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).

Modeling gene regulatory networks has become a problem of great interest in biology and medical research. Most common methods for learning regulatory dependencies rely on observations in the form of gene expression data. In this dissertation, computational models for gene regulation have been developed based on constrained regression by integrating comprehensive gene expression data for M. tuberculosis with genome-scale ChIP-Seq interaction data. The resulting models confirmed predictive power for expression in independent stress conditions and identified mechanisms driving hypoxic adaptation and lipid metabolism in M. tuberculosis. I then used the regulatory network model for M. tuberculosis to identify factors responding to stress conditions and drug treatments, revealing drug synergies and conditions that potentiate drug treatments. These results can guide and optimize design of drug treatments for this pathogen. I took the next step in this direction, by proposing a new probabilistic framework for learning modular structures in gene regulatory networks from gene expression and protein-DNA interaction data, combining the ideas of module networks and stochastic blockmodels. These models also capture combinatorial interactions between regulators. Comparisons with other network modeling methods that rely solely on expression data, showed the essentiality of integrating ChIP-Seq data in identifying direct regulatory links in M. tuberculosis. Moreover, this work demonstrates the theoretical advantages of integrating ChIP-Seq data for the class of widely-used module network models. The systems approach and statistical modeling presented in this dissertation can also be applied to problems in other organisms. A similar approach was taken to model the regulatory network controlling genes with circadian gene expression in Neurospora crassa, through integrating time-course expression data with ChIP-Seq data. The models explained combinatorial regulations leading to different phase differences in circadian rhythms. The Neurospora crassa network model also works as a tool to manipulate the phases of target genes.

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 ...

Made available in DSpace on 2014-12-02T11:16:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-18Bitstream added on 2014-12-02T11:21:06Z : No. of bitstreams: 1 000773336_20190218.pdf: 311514 bytes, checksum: 3013fb73613f9ad6df44d3e85824afe7 (MD5) / O fungo filamentoso Neurospora crassa é um organismo modelo amplamente utilizado para estudos de diversos aspectos da biologia dos eucariotos. Nosso laboratório tem utilizado este fungo para estudar os mecanismos moleculares e bioquímicos envolvidos na regulação do metabolismo de glicogênio. A avaliação de uma coleção de linhagens mutantes individualmente nocauteadas em genes que codificam fatores de transcrição permitiu identificar várias proteínas potencialmente envolvidas na regulação do metabolismo de glicogênio do fungo N. crassa. Dentre as proteínas, o fator de transcrição FLBC foi identificado como uma possível proteína regulatória do metabolismo de glicogênio. A linhagem mutante apresentou alterações no perfil de acúmulo de glicogênio e na expressão do gene gsn em relação à linhagem selvagem, quando submetidas a estresse térmico. Estudos preliminares realizados anteriormente em nosso laboratório mostraram severas alterações morfológicas na linhagem flbCKO, como reduzida capacidade de conidiação, com a presença de poucos microconídios, formação de hifas aéreas reduzidas, morfologia das extremidades das hifas alterada, aspecto e morfologia alterados das colônias e produção acentuada do pigmento melanina, indicando que a proteína FLBC desempenha importante papel no desenvolvimento do fungo. O fator de transcrição FLBC, objeto de estudo deste trabalho, é uma proteína homóloga às proteínas FlbC e FLE1 dos fungos Aspergillus nidulans e Podospora anserina, respectivamente, ambas envolvidas nos processos de conidiação e desenvolvimento dos fungos. O objetivo deste trabalho foi realizar uma caracterização funcional detalhada deste fator de transcrição em N. crassa. Análises de expressão gênica mostraram níveis elevados do transcrito flbC durante o crescimento vegetativo do fungo e após a indução do desenvolvimento assexual. A linhagem nocaute mostrou uma desregulação... / The filamentous fungus Neurospora crassa is a model organism widely used for studies of several aspects of the biology in eukaryotes. We have been studying the molecular and biochemical mechanisms involved in glycogen metabolism regulation in this fungus. The screening of a knocked-out strains set in genes encoding transcription factors has identified many proteins likely involved in the regulation of glycogen metabolism in the fungus N. crassa. Among the proteins, the transcription factor FLBC was identified as a regulatory protein of the glycogen metabolism. The mutant strain showed changes in the glycogen accumulated and in the gsn gene expression when compared to the wild-type strain under heat stress. Preliminary studies carried out in our laboratory showed severe morphological changes in the strain flbCKO, such as reduced ability to conidiate, presence of a few microconidia, reduced production of aerial hyphae, altered morphology of hyphae tips, changes in the colonies morphology and high production of the pigment melanin, suggesting that FLBC plays an important role in the fungus development. The transcription factor FLBC, object of study in this work, is the Aspergillus nidulans and Podospora anserina FlbC/FLE1 homologous protein, respectively, both involved in conidiation and fungal growth. The purpose of this study was to perform functional characterization of this transcription factor in N. crassa. Analysis of gene expression showed high levels of the transcript flbC during vegetative growth and after induction of asexual development. The knockout strain presented a misregulation in the accumulation of reserve carbohydrate (glycogen and trehalose) during vegetative growth accumulating higher levels of both carbohydrates as compared to the wildtype strain. Analysis of gene expression in the strains wild-type and flbCKO showed that gdn (encoding for debranching enzyme) and gpn (encoding glycogen phosphorylase) genes are...

