The diptericidal $\textit{Bacillus thuringiensis}$ (Bt) ssp. $\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\textit{am1}$ and $\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\delta$-endotoxins (Cry19A from Bt ssp. $\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\textit{240A}$ from Bti and an insertion sequence associated with the $\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\alpha$/$\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\alpha$/$\beta$-type SASP from $\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\textit{Bacilli}$.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-07-17T18:32:04Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação (Marcela) -30-01-2017-Corrigida-final -.pdf: 2307445 bytes, checksum: aa95c996c63ff24561a5b4cdf558b62c (MD5) / Made available in DSpace on 2017-07-17T18:32:04Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação (Marcela) -30-01-2017-Corrigida-final -.pdf: 2307445 bytes, checksum: aa95c996c63ff24561a5b4cdf558b62c (MD5) Previous issue date: 2014-03-13 / CNPQ / A exploração mineral é considerada como uma das atividades que mais degradam o meio ambiente, considerando os diversos impactos que gera. Técnicas moleculares, que têm grande potencial para estudos de diversidade de fungos, podem ser utilizadas para monitorar impactos ambientais e recuperação de áreas degradadas. Este trabalho teve como objetivo avaliar molecularmente as comunidades microbianas do solo de áreas nativa e mineradas e suas implicações na dinâmica da regeneração da vegetação de dunas litorâneas. Foram analisados os solos no período de estiagem e de chuva dos anos de 2009, 2010, 2011 e 2012 em seis áreas: duas matas de restinga controle e uma restinga arbustiva (matas naturais) e três dunas revegetadas (em 1989, 2001 e 2009). Foram feitas análises pela técnica de DGGE das comunidades de bactérias e fungos e pelo método de quantificação do ergosterol foi analisada a biomassa de fungos. Observou-se que a concentração de ergosterol no período de chuva foi maior em relação ao período de estiagem em todas as áreas de coleta. A comunidade de bactérias não apresentou diferenças significativas na diversidade, porém, a comunidade de fungos apresentou mudanças, com incremento do número de bandas 84 para 268 ao longo do estudo. O uso do solo de cobertura nas áreas mineradas proporciona boas condições para a recuperação da microbiota, porém, isso ocorre em função do tempo de revegetação. Conforme evidenciam as análises da biomassa de fungos e da estrutura molecular das comunidades microbianas evidenciaram isso. / Mineral exploitation is regarded as one of the activities that mostly degrade the environment, considering the different impacts it generates. Molecular techniques, which have great potential for studies of fungal diversity, can be used to monitor environmental impacts and reclamation. This study aimed to molecularly assess soil microbial communities of cover soil of native and mined areas and their implications on the dynamics of regeneration of the vegetation of coastal dunes. Soils were analyzed during the dry and rainy seasons of the years 2009, 2010, 2011 and 2012 in six areas: two forests and one shrub Restinga (natural forests) and three revegetated dunes (in 1989, 2001 and 2009). DGGE analyzes of the bacterial and fungal communities were performed and the quantification of ergosterol was carried out to access fungal biomass. It was observed that the concentration of ergosterol in the rainy season was higher than in the dry season in all sampling areas. The bacterial community showed no significant differences in diversity, however, the fungal community showed significant changes, with the increase of the number of DGGE bands from 84 to 268 throughout the study. The use of the cover soil in mined areas promotes good conditions for the recovery of soil microbial communities; however, recovery happens over time along with revegetation. Analyzes of the fungal biomass and the molecular structure of microbial communities showed that.

The theory of evolution is the central combing theory that brings together all the tenets of biology that bring about a solid understanding of life. It is also one of the most controversial scientific theories of all time and is widely rejected by about 40% of the general public in the United States. One of the biggest reasons for low evolution acceptance is the perceived conflict between evolution and religious beliefs. Educators and researchers have been working hard to improve how we teach evolution in the classroom; some methods, such as focusing on evolution knowledge, have shown to be mixed. Sometimes it works; other times, it does not, especially with highly religious students. Other methods have shown promising, such as using culturally competent approaches when teaching highly religious students. This is where we have focused our research on reconciling religious beliefs and evolutionary theory. First, in Chapter 1, we strengthened our understanding of the theory of evolution by studying a genus of damselfly in the South Pacific. Through our molecular analyses, we described a new genus, Nikoulabasis. In Chapter 2, we studied a unique of teaching evolution to religious students. We highlighted three of the most compelling reasons students changed their minds about evolution and gave suggestions for improving evolution education in the classroom. In Chapter 3, we created and validated the predictive Factors of Evolution Acceptance and Reconciliation (pFEAR) survey tool as a way for educators to better understand what worldview factors influence students' evolution acceptance. This chapter also gives suggestions to educators on how to use the pFEAR in their classrooms. In Chapter 4, we reviewed the literature to determine the influence of popular media on students learning evolution. Evolution misconceptions come from various sources such as social interactions, religious settings, textbooks, and even teachers. But few studies looked at the influence of popular media on evolution misconceptions. In Chapter 5, we viewed and watched student mentioned evolution references and determined the accuracy with which they depicted evolution. Of the 99 references we viewed, 94% of them depicted evolution accurately.

