da região ITS1-5.8S-ITS2 revelou uma comunidade rica representada por doze famílias diferentes do filo Ascomicota. Alguns dos isolados demonstraram alta similaridade com gêneros que ocorrem comumente como endófitos em plantas de clima tropical, como Cladosporium, Eppicoccum, Fusarium, Guignardia, Pestalotiopsis e Xylaria. A análise de variância molecular (AMOVA) indicou que as flutuações observadas na composição dos haplótipos estão relacionadas tanto ao cultivar transgênico quanto a aplicação do herbicida. Enquanto a aplicação do herbicida induziu mudanças rápidas e transientes na comunidade de fungos, as plantas transgênicas induziram mudanças mais lentas que foram mantidas ao longo do tempo. Uma abordagem independente de cultivo baseada em bibliotecas de 18S ambiental revelou a presença de clones com seqüências similares a gêneros das famílias Ustilaginaceae e Filobasidiaceae, e das ordens Sporidiobolales e Tremellales, todos do filo Basidiomicota. Os resultados aqui demonstrados representam o primeiro relato sobre a composição de fungos endofíticos associados a plantas de cana-de-açúcar e também representam um passo importante para o entendimento dos efeitos que plantas transgênicas e seu manejo podem induzir sobre a comunidade de fungos endofíticos. / The diversity of fungal endophytic community associated with transgenic imazapyr-tolerant sugarcane plants and its non-transgenic lines was evaluated by isolation and ARDRA (Amplified rDNA Restriction Analysis). Transgenic and nontransgenic cultivars and their crop management (herbicide application or manual weed control) were considered in order to assess the possible non-target effects of genetically modified (GM) sugarcane on the fungal endophytic community. A total of fourteen ARDRA haplotypes were observed in the endophytic community of sugarcane. ITS1- 5.8S-ITS2 sequencing revealed a rich community represented by twelve different families from the Ascomycota phylum. Some of the isolates showed a high sequence similarity with genera that commonly occur as endophytes in plants from tropical climates, such as Cladosporium, Eppicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of Molecular Variance (AMOVA) indicated that fluctuations observed in haplotypes composition were related to both transgenic cultivar and herbicide application. While herbicide applications induced quickly transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. A cultivation-independent approach based on libraries of environmental 18S revealed the presence of clones with high sequence similarity with genera from Ustilaginaceae and Filobasidiaceae families and Sporidiobolales and Tremellales orders, all from Basidiomycota phylum. The results demonstrated here represent the first draft on the composition of fungal endophytes associated with sugarcane plants and also represent an important step to understand the effects that transgenic plants and their crop management may induce on fungal endophytic community.

Wheat (genome AABBDD) is one of the essential crops, offering approximate 20% of human calorie consumption worldwide. Allopolyploidization of three diploid ancestors led to hexaploid wheat with narrowed genetic variation. Chromosome engineering is an applicable approach to restore the evolutionarily-omitted genetic diversity by homoeologous chromosomes recombination between wheat and its relatives. Two diploid relatives of wheat, Thinopyrum elongatum (genome EE) and Aegilops speltoides (genome SS), containing favorable genes, are used as gene resources for alien introgression and genome diversification in wheat. An advanced and effective experiment procedure was developed and applied for the production, recovery, detection, and characterization of homoeologous recombinants. Meanwhile, a novel recombinant chromosome recovery strategy was exploited with improved efficiency and accuracy. In this study, recombinants of wheat chromosomes 3B and 7B with their homoeologous chromosomes in Th. elongatum and Ae. speltoides (i.e. 3B-3E, 7B-7E, and 7B-7S) were produced and detected. Totolly, 81 3B-3E recombinants and four aberrations involving in distinct chromosomal regions were developed in three recombination cycles by fluorescent genomic in situ hybridization (FGISH). The secondary and tertiary recombination breakpoints occurred toward the proximal regions comparing to the primary recombination under this advanced recombination procedure. A novel recovery strategy was used to recover 7B-7E and 7B-7S homoeologous recombinants by chromosome-specific markers and FGISH verification. Marker-based pre-screening and subsequent FGISH verification identified 29 7B-7E and 61 7B- 7S recombinants, seven 7B-7E and four 7B-7S Robertsonian translocations, one 7E and five 7S telocentric chromosomes, and three 7S deletions. All the recombinants and aberrations were genotyped by high-throughput wheat 90K single nucleotide polymorphism (SNP) assay and the recombination breakpoints were physically mapped to wheat chromosome 3B or 7B according to their FGISH patterns, SNP results, and wheat reference genome sequence. Chromosome 3B was physically partitioned into 38 bins with 429 SNPs. Meanwhile, 44 distinct bins were resolved for chromosome 7B with 523 SNPs. A composite bin map was constructed for chromosomes 3B and 7B, respectively, with a comprehensive analysis of FGISH and SNPs results. In summary, this project provides a unique physical framework for further wheat genome studies and diversifies the wheat genome for germplasm development in wheat breeding.

