The specificity of fungal isolates from endemic Western Australian orchid species and hybrids in the tribe Diurideae was investigated using symbiotic seed germination and analysis of the fungal DNA by amplified fragment length polymorphism (AFLP). The distribution of the fungal isolates in the field was also assessed using two different seed baiting techniques. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. Two groups of orchids in the tribe Diurideae were studied. Firstly, a number of Caladenia species, their natural hybrids and close relatives from the southwest of Western Australia were selected because orchid species from the genus Caladenia are considered to have among the most specific mycorrhizal relationships known in the orchid family – an ideal situation for the investigation of mycorrhizal specificity. Secondly, species of Drakaea and close relatives, from the southwest of Western Australia and elsewhere in Australia, which are never common in nature and occur in highly specialised habitats, were selected to investigate the influence of habitat on specificity. Seed from the common species Caladenia arenicola germinated on fungal isolates from adult plants of both C. arenicola and its rare and endangered relative C. huegelii, while seed from C. huegelii only germinated on its own fungal isolates. The AFLP analysis grouped the fungal isolates into three categories: nonefficaceous fungi, C. huegelii type fungi, and C. arenicola type fungi. The group of C. huegelii type fungi included some fungal isolates from C. arenicola. An analysis of the AFLP fingerprints of C. arenicola fungal isolates from different collection locations showed that some, but not all, populations were genetically distinct, and that one population in particular was very variable. Despite being thought to have very specific mycorrhizal relationships, Caladenia species hybridise frequently and prolifically in nature, often forming self-perpetuating hybrid lineages. Five natural hybrids within Caladenia and its closest relatives were investigated. Symbiotic cross-germination studies of parental and hybrid seed on fungi from the species and the naturally occurring hybrids were compared with AFLP analyses of the fungal isolates to answer the question of which fungi the hybrids use. The germination study found that, while hybrid seeds can utilise the fungi from either parental species under laboratory conditions, it is likely that the natural hybrids in situ utilise the fungus of only one parental species. Supporting these observations, the AFLP analyses indicated that while the parental species always possessed genetically distinct fungal strains, the hybrids may share the mycorrhizal fungus of one parental species or possess a genetically distinct fungal strain which is more closely related to the fungus of one parental species than the other. The work on Caladenia hybrids revealed that C. falcata has a broadly compatible fungus that germinated seeds of C. falcata, the hybrid C. falcata x longicauda, and species with different degrees of taxonomic affinity to C. falcata. In general, germination was greater from species that were more closely related to C. falcata: seeds from Caladenia species generally germinated well on most C. falcata isolates; species from same subtribe (Caladeniinae) germinated well to the stage of trichome development on only some of the fungal isolates and rarely developed further; and seeds from species from different subtribes (Diuridinae, Prasophyllinae, Thelymitrinae) or tribes (Orchideae, Cranichideae) either germinated well to the stage of trichome development but did not develop further, or did not germinate at all. The AFLP analysis of the fungal isolates revealed that the fungi from each location were genetically distinct. In situ seed baiting was used to study the introduction, growth and persistence of orchid mycorrhizal fungi. A mycorrhizal fungus from Caladenia arenicola was introduced to sites within an area from which the orchid and fungus were absent, adjacent to a natural population of C. arenicola. In the first growing season, the fungus grew up to 50 cm from its introduction point, usually persisted over the summer drought into the second season and even into the third season, stimulating germination and growth to tuber formation of the seeds in the baits. Watering the inoculated areas significantly increased seed germination. Mycorrhizal relationships in Drakaeinae were less specific than in Caladeniinae. A study of the species Spiculaea ciliata revealed that this species, when germinated symbiotically, develops very rapidly and has photosynthetic protocorms, unlike all other members of the Drakaeinae. An AFLP analysis of the fungal isolates of this species grouped the isolates according to whether they had been isolated from adult plants or reisolated from protocorms produced in vitro. Isolates were genetically distinct when compared before germination and after reisolation. A cross-species symbiotic germination study of seeds of three Drakaea species and one Paracaleana species against fungal isolates from the same species and several other Drakaeinae species revealed lower specificity in this group than previously thought. A number of fungal isolates from Drakaea and Paracaleana species germinated two or more seed types, while all seed types germinated on fungal isolates from other species and the seed of Drakaea thynniphila germinated to some extent on every fungal isolate tested. An AFLP analysis of the Drakaeinae fungal isolates supported this information, revealing little genetic differentiation between the fungi of different orchid species. An ex situ seed baiting technique was used to examine the role of mycorrhizal fungi in microniche specialisation in the narrow endemic Drakaea. Soil samples from within and outside two Drakaea populations were tested for germination of the relevant seed types. In both cases, germination was significantly higher on soil samples from within than outside the populations, suggesting that the relevant mycorrhizal fungi may be restricted to the same microniches as the Drakaea species. The presence of similar fungi at distant, disjunct locations may be related to the extreme age and geological stabilityof the Western Australian landscape. The information from these investigations is essential for developing protocols for reintroduction and translocation of orchid species. It appears that the mycorrhizal relationships in these groups of orchids are not as specific as was previously thought. For reintroduction work, a broad sampling strategy is necessary, as it cannot be assumed that the same orchid species has the same fungus at different locations. A broadly compatible fungus may be of considerable utility in conservation work, such as in situations where a specific fungus appears to have poor saprophytic competence or where soil conditions have been altered. Seed baiting studies provide additional data on fungal distribution in situ. In general, molecular data do not provide information about efficacy or fungal distribution, so research programs that combine symbiotic germination studies with seed baiting investigations and genetic analyses of the fungi will provide the maximum benefit for designing more effective conservation programs.