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-01-30Bitstream added on 2014-06-13T18:09:18Z : No. of bitstreams: 1 avaca_js_me_araiq.pdf: 1586941 bytes, checksum: 07a6c5702a63cebb17218e18a8aa2449 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / 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. / 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.

Orientador: Maria Célia Bertolini / Banca: Cleslei Fernando Zanelli / Banca: Iran Malavazi / Resumo: O fungo Neurospora crassa é um organismo amplamente utilizado como organismo modelo na compreensão de aspectos fundamentais da biologia dos eucariotos. Nosso laboratório tem utilizado este fungo para estudar os mecanismos bioquímicos e moleculares envolvidos na regulação do metabolismo do glicogênio. A avaliação de uma coleção de linhagens mutantes individualmente nocauteadas em genes que codificam fatores de transcrição permitiu identificar várias proteínas potencialmente envolvidas na regulação do metabolismo do glicogênio neste organismo modelo. As linhagens mutantes selecionadas apresentaram alterações no perfil de acúmulo de glicogênio e expressão do gene que codifica a enzima glicogênio sintase (gsn) durante a situação de estresse térmico. Entre estas, as linhagens mutantes nas ORFS NCU03043, NCU01629 e NCU04731, anotadas como proteínas hipotéticas no banco de dados do genoma do fungo, foram selecionadas para o presente estudo. Através de análises de Blast, a proteína codificada pela ORF NCU04731 mostrou ser homóloga ao grupo de fator de transcrição SREBPs (Sterol Regulatory Element Binding Protein) de mamíferos, que atuam como principal regulador da síntese de colesterol. Estas proteínas possuem domínio transmembrana e são ativadas após clivagem. Uma proteína ortóloga a SREBP (Sre1) foi identificada em Schizosaccharomyces pombe, entretanto, enquanto a habilidade de resposta a esteróis é conservada, as SREBPs de fungo regulam genes envolvidos na resposta transcricional à hipóxia, sendo necessárias para o crescimento em baixas concentrações de oxigênio. A proteína codificada pela ORF NCU03043 mostrou homologia a proteínas FlbC e FLE1 de fungos, as quais estão envolvidas na conidiação e desenvolvimento asexual. A linhagem flbCKO de N. crassa apresentou defeitos na progressão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The fungus Neurospora crassa has been widely used as a model organism for fundamental aspects of eukaryotic biology. We have been studying the biochemical and molecular mechanisms involved in glycogen metabolism regulation in this fungus. The screening of a set of knocked-out strains in genes encoding transcription factors was previously performed as an attempt to identify transcription factors regulating glycogen metabolism in N. crassa. Mutant strains presenting changes in glycogen accumulation and glycogen synthase gene (gsn) expression under normal growth temperature and under heat shock stress were selected. Among them, the mutant strains in the ORFs NCU03043, NCU01629 and NCU04731, annotated as hypothetical proteins in the fungus genome database were studied in the present work. By Blast analysis, the proteins NCU04731 showed homology to a group of mammalian transcription factors SREBPs (Sterol Regulatory Element Binding Protein), which act as regulators of cholesterol synthesis and requiring cleavage from the membrane for activation. A SREBP orthologue was identified in Schizosaccharomyces pombe (Sre1), however while the ability to respond to sterol is conserved, fungal SREBPs regulates genes involved in the transcriptional response to hypoxia and are required for growth under low-oxygen conditions. The proteins encoded by the ORF NCU03043 showed homology to the fungal proteins FlbC and FLE1 involved in conidiation and asexual development. The N. crassa flbCKO strain presented defects in cell cycle progression and morphology. FLBC protein is necessary for proper induction of conidiation and is important for growth and development in N. crassa. flbC gene expression was highly induced in the early times of conidia germination confirming the importance of this protein to the fungus... (Complete abstract click electronic access below) / Mestre