Yes / Resinous substances were highly prized in the ancient world for use in ritual contexts. Details gleaned from classical literature indicate that they played a significant role in Roman mortuary rites, in treatment of the body and as offerings at the tomb. Outside of Egypt, however, where research has shown that a range of plant exudates were applied as part of the mummification process, resins have rarely been identified in the burial record. This is despite considerable speculation regarding their use across the Roman Empire. Focusing on one region, we investigated organic residues from forty-nine late Roman inhumations from Britain. Using gas chromatographyemass spectrometry and the well-attested biomarker approach, terpenic compounds were characterized in fourteen of the burials analysed. These results provided direct chemical evidence for the presence of exudates from three different plant families: coniferous Pinaceae resins, Mediterranean Pistacia spp. resins (mastic/terebinth) and exotic Boswellia spp. gum-resins (frankincense/olibanum) from southern Arabia or beyond. The individuals accorded this rite had all been interred with a package of procedures more elaborate than the norm. These findings illuminate the multiplicity of roles played by resinous substances in Roman mortuary practices in acting to disguise the odour of decomposition, aiding temporary soft-tissue preservation and signifying the social status of the deceased. Nevertheless, it was their ritual function in facilitating the transition to the next world that necessitated transportation to the most remote outpost of the late Roman Empire, Britain. / R.C.B is supported by a PhD studentship from the Art and Humanities Research Council (43019R00209).

No description available.

Le syndrome de Usher est une maladie génétique associant surdité congénitale et rétinopathie pigmentaire (RP), auxquelles peuvent s'ajouter des troubles vestibulaires. Les différences phénotypiques, distinguées en 3 types cliniques, s'accompagnent d'une hétérogénéité génétique impliquant au moins 10 gènes. Identifier et caractériser les causes moléculaires grâce aux outils d'analyses génétiques disponibles permet d'améliorer la compréhension des mécanismes physiopathologiques à l'origine des symptômes du syndrome de Usher. Dans ce cadre, nous nous sommes inscrits dans une recherche d'exhaustivité des études moléculaires. Dans un premier temps, nous avons ainsi mis en place l'analyse et défini le spectre mutationnel des gènes minoritairement impliqués dans le type II (GPR98 et DFNB31). Nous avons également développé différents outils, notamment pour l'analyse de variants altérant le mécanisme d'épissage ou touchant les régions promotrices des gènes USH2.Ces travaux permettent d'obtenir un taux de détection des altérations conduisant au syndrome de Usher type 2 de 90 %. Ce taux est maintenant similaire à celui observé pour le type 1, qui constituait jusqu'ici la référence.Nous avons, dans un second temps, développé le séquençage nouvelle génération (NGS) appliqué à l'exome Usher. L'objectif de cette analyse était de tester la faisabilité et l'efficacité de cette approche, en vue de son éventuelle utilisation en diagnostic moléculaire. La définition des critères de qualité et la mise en place de la priorisation des variants ont été réalisées sur un groupe contrôle. L'étude a ensuite été étendue sur une cohorte de patients. Les résultats obtenus montrent qu'une utilisation en diagnostic est possible mais restera dépendante de l'amélioration de la technique du séquençage, de son analyse et des outils bioinformatiques pour interpréter le volume de données ainsi généré. / Usher syndrome is a genetic disorder combining sensorineural hearing loss (HL) and retinitis pigmentosa (RP). Some patients will also exhibit vestibular areflexia (VA). Clinical and genetic heterogeneity is recognized as the 3 clinical subgroups, defined mainly on the degree of HL and VA, can be caused by mutations in one of the 10 known genes. It is important to use all accessible genetic tools to identify and characterize molecular origin in order to improve the knowledge of the physiopathological mechanisms causing Usher Syndrome.In this context, we have developed an exhaustive approach. In a first step, we have implemented the analysis and established the mutational spectrum of the 2 minor USH2 genes (GPR98 and DFNB31). In addition, we have developed several tools, in particular to study variants susceptible to alter splicing or lying in the promoter regions of the USH2 genes.Thanks to this work, the USH2 mutation detection rate has now been raised to 90%, similar to that of USH1.We have then designed a targeted exome of the Usher genes to be sequenced using the GS Junior system (Roche 454). The aim of the study was to test the feasibility of this new technics for a possible transfer to diagnostic facilities. Quality criteria and variant priorization were set up on a control cohort (previously studied in one of the USH gene). The study has then been extended on a patient cohort. Our results indicate that NGS Usher-exome can be used in molecular diagnostics but improvement of the reliability of the sequencing technology, bioinformatics tools and dedicated databases is essential.

