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Orientador: Marcos Roberto de mattos Fontes / Coorientador: Agnes Alessandra Sekijima Takeda / Banca: Maria Célia Bertollini / Banca: João Renato Carvalho Muniz / Resumo: 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 ... / Abstract: 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 ... / Mestre

Mutation of repetitive DNA by repeat-induced point mutation (RIP) is a process that occurs in many filamentous fungi of the Ascomycota during the sexual cycle. Concurrently, direct DNA repeats are often deleted by homologous recombination at high frequency during the sexual cycle. Thus, the processes of RIP and deletion compete to either mutate or remove repetitive DNA from the genome of filamentous fungi during sexual cycles. Both processes contribute to genome streamlining by controlling proliferation of transposable elements and by limiting expansion of gene families. While the genetic requirements for deletion by homologous recombination are well known, the mechanism behind the specific detection and mutation of repetitive DNA by RIP has yet to be elucidated as only a single gene essential for RIP, rid, has been identified. We have developed Fusarium graminearum as a model organism for the study of RIP by showing that it mutates repetitive DNA frequently during the sexual cycle and that the mutations due to RIP are dependent on rid. Further, we have sequenced a genetic mapping strain of F. graminearum (00-676-2) and identified 62,310 single nucleotide polymorphisms (SNPs) compared to the reference strain (PH-1). The SNP map will be useful for quickly mapping new mutants by bulk segregant analysis and high-throughput sequencing for which bioinformatic tools were specifically developed. The groundwork has thus been laid for identification of novel RIP mutants in F. graminearum, which being homothallic has a major advantage for identification of recessive mutations. We used a forward genetics approach to shed light on the mechanism of RIP in Neurospora crassa. Two rrr mutants that dominantly r��educe R��IP and r��ecombination were characterized and identified as different mutated alleles of the same gene, rrr-1[superscript L496P] and rrr-1[superscript G325N] by bulk segregant analysis and high-throughput sequencing. Bioinformatic characterization suggests RRR-1 belongs to a previously uncharacterized group of dynamin-like proteins, which are generally involved in membrane fission and fusion. RRR-1-GFP localizes to the nuclear membrane, but not DNA, suggesting it affects RIP and recombination frequency indirectly by altering nuclear membrane dynamics during sexual development and thereby altering temporal aspects of RIP and recombination. We used a reverse genetics approach to determine whether high frequency RIP and homologous recombination of repetitive DNA during the sexual cycle are linked mechanistically or spatio-temporally. We tested strains where genes important for deletion by homologous recombination were knocked out and found all to be completely RIP competent except mre11, which, while sterile in homozygous deletion crosses, displayed lower RIP frequency in heterozygous crosses. This suggests that mre11 has roles in homologous recombination as well as non-homologous end joining may be important for RIP. Collectively, this work developed methods for efficiently mapping mutations and identified a novel protein that reduces RIP and recombination frequency but did not identify any mechanistic link between the two processes. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 10, 2012 - Dec. 10, 2014

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.

Calcium is a highly versatile intracellular signal molecule that can regulate numerous different cellular functions. In filamentous fungi there is evidence for it being involved in regulating various processes, including spore germination, hyphal tip growth, hyphal branching and conidiation. During colony initiation in the filamentous fungus Neurospora crassa, conidia form germ tubes which are involved in colony establishment, and conidial anastomosis tubes (CATs) which are involved in generating fused networks of conidial germlings. The primary research aim of this thesis was to analyze the role of Ca2+-signalling and homeostasis during colony initiation in N. crassa. Removal of Ca2+ from the growth medium showed that external Ca2+ was necessary for CAT fusion and, more specifically, was required for CAT chemoattraction. Two L-type Ca2+ channel blockers (verapamil and diltiazem) with different modes of action were found to inhibit both conidial germination and CAT fusion in wild type strains and CAT fusion was shown to be more sensitive to these two drugs. These channel blockers were additionally found to inhibit Ca2+ uptake by conidial germlings of the wild type expressing the aequorin Ca2+ reporter. However, the channel blockers also, unexpectedly, raised the cytosolic free Ca2+ ([Ca2+]c) resting level in these germlings suggesting that they did not just inhibit L-type Ca2+ activity. The morphological phenotypes (conidial germination, hyphal extension rate, conidiation and hyphal branching) of 22 mutants defective in different components of their Ca2+-signalling and homeostasis machinery were characterized in order to identify their possible roles of Ca2+ during colony initiation and development. The ∆cch-1 mutant lacking the CCH-1 L-type Ca2+ channel gene exhibited a reduction in CAT fusion. CAT fusion was decreased even further in a double mutant (∆cch-1∆mid-1) suggesting that that the CCH-1 and MID-1 proteins operate in combination during this process. Increased extracellular Ca2+ partially restored the phenotypes of the ∆cch-1, ∆mid-1 smco-1 and ∆cch-1∆mid-1 mutants which is consistent with CCH-1 and MID-1 being involved in Ca2+ uptake from the external medium. Calcium signatures following mechanical perturbation were successfully measured in populations of conidial germlings using aequorin expressed in the wild type and in deletion mutants (∆cch-1, ∆yvc-1, ∆fig-1) lacking different Ca2+ channels. The removal of external Ca2+ completely abolished the [Ca2+]c increase in response to mechanical perturbation and CCH-1 was found to partly contribute to this increase in [Ca2+]c. Various Ca2+-sensitive dyes (Oregon green 488, Fluo-4 and Calcium Green-1) were also tested to determine if they can be used to image [Ca2+]c at the single cell and subcellular levels. Only Fluo-4 allowed the measurement of [Ca2+]c in individual cells but the changes in dye fluorescence in response to changes in [Ca2+]c were too small to be useful for imaging [Ca2+]c dynamics at the subcellular level. The other two dyes underwent rapid compartmentalization in organelles when loaded into germlings. The plant antifungal proteins (defensins), MsDef1, MtDef4 and PAF were all found to disrupt Ca2+ signaling/homeostasis in conidial germlings of N. crassa. They all inhibited the [Ca2+]c increase and raised the resting level of [Ca2+]c in response to mechanical perturbation. Analysis of an aequorin expressing mutant that was defective in glucosylceramide synthase (∆gcs) showed that the effects of MsDef1 (but not MtDef4) on [Ca2+]c were mediated by the sphingolipid glucosylceramide. All of the defensins tested were found to exhibit different potencies with regard to their inhibitory effects on conidial germination and CAT fusion.

