Cianobactérias são micro-organismos que realizam fotossíntese oxigênica e têm uma distribuição cosmopolita. Algumas cianobactérias são capazes de realizar fotossíntese e fixação biológica de nitrogênio (FBN), dois dos mais importantes processos na natureza, simultaneamente. As cianobactérias da ordem Nostocales são capazes realizar uma separação espacial destes dois processos por meio da formação de células especializadas, os heterócitos, onde acontece a fixação de nitrogênio, restringindo a fotossíntese às células vegetativas. Embora tenham importância ecológica, econômica e social, as cianobactérias foram muito pouco estudas com enfoque genômico. Este trabalho teve como objetivo a caracterização dos agrupamentos gênicos envolvidos na fixação biológica de nitrogênio e na diferenciação de heterócitos de três isolados brasileiros de cianobactérias da ordem Nostocales. Para este fim, culturas das linhagens Sphaerospermopsis torquesreginae ITEP-024, Nostoc sp. CENA67 e Fischerella sp. CENA161 foram sequenciadas com a plataforma MiSeq e então foi realizada a montagem ab initio do genoma com as leituras obtidas. Além desses três isolados, os genomas de outras 31 linhagens disponíveis no banco de dados GenBank foram recuperados e utilizados para comparação. A anotação dos genes foi realizada por meio do alinhamento de sequências nucleotídicas já conhecidas de outras linhagens contra os genomas dessas linhagens, utilizando a ferramenta BLASTn, e por meio do servidor antiSMASH. Além disso, análises filogenéticas foram realizadas a partir dos genes anotados. O sequenciamento dos genomas das três linhagens apresentou altos valores de qualidade de bases e elevada cobertura genômica. A anotação dos agrupamentos envolvidos na FBN revelou a presença de um total de 22 genes envolvidos na síntese da Mo-nitrogenase, sendo 19 presentes em todas as linhagens. Os isolados brasileiros apresentaram sintenia com outras linhagens próximas filogeneticamente, apresentando variação apenas na presença de regiões de excisão e do gene glbN. A linhagem CENA161 apresentou um agrupamento gênico completo para síntese da V-nitrogenase, assim como apenas outras 5 linhagens em toda a ordem. Foram encontradas nas três linhagens sequenciadas os genes devACB, relacionados à formação da camada polissacarídica do heterócito. Os genes hglEGDCA e hetM, que estão relacionados à formação da camada glicolipídica de heterócitos, foram encontrados completos nas linhagens ITEP-024 e CENA67, mas apenas parcialmente na CENA161. Genes reguladores do processo de diferenciação também foram acessados nas três linhagens brasileiras, entretanto apenas os reguladores globais do processo formam encontrados em CENA161. As análises filogenéticas mostraram que o gene nifH não reflete a filogenia do táxon e não é um bom marcador filogenético. Entretanto, a análise de todo o agrupamento refletiu o padrão filogenético de acordo com a taxonomia. Os resultados contribuem para a melhor compreensão dos aspectos genéticos e evolutivos do processo de FBN em cianobactérias. / Cyanobacteria are oxygenic photosynthetic microorganisms that have a worldwide distribution. Some cyanobacteria are capable of photosynthesis and biological nitrogen fixation (BNF), two of the most important processes in nature, simultaneously. Cyanobacteria from the Nostocales order perform a spatial separation of these processes through the formation of specialized cells, heterocytes, where nitrogen fixation is carried out, while photosynthesis is limited to vegetative cells. Although cyanobacterial have ecological, economic and social importance, they have been understudied with genomic approaches. This study aimed to characterize gene clusters involved in nitrogen fixation and heterocytes differentiation from three Brazilian strains of cyanobacteria from Nostocales order. For this purpose, nonaxenic cultures of the strains Sphaerospermopsis torque-reginae ITEP-024, Nostoc sp. CENA67 and Fischerella sp. CENA161 were sequenced with the Illumina MiSeq platform and ab initio genome assembly was performed. In addition to these strains, genomes from 31 strains available in the GenBank database were retrieved and used for comparison. Gene annotation was performed through alignments between known genes present in other cyanobacteria and the strains genomes, using the BLASTn tool, and through the antiSMASH server. Furthermore, phylogenetic analyses were performed on the annotated genes. The genome sequencing showed high bases quality values and genomic coverage. The annotation of clusters involved in