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.

Les ribozymes sont des ARN catalytiques fréquemment exploités pour le développement d’outils biochimiques et d’agents thérapeutiques. Ils sont particulièrement intéressants pour effectuer l’inactivation de gènes, en permettant la dégradation d’ARNm ou d’ARN viraux associés à des maladies. Les ribozymes les plus utilisés en ce moment pour le développement d’agents thérapeutiques sont les ribozymes hammerhead et hairpin, qui permettent la reconnaissance spécifique d’ARN simple brin par la formation de structures secondaires stables. In vivo, la majorité des ARN adoptent des structures secondaires et tertiaires complexes et les régions simples brins sont parfois difficiles d’accès. Il serait intéressant de pouvoir cibler des ARN repliés et un motif d’ARN intéressant à cibler est la tige-boucle d’ARN qui peut être importante dans le repliement global des ARN et pour accomplir des fonctions biologiques. Le ribozyme VS de Neurospora fait la reconnaissance de son substrat replié en tigeboucle de façon spécifique par une interaction kissing-loop, mais il n’a jamais été exploité pour faire la reconnaissance d’un ARN cible très différent de son substrat naturel. Le but des travaux présentés dans cette thèse est de déterminer si le ribozyme VS possède l’adaptabilité nécessaire pour l’ingénierie de ribozymes qui clivent des ARN cibles différents du substrat naturel. Dans le cadre de cette thèse, le ribozyme VS a été modifié pour l’adapter à différents substrats et des études de cinétiques ont été réalisées pour évaluer l’impact de ces modifications sur l’activité de clivage du ribozyme. Dans un premier temps, le ribozyme a été modifié pour faire la reconnaissance et le clivage de substrats possédant différentes longueurs de tiges Ib. Le ribozyme a été adapté avec succès à ces substrats de différentes longueurs de tige Ib, avec une activité qui est similaire à celle du ribozyme avec un substrat sans modification. Dans un deuxième temps, c’est l’interaction kissing-loop I/V du ribozyme qui a été substituée de façon rationnelle, dans le but de savoir si un ribozyme VS mutant peut reconnaitre et cliver un substrat ayant une boucle différente de celle de son substrat naturel. L’interaction kissing-loop I/V a été substituée pour les interactions kissing-loop TAR/TAR* de l’ARN du VIH-1 et L22/L88 de l’ARN 23S de Deinococcus radiodurans. La réaction de iii clivage des ribozymes comportant ces nouvelles interactions kissing-loop est toujours observée, mais avec une activité diminuée. Finalement, la sélection in vitro (SELEX) de ribozymes a été effectuée pour permettre un clivage plus efficace d’un substrat mutant avec une nouvelle boucle. Le SELEX a permis la sélection d’un ribozyme qui clive un substrat avec une boucle terminale mutée pour celle de l’ARN TAR du VIH-1 et cela avec une activité de clivage très efficace. L’ensemble de ces études démontre que le ribozyme VS peut être modifié de diverses façons pour la reconnaissance spécifique de différents substrats, tout en conservant une bonne activité de clivage. Ces résultats montrent le grand potentiel d’ingénierie du ribozyme VS et sont prometteurs pour la poursuite d’études d’ingénierie du ribozyme VS, en vue du clivage d’ARN cibles repliés en tige-boucle complètement différents du substrat naturel du ribozyme VS. / Ribozymes are catalytic RNAs frequently exploited for the development of biochemistry tools and therapeutic agents. They are particularly interesting in gene inactivation strategies for the degradation of mRNA and viral RNA genome. Currently, the most common ribozymes used in the development of therapeutic agents are the hammerhead and hairpin ribozymes, which can specifically recognize and cleave target single-stranded RNAs through the formation of stable secondary structures. In vivo, single-stranded RNAs can be difficult to target because most RNA adopt complex secondary and tertiary structures. It could be worthwhile to target folded RNA motifs, and an interesting target is the stem-loop structure because stem-loops are important for the overall folding of RNA molecules and they can perform important biological functions. The Neurospora VS ribozyme recognizes its stem-loop folded substrate via a specific kissing-loop interaction, but it has never been exploited for the recognition of target RNA very different from its natural substrate. The goal of the work presented in this thesis is to determine whether the VS ribozyme possesses the necessary adaptability for engineering ribozymes that target RNAs different from its natural substrate. For this thesis, the VS ribozyme was adapted for the recognition of different substrates and kinetic studies were performed to evaluate the effect of these modifications on the cleavage activity. In a first study, the VS ribozyme was modified to recognize and cleave substrates with different stem Ib lengths. The VS ribozyme was successfully adapted to theses substrates of different lengths with a cleavage activity similar to the unmodified ribozyme and substrate. In a second study, the I/V kissing-loop interaction was substituted by rational design, in order to establish if the VS ribozyme variants could recognize and cleave a substrate with a different loop than its natural substrate. The I/V kissing-loop interaction was substituted for the HIV-1 TAR/TAR* and the Deinococcus radiodurans RNA large rRNA subunit L22/L88 kissing-loop interactions. The cleavage reaction was observed for the ribozymes with these new interactions, but with reduced activity. Finally, in vitro selection (SELEX) was used to enable more efficient cleavage of a mutant substrate with a new loop. SELEX experiments enabled v the selection of ribozyme variants that cleave a substrate with a terminal loop mutated to that of the HIV-1 TAR RNA with a very efficient cleavage activity. All of the studies presented in this thesis show that the VS ribozyme can be modified in various ways for the specific recognition of different substrates, while maintaining efficient cleavage activity. These results demonstrate the great potential of VS ribozyme engineering and are very promising for further engineering studies of VS-derived ribozymes for the cleavage of stem-loop folded target RNA completely different from its natural VS ribozyme substrate.