Dentro do gênero Adesmia, as técnicas moleculares ainda não foram empregadas na caracterização de germoplasma e na análise da diversidade genética das espécies brasileiras que compôem o gênero. Portanto os objetivos deste trabalho foram: caracterizar, com a utilização de marcador molecular do tipo RAPD, as espécies brasileiras do gênero Adesmia DC; com base nestas informações estabelecer relações de diversidade genética entre as espécies e os acessos analisados; relacionar dados de diversidade com dados morfológicos e de reprodução.

Made available in DSpace on 2016-06-02T20:20:29Z (GMT). No. of bitstreams: 1 2739.pdf: 371228 bytes, checksum: c62273cf2751279fd8df20ebbb145ad4 (MD5) Previous issue date: 2009-07-23 / Universidade Federal de Minas Gerais / Two genetic linkage maps were constructed referent to two full-sib families (G1 and G2) of the marine shrimp species Litopenaeus vannamei, and the integration of these maps was accomplished. The mapping was based on polymorphic markers derived from nine AFLP (Amplified Fragment Length Polymorphism) primers. A total of 103 and 59 segregating polymorphic markers were used in the construction of the maps for families G1 and G2, respectively. The G1 family map consisted of 14 linkage groups, with estimated genome coverage of approximately 350 cM. The G2 map presented 4 linkage groups, with genome coverage of around 300 cM. The integration of the maps evidenced eight AFLP anchor markers in a same linkage group (LG1) in both families, to which 66 markers were allocated, leading to a well saturated linkage group, with estimated genome coverage of 874.16 cM. / Dois mapas genéticos de ligação foram construídos referentes a duas famílias de irmãos completos (G1 e G2) da espécie de camarão marinho Litopenaeus vannamei e foi realizada a integração desses mapas. O mapeamento foi baseado em marcadores polimórficos derivados de nove primers AFLP (Polimorfismo no Comprimento de Fragmentos Amplificados). Um total de 103 e 59 marcadores polimórficos segregantes foram utilizados respectivamente para a construção dos mapas das famílias G1 e G2. O mapa da família G1 consistiu de 14 grupos de ligação, com cobertura estimada do genoma de aproximadamente 350 cM. O mapa da G2 apresentou 4 grupos de ligação, com cobertura do genoma de aproximadamente 300 cM. A integração dos mapas evidenciou 8 marcadores âncoras AFLP em um mesmo grupo de ligação (GL1) em ambas as famílias, no qual foram alocados 66 marcadores, o que conduz um grupo de ligação bem saturado, com estimativa de cobertura do genoma de 874,16 cM.