the BNF revealed the presence of a total of 22 genes involved in the synthesis of Mo-nitrogenase, among 19 were present in all strains. Brazilian isolates showed synteny with phylogenetically related strains, with variations only in the presence of excision regions and glbN gene. The CENA161 strain showed a complete gene cluster for synthesis of V-nitrogenase, present in only 5 other strains in this order. devACB genes, related to the heterocyte polysaccharide layer formation, were found in the three strains sequenced. The hglEGDCA and hetM genes, related to the formation of heterocyte glycolipid layer, were complete in ITEP-024 and CENA67 strains, but only partial in CENA161. Gene regulation of the heterocyte differentiation process have also been accessed in the three Brazilian strains, but only the general process regulatory genes were found in CENA161. Phylogenetic analysis showed that the gene nifH does not reflect the phylogeny of this group and should not be considered a good phylogenetic marker. However, the complete genes cluster analysis reflected the patterns of phylogenetic grouping according to taxonomy. The results contribute to a better understanding of the genetic and evolutionary aspects of the BNF process in cyanobacteria.

Cianobactérias são micro-organismos que realizam fotossíntese oxigênica e têm uma distribuição cosmopolita. Algumas cianobactérias são capazes de realizar fotossíntese e fixação biológica de nitrogênio (FBN), dois dos mais importantes processos na natureza, simultaneamente. As cianobactérias da ordem Nostocales são capazes realizar uma separação espacial destes dois processos por meio da formação de células especializadas, os heterócitos, onde acontece a fixação de nitrogênio, restringindo a fotossíntese às células vegetativas. Embora tenham importância ecológica, econômica e social, as cianobactérias foram muito pouco estudas com enfoque genômico. Este trabalho teve como objetivo a caracterização dos agrupamentos gênicos envolvidos na fixação biológica de nitrogênio e na diferenciação de heterócitos de três isolados brasileiros de cianobactérias da ordem Nostocales. Para este fim, culturas das linhagens Sphaerospermopsis torquesreginae ITEP-024, Nostoc sp. CENA67 e Fischerella sp. CENA161 foram sequenciadas com a plataforma MiSeq e então foi realizada a montagem ab initio do genoma com as leituras obtidas. Além desses três isolados, os genomas de outras 31 linhagens disponíveis no banco de dados GenBank foram recuperados e utilizados para comparação. A anotação dos genes foi realizada por meio do alinhamento de sequências nucleotídicas já conhecidas de outras linhagens contra os genomas dessas linhagens, utilizando a ferramenta BLASTn, e por meio do servidor antiSMASH. Além disso, análises filogenéticas foram realizadas a partir dos genes anotados. O sequenciamento dos genomas das três linhagens apresentou altos valores de qualidade de bases e elevada cobertura genômica. A anotação dos agrupamentos envolvidos na FBN revelou a presença de um total de 22 genes envolvidos na síntese da Mo-nitrogenase, sendo 19 presentes em todas as linhagens. Os isolados brasileiros apresentaram sintenia com outras linhagens próximas filogeneticamente, apresentando variação apenas na presença de regiões de excisão e do gene glbN. A linhagem CENA161 apresentou um agrupamento gênico completo para síntese da V-nitrogenase, assim como apenas outras 5 linhagens em toda a ordem. Foram encontradas nas três linhagens sequenciadas os genes devACB, relacionados à formação da camada polissacarídica do heterócito. Os genes hglEGDCA e hetM, que estão relacionados à formação da camada glicolipídica de heterócitos, foram encontrados completos nas linhagens ITEP-024 e CENA67, mas apenas parcialmente na CENA161. Genes reguladores do processo de diferenciação também foram acessados nas três linhagens brasileiras, entretanto apenas os reguladores globais do processo formam encontrados em CENA161. As análises filogenéticas mostraram que o gene nifH não reflete a filogenia do táxon e não é um bom marcador filogenético. Entretanto, a análise de todo o agrupamento refletiu o padrão filogenético de acordo com a taxonomia. Os resultados contribuem para a melhor compreensão dos aspectos genéticos e evolutivos do processo de FBN em cianobactérias. / Cyanobacteria are oxygenic photosynthetic microorganisms that have a worldwide distribution. Some cyanobacteria are capable of photosynthesis and biological nitrogen fixation (BNF), two of the most important processes in nature, simultaneously. Cyanobacteria