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No description available.

It is usually assumed that the reduction of nitrite by nitrite reductase results in the formation of ammonia. The purpose of this investigation was to enquire whether more than one nitrite reductase activity is responsible for in vivo nitrite reduction. An assay system which measures the production of ammonia, as a result of nitrite reductase is described. The reduction of nitrite by nitrite reductase did not result in the formation of stoichiometric amounts of ammonia. Nitrite non-utilizing mutants showed that nitrite reduction could occur in vitro with no subsequent formation of ammonia or could result in the formation of essentially stoichiometric amounts of ammonia. Sedimentation velocity gradient centrifugation resulted in the separation of at least two peaks of nitrite reductase activity. A model is described which accounts for the results in terms of two nitrite reductase activities, necessary for in vivo nitrite reduction. / Thesis / Master of Science (MSc)

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

Esponjas e micro-organismos isolados do meio marinho constituem grupos biológicos que apresentam produção e/ou acúmulo de substâncias do metabolismo secundário com ações em diversos sistemas biológicos. Neste trabalho foi realizada a investigação química das esponjas marinhas Aiolochroia crassa e Dysidea robusta e do meio de cultura de crescimento do fungo Aspergillus sydowii, isolado da esponja Dragmacidon reticulatum, com o objetivo de ampliar o conhecimento sobre a química destes organismos. O extrato bruto da esponja Aiolochroia crassa apresentou atividade antiviral e foi submetido à separação de seus componentes por diferentes técnicas de fracionamento. Foram obtidos dois compostos puros identificados como os alcaloides derivados da bromotirosina, purealidina L e a 3-bromo-N,N,N,O-trimetiltirosina. Já o extrato aquoso da esponja Dysidea robusta foi submetido a sucessivas separações por CLAE-UV até a purificação de uma fração, que forneceu uma mistura de pirazinas isoméricas, a palitazina e a isopalitazina. O fungo Aspergillus sydowii cultivado em meio líquido forneceu extrato que apresentou atividade anticancerígena. Uma sequência de separações por CLAE-UV levou ao isolamento de três compostos puros: o ácido sidowico e dois compostos adicionais cujas estruturas ainda não foram identificadas. / Sponges and micro-organisms isolated from the marine environment are groups that have biological production and/or accumulation of substances of secondary metabolism with shares in various biological systems. This work was conducted chemical research of marine sponges Dysidea robust and Aiolochroia crassa and culture medium for growth of the fungus Aspergillus sydowii isolated sponge Dragmacidon reticulatum, with the goal of increasing knowledge about the chemistry of these organisms. The crude extract of the sponge Aiolochroia crassa showed antiviral activity and was subjected to separation des components by different fractionation techniques. Two pure compounds obtained were identified as derivatives of alkaloids bromotyrosine, purealidin L and 3-bromo-N,N,N,O-trimethyltyrosine. The aqueous extract of the sponge Dysidea robusta underwent successive separations by HPLC-UV to the purification of a fraction, which provided a mixture of isomeric pyrazines, the palythazine and the isopalythazine. The fungus Aspergillus sydowii cultivated in liquid medium has provided that extract showed anticancer activity. A sequence of separations by HPLC-UV allowed the isolation of three pure compounds: sydowico acid and two additional compounds whose structures have not yet been identified.

Esponjas e micro-organismos isolados do meio marinho constituem grupos biológicos que apresentam produção e/ou acúmulo de substâncias do metabolismo secundário com ações em diversos sistemas biológicos. Neste trabalho foi realizada a investigação química das esponjas marinhas Aiolochroia crassa e Dysidea robusta e do meio de cultura de crescimento do fungo Aspergillus sydowii, isolado da esponja Dragmacidon reticulatum, com o objetivo de ampliar o conhecimento sobre a química destes organismos. O extrato bruto da esponja Aiolochroia crassa apresentou atividade antiviral e foi submetido à separação de seus componentes por diferentes técnicas de fracionamento. Foram obtidos dois compostos puros identificados como os alcaloides derivados da bromotirosina, purealidina L e a 3-bromo-N,N,N,O-trimetiltirosina. Já o extrato aquoso da esponja Dysidea robusta foi submetido a sucessivas separações por CLAE-UV até a purificação de uma fração, que forneceu uma mistura de pirazinas isoméricas, a palitazina e a isopalitazina. O fungo Aspergillus sydowii cultivado em meio líquido forneceu extrato que apresentou atividade anticancerígena. Uma sequência de separações por CLAE-UV levou ao isolamento de três compostos puros: o ácido sidowico e dois compostos adicionais cujas estruturas ainda não foram identificadas. / Sponges and micro-organisms isolated from the marine environment are groups that have biological production and/or accumulation of substances of secondary metabolism with shares in various biological systems. This work was conducted chemical research of marine sponges Dysidea robust and Aiolochroia crassa and culture medium for growth of the fungus Aspergillus sydowii isolated sponge Dragmacidon reticulatum, with the goal of increasing knowledge about the chemistry of these organisms. The crude extract of the sponge Aiolochroia crassa showed antiviral activity and was subjected to separation des components by different fractionation techniques. Two pure compounds obtained were identified as derivatives of alkaloids bromotyrosine, purealidin L and 3-bromo-N,N,N,O-trimethyltyrosine. The aqueous extract of the sponge Dysidea robusta underwent successive separations by HPLC-UV to the purification of a fraction, which provided a mixture of isomeric pyrazines, the palythazine and the isopalythazine. The fungus Aspergillus sydowii cultivated in liquid medium has provided that extract showed anticancer activity. A sequence of separations by HPLC-UV allowed the isolation of three pure compounds: sydowico acid and two additional compounds whose structures have not yet been identified.

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.