In this thesis work, I have used a comparative genomics approach to study a fungal model organism, Neurospora tetrasperma. My specific focus has been on genomic introgression, intron evolution, chromosomal structural rearrangements and codon usage. All of the studies are based on large-scale dataset generated by next-generation sequencing technology (NGS), combined with other techniques, such as Optical Mapping. In the introgression study, we detected large-scale introgression tracts in three N. tetrasperma lineages, and the introgression showed allele-specific and chromosomal-specific pattern. In the study of introns, we found indications of mRNA mediated intron loss and non-homologous end joining (NHEJ) mediated intron gains in N. tetrasperma. We found that selection is involved in shaping intron gains and losses, and associated with intron position, intron phase and GC content. In the study of chromosomal structural rearrangements, we found a lineage specific chromosomal inversion pattern in N. tetrasperma, which indicates that inversions are unlikely to associate with the origin of the suppressed recombination and the mating system transition in N. tetrasperma. The result suggests inversions are the consequences, rather than the causes, of suppressed recombination on the mating-type chromosome of N. tetrasperma. In the final study, analyses of codon usage indicated that the region of suppressed recombination in N. tetrasperma is subjected to genomic degeneration, and selection efficiency has been much reduced in this region.

Jordens befolkning förväntas ha överstigit 9 miljarder till 2050, vilket leder till en ökning av matbehov med 70%. Ungefär en tredjedel av all producerad mat slängs idag, speciellt grönsaker, bröd och annan mat med kort hållbarhetsdatum. Det är väldigt viktigt att hitta sätt att minska och återanvända avfallet om miljöpåverkan ska kunna minskas. Brödrester är en stor del av Sveriges totala matavfall och använt spannmål (BSG) står för den största delen av biprodukter från bryggningsindustrin. De kan tillsammans användas som substrat för att fermentera en svampburgare som är rik på näring och protein. Denna studien syftar till att bestämma vilken filamentös svamp, N. intermedia eller R. oryzae, som ger de bästa resultaten med avseende på konsistens, proteininnehåll, smak och utseende. Detta gjordes genom textur- och proteinanalys på svampburgarna. Ingen av svamparna utmärkte sig med märkvärt bättre resultat gällande konsistensen, men resultaten för studien hade en del osäkerheter i resultatet på grund av otillräcklig tillväxt. Med avseende på smak var N. intermedia att föredra, då den hade en sötare och mer behaglig smak. Vad gäller utseende så ser båda burgarna väldigt aptitliga ut, och de anses ha stor potential som köttsubstitut, men vidare forskning inom området krävs. / Earth’s population is expected to have exceeded 9 billion people by 2050, leading to a 70% increase in food demand. Today, approximately one quarter to one third of all food produced goes to waste, especially vegetables, bread and other foods with short shelf life. Finding ways to reduce and reuse the waste is very important if the impact on the environment is to be decreased. Stale bread accounts for a large part of Sweden’s total food waste and brewers spent grain (BSG) is the brewing industry’s major by-product. Combined, they can be used as a substrate to produce a fungal fermented burger, rich in nutrients and protein. This study aims to determine which fungus, N. intermedia and R. oryzae, provides the best results regarding texture, protein content, taste and appearance. This was done by performing texture analysis and protein analysis on the fermented burgers. None of the fungi showed significantly better results than the other regarding texture. However, the results from this study have a few uncertainties due to lack of growth. The fungus that was preferred when considering taste was N. intermedia, which had a sweeter and more pleasant taste in general. Regarding the appearance of the fermented burgers, they are very different but they both are equally as appealing to the eye. The fermented burgers show great potential as meat substitute, but extended research is required.