Among viral diseases affecting cultivated tomato, Tomato yellow leaf curl disease (TYLCD) is one of the most devastating. This disease is caused by a complex of viruses of which Tomato yellow leaf curl virus (TYLCV) is regarded as the most important species. Current control strategies to fight viral diseases in tomato are mainly based on genetic resistance derived from wild relatives. In the present thesis, resistance derived from S. chilense and S. peruvianum has been exploited in breeding for resistance to TYLCD. In a previous study, TYLCV-resistant breeding lines derived from LA1932, LA1960 and LA1971 S. chilense accessions were developed. Therefore, the first objective of this thesis was to study the genetic control of the resistance derived from these accessions. With this aim, response to viral infection was assayed in segregating generations derived from the aforementioned resistant lines. The results obtained were compatible with a monogenic control of resistance. Resistance levels were higher in LA1960- and LA1971-derived F2 generations, as shown by slighter symptoms in the resistant plants and a higher number of asymptomatic plants compared with the results obtained in the LA1932-derived F2 generation. It is noteworthy that the level of resistance present in our materials is comparable to or even higher than the levels found in tomato lines homozygous for Ty-1. The response in plants heterozygous for the resistance gene was comparable to the response in homozygous plants for all three sources employed. This implies that the resistance genes derived from all three sources seem to be almost completely dominant. This effect was stronger for LA1971-derived resistance. The results were similar when comparing viral accumulation, as was expected, since a positive correlation was found in these families between viral accumulation and symptom scores. This has important implications in breeding, since the resistance will be used mostly for hybrid development. Our second objective was to map the loci associated with the major resistance genes identified. A total of 263 markers were screened, 94 of them being polymorphic between both species. Recombinant analysis allowed the resistance loci to be localized on chromosome 6, in a marker interval of 25 cM. This interval includes the Ty-1/Ty-3 region, where two S. chilense-derived TYLCD resistance loci were previously mapped. In order to test if the resistance genes identified in our populations were allelic to Ty-1 and Ty-3, further fine mapping was carried out. A total of 13 additional molecular markers distributed on chromosome 6 allowed 66 recombinants to be identified, and the resistance region to be shortened to a marker interval of approximately 950 kb, which overlaps with the Ty-1/Ty-3 region described previously by other authors. Therefore, the results obtained indicate that closely linked genes or alleles of the same gene govern TYLCV resistance in several S. chilense accessions. The third objective of the present thesis was to start the construction of a set of introgression lines (ILs) derived from Solanum peruvianum accession PI 126944 into the cultivated tomato genetic background. Once this collection of ILs is developed, it will represent a powerful tool for exploiting the resistance to different pathogens found in this particular accession in addition to other possible characters of interest. The starting plant material consisted of several segregating generations that were derived from two interspecific hybrids previously obtained by our group. Many crosses and embryo rescue were required to obtain subsequent generations due to the high sexual incompatibility that exists between tomato and PI 126944. Several mature fruits from the most advanced generations produced a few viable seeds, although embryo rescue was also employed to obtain progeny. As only a few plants were obtained by direct backcrossing, additional crosses were made in order to increase the number of descendants. A high degree of incompatibility was also found in crosses between sib plants. A total of 263 molecular markers were tested in some generations, 105 being polymorphic between tomato and PI 126944. Available generations were genotyped with these polymorphic markers in order to determine which alleles of S. peruvianum were already introgressed. On average, 79, 78 and 84 % of the S. peruvianum genome was represented in the pseudo-F2, pseudo-F4 and pseudo-F5 generations, respectively, for the markers analyzed. A reduction in the S. peruvianum genome was observed in more advanced generations, such as BC1 (56 %), pseudo-F2-BC1 (60 %) and pseudo-F3-BC1 (70 %). A greater reduction was observed in the pseudo-F3-BC2 generation (33 %). As a consequence of the reduction in the S. peruvianum genome, a loss of incompatibility was observed in some cases. The S. peruvianum genome was almost completely represented among the different plants of the most advanced generations. An evaluation for resistance to TYLCD and Tomato spotted wilt virus (TSWV) was carried out in some of the advanced generations, some of which were resistant to one or both viruses. In conclusion, we have conducted a successful and deeper exploitation of two wild species with proved resistance to TYLCD, S. chilense and S. peruvianum, identifying and fine mapping new genes of resistance. / Julián Rodríguez, O. (2014). Exploitation of Solanum chilense and Solanum peruvianum in tomato breeding for resistance to Tomato yellow leaf curl disease [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36867

