A regulação da expressão gênica é vital para todos os organismos se adaptarem rapidamente às mudanças ambientais. Estes mecanismos adaptativos são altamente complexos e a maioria deles não está completamente esclarecida. O fosfato inorgânico (Pi), um nutriente essencial para todos os organismos, é requerido em importantes processos celulares como a biosíntese de ácidos nucléicos e a sinalização metabólica. O sistema de aquisição de Pi no fungo filamentoso Neurospora crassa inclui pelo menos quatro genes regulatórios: nuc-2, preg, pgov e nuc-1. Em condições limitantes de Pi, NUC- 2, uma proteína com domínio de repetição de anquirina, inibe o funcionamento do complexo PREG-PGOV, ativando assim o fator de transcrição NUC-1 e a expressão de genes envolvidos na captação de fosfato, como fosfatases, fosfato permeases e nucleases. Visando entender a funcionalidade do gene nuc-2 na regulação da expressão gênica em resposta aos níveis de Pi exógeno, foram construídas duas bibliotecas de subtração de cDNA entre as linhagens selvagem St.L.74A e nuc-2A de N. crassa, cultivadas em Pilimitante. Obtivemos 52 transcritos induzidos e 16 reprimidos pela proteína NUC-2. A categorização funcional destas sequências revelou genes envolvidos em diversos processos celulares, como transporte, regulação transcricional, transdução de sinal, metabolismo, síntese protéica e desenvolvimento. Entre os genes modulados negativamente pela proteína NUC-2, foi identificado um gene que codifica a proteína MAK-2 (mitogen-activated protein kinase-2), envolvida em vias de sinalização intracelular. O papel funcional deste gene no monitoramento do Pi extracelular foi avaliado por microarranjos de oligonucleotídeos, comparando as linhagens selvagem e mutante mak-2, cultivadas em baixa concentração de Pi. Foram identificados 4.214 genes regulados pela proteína MAK- 2, dentre eles a ciclina codificada pelo gene preg. Além disto, genes regulados em função da concentração de Pi foram identificados, mostrando o envolvimento de 3.174 transcritos. Os resultados obtidos neste trabalho revelam novos aspectos moleculares envolvidos na adaptação à disponibilidade de Pi extracelular, sugerindo que o gene mak-2 constitui um novo componente da via de sinalização e monitoramento de fosfato em N. crassa. / Gene expression regulation is crucial for all organisms to rapidly adapt to environmental changes. These adaptive mechanisms are highly complex and most of them have not been completely elucidated. The inorganic phosphate (Pi), an essential nutrient for all organisms, is required for important cellular processes, such as nucleic acids biosynthesis and metabolic signaling. The Pi acquisition system in the filamentous fungus Neurospora crassa includes at least four regulatory genes: nuc-2, preg, pgov and nuc-1. Under limiting Pi conditions, NUC-2, an ankyrin-like repeat protein, inhibits the functioning of the PREG-PGOV complex, allowing the activation of the transcription factor NUC-1 and the expression of genes involved in phosphate acquisition, such as phosphatases, phosphate permeases and nucleases. Aiming at a better comprehension of the nuc-2 functionality in gene expression regulation in response to exogenous Pi levels, two cDNA subtraction libraries were constructed comparing N. crassa wild type St.L.74A and nuc-2A strains, grown under Pi starvation. We obtained 52 NUC-2 up- and 16 downregulated genes. Functional categorization of these sequences revealed genes involved in several cellular processes, such as cellular transport, transcriptional regulation, metabolism, protein synthesis and development. Among the NUC-2 negatively modulated genes, we identified the MAK-2 (mitogen-activated protein kinase-2) protein coding gene, involved in the intracellular signaling pathway. The functional role of this gene in the extracellular Pi sensing was evaluated by oligonucleotide microarrays, comparing wild type and mak-2 strains responses under Pi starvation. We identified 4.214 MAK-2 regulated genes, among them the cyclin coding gene, preg. Furthermore, 3.174 genes regulated in response to Pi levels were identified. In a nutshell, the results obtained in this work reveal novel molecular aspects of the adaptation to extracellular Pi availability, suggesting that the mak-2 gene constitutes a novel component of the N. crassa phosphate sensing and signaling pathway.