During the evolution of sex, different sexual conflicts arise. Sexual conflicts reduce fitness of the opposite sex. That is why several mechanisms have evolved to resolve them, which leads to rapid and unpredictable co-evolution of male and female traits involved in reproduction. This rapid co-evolution of male and female reproductive traits driven by sexual conflict can further lead to reproductive isolation resulting in speciation.             I used the hermaphroditic fungus Neurospora crassa, which has two mating types, as a model organism. Mating types are proxy to sex, because both are needed for sexual reproduction, but they are not limited to either sex role. However by using male pheromone knock-out lines, I created an evolutionary setup, where either mating type is forced to adapt to its restricted sex role. After 21 sexual generations of adaptive co-evolution, I tested if mating types had adapted to the assigned sex by measuring fitness (production of sexual spores called ascospores). I used three evolutionary setups (lines): Δccg4 lines, where mat A is female and mat a is adapted to the male role, Δmfa1 lines, where conversely mat A is adapted to the male role and mat a is female, and wild-type lines used as controls, where both mating types have maintained and adapted to both sex roles. And discovered one Δccg4 line, which indeed adapted to the newly assigned sex roles. At generation 15 and 21 I obtained mixed results for the presence of sexual conflict by correlating male and female fitness in hermaphroditic partner mat a in this line, however I found a sexual conflict also in the asexual growth, where male role is associated with increased, but female role with decreased mycelium growth rate. This work will further allow to study genomic mechanisms underlying this adaptation.

Nitrilases are enzymes able to convert toxic nitriles to corresponding carboxylic acids or amides. Thus they might be used in the detoxification of dyes, herbicides and pharmaceutical intermediates and byproducts. They can be used also for enzymatic syntheses of carboxylic acids not available by standard procedures. The aim of this diploma thesis is a recombinant expression of nitrilases from Neurospora crassa and the optimization of their purification. Cells of E. coli (BL 21 Gold) were utilized as an expression system. The purification was performed by ion-exchange chromatography, chelation chromatography and gel filtration - all under reducing conditions. Purified enzymes were studied by sedimentation analysis in an analytical ultracentrifuge. They were also used for searching of optimal conditions for their crystallization. Keywords: nitrilase, Neurospora crassa, recombinant expression

Orientador: Maria Célia Bertolini / Co-orientador: Fernanda Barbosa Cupertino / Banca: Ian Malavazi / Banca: Márcia Eliana da Silva Ferreira Balieiro / Resumo: 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... / Abstract: 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... / Mestre

Orientador: Maria Célia Bertolini / Banca: Cleslei Fernando Zanelli / Banca: Ana Paula Ulian de Araújo / Banca: Maria Teresa Marques Novo Mansur / Banca: Marcos Roberto de Mattos Fontes / Resumo: O fungo Neurospora crassa tem sido amplamente usado como um organismo modelo para os aspectos fundamentais da biologia dos eucariotos. Neste trabalho, foi investigado o papel funcional de uma ciclina de N. crassa (PCL-1), codificada pela ORF NCU08772 e ortóloga a Pcl10 de Saccharomyces cerevisiae. Na levedura, a proteína Pcl10, em conjunto com a proteína quinase dependente de ciclina Pho85, fosforila a enzima glicogênio sintase, a enzima regulatória da síntese de glicogênio. A fosforilação resulta na inativação da enzima e, portanto, em diminuição do acúmulo de glicogênio. A linhagem pcl-1 de N. crassa apresentou um atraso na germinação dos conídios e um retardo na progressão do ciclo celular quando comparado com a linhagem selvagem, sugerindo que esta ciclina pode regular o desenvolvimento e a divisão celular. Além disto, a linhagem nocauteada acumulou níveis mais elevados de glicogênio que a linhagem selvagem indicando o papel na regulação do metabolismo deste carboidrato. A fosforilação da enzima glicogênio sintase de N. crassa (GSN) foi analisada na linhagem nocaute através de análises de atividade enzimática, e os resultados mostraram que a GSN apresentou baixo índice de fosforilação, portanto alta atividade durante o crescimento, um resultado que pode explicar o alto acúmulo de glicogênio observado. Este resultado foi confirmado por análise de 2D-PAGE seguida por western blot, utilizando anticorpos anti-GSN. GSN apresentou na linhagem mutante isoformas m... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor