Orientador: Maria Célia Bertolini / Banca: Heloisa Sobreiro Selistre de Araujo / Banca: Ana Paula Ulian de Araujo / Resumo: Neste trabalho foi realizado o isolamento do cDNA e do gene que codifica a proteína semelhante à Pho85p de Saccharomyces cerevisiae, em Neurospora crassa. Um fragmento de 1,1 kb correspondendo à seqüência nucleotídica do gene em N. crassa foi amplificado e confirmado por sequenciamento, o qual foi utilizado no rastreamento de uma biblioteca de cDNA de N. crassa, resultando em um plasmídeo contendo a ORF que codifica a Ncpho85 e as seqüências upstream e downstream à ORF. Este inserto foi seqüenciado, a ORF isolada apresentou 1014 bp, a região upstream contém 304 bp e a downstream 561 nucleotídeos. Após sequenciamento do cDNA, a seqüência polipeptídica da proteína foi comparada com outras proteínas semelhantes à Pho85p de fungos miceliares, leveduriformes além de S. cerevisiae. Esta ORF isolada foi utilizada no rastreamento de uma biblioteca genômica de N. crassa, possibilitando o isolamento de um plasmídeo contendo a seqüência genômica, o qual foi submetido a sequenciamento. No inserto foi possível confirmar a presença do intron (de 89 bp) no nucleotídeo 69, confirmando os dados existentes no banco de dados de N. crassa. A expressão do gene foi analisada ao longo do crescimento do fungo, em diferentes meios de cultivo, através de experimentos de Northern blot. A presença de duas bandas de hibridização, uma de 2,2 kb e uma 2,7 kb foi observada em todos os experimentos realizados. Um pico de expressão dos dois transcritos ocorreu no tempo de 12 horas, sendo mais evidente para o transcrito de menor tamanho, o qual seria o transcrito do cDNA isolado anteriormente. Resultados preliminares da expressão da proteína ao longo do crescimento do fungo foram obtidos por Western blot. Apenas uma banda da proteína foi observada, a qual apresentou o tamanho aproximado de 40 kDa, próximo ao tamanho da proteína NcPHO85, deduzida a partir da seqüência do cDNA. / Abstract: In this study we performed the isolation of the cDNA and the gene that codes for the Pho85p-like protein from Saccharomyces cerevisiae in Neurospora crassa. A fragment of 1.1 kb corresponding to the nucleotide sequence of the gene from N. crassa was amplified by PCR and confirmed by DNA sequencing. This fragment was used to screen a N. crassa cDNA library resulting in a plasmid containing the ORF plus and the upstream and downstream regions. The insert from the plasmid was sequenced and the ORF showed to have 1014 bp, whereas the upstream and downstream regions had 304 bp and 561 bp, respectively. After sequencing the cDNA, the polypeptide sequence was compared to the same protein from mycelial fungi and yeasts. The isolated ORF was used to screen a N. crassa genomic library leading to the isolation of a plasmid containing the genomic sequence. After DNA sequencing the insert showed the presence of an intron (89 bp) starting at nucleotide 69. This result was in agreement with the information from the N. crassa database. Gene expression analysis during the vegetative growth in different media was performed by Northern blot. Two hybridization bands were observed in the experiments, one of 2.2 kb and another one of 2.7 kb. The maximum expression of both transcripts happened at 12 h of growth, this was more pronounced for the smaller transcript, which probably is the transcript of the isolated cDNA. Preliminary results of protein expression during vegetative growth were obtained by Western blot analysis. Only one band with approximately 40 kDa was observed, the expected size for the NcPHO85 protein sequence deduced from the cDNA sequence. In order to evaluate the role of this protein in glycogen metabolism and in other cellular functions in N. crassa we started the gene inactivation. / Mestre

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This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor.

Thesis (M.Sc.) - Carleton University, 2002. / Includes bibliographical references (p. 88-93). Also available in electronic format on the Internet.

Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 137-145). Also available in electronic format on the Internet.

Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 79-85). Also available in electronic format on the Internet.

Thesis (M.Sc.) - Carleton University, 2008. / Includes bibliographical references (p. 70-75). Also available in electronic format on the Internet.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-10-11Bitstream added on 2014-06-13T19:03:39Z : No. of bitstreams: 1 jesus_gj_dr_rcla.pdf: 4895578 bytes, checksum: 6970fd46e2b766b5810c294018cd18da (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho utilizou Neurospora crassa 74A em sua forma paramorfogênica, para avaliar o processo inicial da remoção da cor de efluentes aquáticos, que se desenvolveu em duas etapas; a biosorção e a biodegradação. Nos espectros de UV-Vis foi possível estabelecer o grau de remoção do corante por biosorção, seguido, após um tempo mais longo de contato, do início de processo biodegradativo; em que ficou caracterizado, principalmente, o aparecimento de bandas, indicando a presença de aminas primárias, após 120 horas de contato. Deste modo, pode-se deduzir que o fungo após esgotar sua fonte de carbono decorrente do inóculo, recorreu à fonte de carbono alternativo, vindo do substrato mais próximo, ou seja, das moléculas de corantes. No estudo da tolerância ficou evidenciado que todos os corantes estudados apresentaram fatores que provocaram uma diminuição da produção de biomassa, contudo, foi possível com a determinação da Concentração Efetiva (EC50), que caracteriza a inibição ou mortalidade de 50% dos organismos testados, para saber o grau de toxicidade destes corantes. Os mais tóxicos foram Procion Red MX-5B em pH 2,50; seguido do Acid Red 151 em pH 2,50. Menos tóxicos foram Direct Red 23 no pH 6,50 e Erythrosine B no pH 4,50. / In this research Neurospora crassa 74A in its paramorphogenic form was used to evaluate the initial process of the dye removal from water effluents, which was carried out in two steps; biosorption and biodegradation. It was possible to establish in the UV-Vis spectra a dye removal degree through biosorption, soon after a longer contact time between the microorganism and the dye, beginning the biodegradative process. In which it was mainly characterized the band appearance, pointing out the presence of primary amines after 120-hours contact. Thus, it can be deduced that the fungus after exhausting its carbon source due to the innoculum, it came after the alternative carbon source, coming from nearer substrate, that its, the dye molecules. The tolerance study showed that all dyes presented factors, which induced a decrease in the biomass production; however, it was possible to know the toxicity of the dyes because of the determination of the EC50. The most toxics were (Procion Red MX-5B at pH 2.50); followed by (Acid Red 151 at pH 2.50). The less toxics were (Direct Red 23 at pH 6.50) and (Erythrosine B at pH 4.50).