A proteína serina/treonina fosfatase do tipo 1 (PP1) é a principal serina/treonina fosfatase envolvida na regulação de diversos processos tais como metabolismo, crescimento e divisão celular, síntese protéica e processamento de RNA. A holoenzima PP1 é constituída de uma subunidade catalítica conservada (PP1c) e subunidades reguladoras variáveis. Em mamíferos já foram identificados dezenas de polipeptídeos que associam-se direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre as proteínas que se associam a PP1c, muitas têm função inibitória como o inibidor-1 (I-1) e o inibidor-2 (I-2). A partir de extratos de micélios de Neurospora crassa foi purificada uma proteína, denominada INc-1, que atua in vitro como inibidor da atividade de fosforilase fosfatase de PP1c e constitui-se no primeiro exemplo de subunidade reguladora da PP1 descrito em fungos filamentosos. INc-1 apresenta diversas características bioquímicas comuns ao I-2 de mamíferos. Seqüências parciais de aminoácidos de três fragmentos proteolíticos obtidos de INc-1 permitiram a identificação de uma ORF (fase aberta de leitura) no genoma de N. crassa que provavelmente codifica INc-1. A análise dessa ORF mostrou que a sequência de aminoácidos do INc-1 é similar a do I-2, especialmente em regiões supostamente envolvidas em sua interação com a PP1c. Neste trabalho descrevemos a clonagem e a expressão em bactérias da sequência codificadora de INc-1. A atividade inibidora de PP1c de duas isoformas recombinantes purificadas, INc-1L e INc-1, foram avaliadas e comparadas. A forma denominada INc-1L apresenta em sua região aminoterminal um segmento de 38 aminoácidos derivado da retenção de um íntron, sem alterar a fase de leitura. Ambas proteínas recombinantes exibiram efeito inibidor sobre a atividade de fosforilase fosfatase de PP1c recombinante, sendo que a IC50 determinada para INc-1L foi de ~50nM e para INc-1 foi de ~11nM, sugerindo que a retenção do segmento de aminoácidos codificado pelo íntron na isoforma INc-1L diminui seu potencial inibitório. Verificamos também que o mRNA de INc-1 é expresso durante o crescimento vegetativo de N.crassa, apresentando níveis máximos na fase exponencial. / Type 1 protein serine/threonine phosphatases (PP1) play important roles in the regulation of many cellular functions including metabolism, cell growth and division, protein synthesis and pre-mRNA splicing. PP1 holoenzyme consists of one highly conserved catalytic subunit (PP1c) and variable regulatory subunits. A number of proteins that interact with PP1c have been described in mammals and the respective holoenzymes present distinct substrate specificity and/or different subcelular localization. Among the proteins that interact with PP1c, there are many with inhibitory effect such as inhibitor-1 (I-1) and inhibitor-2 (1-2). It has been demonstrated that a protein denominated INc-1, purified from Neurospora crassa extracts, specifically inhibits PP1c and has biochemical properties that resemble those of mammalian I-2. INc-1 is the first example of a PP1c regulatory subunit in filamentous fungi. Partial amino acid sequences of INc-1 led to the identification of an ORF (open reading frame) in Neurospora crassa genome which appears to encode INc-1. This ORF shows similarity with mammalian I-2 mainly in regions mapped as sites for interaction with PP1c. In this work we report the cloning and bacterial expression of the coding sequence for INc-1. The PP1c inhibitory activities of two recombinant isoforms, named INc-1L and INc-1, were compared. INc-1L aminoacid sequence presents an in frame segment of 38 residues encoded by an non-processed intron. 80th recombinant proteins showed inhibitory effect against phosphorylase phosphatase activity of recombinant PP1c, with IC50 of ~50nM for INc-1L and ~11nM for INc-1, suggesting that retention of the 38 residue segment decrease the inhibitory potential of INc-1L. We have also verified that INc-1 mRNA is expressed during N.crassa vegetative growth with maximum level at the exponential phase.