from the Nostocales order perform a spatial separation of these processes through the formation of specialized cells, heterocytes, where nitrogen fixation is carried out, while photosynthesis is limited to vegetative cells. Although cyanobacterial have ecological, economic and social importance, they have been understudied with genomic approaches. This study aimed to characterize gene clusters involved in nitrogen fixation and heterocytes differentiation from three Brazilian strains of cyanobacteria from Nostocales order. For this purpose, nonaxenic cultures of the strains Sphaerospermopsis torque-reginae ITEP-024, Nostoc sp. CENA67 and Fischerella sp. CENA161 were sequenced with the Illumina MiSeq platform and ab initio genome assembly was performed. In addition to these strains, genomes from 31 strains available in the GenBank database were retrieved and used for comparison. Gene annotation was performed through alignments between known genes present in other cyanobacteria and the strains genomes, using the BLASTn tool, and through the antiSMASH server. Furthermore, phylogenetic analyses were performed on the annotated genes. The genome sequencing showed high bases quality values and genomic coverage. The annotation of clusters involved in the BNF revealed the presence of a total of 22 genes involved in the synthesis of Mo-nitrogenase, among 19 were present in all strains. Brazilian isolates showed synteny with phylogenetically related strains, with variations only in the presence of excision regions and glbN gene. The CENA161 strain showed a complete gene cluster for synthesis of V-nitrogenase, present in only 5 other strains in this order. devACB genes, related to the heterocyte polysaccharide layer formation, were found in the three strains sequenced. The hglEGDCA and hetM genes, related to the formation of heterocyte glycolipid layer, were complete in ITEP-024 and CENA67 strains, but only partial in CENA161. Gene regulation of the heterocyte differentiation process have also been accessed in the three Brazilian strains, but only the general process regulatory genes were found in CENA161. Phylogenetic analysis showed that the gene nifH does not reflect the phylogeny of this group and should not be considered a good phylogenetic marker. However, the complete genes cluster analysis reflected the patterns of phylogenetic grouping according to taxonomy. The results contribute to a better understanding of the genetic and evolutionary aspects of the BNF process in cyanobacteria.

Genomic imprinting is the epigenetic modification of loci, primarily by DNA methylation, which results in parent-of-origin-specific monoallelic expression of a small subset of genes. In plants, imprinting occurs during endosperm development and a balance of maternally- and paternally-expressed imprinted genes is essential for normal seed development. Dependence on DNA methylation for imprinting highlights the potential to manipulate seed development, and consequently seed size, by altering DNA methyltransferase activity. DNA METHYLTRANSFERASE 1 (MET1) is the primary plant maintenance DNA methyltransferase and plays a significant role in imprinting. However, no evaluation of the potential role for other MET1 family members in genomic imprinting has been reported. The current model for the control of imprinting in plants suggests that maintenance DNA methyltransferases are required throughout development, yet the tissue-specific requirement of these enzymes is unconfirmed as analysis has relied solely on constitutive DNA methyltransferase mutants. To address these problems and to evaluate the potential to alter seed size, the work reported in this thesis investigated the potential involvement of putative maintenance DNA methyltransferases MET2a, MET2b and MET3 and the tissue-specific role of MET1 in imprinting. Imprinting was not significantly altered in met2a-1, met2b-1 and met3-1 mutants, indicating that MET1 is the sole DNA methyltransferase required for imprinting. Transcriptional analysis suggested MET1 is expressed throughout floral organ development and in the male and female gametophyte generation indicating that MET1 is potentially available to maintain imprinting-dependent methylation in these tissues. Tools to suppress MET1 tissuespecifically were developed to investigate the tissue-specific requirement of MET1 for imprinting. Analysis indicates that such tools could also be used to alter seed size by manipulating imprinting in commercially important species. Further work is needed to validate this approach.