Despite the existence of various molecular marker systems there are still limitations in distinguishing between closely related species based on molecular divergence, especially when hybridization events have occurred in the past. The characterisation of plant small nucleolar RNA (snoRNA) genes and their organisation into multigene clusters provides a potential nuclear marker system which could help in resolving the phylogenetic history of plants and might be applicable in DNA barcoding. Using closely and distantly related Senecio species, I investigated a combination of fragment length and sequence variation of snoRNA genes/snoRNA gene clusters to assess the utility of this marker system for barcoding and resolving species relationships. SnoRNA gene and gene cluster sequences identified in Arabidopsis thaliana were used to find homologues in other species and subsequently used for the design of universal primers. Most of the universal primer pairs designed were successful in amplifying snoRNA fragments in most Senecio species and fragment length variation between and within species could be detected. Furthermore, the combination of some fragment length datasets produced by different primer pairs enabled the separation of species and the detection of reticulate evolution indicating a high potential of snoRNA gene/gene cluster fragment length polymorphisms (SRFLPs) for phylogenetic reconstructions in Senecio and other plant genera. Most of the examined gene clusters showed a similar gene order in Senecio and Arabidopsis. However, the majority of these clusters appeared to exhibit more copies in Senecio, some of which were distinguishable by a combined sequencing/fragment profiling approach, and shown to be putative single copy regions with the potential to be used as co-dominant markers. However, a high number of paralogues and possible differences in copy number between species excludes these regions from being used in DNA barcoding. This is because specific primers would have to be developed for specific copies which would preclude development of a universal application for barcoding. None of the regions showed enough sequence variation to delimit distinctly closely related Senecio species and were therefore also considered to be unsuitable for DNA barcoding. Although most snoRNA genes and gene clusters might be inapplicable for DNA barcoding, they are likely to be valuable for phylogenetic studies of species groups, genera and families. On this scale, specific primers might act universally and the number of paralogous copies is likely to be equal across the species group of interest.

Un inventaire des Tetrany chidae du Brésil a été réalisé dans 15 États ainsi que dans le District Fédéral, avec 550 échantillons de 120 espèces végétales collectés. Des infestations par des acariens tétranychidés ont été confirmées dans 207 de ces échantillons. Vingt-deux espèces appartenant à sept genres de la sous-familles des Bryobiinae et Tetranychinae ont été identifiées chez 58 hôtes différents. Trente-six nouvelles plantes hôtes pour 11 espèces ont été répertoriées au Brésil, en Amérique du sud ou dans le monde. De nouvelles localités ont été enregistrées au Brésil pour quatre tétranychides et une espèce a également été signalée pour la première fois en Amérique du sud. Quatre nouvelles espèces ont été découverte: deux appartenant au genre Oligonychus Berlese et deux appartenant aux genres Monoceronychus McGregor et Schizotetranychus Tragardh. La contribution à la systématique du genre Tetranychus a consisté à analyser conjointement des substuences d´ADN ribosomique (ITS) et mitochondrial (COI) de femelles de T.urticae, T. cinnabarinus (synonyme potentiel de T. urticae) et d'espèces très proches appartenant également au groupe Tetranychus sensu stricto. Cette étude a mis en évidence des incohérences ce qui nous a amené à remettre en question la fiabilité des données moléculaires concernant le groupe Tetranychus s. str. disponibles dans Genbank. Des données sur la variabilité génétique, la phylogénie et la structure de populations de T. urticae au Brésil et dans le monde sont ensuite présentées. Les