In eukaryotes, the defined loci on each chromosome, the centromeres, accomplish the critical task of correct cell division. In some organisms, centromeres are composed of a euchromatic central core region embedded in a stretch of heterochromatin and the inheritance and maintenance of centromeres are controlled by dynamic epigenetic phenomena. Although the size of centromeres differs between organisms, its organization, and the placement of euchromatic and heterochromatic regions is conserved from the fission yeast, Schizosaccharomyces pombe, to humans, Homo sapiens. However, relatively little is known about centromeres in the filamentous fungi from the Ascomycota, representing the largest group of fungi and fungal pathogens. Further, studies from humans, flies, yeast and plants have shown that the inheritance of centromeres is not strictly guided by centromeric DNA content, which is highly AT-rich, repetitive and constantly evolving. Therefore, it is difficult to align ans assemble the sequenced contigs of centromeric regions of higher eukaryotes, including most filamentous fungi. A genetic technique, tetrad (or octad) analysis has helped to map the centromeres of the filamentous fungus Neurospora crassa early on. The research presented in this dissertation used N. crassa as a model to focus on characterizing different features of centromeres with an emphasis on the centromere-specific histone H3 (CenH3) protein. Data included here represent the first study on centromere-specific proteins in Neurospora, and demonstrate that the central core of the centromeres are heterochromatic, showing enrichment of silent histone marks, which is in contrast to the centromere arrangement in fission yeast. The CenH3 protein, whose deposition on the genome licenses formation or maintenance of centromeres, shows highly divergent N-terminal regions and a conserved histone fold domain (HFD) in all eukaryotes. This bipartite nature of CenH3 is also observed in the Ascomycota, which provides an opportunity for functional complementation assays by replacing Neurospora CenH3 (NcCenH3) with CenH3 genes from other species within the Ascomycota. The results from this experimental approach provide good measures for (1) determining the specific regions of CenH3 required for the assembly of centromeres during meiotic and mitotic cell divisions and (2) analyzing the resistance to changes in the organization of centromeres in N. crassa. The genetic analysis showed that the divergent N-terminal region is essential for the proper assembly of centromeres, and that the conserved carboxy-terminus of CenH3 is important for the process of meiosis but not mitotic cell division. ChIP-seq analyses suggest that the observed loss of Podospora anserina CenH3 (PaCenH3- GFP) from certain N. crassa centromeres does not result in obvious phenotypic defects, e.g. diminished growth or evidence for aneuploidy. Further, the low enrichment of PaCenH3-GFP at certain centromeres is possibly predetermined during meiosis, which results in irreversible and progressive decreases in enrichment. It remains to be determined if this process is random as far as selection of centromeres is concerned. Together the results presented here suggest that during meiosis more stringent structural requirements for centromere assembly apply and that these are dependent on CenH3, and that depletion of CenH3 from centromeres does not critically affect mitosis in the asynchronously dividing nuclei of Neurospora hyphae. / Graduation date: 2013

A proteína serina/treonina fosfatase do tipo 1 (PP1) é a principal serina/treonina fosfatase envolvida na regulação de diversos processos tais como metabolismo, crescimento e divisão celular, síntese protéica e processamento de RNA. A holoenzima PP1 é constituída de uma subunidade catalítica conservada (PP1c) e subunidades reguladoras variáveis. Em mamíferos já foram identificados dezenas de polipeptídeos que associam-se direta ou indiretamente a PP1c, gerando holoenzimas com localizações celulares e especificidades distintas. Entre as proteínas que se associam a PP1c, muitas têm função inibitória como o inibidor-1 (I-1) e o inibidor-2 (I-2). A partir de extratos de micélios de Neurospora crassa foi purificada uma proteína, denominada INc-1, que atua in vitro como inibidor da atividade de fosforilase fosfatase de PP1c e constitui-se no primeiro exemplo de subunidade reguladora da PP1 descrito em fungos filamentosos. INc-1 apresenta diversas características bioquímicas comuns ao I-2 de mamíferos. Seqüências parciais de aminoácidos de três fragmentos proteolíticos obtidos de INc-1 permitiram a identificação de uma ORF (fase aberta de leitura) no genoma de N. crassa que provavelmente codifica INc-1. A análise dessa ORF mostrou que a sequência de aminoácidos do INc-1 é similar a do I-2, especialmente em regiões supostamente envolvidas em sua interação com a PP1c. Neste trabalho descrevemos a clonagem e a expressão em bactérias da sequência codificadora de INc-1. A atividade inibidora de PP1c de duas isoformas recombinantes purificadas, INc-1L e INc-1, foram avaliadas e comparadas. A forma denominada INc-1L apresenta em sua região aminoterminal um segmento de 38 aminoácidos derivado da retenção de um íntron, sem alterar a fase de leitura. Ambas proteínas recombinantes exibiram efeito inibidor sobre a atividade de fosforilase fosfatase de PP1c recombinante, sendo que a IC50 determinada para