Staphylococcus aureus and methicillin resistant Staphylococcus aureus (MRSA) are important pathogens of humans and livestock, including dairy cows. In livestock it represents a concern for public health as it may act as a reservoir that can infect humans and provide a source of transferrable resistance genes. The divergent mecA gene, mecC, was first identified in dairy cows in the UK, and has since been detected in many species in continental Europe. However, limited data exists on the epidemiological characteristics or the transmission dynamics of bovine mecC MRSA in the UK. S. aureus Mobile Genetic Elements (MGEs) often encode virulence and antimicrobial resistance genes that can contribute to the pathogenicity of the strain. Acquisition or loss of MGEs and variations within the core genome are reported to play an important role in S. aureus host adaptation although the mechanisms underlying S. aureus host specificity are not fully understood. This study was undertaken to determine the antimicrobial resistance and bovine associated virulence features of S. aureus isolated from bovine milk. The first two chapters of this thesis comprise of an introduction and a description of the methods used. Chapter 3 describes bovine associated features in bulk tank milk S. aureus isolates. SaPI borne-vwb and sec_bovine were widespread. Further, genome analysis detected six new SaPIs harbouring these genes and a class of site-specific integrases that appear to be associated with ruminant derived isolates. Examination of the fnbpA gene identified host specific variations that may be used to distinguish bovine from human isolates. Chapter 4 presents data on the epidemiological characteristics of mecC MRSA at the cow level within a dairy herd. Data from a longitudinal study undertaken suggested that mecC MRSA infections were transient and may not be associated with clinical disease. The prevalence varied between 1.14%-5.07% during the one-year study period, while SNP based whole genome phylogeny revealed four possible chains of transmission. Chapter 5 details a survey conducted to detect the prevalence of MRSA in UK retail meat. No mecC MRSA were found however CC398 were found and compared to CC398 isolates from the bulk tank milk study. A phylogenetic study provided no evidence of a common lineage of livestock association MRSA colonising both cattle and pigs in the UK. Chapter 6 describes the characterisation of a novel Streptococcus species isolated from a mastitic cow from New Zealand. Genome sequencing and phenotypic studies revealed this isolate, originally thought to be Streptococcus uberis to be most closely related to Streptococcus pseudoporcinus.

Comparative genomic studies have identified noncoding regions of the genome which are often more highly conserved between species than protein-coding sequences. One possible explanation for this conservation of non-coding sequences is some form of selective constraint since sequence conservation at great evolutionary depths is a preliminary indication of functional constraint. Here, I consider nearly 2500 putative regulatory elements, termed Conserved Noncoding Elements (CNEs), that are conserved across seven vertebrate species (human, macaque, mouse, chicken, frog, zebrafish and fugu). I distinguish between CNEs that show accelerated rates of evolution and those that have remained more constrained throughout evolution, and identify CNEs that show higher than expected substitution rates in the human lineage that may be potential candidates of adaptive evolution. However, it is not trivial to demonstrate the action of selection on such sequences. It is relatively easier in the case of protein-coding DNA, since selection would be predicted to result in different rates of substitution for synonymous and non-synonymous sites. Hence, I use the same seven species to define phylogenetically invariant positions in CNEs in contrast to those that have at least one substitution and analyse them independently to determine if there is a positive correlation between evolutionary conservation and the strength of purifying selection at individual sites. In the 1000 Genomes, but not the HapMap, data I find a significant excess of rare derived alleles in CNEs relative to coding sequences. This excess of rare alleles can be best explained if selection is relatively consistent across sites, with most mutations resulting in a similar reduction in fitness. Finally, I explore patterns of variation in the allele-frequencies within human populations, however do not detect any significant differences in the underlying distribution of negatively selected variants among human populations.