Det pågår ständigt forskning på att göra bioetanolen till ett mer konkurrenskraftigt bränsle gentemot fossila bränslen. Flera försök till att optimera processen både ekonomiskt och energimässigt görs i flera länder världen över. I detta examensarbete har den filamentösa svampen Neurospora intermedia odlats i tunndrank som är en restprodukt från bioetanolsproduktionen. Olika utspädningar på tunndranken har använts för att se vilken som producerar mest bioetanol. Enligt detta examensarbete produceras det mest bioetanol när N. intermedia odlas i 50 % utspädd tunndrank. När den optimala utspädningen hade hittats prövades det vilken tid under odlingen som N. intermedia producerade den högsta koncentrationen bioetanol. Efter 39 timmar hade det producerats 4,34 g/l bioetanol och det var även vid denna tidpunkt som biomassan hade de bästa protein- och RNA-innehållen för att kunna torka biomassan och producera djurfoder till lantbruket.

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.

The aim of this thesis was to study the evolution of reproductive systems and reproductive traits in the fungal genus Neurospora. More specifically, I have investigated the evolutionary forces shaping the genes involved in sexual reproduction, focusing on mating-type (mat) and pheromone receptor (pre) genes. Neurospora contains species exhibiting three different mating systems, i.e., heterothallism (self-incompatibility), homothallism (self-compatibility) and pseudohomothallism (partial self-incompatibility). First, a robust phylogeny of Neurospora was established. The phylogenetic analyses revealed multiple independent transitions in reproductive life style during the evolutionary history of the genus. We argued for a heterothallic ancestor of the genus, although our subsequent ancestral reconstruction analyses favored a homothallic ancestor. To be able to settle the ancestral mating system, we zoomed in on the structural architecture of the mat-locus in four homothallic species of Neurospora, thought to have arisen from independent transitions. Our results led us to suggest two different genetic mechanisms (translocation and unequal crossover) to explain the transitions in mating system from heterothallism to homothallism. We pointed out that the mating-system transitions in Neurospora are unidirectional, and suggested that transposable elements might be driving the transitions. In conclusion, we suggest a heterothallic ancestor for Neurospora, and that at least six transitions to homothallism and two transitions to pseudohomothallism have occurred in its evolutionary history. Further, we used the phylogeny of Neurospora as a framework to test if the evolution of pre-genes (pre-1 and pre-2) in hetero- and homothallic Neurospora is dependent on mating systems and/or even the homothallic clades themselves (i.e., mating-system and/or switch-dependent). The molecular evolution results suggest that pre-1 and pre-2 are overall functional in both homothallic and heterothallic Neurospora. The molecular evolution of pre-1 seems to be independent of mating-system or homothallic clade, and we detected signs for positive selection in the C-terminal tail. For pre-2 we found no support for mating-system dependent evolution, but indications for switch-dependent evolution. In this study we also included expression analyses of both pre- as well as mat-genes, with the prospect to assess functionality and regulation. During this thesis work, we also performed a phylogenetic study were we found that reproductive genes might be more permeable to introgression than other genes, which is in contrast to theoretical expectations. In the last study, we confirmed the co-existence of two alternative splice variants of the pheromone receptor gene pre-1 in Neurospora crassa, and performed expression profiles studies using quantitative RT-PCR. I hope this thesis work will further strengthen Neurospora as a model for research in evolutionary genetics.

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.

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.