résultats indiquent la présence significative d'une structuration génétique des populations de T. urticae par rapport à la localisation géographique. L´effet de la plante hôte n´est pas observé. La diversité haplotypique, inférée à partir des ces deux régions du génome (ITS et COI) est plus élevée dans les pays du pourtour méditerranéen. On a constaté la présence de deux haplotypes mitochondriaux (COI) au Brésil, l´un partagé avec la France, l´Espagne et les îles Canaries, et l´autre avec le Japon. / In this study we performed a survey of Tetranychidae mites from Brazil, including 15 States and the Federal District. A total of 550 samples of 120 different plant species were collected. Tetranychid mite infestations were confirmed in 207 samples, and 22 species belonging to seven genera of the Bryobiinae and Tetranychinae subfamilies were identified on 58 different host plants. Thirty-six new hosts were recorded in Brazil, South America and worldwide for eleven species. New localities were registered for four tetranychid genera and a new record to South America was confirmed. Four species were identified as new for science: two belonging to the Oligonychus Berlese genus and two belonging to the Monoceronychus McGregor and Schizotetranychus Tragardh genera. We analyzed and evaluated the identity of 105 Genbank accessions of ITS2 rDNA and 138 COI mtDNA sequences which were deposited as T. urticae and as fourteen other taxa morphologically close ly related to Tetranychus sensu stricto. Among the deposited sequences in the Genbank, numerous cases of apparently mistaken identities were identified in the group Tetranychus s. str., especially between T. urticae, T. cinnabarinus, T. kanzawai and T. truncatus. New sequences of ITS and COI were obtained from individuals collected in Brazil and some localities of the Palearctic Region (France, continental Spain, Canary Islands, Greece, Syria, Tunisia, Poland and Norway plus one from Canada). While significant differences were detected on population genetic structure of the analyzed samples according to the geographic region, any effect of the host plant was observed. Haplotype diversity inferred from both ITS and COI sequences was higher in samples from the Mediterranean basin. ITS sequences obtained from Brazil samples were homogenous, and two COI haplotypes were found, one of them also present in France, Spain and the Canary Islands and the other in Japan.

Le cancer colorectal est une affection multifactorielle qui conjugue des anomalies épigénétiques et des altérations moléculaires à une déstabilisation du génome. En dehors de deux processus bien établis, l’instabilité des microsatellites et l’instabilité des chromosomes, la déstabilisation du génome peut être consécutive à la réexpression d’éléments génétiques mobiles, HERV et LINEs-­‐1. Les HERVs ont un potentiel de modulation de l’immunité qui pourrait contrôler les réactions de l’hôte dans le processus de la carcinogenèse. Compte tenu du rôle que pourrait jouer les HERV, nous avons voulu déterminer la relation entre l’infiltrat lymphocytaire intratumoral et les différentes caractéristiques moléculaires des tumeurs. L’infiltrat lymphocytaire intratumoral a été évalué pour trois marqueurs (CD3, CD8 et CD45RO). Les marqueurs moléculaires analysés ont été : les mutations des gènes B-­‐raf, k-­‐ras et Pi3K, le statut des microsatellites, la méthylation de MLH1 et des LINEs-­‐1, un polymorphisme de C-­‐met et de HLA-­‐G. Ces marqueurs ont été comparés à la prévalence de la délétion du locus Apobec 3 pour une cohorte de 135 patients porteurs de cancer colorectal. / Colorectal cancer is a multifactorial disease associated to epigenetic abnormalities and molecular alterations inducing a destabilization of the cellular genome. Besides two well-­established processes, microsatellite instability and chromosome instability, the destabilization of the genome may follow mobilisation of endongenised mobile genetic elements, such as HERV or LINEs-­‐1. HERV have a potential to modulate immune response, which suggests that they could contribute to the process of carcinogenesis. We thus wanted to determine the relationship between intratumoral lymphocytic infiltration and the different molecular characteristics of tumours, taking into account factors that could influence the expression of HERV, such as the frequently observed deletion of a locus on 22q13 containing the Apobec 3 gene family. The intratumoral lymphocytic infiltration was assessed for three markers (CD3, CD8 and CD45RO). The molecular markers analysed were: the mutations of B-­‐raf, K-­‐ras and Pi3K genes, the instability status of microsatellites, the methylation of MLH1 and LINEs-­‐1, a polymorphism in C-­‐met and in HLA-­‐G. These markers were linked to the deletion of the locus containing Apobec 3 genes, in a cohort of 135 colorectal cancer patients.