Made available in DSpace on 2016-06-02T20:21:20Z (GMT). No. of bitstreams: 1 1814.pdf: 769331 bytes, checksum: eea2f2d52c718aafb2beca6a1ffc226b (MD5) Previous issue date: 2008-04-23 / Universidade Federal de Sao Carlos / Genetic studies have been performed for an endemic species from São Francisco River basin, Prochilodus argenteus, which has a great importance in the artisanal and subsistence fishing in the region. The linkage mapping and the genetic variability studies were made with AFLP (Amplified Fragment Length Polymorphism) dominants markers and with 189 specimens from a F1 cross, that was also used for restocking the upstream area from Três Marias hydroelectric dam. All the analyses were carried out with 15 primer pairs combinations and the linkage map was made using the pseudo-testcross mapping strategy. Forty six heterozygous marks were found for the genitors, with mendelian segregation of 1:1. The female genitor map had 3 linkage groups and the lenght of the analysed genome was 128,45 cM, the male genitor map had the same number of linkage groups and the total length of 192,67 cM. Common markers for both genitors, with mendelian segregation ratio of 3:1, served as bridge between the maps, for the construction of an integrated map. This map had 9 linkage groups, and the total map length was 442,08 cM. Additionally, the genetic variability was assessed and an expected heterozygosity mean was of 0,32082, with a Jaccard s similarity coefficient of 0,72564 + 0,00451. These preliminary values show that the cultured sample has a higher similarity coefficient than that obtained for the wild populations. Hence, the present results suggest that genetic studies and management restocking practices should be simultaneously performed for the maintenance of the genetic patrimony of this species at the São Francisco River basin. The results also showed that AFLP marks were suitable and effective to identify linkage marks in Prochilodus argenteus and for genetic variability studies in cultivar samples. / Estudos genéticos foram realizados em uma espécie endêmica da bacia do rio São Francisco, Prochilodus argenteus, a qual possui grande importância na pesca artesanal e de subsistência da região. Os estudos do mapa de ligação e de variabilidade genética foram realizados com o uso do marcador dominante AFLP (Polimorfismo de Comprimento de Fragmentos Amplificados) e com 189 indivíduos de um cruzamento F1, utilizado também para o repovoamento do rio São Francisco, à montante da barragem de Três Marias (MG). Todas as análises foram realizadas com 15 combinações de primers. Para a construção dos mapas de ligação foi utilizada a abordagem pseudocruzamento teste. Os primers utilizados geraram 46 marcas heterozigóticas para os genitores, com segregação mendeliana de 1:1. O mapa referente ao genitor feminino apresentou 3 grupos de ligação e o comprimento do genoma analisado foi de 128,45 cM, e o mapa do genitor masculino também consistiu em 3 grupos de ligação com comprimento total de 192,67 cM. Marcadores comuns aos dois genitores, com segregação mendeliana de 3:1, foram utilizados como pontes na integração dos mapas. O mapa integrado foi formado por 9 grupos de ligação, o que correspondeu a 442,08 cM de genoma analisado. Adicionalmente, a variabilidade genética foi estudada por meio da média da heterozigosidade esperada (He), a qual foi de 0,32082 e pela análise do coeficiente de similaridade de Jaccard, que foi igual a 0,72564 + 0,00451. Estes valores, ainda que preliminares, mostraram que essa amostragem cultivada possui o coeficiente de similaridade maior quando comparado com os de populações selvagens. Desta forma, sugere-se no presente trabalho que estudos genéticos devam ser realizados juntamente com a prática de repovoamento de rios, visando conservar o patrimônio genético desta espécie na bacia do São Francisco. Os resultados mostraram também que os marcadores AFLP foram adequados e eficientes para a identificação de marcas ligadas em Prochilodus argenteus e no estudo da variabilidade genética de amostras cultivadas.

Dentro do gênero Adesmia, as técnicas moleculares ainda não foram empregadas na caracterização de germoplasma e na análise da diversidade genética das espécies brasileiras que compôem o gênero. Portanto os objetivos deste trabalho foram: caracterizar, com a utilização de marcador molecular do tipo RAPD, as espécies brasileiras do gênero Adesmia DC; com base nestas informações estabelecer relações de diversidade genética entre as espécies e os acessos analisados; relacionar dados de diversidade com dados morfológicos e de reprodução.