La caractérisation moléculaire multidimensionnelle des tumeurs et des tumeurs gliales en particulier est une étape importante pour l’identification de biomarqueurs (diagnostique, pronostique, théranostique et/ou de prédisposition), pour l’identification de cibles thérapeutiques et pour une meilleure compréhension de l’oncogénèse moléculaire.Nos travaux ont permis de confirmer et de consolider certaines données de la littérature comme par exemple : (i) la valeur pronostique favorable de la codélétion 1p/19q, (ii) la valeur pronostique favorable de la mutation IDH, (iii) le caractère mutuellement exclusive des mutations TP53 et de la codélétion 1p/19q et (iv) la rareté des altérations génétiques du PDGFRA dans les gliomes de bas grade (GDBG). De manière plus originale, nous avons identifié plusieurs sous-groupes génomiques de GDBG pertinents sur le plan clinico-biologique, notamment au sein des GDBG non 1p/19q codélétés : (i) 19q-délété ; (ii) 11p-délété, (iii) 7-gagné, (iv) 19-gagné et (v) inclassés. La perte du bras chromosomique 19q annule la valeur pronostique favorable de la mutation IDH dans les GDBG non 1p/19q codélétés. Nous avons également identifié des mutations géniques originales dans les GDBG (i.e. mutation TEP1 et RNF40) qui renforcent le rôle des télomères et du remodelage de la chromatine au sein des GDBG.Enfin, nous nous sommes concentrés sur la caractérisation des GDBG 11p-délétés qui sont de phénotype majoritairement astrocytaire et de moins bon pronostic. Ces GDBG surexpriment des gènes des cellules immunitaires (les GIM -Glioma infiltrating microglia-, les macrophages de type 1, les macrophages de type 2) et sont infiltrés par des cellules macrophagiques et microgliales. Ce microenvironnement dérégulé peut constituer une cible thérapeutique au sein des GDBG 11p-délétés. En conclusion, nos travaux participent à la dissection clinico-moléculaire des GDBG et à préciser la biologie d’un sous-type de GDBG caractérisé la perte du bras chromosomique 11p. / Multildimensional molecular characterization of tumors and more specifically of gliomas is of pivotal importance to identify: (i) new biomarkers (i.e. diagnostic, prognostic, theranostic or predisposing), (ii) new therapeutic targets and (iii) to improve our understanding of molecular oncogenesis.Our work has confirmed and consolidated previous data published in the literature, for example that: (i) 1p/19q co-deletion is associated with better prognosis, (ii) IDH mutation is associated with better prognosis, (iii) TP53 mutations and 1p/19q codeletion are mutually exclusive and (iv) PDGFRA is rarely altered, at genomic level, in low-grade gliomas (LGG).More originally, we have identified several genomic groups, with clinical and biological relevances, in LGG and more specifically in LGG without 1p/19q co-deletion: (i) 19q-deleted, (ii) 11p-deleted, (iii) 7-gained, (iv) 19-gained and (v) unclassified. Interestingly, 19q deletion abrogates the positive prognostic value of IDH mutation in LGG without 1p/19q codeletion.We have also identified new recurrent somatic gene mutations in LGG (i.e. TEP1 and RNF40 mutations), supporting the critical role of telomeres and chromatin remodelling in LGG.Finally, we have characterized further 11p-deleted LGG that exhibit mostly astrocytic phenotype and poor prognosis. This subgroup includes LGG overexpressing genes of inflammatory/immune cells (GIM -Glioma infiltrating microglia-, M1 macrophages and M2 macrophages) and infiltrated by macrophagic/microglial cells. This peculiar microenvironment detected in 11p-deleted LGG might be used as a therapeutic target. In conclusion, our work participates to characterize clinico-biological portrait of LGG and to describe a singular genomic subgroup of LGG characterized by 11p loss.