Em mamíferos, a espermatogênese é mantida pela autorrenovação e diferenciação das células-tronco espermatogoniais (SSC). Apesar da grande importância do SSC para a fertilidade masculina, em Bos taurus pouco se sabe sobre a sua identificação e biologia celular. Para roedores, mais de 30 marcadores para células germinativas indiferenciadas já foram descritos. No entanto, ainda não é conhecido um marcador específico apenas para SSC. Quase todos são também expressos por gonócitos, espermatogônias mais diferenciadas ou mesmo células somáticas. Yin Yang 2 (YY2) é um factor de transcrição expresso nas células com a morfologia de gonócitos e SSC, sendo um candidato a marcador de SSC. Assim, a identificação de novos marcadores para SSC e factores que afectam a sua expressão, tais como a idade, são fundamentais para o desenvolvimento da biotecnologia como transgenia e tratamento de infertilidade, nos quais as SSC poderiam ser ferramentas biológicos importantes. Assim, nesta tese temos duas hipóteses principais: 1) a idade do dador afeta a expressão de marcadores moleculares específicos de SSC bovinas assim como potencial de células-tronco dessas células e que as sequências de DNA em que se associa YY2 regulam a expressão génica de SSC em camundongos. Os objetivos específicos, organizados em 4 artigos científicos, foram: identificar a melhor plaqueamento diferencial para enriquecer SSC bovina (artigo 1), verificar se a expressão de marcadores moleculares de SSC bovina difere entre adultos pré-púberes (artigo 2 e 3), identificar novos marcadores específicos para SSC em Bos taurus (artigo 3), verificar que a idade afeta o potencial de célula-tronco de SSC bovinas (artigo 3), descrever YY2 como um marcador específico para SSC em camundongos e verificar se as sequências DNA associadas YY2 são loci de importância para SSC. Assim, definimos o melhor plaqueamento diferencial para o enriquecimento de SSC bovinas, que idade afeta a expressão marcadores já estabelecidos assim como genes específicos do transcriptoma de SSC bovinas e que idade também afeta o seu potencial de células-tronco (ensaio de repopulação). Concluímos também que YY2 é um marcador para SSC de camundongo em cultivo, em animais adultos e que as sequências do genoma que se associam YY2 possivelmente tem capacidade de regulação génica em SSC murina. / Mammalian spermatogenesis is sustained by self-renewal and differentiation of spermatogonial stem cells (SSC). Despite the importance of the SSC for male fertility, in Bos taurus líttle is known about their identification identity and cell biology. For rodents, more than thirty markers for undifferentiated germ cells have already been described. However, none of these represents a marker specific for SSC, as most are also expressed in gonocytes, differentiated spermatogonia or even somatic cells. Yin Yang 2 (YY2) is a transcription factor specifically expressed in cells with the morphology of gonocytes and SSC in the mouse, being a candidate marker for SSC. For the use of SSC as an important biological tool in the development of biotechnology such as transgenesis and the treatment of infertility, it is important to identify new markers for SSC and factors that affect their expression, such as the age of donors. Therefore, the experimental work described in this thesis was based on two main hypothesis: (1) the donor age affects the expression of specific molecular markers in SSC as well their potential as stem cells and 2) YY2 exerts important functions in SSC and genomic targets correspond to loci relevant for gene regulation or genome management in SSC. The specific goals have been organized in 4 manuscripts as follows: optimization of differential plating to enrich for bovine SSC (Article 1), check if the expression of molecular markers of bovine SSCs differs between prepubertal and adult donors (Article 2 and 3), identify new markers specific for SSC in Bos taurus (Article 3), continuation of the initial description of YY2 as a specific marker for SSC in mice and check if sequences bound by YY2 in vivo harbor the capacity to influence gene expression in SSC. As conclusions, we present an optimized differential plating protocol for the enrichment of bovine SSC, we conclude that age effects the expression of SSC markers, the expression of specific genes of bovine SSC and that age also affects their potential as stem cells (measured in repopulation assays). We also contribute to the description of the restricted expression of YY2 in prepuberal and adult SSC in mice and we show that YY2 binding sites represent genomic sequences relevant for control of gene expression.