O gênero Okenia possui cerca de 50 espécies distribuídas ao redor do mundo, o qual claramente necessita de uma profunda revisão taxonômica. No Brasil, até então, são reportadas apenas três espécies: Okenia impexa, O. evelinae -- ambas com localidade-tipo em São Paulo -- e O. zoobotryon que foi originalmente descrita para Bermudas. Entretanto os registros de O. zoobotryon em águas brasileiras são ainda questionáveis. Esta espécie tem sido considerada durante muitos anos uma das mais problemáticas dentro do gênero, com outras como O. evelinae e O. polycerelloides já tendo sido propostas como sinônimas de O. zoobotryon e revalidadas por diferentes autores. Adicionalmente, O. zoobotryon tem sido reportada em diferentes partes do mundo, supostamente apresentando uma distribuição praticamente cosmopolita. Nesse contexto, este estudo apresenta uma análise morfológica e molecular de espécimes previamente identificados como O. zoobotryon procedentes de Bermudas, Austrália e Brasil. Além disso, exemplares de O. evelinae também foram analisados, com o intuito de esclarecer a possível sinonímia entre estas espécies. A análise morfológica revelou que os exemplares da Austrália são de fato uma espécie diferente, recentemente descrita como Okenia harastii, enquanto os espécimes do Brasil pertencem à espécie O. polycerelloides, que é claramente diferente de O. zoobotryon proveniente das Bermudas. Os principais caracteres anatômicos que corroboram a distinção entre as espécies aqui estudadas estão presentes na rádula e nos sistemas reprodutor e nervoso. A coloração é muito diferente entre O. evelinae e O. zoobotryon, porém a morfologia não as separou claramente, o que foi conseguido através de estudos moleculares preliminares. Dessa forma, ao contrário do que se pensava, O. zoobotryon parece estar restrita ao Oceano Atlântico Norte / The genus Okenia has about 50 species distributed around the world, which clearly need a deep taxonomic revision. In Brazil only three species are reported: Okenia impexa, O. evelinae -- both with type locality in São Paulo, Brazil -- and O. zoobotryon. Originally described from Bermuda, the records of the latter on the Brazilian waters are still questionable. In fact this species has been one of the most problematic in the genus. Okenia evelinae and O. polycerelloides have already been considered as synonyms of O. zoobotryon and were revalidated by different authors. Additionally, O. zoobotryon has been reported in different parts of the world, with a supposed cosmopolitan distribution. Thus, this study presents a morphological and molecular analysis of specimens previously identified as O. zoobotryon from Bermuda, Australia, and Brazil. We also studied specimens of O. evelinae in order to clarify the possible synonymy of these species. The morphological analysis revealed that the specimens from Australia are indeed a different species, recently described as Okenia harastii, while the ones from Brazil belong to Okenia polycerelloides, which is clearly distinct from O. zoobotryon. The distinctive anatomical characteristics that justify the separation among the species studied here are present in the radula, and reproductive and nervous systems. The color is very different between O. evelinae and O. zoobotryon, but the morphology did not clearly separate these two taxa. However, preliminary molecular data reveal that they are two distinct species. Thus, contrary to what was thought O. zoobotryon seems to be restricted to the North Atlantic Ocean.

This study provides chemical confirmation for the use of resinous plant exudates in mortuary contexts in Roman Britain. Analysis of amorphous masses, adhering residues and grave deposits using gas chromatography-mass spectrometry has revealed terpenoid biomarkers in sixteen inhumation and two cremation burials. The natural products characterized include European Pinaceae (conifer) resins, Pistacia spp. (mastic/terebinth) resins from the Mediterranean or the Levant and Boswellia spp. (frankincense) gum-resins from southern Arabia or eastern Africa. In addition, traces of a balsamic resin, probably Liquidambar orientalis, have been identified. A correlation between the use of these exotic exudates and interment in substantial, often multiple, containers with high-quality textiles and grave goods was observed. Theoretical consideration of this imported rite illuminates the multiplicity of roles played by resins/gum-resins in the mortuary sphere. The material properties of these highly scented substances speak to the biological reality of the decomposing body and to the socially constructed identity of the individual. On a practical level, they acted as temporary preservatives and masked the odour of decay. As social signifiers, they denoted the status of the deceased and promoted remembrance through conspicuous consumption and sensory impact. Encoded with ritual meaning, they purified the body and facilitated the final rite of passage to the afterlife. The recovery of these resinous traces provides us with new insights into the treatment of the body in the Roman period and establishes fresh links between the remote province of Britannia and the remainder of the Empire.