A música é vista principalmente como arte e lazer e também pode exercer um importante papel na educação de adolescentes, trazendo muitos benefícios. Ela não é apenas agradável para a audição, contribui para o bem estar físico e emocional, desencadeiam sensações que podem afetar o humor, a memória, a função cerebral (córtex pré-frontal, raciocínio), frequência cardíaca, metabolismo e sistema imunológico e percepção da dor. No entanto, além dos benefícios da música, quando o indivíduo se expõe de forma inadequada, o prazer proporcionado pode trazer efeitos desagradáveis para a saúde. A indústria da música amplificada, e principalmente a popularização dos dispositivos sonoros portáteis individuais é crescente entre os jovens, tornando a perda auditiva induzida por elevados níveis de pressão sonora entre crianças e adolescentes uma preocupação evidente. Os riscos, para adquirir a perda auditiva dependem de fatores como número de horas de uso dos fones auriculares ou intensidade. A quantidade de intensidade sonora bem como o tempo que se fica exposto a eles são fatores determinantes da capacidade de prejudicar a audição. Embora estes jovens estejam habituados à exposição à música elevada, não possuem conhecimento sobre os danos auditivos futuros que podem ser causados pela exposição, e quando apresentam esse conhecimento, não sabem exatamente como prevenir estes danos e proteger a sua audição. Dessa forma, o objetivo deste estudo foi: Investigar o nível de informação que os adolescentes têm sobre a música amplificada e a saúde auditiva bem como seus hábitos de escuta, investigar a prevalência de zumbido e alterações auditivas em adolescentes de ambos os sexos, e realizar revisão sistemática sobre os artigos relacionados ao tema. 59 adolescentes participaram deste estudo, responderam a um questionário sobre música amplificada e audição. O THI, foi realizado quando relataram zumbido. Realizaram meatoscopia, imitanciometria, audiometria tonal liminar e de alta frequência e logoaudiometria. Nos resultados, a pergunta formulada na revisão sistemática de literatura foi respondida e evidenciou que embora os jovens tenham uma informação prévia dos riscos da música amplificada e a audição, não é uma informação concreta e não demonstram vontade de mudar seus hábitos de escuta. Nos resultados do estudo clínico, embora não tenha sido encontrada perda auditiva, as curvas audiométricas sugeriram uma futura PAIM, e os questionários mostraram que os adolescentes tem uma informação prévia do assunto, ainda fazem o uso incorreto da música amplificada. Levando a concluir que campanhas efetivas e eficientes com informações completas devem ser feitas, de forma que atinja o interesse dos adolescentes e mude o seu comportamento frente à música amplificada, para que não se crie uma geração de jovens surdos. / The music is mainly seen as art and leisure and can bring many benefits on a teenagers education. It´s not just pleasant for hearing but contributes to the physical and emotional wellbeing, triggering feelings that can affect humor, memory, brain function (prefrontal cortex, reasoning) heart rate, metabolism, immunologic system and pain perception. However, besides of its benefits, when the person is improperly exposed to the music, the pleasure can bring unpleasant health effects. The amplified music industry and mainly by the popularization and by the rise of individual portable sound devices between the young people make the hearing loss, inducted by the exposure on high sound pressure level, an evident concern. The risks to acquire hearing loss are dependent on factors like the number of hours using the earphones and intensity. The quantity of sound intensity as well the period of exposure are determinant factors of the capacity to cause hearing harm. Although young people are accustomed to be exposed to high sound pressure level, they have no knowledge about the future harms that can be caused by exposure, and when they understand the risks, they not exactly know how to prevent those harms and how to protect their hearing. Thus, the objective of this study is to investigate the amount of information young people have about amplified music and hearing health as well to understand their hearing habits, investigate the prevalence of tinnitus and hearing changes on young people of both sexes and perform a systematic review of the studies regarding the theme. 59 teenagers have attended this study, answered a questionnaire about amplified music and hearing. The THI was executed for the subjects that reported tinnitus. They were submitted to meatoscopy, immittanciometry, tonal threshold and high frequency audiometry and logoaudiometry. The question raised during the review of the literature was answered by the results that showed that although young people have knowledge about the risks of amplified music and hearing, the information is not concrete and they show no desire to change their hearing habits. Although no hearing losses were detected, the results of the clinical studies, mainly by the audiometric curves, suggests future PAIM and the questionnaires revealed that the adolescents have prior information about the issue but even so, they still incorrectly use the amplified music which bring us to the conclusion that effectives and efficiency campaigns with complete information should be done to reach teenagers interests with the purpose to change their behavior regarding amplified music to avoid a future generation of deaf adolescents.