A doença vassoura-de-bruxa do cacaueiro (Theobroma cacao), causada pelo basidiomiceto Crinipellis perniciosa, levou a total destruição da lavoura sul-baiana, previamente cultivada principalmente com variedades altamente suscetíveis, tornando o Brasil, um país tipicamente exportador de cacau em importador em poucos anos. A perda da resistência do genótipo ?Scavina 6?, única fonte de resistência reconhecida contra C. perniciosa, está associada à variabilidade genética do patógeno. Diante disto, o objetivo deste estudo foi caracterizar a diversidade genética de isolados de C. perniciosa derivados de tecido infectado de Theobroma cacao (vassoura vede), originalmente coletados na Amazônia (Amazonas, Pará e Rondônia), uma região de ocorrência endêmica do fungo, usando marcadores moleculares. A identificação de relações genéticas entre isolados amazônicos e da Bahia e a possível existência de isolados geográficos também foram objetos deste trabalho. Primeiramente, a confirmação de identidade dos isolados Amazônicos foi conduzida usando amplificação e digestão da região ITS do rDNA e marcadores teloméricos. Todos os isolados avaliados foram confirmados como C. perniciosa. O primer telomérico TeloC1, previamente apresentado para discriminar o biótipo C, permitiu a separação do biótipo C dos biótipos S e L, porém, revelou variabilidade genética nos isolados de Cametá, PA e Cacaulândia, RO. Usando marcadores telomérico amplificado com o primer TeloA1R e ERIC, uma grande diversidade foi encontrada para os isolados da Região Amazônica em comparação àqueles da Bahia. Dentro dos isolados Amazônicos, a maior diversidade foi detectada para os isolados de Rondônia (Ji-Paraná, Cacaulândia e Ariquemes) e Pará (Cametá), áreas com 11 ocorrência endêmica ou de instalação histórica (mais de 300 anos) do cacaueiro, respectivamente. Isolados coletados em municípios localizados na regiãoTransamazônica, tais como Anapú, Altamira, Brasil Novo, Medicilândia e Uruará apresentaram maior similaridade com aqueles de Santarém e municípios relacionados à Belém, como Cametá, Baião e Mocajuba. Estes resultados sugerem que isolados da região Transamazônica pode ter sido originados de Belém ou Santarém, Pará. Na Bahia, houve a formação de dois grupos de isolados como previamente demonstrado. Os marcadores moleculares Microssatélites, ERIC e teloméricos foram eficientes na detecção da variabilidade genética em C. perniciosa. A diversidade genética observada auxiliará na identificação e escolha de regiões com maior diversidade de isolados para serem usados na seleção para resistência à vassoura-de-bruxa em programas de melhoramento do cacau / Witches broom disease of cacao (Theobroma cacao L.), caused by the basidiomycete Crinipellis perniciosa, devastated the producing region of Southern Bahia, previously cultivated mainly with highly susceptible cultivars, forcing Brazil, a typical exporter country to become a cocoa importer. The loss of resistance of the genotype ?Scavina 6?, the unique source of resistance against C. perniciosa has been associated with pathogen genetic variability. Thus, the objective of this study was to characterize the genetic diversity of C. perniciosa isolated derived from infected tissues of T. cacao (green-brooms), originally collected in the Amazon (Amazonas, Pará and Rondônia states), a region with endemic occurrence of the fungus, using molecular markers. The identification of the genetic relationships among the Amazonian and Bahian isolates, and the possible existence of geographical isolates were also objectives of this work. First, the identification confirmation of the Amazonian isolates was conducted using amplification and digestion of the ITS region of the rDNA and telomeric markers. All isolates evaluated were confirmed as C. perniciosa. The telomeric primer TeloC1, previously shown to discriminate the C biotype, allowed the separation of biotype C from biotypes S and L, but it revealed genetic diversity for isolates from Cametá, PA and Cacaulândia, RO. Using another telomeric marker amplified with TeloA1 primer and ERIC, a large genetic diversity was detected for isolates from the Amazon in comparison to Bahian. Within the Amazonian isolates, more diversity was detected for isolates from Rondônia (Ji-Paraná, Cacaulândia and Ariquemes) and Pará (Cametá), areas with endemic occurrence of wild cacao or historical introduction and cultivation (over 300 years), respectively. Isolates colletected at the Transamazônica roadway, such as from Anapú, Altamira, Brasil Novo, Medicilândia and Uruará presented more similarity with those from Santarém and locales nearer to Belém, such as Cametá, Baião and Mocajuba. These results suggested that isolates from the Transamazônica region might have originated from Belém or Santarém, Pará. In Bahia, there were two groups of isolates as previously demonstrated. Microsatellite, ERIC and telomeric markers were efficient in detecting the genetic variability of C. perniciosa. The genetic diversity observed will help in identifying and choosing regions with more diverse isolates to be used to screen for witches? broom resistance in cacao breeding programs