Made available in DSpace on 2015-06-17T19:33:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-24. Added 1 bitstream(s) on 2015-06-18T12:47:41Z : No. of bitstreams: 1 000831499_20170701.pdf: 551757 bytes, checksum: f00c3874e9a2ca554c17723cb987dd9a (MD5) Bitstreams deleted on 2017-07-24T11:34:13Z: 000831499_20170701.pdf,. Added 1 bitstream(s) on 2017-07-24T11:35:17Z : No. of bitstreams: 1 000831499.pdf: 3553272 bytes, checksum: 51518ed089010faa18418207d6328203 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Um dos mecanismos de transporte de macromoléculas do citoplasma para o núcleo celular ocorre através da passagem da macromolécula pelo complexo poro nuclear (CPN), presente no envoltório nuclear, e depende de proteínas transportadoras denominadas Importinas. Neste processo, conhecido como via clássica de importação nuclear, a proteína Importina-α (Impα) reconhece sequências de localização nuclear (NLSs) na proteína a ser transportada para a formação de um complexo junto a Importina-β (Impβ) permitindo o transporte da macromolécula. O objetivo do presente trabalho é o estudo estrutural da Impα proveniente do fungo filamentoso Neurospora crassa (ImpαNc), a fim de reconhecer as regiões que determinam especificidades da proteína, além de comparar seu comportamento nativo e na presença de um peptídeo NLS. Os primeiros experimentos com a ImpαNc permitiram uma caracterização inicial da proteína e seu comportamento em solução. Experimentos de cromatografia analítica de exclusão molecular, comparando duas amostras de ImpαNc: (1) na presença do peptídeo NLS da proteína FEN1 (FEN1 NLS) e (2) sem peptídeo NLS, indicaram a formação de aglomerados na amostra 2 e a conformação, predominantemente, monomodal na amostra 1, sugerindo uma maior estabilidade da proteína na presença de peptídeos NLS. Para aprofundar as informações sobre a ImpαNc, experimentos de cristalização foram conduzidos com a proteína complexada ao peptídeo de NLS clássica e monopartida do SV40 (SV40 NLS), conforme experimentos anteriores sugeriram a maior estabilidade da proteína na presença de NLSs. Um primeiro cristal (ImpαNc-1), obtido na condição 0,2mM fosfato de sódio dibásico dihidratado e 20% (w/v) de polietilenoglicol 3350, foi submetido á difração de raios X e apresentou padrão de difração satisfatório para elucidação da estrutura do complexo a uma resolução de 2,05 Å. Um segundo cristal (ImpαNc-2), obtido na ... / The transport of macromolecules from the cytoplasm to the nucleus occurs by passage through the nuclear pore complex, present in the nuclear envelope. One of that nuclear transport pathway depends on carrier proteins called Importins. In this process, known as classical nuclear import pathway, the Importin-α protein (Impα) recognizes nuclear localization sequences (NLSs) in the protein to be transported to the formation of a complex with Importin-β (Impβ) allowing the transport of macromolecules. The aim of this work is the structural study of the protein Importin-α, from the filamentous fungus Neurospora crassa (ImpαNc) in order to recognize the regions that determine the specificity of the protein and to compare their behavior in its native state and in presence of a NLS peptide. The first experiments with ImpαNc allowed an initial characterization of the protein and its behavior in solution. Experiments of analytical size exclusion chromatography comparing two samples of Impα: (1) in the presence of the NLS peptide of the protein FEN1 ( FEN1 NLS) and, (2) without NLS peptide; indicated the formation of agglomerates in the sample 2 and the conformation predominantly monomodal, in the sample 1, suggesting a greater stability of the protein in the presence of NLS peptides. For further information about the ImpαNc, crystallization experiments were carried out with the peptide complexed to the classic NLS SV40 (SV40 NLS) protein as previous experiments have suggested the increased stability of the protein in the presence of NLSs. A first crystal obtained in the condition 0.2mM dibasic sodium phosphate dihydrate and 20% (w / v) polyethylene glycol 3350 were subjected to xray diffraction and showed satisfactory diffraction pattern for the elucidation of the structure of the complex at a resolution of 2.05 Å. A second crystal (ImpαNc-2) obtained under the condition of 0.2 mM bicine pH 8.5 and 20% PEG 6000, was subjected to x-ray ...