Scrapie ou paraplexia enzoótica dos ovinos é uma doença neurodegenerativa fatal que acomete ovinos e raramente caprinos. A doença é influenciada por polimorfismos nos códons 136, 154 e 171 do gene prnp que codifica a proteína priônica. Os animais podem ser susceptíveis ou resistentes, de acordo com as sequências alélicas observadas nos referidos códons. No Brasil ocorreram apenas casos de animais que foram importados, sendo o país considerado livre da doença. Neste trabalho foi realizada a genotipagem dos diferentes polimorfismos associados ao desenvolvimento do scrapie e a categorização em animais susceptíveis e resistentes. Foram sequenciadas 118 amostras provenientes de ovinos da raça Santa Inês criados em propriedades localizadas no Estado de São Paulo. Destas amostras foram identificados 6 alelos e 11 genótipos (ARQ/ARQ, ARR/ARQ, ARQ/AHQ, ARQ/VRQ, AHQ/AHQ, ARR/ARR, ARR/AHQ, VRQ/VRQ, ARQ/TRQ, TRR/TRR, TRQ/TRQ), dentre os quais o genótipo ARQ/ARQ teve ocorrência de 56,7%. Em nosso estudo foi detectada a presença da tirosina no códon 136, observação rara na medida em quenão existem relatos nacionais e relatos envolvendo a raça Santa Inês descrevendo este polimorfismo. Com os resultados obtidos, foi possível determinar a existência de grande variabilidade genética relacionada à raça Santa Inês no Estado de São Paulo, apesar da variabilidade, apenas 1,69% dos genótipos observados são extremamente resistentes ao scrapie. Estes dados demonstram que a raça nativa Santa Inês pode ser considerada potencialmente susceptível ao scrapie. / Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.

O comportamento materno é essencial para a sobrevivência e desenvolvimento do filhote mamífero. Durante a prenhez, as fêmeas recebem estímulos sensoriais e hormonais capazes de modificar e preparar o cérebro da mãe para o início dos padrões de comportamento materno (por exemplo, aumentando o número neurônios produtores de oxitocina no hipotálamo). Estudos têm identificado o hipotálamo como o principal responsável por estas mudanças, porém outras áreas do cérebro também estão envolvidas no processo do comportamento materno. Peg3, um gene marcado paternalmente expresso, é conhecido por controlar o comportamento materno em camundongos. Fêmeas nocautes para o gene Peg3 falham em aumentar a ingestão de alimentos, na ejeção de leite e em algumas atividades maternais, como placentofagia e construção do ninho. Este estudo teve como objetivo determinar os padrões de metilação da região diferencialmente metilada de Peg3 (Peg3DMR) de animais da raça Nelore de bovinos em diversas áreas do cérebro. Amostras foram coletadas das seguintes áreas: córtex frontal, occipital, temporal e parietal, hipocampo e hipotálamo, num total de 8 animais (4 machos e 4 fêmeas). O padrão de metilação destas amostras foi analisado pelo protocolo COBRA (do inglês, Combined Bisulfite-Restriction Analysis), que combina a modificação do DNA por bissulfito de sódio, amplificação por PCR e digestão por enzima de restrição. Foram encontrados diferentes padrões de metilação entre as amostras, ocorrendo uma predominância de hipometilação entre as amostras do sexo masculino, e padrões mais variados nas amostras do sexo feminino. As variações nos padrões de metilação ocorreram de maneira mais marcante entre as amostras de uma mesma região cerebral de diferentes animais, do que entre as amostras de várias regiões de um mesmo animal. Os resultados indicam que pode haver uma variação no status de imprinting em nível populacional, porém estudos com um número maior de amostras são necessários para a verificação da significância estatística destas variações. / The maternal behavior is essential to survival and development of mammalian offspring. Throughout pregnancy, females receive sensory and hormonal stimuli which promote modifications and prepare the mothers brain to the onset of maternal behavior patterns (for example, by increasing numbers of neurons producing oxytocin in the hypothalamus). Studies have identified the hypothalamus as the main responsible for these changes, but other areas of the brain are also involved in the maternal behavior process. Peg3, an imprinted paternally expressed gene, is known to control maternal behavior in mice. Peg3 knockout females failed in increasing food intake, milk ejection and some maternal activities as placentofagia and nest building. This study aimed to determine the methylation patterns of the differently methylated region of Peg3 (DMR-Peg3) of animals from Nellore cattle breed in several areas of the brain. Samples were collected from the following areas of cattle brain: the frontal, occipital, temporal and parietal cortices, hippocampus and hypothalamus, in a total of 8 animals (4 males and 4 females). The methylation pattern of these samples was analyzed by the protocol COBRA (Combined Bisulfite-Restriction Analysis), which combines DNA modification by sodium bisulfite, PCR amplification and digestion by restriction enzymes. It was found different methylation patterns among the samples. There was a predominance of hypomethylation among male samples, while different patterns were found among the female samples. Variation in the methylation patterns was more markedly observed among samples of the same cerebral region among different animals, then among samples of several regions within an animal. The results suggest that there may be a variation in the imprinting status at a population level, but further assays, with an increased number of samples are needed to verify the statistical significance of this variation.