Doenças tireoideanas são bastante comuns, sendo sua maioria benigna. A relação entre os diversos tipos de doenças tireoideanas, bem como seus aspectos moleculares, são pouco conhecidos. O bócio (hiperplasia), por exemplo, é descrito por alguns como relacionado com carcinoma (tumor maligno) papilífero, enquanto que outros afirmam não haver relação causal entre as duas doenças. A questão mais desafiante, porém, refere-se à distinção entre adenoma (tumor benigno) e carcinoma folicular, que atualmente é feita apenas após à cirurgia, não permitindo tratamento diferenciado para os dois tipos de tumor. Este trabalho buscou identificar genes diferencialmente expressos entre tecido tireoideano normal, bócio, adenoma e carcinoma papilífero utilizando microarrays. Carcinomas foliculares não foram incluídos devido ao número e tamanho reduzidos das amostras. Dois tipos de array foram utilizados: arrays em membranas de nylon, contendo 213 clones obtidos por DDRT-PCR de amostras de tireóide, e arrays em vidro, contendo 3800 clones ORESTES. Experimentos utilizando o primeiro tipo de array identificaram três genes diferencialmente expressos, cuja expressão foi analisada por RT-PCR em 10 amostras de cada tipo de tecido. Dois deles foram capazes de diferenciar carcinomas papilíferos de tecido normal e bócio com 89% de precisão para o tumor maligno e 80% para os tecidos não malignos. Os arrays em vidro foram utilizados para avaliar o perfil de expressão de aproximadamente 10 amostras de cada tipo de tecido tireoideano. Foram identificados 160 clones diferencialmente expressos entre quaisquer dois tipos de tecido, cujas seqüências foram determinadas e comparadas com as dos bancos de dados. Dentre os genes mais interessantes destacam-se o correspondente à ATPase Na/K, cuja expressão está reduzida nos carcinomas em relação a tecidos normais e adenomas, o da proteína PDCD4, envolvida em morte celular programada, mais expresso em adenomas e tecidos normais em comparação com carcinomas e bócios, e os da calgizzarin (S100A11) e da α1-anti-tripsina, ambos mais ativos nos carcinomas do que nos demais tecidos. Todos esses genes já foram descritos como diferencialmente expressos em algum tipo de tumor. Este trabalho levou à padronização da metodologia de microarray em lâminas de vidro em nosso laboratório, bem como à identificação de genes que podem elucidar as alterações envolvidas na formação do bócio, adenoma e carcinoma papilífero. A implantação da técnica de amplificação de mRNA em nosso laboratório viabilizou a utilização de 10 amostras de carcinoma folicular, cuja massa de RNA total era insuficiente para as hibridizações. Essas amostras serão hibridizadas, juntamente com 10 amostras de adenoma, com microarrays contendo 4800 genes humanos conhecidos para a busca de genes diferencialmente expressos, de grande interesse diagnóstico. / Thyroid diseases are very common and are usually benign. The causal relationships among the different types of disease, as well as their molecular aspects, are not well understood. The goiter (hyperplasia), for instance, is described by some as related to papillary carcinoma (a malignant tumor), while others say there is no causal relationship between the two diseases. The most defying question, however, concerns the distinction between adenoma (benign tumor) and follicular carcinoma, which is currently made only after surgery, not allowing distinct treatments for the two kinds of tumor. This work aimed to identify differentially expressed genes among normal thyroid tissue, goiter, adenoma and papillary carcinoma using microarrays. Follicular carcinomas were not included due to the reduced number and size of the samples. Two kinds of array were used: arrays in nylon membranes, with 213 clones isolated from thyroid samples by differential display (DDRT-PCR); and glass slide arrays containing 3800 ORESTES clones.Experiments using the first type of array identified three differentially expressed genes, whose expression was analyzed by RT-PCR in 10 samples of each kind of tissue. Two of these genes were able to differentiate papillary carcinomas from goiters and normal tissues with precisions of 89% for the malignant tumor and 80% for the non-malignant tissues. Glass slide arrays were used to evaluate gene expression profile of approximately 10 samples of each type of thyroid tissue. 