INc-1L foi de ~50nM e para INc-1 foi de ~11nM, sugerindo que a retenção do segmento de aminoácidos codificado pelo íntron na isoforma INc-1L diminui seu potencial inibitório. Verificamos também que o mRNA de INc-1 é expresso durante o crescimento vegetativo de N.crassa, apresentando níveis máximos na fase exponencial. / Type 1 protein serine/threonine phosphatases (PP1) play important roles in the regulation of many cellular functions including metabolism, cell growth and division, protein synthesis and pre-mRNA splicing. PP1 holoenzyme consists of one highly conserved catalytic subunit (PP1c) and variable regulatory subunits. A number of proteins that interact with PP1c have been described in mammals and the respective holoenzymes present distinct substrate specificity and/or different subcelular localization. Among the proteins that interact with PP1c, there are many with inhibitory effect such as inhibitor-1 (I-1) and inhibitor-2 (1-2). It has been demonstrated that a protein denominated INc-1, purified from Neurospora crassa extracts, specifically inhibits PP1c and has biochemical properties that resemble those of mammalian I-2. INc-1 is the first example of a PP1c regulatory subunit in filamentous fungi. Partial amino acid sequences of INc-1 led to the identification of an ORF (open reading frame) in Neurospora crassa genome which appears to encode INc-1. This ORF shows similarity with mammalian I-2 mainly in regions mapped as sites for interaction with PP1c. In this work we report the cloning and bacterial expression of the coding sequence for INc-1. The PP1c inhibitory activities of two recombinant isoforms, named INc-1L and INc-1, were compared. INc-1L aminoacid sequence presents an in frame segment of 38 residues encoded by an non-processed intron. 80th recombinant proteins showed inhibitory effect against phosphorylase phosphatase activity of recombinant PP1c, with IC50 of ~50nM for INc-1L and ~11nM for INc-1, suggesting that retention of the 38 residue segment decrease the inhibitory potential of INc-1L. We have also verified that INc-1 mRNA is expressed during N.crassa vegetative growth with maximum level at the exponential phase.

The primary research aims of this thesis were to analyse the mechanism of Ca2+/calmodulin (CaM) signalling during conidial germination and conidial anastomosis tube (CAT)-mediated fusion in Neurospora crassa. Ca2+ is an ubiquitous signalling molecule that regulates many important processes in filamentous fungi including spore germination, hyphal growth, mechanosensing, stress responses, circadian rhythms, and the virulence of pathogens. Transient increases in cytosolic free calcium ([Ca2+]c) act as intracellular signals. As the primary intracellular Ca2+ receptor, calmodulin (CaM) converts these Ca2+ signals into responses by regulating the activities of numerous target proteins. Ca2+-free medium, antagonists of L-type Ca2+ channels, CaM and calcineurin were found to inhibit CAT fusion. In addition, my results showed that CAT chemotropism is dependent on extracellular Ca2+. 65 genes were identified as likely components of the Ca2+ signalling machinery of N. crassa based on a comparative genomic analysis of S. cerevisiae, A. fumigatus and C. albicans. Deletion mutants of 29 of these genes were characterized in relation to their possible roles during colony initiation and development. Four of these mutants (Δcna-1, Δcnb-1, Δcamk-1, Δplc-2, and Δrgs-1), which were homokaryons, exhibited strong morphological phenotypes associated with CAT fusion. To identify the protein machinery involved in Ca2+/CaM signalling during colony initiation, proteins that directly or indirectly interacted with CaM were isolated from germlings by immunoprecipitation and analyzed by mass spectroscopy. A total of 286 putative Ca2+/CaM-interacting proteins were identified in this way and 30 of these proteins contained CaM-binding motifs. This proteomics analysis provided evidence for Ca2+/CaM signalling playing a role in regulating the activity of a wide range of proteins including MAP kinases in the cell integrity pathway, Ras/Rho signalling pathway, and microtubule and actin cytoskeletal proteins. GFP labelled CaM localized as dynamic spots associated with the plasma membrane and cytoplasm in both germ tubes and CATs. Significant CaM accumulation was observed in the tips of CATs growing towards each other, around fusion pores at sites of CAT fusion, and at developing septa in germ tubes. CaM localization was influenced by the actin and microtubule cytoskeleton during the colony initiation. Inhibition of F-actin polymerization with latrunculin-A suppressed the pronounced accumulation of CaM at growing germ tube and CAT tips. The movement of CaM associated with spindle pole bodies was prevented by treatment with the microtubule polymerization inhibitor benomyl. The absence of myo-5 resulted in reduced CAT fusion and the lack recruitment of CaM at growing tips indicating a role for the motor protein, myosin-5, in these processes. Finally, by expressing the genetically encoded Ca2+ sensor GCaMP6s under the control of tef-1 promoter in N. crassa, I have been able to image [Ca2+]c dynamics in this fungus for the first time. Using this I have been able to detect localized [Ca2+]c spikes and waves in conidia, germ tubes and CATs. However, I obtained no clear evidence for localized [Ca2+]c changes being associated with CAT chemotropism or fusion.