Senescence, the progressive loss of growth potential culminating in death, is common among Kauaian strains of Neurospora intermedia. Senescence is initiated by the insertion of kalilo DNA into the mitochondrial DNA. Mitochondrial DNA molecules carrying the insert accumulate and death occurs when the insert is equimolar with the mitochondrial DNA. The inserted form of kalilo DNA is referred to as mtlS-kalDNA. Studies on the somatic transmission of mtlS-kalDNA in ascospore series have revealed that kalilo DNA is capable of assuming new locations within the mitochondrial DNA. It is proposed that these novel insertions originate from intramitochondrial movement and an autonomous form of kalilo DNA, mtFF-kalDNA, is predicted to be an intermediate in movement. Novel insertion of kalilo DNA appears to depend on the form of mtlS-kalDNA transmitted sexually. If a mutagenic insert is transmitted, senescence is initiated at the onset of vegetative growth of the ascospores and no novel insertions are detected. The lifespans of these ascospores are quite short, death occurring in 10 subcultures or less. Transmission of a nonmutagenic insert delays the onset of senescence until either a novel insertion or a rearrangement of the transmitted insert occurs. The lifespans of these ascospores usually exceed 10 subcultures and are variable. Information obtained from tetrad analysis has revealed that novel insertion of kalilo DNA may also be under the influence of the host genome. A senescent Kauaian strain was identified which shows some but not all characteristics of kalilo senescence. In this strain and its derivatives, the behaviour of mtlS-kalDNA is erratic and in, some cultures the characteristic mitochondrial biochemical deficiencies, normally accompanying kalilo senescence, are not observed. It is suspected that kalDNA is not responsible for senescence in this strain and its derivatives but rather some other unknown factor is affecting the normal growth patterns of these cultures. Kauaian strains were surveyed for the presence of dsRNA to determine whether kalDNA has a viral origin. Only one senescent strain contains detectable amounts of dsRNA which was not homologous with a kalDNA probe. The survey identified six nonKauaian strains which contain dsRNA and seven dsRNA species were delineated. Although the presence of dsRNA is not relevant to kalilo senescence, analysis of dsRNA in a genetically-well defined organism like Neurospora may give insight into the significance of dsRNA in fungi in general. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Circadian clocks are internal timekeepers that aid survival by allowing organisms, from photosynthetic cyanobacteria to humans, to anticipate predictable daily changes in the environment and make appropriate adjustments to their cellular biochemistry and behaviour. Whilst many of the molecular cogs and gears of circadian clocks are known, the complex interactions of clock components in time and space that generate a reliable internal measure of external time are still under investigation. Computational modelling has aided our understanding of the molecular mechanisms of circadian clocks, nevertheless it remains a major challenge to integrate the large number of clock components and their interactions into a single, comprehensive model that is able to account for the full breadth of clock properties. An important property of circadian clocks is their ability to maintain a constant period over a range of temperatures. Temperature compensation of circadian period is the least understood characteristic of circadian clocks. To investigate possible mechanisms underlying temperature compensation, I first constructed a comprehensive dynamic model of the Neurospora crassa circadian clock that incorporates its key components and their transcriptional and post-transcriptional regulation. The model is based on a compilation of published and new experimental data and incorporates facets of previously described Neurospora clock models. Light components were also incorporated into the model to test it and to reproduce our knowledge of light response of the clock. Also, experiments were carried out to investigate the unknown mechanisms of light response, such as the molecular mechanisms supporting the correct timing of conidiation after light to dark transfer. The model accounts for a wide range of clock characteristics including: a periodicity of 21.6 hours, persistent oscillation in constant conditions, resetting by brief light pulses, and entrainment to full photoperiods. Next, I carried out robustness tests and response coefficient analysis to identify components that strongly influence the period and amplitude of the molecular oscillations. These data measure the influence of the parameters in the model and were beneficial for making and testing predictions in the model. Thermodynamic properties were then introduced into reactions that experimental observations suggested might be temperature sensitive. This analysis indicated that temperature compensation can be achieved if nuclear localisation of a key clock component, FRQ, decreases with increasing temperature. Experiments have been carried out to validate this hypothesis and simulations were made to explore other possible mechanisms. However, from my experimental data and modelling results, the restriction of FRQ nuclear localisation might not be the only mechanism required to achieve temperature compensation. In conclusion, temperature compensation is most likely a complex property and may involve a combination of multiple mechanisms regulating clock component activity over a range of temperatures.

No description available.