160 clones differentially expressed between any two tissues were identified, and their sequences were determined and compared with databases. Among the most interesting genes are Na/K ATPase gene, whose expression is reduced in carcinomas compared to normal tissues and adenomas, the gene corresponding to PDCD4 protein, involved in program cell death, with elevated expression in adenomas and normal tissues than carcinomas and goiters, and the genes of calgizzarin (S100A11) and α1-antitrypsin, both more active in carcinomas than the other tissues. All these genes have already been described as differentially expressed in at least one type of human cancer. This work led to the standardization of glass slide microarray technology in our laboratory, and to the identification of genes that may clarify the alterations involved in the formation of goiter, adenoma and follicular carcinoma. The implementation of mRNA amplification technique in our laboratory allowed the utilization of 10 samples of follicular carcinoma, whose mass was insufficient for microarray hybridizations. These samples will be hybridized along with 10 samples of adenomas, with microarrays containing 4800 known human genes to search for differentially expressed genes, of great diagnostic interest.

A raiva é uma doença aguda, progressiva e infecciosa do sistema nervoso central de mamíferos, causada pelo vírus da raiva (RABV). Embora possa ser prevenida por vacina, continua sendo um grave problema de saúde pública, além de ser responsável pela morte de seres humanos e muitos outros animais, incluindo os de interesse econômico. Este estudo teve como objetivo avaliar a relação entre polimorfismos dos genes que codificam as proteínas P e L de amostras de RABV pertencentes a variantes antigênicas 2 e 3 e períodos de incubação e títulos em camundongos. Para isso, foram selecionadas amostras isoladas de diferentes reservatórios de raiva de mamíferos das Ordens Carnivora e Chiroptera e amostras de bovinos, de áreas endêmicas para o vírus da raiva. As sequências obtidas foram utilizadas para a construção de árvores filogenéticas para procurar os padrões de segregação de linhagens. Os resultados mostraram que não houve marcadores ou polimorfismos que explicam as variações nos períodos de incubação e de letalidade entre cepas pertencentes a variantes antigênicas 2 e 3. Esta informação pode ser usada para discussões sobre a importância de reservatórios de raiva, a dinâmica do vírus da manutenção e evolução das diferentes formas desta zoonose entre os animais infectados, contribuindo para um estudo mais aprofundado sobre a busca de marcadores moleculares para patogênese. / Rabies is an acute, progressive and infectious disease of the central nervous system of mammals, caused by Rabies virus (RABV). Although preventable by vaccine, it remains a serious public health problem, and is responsible for the death of humans and many other animals, including those of economic interest. This study aimed to assess the relationship between polymorphisms in genes encoding the P and L proteins of RABV samples belonging to antigenic variants 2 and 3 and incubation periods and titers in mice. For this, samples isolated from different mammalian rabies reservoirs of the Orders Carnivora and Chiroptera and samples of cattle from endemic areas for rabies virus were selected. The sequences obtained were used to construct phylogenetic trees to search for the segregation patterns of strains. The results showed that there were no markers or polymorphisms that explain variations in incubation periods and lethality amongst strains belonging to antigenic variants 2 and 3. This information might be used for discussions about the importance of rabies reservoirs, the dynamics of the virus maintenance and evolution of the different forms of this zoonotic disease among infected animals, contributing to further study about the search for molecular markers for pathogenesis.