Fenilcetonúria (PKU) é uma doença autossômica recessiva caracterizada pela deficiência da enzima fenilalanina hidroxilase (PAH). Mutações no gene da PAH são responsáveis por PKU e uma grande heterogeneidade alélica é observada em pacientes em todo o mundo. Até o momento, mais de 500 diferentes alterações no gene PAH são encontradas no PAHdb. A maioria dessas mutações são raras e/ou específicas nas populações estudadas. O espectro de mutações de PKU em pacientes do sul do Brasil foi definido anteriormente e 6 mutações (IVS2+5G>C, p.I65T, p.R261X, p.R261Q, p.R408W e IVS12+1G>A) foram identificadas como responsáveis por 63,6% dos alelos mutantes. Este trabalho apresenta uma estratégia baseada no sistema TaqMan® (Applied Biosystems) para uma identificação rápida e específica das mutações listadas acima. Esta estratégia foi aplicada a uma amostra populacional que anteriormente foi caracterizada por diferentes técnicas. Os testes mostraram-se altamente sensíveis e são potencialmente aplicáveis à identificação de amostras de sangue impregnadas em papel filtro, podendo ser utilizados na triagem neonatal. / Phenylketonuria (PKU) is an autossomal recessive disease characterized by phenylalanine hydroxylase (PAH) deficiency. Mutations in the PAH gene are responsible for PKU, and an extensive allelic heterogeneity was observed in patients worldwide. Up to date, more than 500 different alterations can be found in PAHdb, the majority being rare mutations and/or specific to a genetic background. Our group was able to define mutation spectrum in PKU patients in Southern Brazil, and identified six mutations, named IVS2+5G>C, p.I65T, p.R261X, p.R261Q, p.R408W and IVS12+1G>A, that are responsible for 63.6% of mutant alleles. This work introduced a strategy, based in the TaqMan® System (Applied Biosystems), for a fast and specific identification of the above mutations. This strategy was applied to a sample population that was previously characterized by different techniques. Assays were shown to be highly sensitive and are potentially applicable to blood spots in order to be used in neonatal screening.

Fenilcetonúria (PKU) é uma doença autossômica recessiva caracterizada pela deficiência da enzima fenilalanina hidroxilase (PAH). Mutações no gene da PAH são responsáveis por PKU e uma grande heterogeneidade alélica é observada em pacientes em todo o mundo. Até o momento, mais de 500 diferentes alterações no gene PAH são encontradas no PAHdb. A maioria dessas mutações são raras e/ou específicas nas populações estudadas. O espectro de mutações de PKU em pacientes do sul do Brasil foi definido anteriormente e 6 mutações (IVS2+5G>C, p.I65T, p.R261X, p.R261Q, p.R408W e IVS12+1G>A) foram identificadas como responsáveis por 63,6% dos alelos mutantes. Este trabalho apresenta uma estratégia baseada no sistema TaqMan® (Applied Biosystems) para uma identificação rápida e específica das mutações listadas acima. Esta estratégia foi aplicada a uma amostra populacional que anteriormente foi caracterizada por diferentes técnicas. Os testes mostraram-se altamente sensíveis e são potencialmente aplicáveis à identificação de amostras de sangue impregnadas em papel filtro, podendo ser utilizados na triagem neonatal. / Phenylketonuria (PKU) is an autossomal recessive disease characterized by phenylalanine hydroxylase (PAH) deficiency. Mutations in the PAH gene are responsible for PKU, and an extensive allelic heterogeneity was observed in patients worldwide. Up to date, more than 500 different alterations can be found in PAHdb, the majority being rare mutations and/or specific to a genetic background. Our group was able to define mutation spectrum in PKU patients in Southern Brazil, and identified six mutations, named IVS2+5G>C, p.I65T, p.R261X, p.R261Q, p.R408W and IVS12+1G>A, that are responsible for 63.6% of mutant alleles. This work introduced a strategy, based in the TaqMan® System (Applied Biosystems), for a fast and specific identification of the above mutations. This strategy was applied to a sample population that was previously characterized by different techniques. Assays were shown to be highly sensitive and are potentially applicable to blood spots in order to be used in neonatal screening.