Conidiation is a common and critical asexual reproductive mode in fungi. The ascomycetes, the largest group in the kingdom Fungi undergo conidiation. The wide array of morphological difference in a conidiophore and conidial size, shape, and cellular organization demonstrates the importance of evolution in driving the morphological and functional diversity. An important unanswered question is how these conidiation processes evolve. We hypothesized that a conidiation regulatory pathway was present in the ancestral species, and became specialized in the extant species to lead to morphological and functional diversity. To address this hypothesis we assessed the conserved function of conidiation regulators in two distantly related ascomycetes, Aspergillus nidulans and Neurospora crassa. Using sequence similarity analysis, N. crassa orthologs were characterized to seven main conidiation regulatory genes in A. nidulans (fluG, flbC, flbD, abaA, wetA, medA, and stuA). Expression of the N. crassa orthologs complemented defective conidiation in the A. nidulans fluG, flbD, wetA, medA, and stuA mutants. In contrast, abaA and flbC and the N. crassa orthologs did not share conserved biochemical function. Taken in context of other recent studies of conidiation regulators, there are four distinct evolutionary patterns: (i) Non-homologous genes with analogous roles in conidiation (‘brlA’ and ‘fl’), (ii) Orthologs with retained biochemical function that lack analogous role in conidiation (‘fluG’, ‘flbD’, and ‘wetA’), (iii) Orthologs with retained biochemical function and analogous roles in conidiation (‘medA’ and ‘stuA’), and (iv) Orthologs with biochemical function not conserved but with analogous roles in conidiation (‘abaA’ and ‘flbC’). These studies set the stage for long-term studies of how evolution proceeded during the evolution of conidiation at different levels of phylogenetic diversity. An understanding of how evolutionary mechanisms shape the dynamics of developmental pathways will be significant for our understanding of fungal evolution of other novel adaptations such as pathogenesis.