This aim of this research was to elucidate the genotypic diversity of the mycotoxigenic species Fusarium proliferatum associated with diverse hosts and distributed in wide geographic locations to gain new insights into the biology of this emerging pathogen. This study developed a novel molecular genetic marker FG1056. Multilocus typing of F. proliferatum isolates (52) using F. verticillioides (2) and F. oxysporum (3) as references was carried out with FG1056 and a set of known genetic markers (ITS, TEF1, CAL and FUM1). This distinguished up to 10 genetic groups, 2 clusters and 23 haplotypes among the F. proliferatum isolates. FG1056 marker showed the highest number of SNPs (169), informative sites (89) and haplotypes (23) relative to other markers used and was comparable to the multi locus typing. Varying patterns of relationships were observed between isolates represented in the genetic groups and their host and geographic origin. Considerable biological variability was recorded among the F. proliferatum isolates in morphology, growth, sporulation and most notably fumonisin production (up to 140-fold differences) with reference to variable temperature, water activity and duration. De novo genome assemblies with the size ranging from 43.96 - 50 Mb have been developed for four diverse F. proliferatum isolates. In silico analysis led to the identification of 12,980 genes common to all isolates and up to 134 genes potentially unique to an isolate. Using these resources, FUM gene cluster (~45.3 Kb) was identified for the first time in F. proliferatum. Order and orientation of the 16 FUM genes and the complete flanking genes (MSF1 and ZCB1 at 5’; ANK1 and GAT1 at 3’) have been determined. This study has provided new insights into the genetic and biological diversity of F. proliferatum and also developed new genetic and genomic resources, which will serve as a solid platform for further research particularly to understand the regulation of fumonisins production in the laboratory and in the field.

Scrapie ou paraplexia enzoótica dos ovinos é uma doença neurodegenerativa fatal que acomete ovinos e raramente caprinos. A doença é influenciada por polimorfismos nos códons 136, 154 e 171 do gene prnp que codifica a proteína priônica. Os animais podem ser susceptíveis ou resistentes, de acordo com as sequências alélicas observadas nos referidos códons. No Brasil ocorreram apenas casos de animais que foram importados, sendo o país considerado livre da doença. Neste trabalho foi realizada a genotipagem dos diferentes polimorfismos associados ao desenvolvimento do scrapie e a categorização em animais susceptíveis e resistentes. Foram sequenciadas 118 amostras provenientes de ovinos da raça Santa Inês criados em propriedades localizadas no Estado de São Paulo. Destas amostras foram identificados 6 alelos e 11 genótipos (ARQ/ARQ, ARR/ARQ, ARQ/AHQ, ARQ/VRQ, AHQ/AHQ, ARR/ARR, ARR/AHQ, VRQ/VRQ, ARQ/TRQ, TRR/TRR, TRQ/TRQ), dentre os quais o genótipo ARQ/ARQ teve ocorrência de 56,7%. Em nosso estudo foi detectada a presença da tirosina no códon 136, observação rara na medida em quenão existem relatos nacionais e relatos envolvendo a raça Santa Inês descrevendo este polimorfismo. Com os resultados obtidos, foi possível determinar a existência de grande variabilidade genética relacionada à raça Santa Inês no Estado de São Paulo, apesar da variabilidade, apenas 1,69% dos genótipos observados são extremamente resistentes ao scrapie. Estes dados demonstram que a raça nativa Santa Inês pode ser considerada potencialmente susceptível ao scrapie. / Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.