Dentro do gênero Adesmia, as técnicas moleculares ainda não foram empregadas na caracterização de germoplasma e na análise da diversidade genética das espécies brasileiras que compôem o gênero. Portanto os objetivos deste trabalho foram: caracterizar, com a utilização de marcador molecular do tipo RAPD, as espécies brasileiras do gênero Adesmia DC; com base nestas informações estabelecer relações de diversidade genética entre as espécies e os acessos analisados; relacionar dados de diversidade com dados morfológicos e de reprodução.

Made available in DSpace on 2014-06-12T15:04:52Z (GMT). No. of bitstreams: 2 arquivo4510_1.pdf: 1312401 bytes, checksum: 103b70bf03d8851fef2766e8bd0290f9 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Paracoccidioides brasiliensis é um fungo dimórfico e agente etiológico da paracoccidioidomicose, uma micose sistêmica de evolução aguda ou crônica que se não diagnosticada e tratada a tempo pode ser fatal. Um método molecular para caracterização e detecção de P. brasiliensis foi desenvolvido a partir da clonagem e do sequenciamento de um fragmento de DNA de ~750 pb, obtido por RAPD (Random Amplified Polymorphic DNA), presente em isolados virulentos e ausente em isolados avirulentos deste fungo. Uma região interna do fragmento de DNA seqüenciado foi usada para desenhar primers que posteriormente foram utilizados em uma reação de hemi-nested PCR em tubo único. A reação de PCR específica foi capaz de amplificar DNA de três isolados de P. brasiliensis reconhecidamente virulentos e três isolados recentemente obtidos de pacientes com paracoccidioidomicose. A especificidade desta PCR foi confirmada pela ausência de produtos amplificados com DNA genômico de isolados de Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Sporothrix schenckii, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, Mycobacterium tuberculosis, Leishmania braziliensis, Trypanosoma cruzi, Schistosoma mansoni, DNA genômico humano (leucócitos) e de isolados de P. brasiliensis reconhecidamente avirulentas. A amplificação de cDNA de um isolado virulento sugere tratar-se de um gene expresso. A detecção específica de isolados virulentos de P. brasiliensis sugere ser este um candidato a marcador de virulência para este fungo. O potencial diagnóstico da PCR específica foi verificado com DNA extraído de aspirado de linfonodo de um paciente com paracoccidioidomicose

>Magister Scientiae - MSc / The class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint. / The National Research Foundation and the Norwegian Research Council

Enzymes, as nature’s catalysts, show remarkable abilities that can revolutionize the chemical, biotechnological, bioremediation, agricultural and pharmaceutical industries. However, the narrow range of stability of the majority of described biocatalysts limits their use for many applications. To overcome these restrictions, extremozymes derived from microorganisms thriving under harsh conditions can be used. Extremophiles living in high salinity are especially interesting as they operate at low water activity, which is similar to conditions used in standard chemical applications. Because only about 0.1 % of all microorganisms can be cultured, the traditional way of culture-based enzyme function determination needs to be overcome. The rise of high-throughput next-generation-sequencing technologies allows for deep insight into nature’s variety. Single amplified genomes (SAGs) specifically allow for whole genome assemblies from small sample volumes with low cell yields, as are typical for extreme environments. Although these technologies have been available for years, the expected boost in biotechnology has held off. One of the main reasons is the lack of reliable functional annotation of the genomic data, which is caused by the low amount (0.15 %) of experimentally described genes. Here, we present a novel annotation algorithm, designed to annotate the enzymatic function of genomes from microorganisms with low homologies to described microorganisms. The algorithm was established on SAGs from the extreme environment of selected hypersaline Red Sea brine pools with 4.3 M salinity and temperatures up to 68°C. Additionally, a novel consensus pattern for the identification of γ-carbonic anhydrases was created and applied in the algorithm. To verify the annotation, selected genes were expressed in the hypersaline expression system Halobacterium salinarum. This expression system was established and optimized in a continuously stirred tank reactor, leading to substantially increased cell amounts and protein yields. The resulting gene expression products were assessed for function in vivo and/or in vitro. Our functional evaluation of the tested genes confirmed our annotation algorithm. Our developed strategy offers a general guide for using SAGs as a source of scientific and industrial investigations into “microbial dark matter” and may help to develop new catalysts, applicable for novel reactions in green chemistry.