In contrast to plant and animal cells, the fungal cells are multinucleate. A consequence of their multinucleate condition is heterokaryosis — the occurrence of genetically different nuclei in a common cytoplasm. In nature this condition occurs because of spontaneous mutations in the haploid nuclei in the coenocytic mycelium. Inspite of heterokaryosis being a fundamental aspect of fungal biology, the behaviour and dynamics of nuclei in fungal mycelium are little understood. This study was prompted by the following questions: (1) Why does a fungus need so many nuclei? (2) Are they all active simultaneously? (3) Does the proportion of the different nuclear types in fungal mycelium alter in response to change in conditions of growth? (4) Is the activity of an enzyme related to the dose of nuclei containing the encoding gene? Experimental approach. The approach taken was to generate heterokaryons in which one of the nuclear types carries a mutant allele for a specific enzyme while the other nuclear type carries the functional allele, introduced by transformation. Because in filamentous fungi, the transforming DNA commonly integrates randomly into the chromosomal DNA, the transformants would be genetic 'variants' in which the ratios of transformed to non-transformed nuclei might be controlled differently. The transformants could thus be useful in investigating the relationship between the frequency of transformed nuclei and the activity of encoded enzyme. In addition the transformants might be useful for studying nuclear behaviour. The availability of developmental information, genetic and molecular methodology, and biochemical mutant in Neurospora crassa made this fungus a material of choice for this investigation. Strain construction. A histidinol dehydrogenase (his-3) mutant strain was used into which an albino colour marker and a biochemical marker, inositoL were introduced by crossing. The latter two markers served as check against possible laboratory contamination. In addition, a gene mem, was introduced into the strain. In the mem genetic background, the strain has a wild-type morphology on agar medium but when grown in liquid shake culture it produces uninucleate microconidia that are useful in estimating nuclear ratio. Protoplasts of a constructed strain (his-3 al-1; mem; inl) were transformed with a plasmid containing the wild-type his-3 allele, thereby converting the original strain into a heterokaryotic strain having a mixture of transformed (his-3+t) and untransformed (his-3) nuclei. [The superscript +/ is used here to denote an his~3+ allele ectopically introduced by transformation]. Integration of plasmid DNA sequence in three selected transformants, 2T5, 3T3 and 4T12, was confirmed by genomic Southern analysis using the vector DNA as probe. The exponential growth rate of all three transformants was similar (~0.08mgh"1). Nuclear ratio. Assuming a uniform distribution of nuclei in mycelium, and a correspondence between nuclear ratio in mycelium and conidia, the ratio his-3* {: his-3 was estimated by plating microconidia. In transformant 3T3, the nuclear ratio was 7:1. In 2T5, all nuclei were his-3n. Transformant 4T12 did not produce microconidia. The nuclear ratio in this transformant was therefore estimated by macroconidial plating and found to be 1:5, in favour of his-3 nuclei. Behaviour of transformants in vegetative and sexual phase. Although the transformants had originally been selected for the expression of his-3+T gene, a majority of macroconidia produced in cultures of 3T3 and 2T5 required histidine to trigger their germination. This condition, referred to as cphenotypic lag', led to a gross underestimation of the proportion of prototrophic macroconidia by the direct plating method and biased the estimation of nuclear ratios. Therefore nuclear ratio was estimated by first germinating macroconidia on histidine supplemented medium before testing colonies in histidine dropout slants and comparing the numbers of auxotrophic and prototrophic mycelia. Phenotypic lag was not observed in 4T12. The variation in the degree of expression of phenotypic lag among the transformants was ascribed to transgene position effect. The transformants differed also in meiotic instability of the transforming DNA — the transforming DNA in 3T3 was passed through unchanged but it was deleted or modified in4T12and2T5. Experimental alteration of nuclear ratio. The transformants differed with respect to the self-adjusted ratio of transformed to non-transformed nuclei and also to the degree to which their nuclear ratio could be altered by nutritional manipulation of the growth medium, i.e., by growing the transformants in the presence or absence of histidine in the medium. In 3T3, the proportion of his-3+t nuclei progressively decreased by 3.5-fold in the sixth subculture on histidine medium. The change in 4T12 was even more striking: in the sixth serial subculture, the proportion of his-3+t nuclei decreased from 17-20% to -0.05%.However, when it was propagated again in medium that lacked histidine, the frequency of his-3+t nuclei was immediately restored to original level (-17%). That drastic alterations in nuclear ratio occurred upon nutritional manipulation was verified by Southern analysis. The intensity of signal specific for transformed DNA (nuclei) in cultures grown without histidine supplement was strong, but barely detectable in cultures grown with histidine. The signal reappeared when 4T12 was propagated in medium lacking histidine. Histidine induced change in nuclear ratio in 4T12 was further confirmed by three tests: (i) inoculum test using conidia, (ii) hyphal tip analysis, and (iii) genetic test using colour markers. Nuclear ratio and enzyme activity. Because in 4T12 changes in nuclear ratio could be manipulated, this transformant was used to investigate whether the proportion of his-3+t nuclei is correlated with the levels of encoded enzyme, histidinol dehydrogenase. Surprisingly, the specific activity of histidinol dehydrogenase was the same regardless of the percentage of his-3+t nuclei. This observation suggested that the physiological demand of a metabolite may be satisfied with only a few nuclei carrying the relevant gene. Or in other words the majority of nuclei in the coenocytic mycelium may, perhaps, not be active simultaneously. Silencing of transforming DNA in nuclei. Two experiments were done to test the possibility that in a majority of nuclei, the transforming DNA is selectively silenced by methylation of cytosine: (1) Southern analysis of chromosomal DNA digested with isoschizomers, and (2) Reactivation by growth of transformants in presence of 5-azacytidine, an inhibitor of methylation. The results suggested that a majority of transformed nuclei may, perhaps, be inactive. The results of Northern analysis suggested that the amount of his-3+t transcript was correlated (but 5-azacytidine experiment indicated that only few his-3+t nuclei may be active) with the proportion of his-3+t nuclei, but not histidinol dehydrogenase activity. The above results suggested that expression of his-3+t gene was controlled both at the levels of transcription and posttranscription. Nuclear selection. To study competition between nuclei containing mutant (his-3) nuclei and prototrophic nuclei containing his-3+ gene at its normal chromosomal location or at the ectopic location, heterokaryons were synthesized using strains in which the nuclear types had been marked by non-allelic genetic colour markers, al-1 and al-2. The results suggested that in heteronuclear mixture, the replication rate of the transformed nuclei is affected as compared to the nuclei having the gene in normal chromosomal location. Major contributions. This study generated (his-3 + his-3+) heterokaryons by transformation. The behaviour of transformants differed in some respects both in the vegetative and sexual phases. It was demonstrated that nuclear ratio could be experimentally altered. However, there was no correlation between nuclear ratio and enzyme activity. The observations imply asynchronous division rate among nuclei and raise the possibility that not all nuclei in the coenocytic mycelium are active simultaneously.

The garlic extract ajoene is considered to have antimicrobial and antitumor effects against a variety of cell types, and it is suggested to have the potential to be used as an antifungal or antitumor drug clinically. The underlying mechanism of its inhibitory effects is still uncertain. In this project, the effects of ajoene on the growth of fungal and oomycete cells were studied on Candida albicans, Neurospora crassa and Achlya bisexualis. Endometrial cancer is the most common gynecologic cancer. A 3D spheroid model of endometrial cancer cells were for the first time used to investigate the antitumor effects of ajoene and selected antitumor agents. Ajoene was extracted from fresh garlic by chromatographic methods and the outcome of the extractions was verified with Mass spectrometry and NMR spectroscopy. Ajoene was then tested on the yeast form or germ tubes of C. albicans, and the cell division and germ tube formation was analyzed. N. crassa and A. bisexualis were treated with ajoene on plates or on glass slides to measure the hyphae radial extension or individual hyphal extension. 3D endometrial adenocarcinoma cell (Ishikawa) spheroids were treated with ajoene, paclitaxel, targeted drugs everolimus, sorafenib, gefitinib and canertinib alone or in combinations. The growth activity, metabolic activity, cell proliferation, apoptotic activity and the cytoskeletons were analyzed after the treatments. Cell division of C.albicans was inhibited by ajoene at 5µg/ml or higher concentrations. The length of C.albicans germ tubes was significantly shorter in ajoene treated groups than the untreated ones. Radial extension and individual hyphal extension of N. crassa and A. bisexualis were both inhibited by ajoene. Ajoene did not show any antitumor effects on the 3D cell model of Ishikawa cells. No synergistic effect was detected between ajoene and paclitaxel or ajoene and everolimus. The targeted drugs Canertinib and everolimus showed an inhibitory effect on growth activity of the spheroids, but no synergy with paclitaxel. In conclusion, ajoene was able to inhibit various forms of fungal and oomycete growth, but any antitumor activity of ajoene did not show on 3D culture of endometrial cancer cells.

Thesis (Ph. D.)--Illinois State University, 1980. / Title from title page screen, viewed Feb. 17, 2005. Dissertation Committee: Herman Brockman (chair), Arlan Richardson, David Weber, Alan Katz, Brian Wilkinson. Includes bibliographical references (leaves 150-166) and abstract. Also available in print.

Thesis (Ph. D.)--Illinois State University, 1991. / Title from title page screen, viewed December 8, 2005. Dissertation Committee: Herman E. Brockman (chair), Radheshym K. Jayaswal, Alan J. Katz, David F. Weber, Brian J. Wilkinson. Includes bibliographical references (leaves 113-125) and abstract. Also available in print.