Thèses sur le sujet « Funori »

Dopo alcuni risultati preliminari di algebra commutativa, la tesi introduce e sviluppa lo studio del funtore di tensorizzazione e del funtore Hom, e dei loro derivati Tor e Ext su moduli generici e con esempi di calcolo su Z.

Le micotossine sono metaboliti secondari prodotti da alcuni funghi filamentosi, che possono contaminare piante coltivate o alimenti e mangimi immagazzinati; tra questi i più importanti funghi micotossigeni coinvolti nella contaminazione alimentare, appartengono a tre generi: Aspergillus, Fusarium e Penicillium. Più di 300 micotossine sono state identificate e questi metaboliti secondari possono essere dannosi per la salute umana ed animale quando ingeriti (Bennett and Klich, 2003). Le principali micotossine contaminati di alimenti e mangimi sono: aflatossine, ocratossina, fumonisine, tricoteceni e zearalenone. Le aflatossine rappresentano la classe più importante di micotossine, comunemente presenti nel mais e in altri cereali, i principali funghi responsabili della loro produzione sono Aspergillus flavus e A. parasiticus (Shepard, 2008). Invece i cereali, il caffè, il cacao, il vino, la birra e gli alimenti di origine animale sono spesso contaminati dall'ocratossina A, prodotta principalmente da A. ochraceus (Van der Merwe et al., 1965), A. carbonarius, Penicillium verrucosum e P. nordicum. Fusarium verticillioides e F. proliferatum producono fumonisine, che sono spesso rilevate nel mais e nei sottoprodotti (Dutton, 1996). I trichoteceni, che si trovano spesso nei cereali, in particolare nel frumento e nel mais, sono divisi in quattro gruppi, i due principali sono: il tipo A con le tossine T2 e HT-2, prodotte da F. langhsetiae e F. sporotrichioides (Van der Fels-Klerk e Stratakou, 2010); il tipo B con il deossinivalenolo (DON) e il nivalenolo (NIV) prodotti da F. graminearum e F. culmorum (Placinta et al., 1999; Turner, 2010). Inoltre, la contaminazione da DON si trova frequentemente in associazione con un'altra micotossina prodotta dagli stessi funghi, lo zearalenone (ZEA) (Logrieco et al., 2002a). Queste specie sono responsabili di infezioni che si verificano sia nel campo che durante la conservazione post-raccolta, in particolare quando i cereali sono immagazzinati in condizioni inadeguate (ad esempio alte temperature e alta umidità). Una grande varietà di effetti tossici negli animali e nell'uomo è stata osservata a causa dell'ingestione di cibo contaminato da micotossine, quali: immunosoppressione, effetti cancerogeni, genotossici, teratogeni o mutageni (Peraica et al., 1999; Richard, 2007). La contaminazione da micotossine è diventata una preoccupazione per la salute pubblica con gravi implicazioni economiche ed etiche. Dal momento che non è completamente possibile impedire la produzione di micotossine, le autorità nazionali e internazionali hanno adottato limiti regolatori e linee guida per monitorare i livelli di micotossine in vari prodotti alimentari e nei mangimi (EC 2006a e 2006b; Commision Recommendation 2013/165/UE). Diversi metodi fisici, chimici e biologici sono stati raccomandati per la detossificazione di alimenti e mangimi contaminati da micotossine. Tuttavia, solo alcuni di essi sono stati accettati per l'uso pratico. Molti specialisti pensano che l'approccio migliore per la decontaminazione da micotossine debba essere la degradazione biologica, dando la possibilità di rimuovere micotossine in blande condizioni, senza usare sostanze chimiche dannose e senza perdite significative nel valore nutritivo e nell’appetibilità di alimenti o mangimi detossificati. A seconda della loro modalità di azione, questi additivi per mangimi possono agire legando micotossine alla loro superficie (adsorbimento) o degradandoli o trasformandoli in metaboliti meno tossici (biotrasformazione). L'efficacia del legante di queste sostanze si basa sulle proprietà sia del legante che della micotossina. La biotrasformazione può essere ottenuta da enzimi che degradano le micotossine o da microrganismi (funghi e batteri) che producono tali enzimi. Vari adsorbenti inorganici, alluminosilicati e carboni attivi, sono stati testati e utilizzati come leganti micotossine (MB). Un'alternativa interessante agli adsorbenti inorganici per la detossificazione delle micotossine è l'uso di leganti organici, come i componenti della parete cellulare del lievito, i batteri dell'acido lattico, i conidi degli Aspergilli. Questi MB sono utilizzati l’alimentazione animale al fine di ridurre l'assorbimento delle micotossine dal tratto gastrointestinale e la loro distribuzione al sangue e agli organi bersaglio, prevenendo o riducendo le micotossicosi nel bestiame. Recentemente, l'uso di tali sostanze come additivi per mangimi tecnologici è stato ufficialmente autorizzato dall'Unione europea (Commission Regulation 2015/786). Enzimi ligninolitici, come le laccasi, dai funghi del marciume bianco, come Pleurotus spp. che catalizzano l'ossidazione di un ampio spettro di composti fenolici e di ammine aromatiche utilizzando l’ossigeno molecolare come accettore di elettroni, che viene quindi ridotto ad acqua (Reinhammar e Malstrom, 1981). Aggiungendo alla reazione l’appropriato mediatore redox può estendere l'attività degli enzimi laccasi a substrati non fenolici, come le micotossine. Questa tesi di dottorato è organizzata in sei capitoli e un allegato, in cui sono descritte le seguenti attività. Nel Capitolo 1 sono stati raccolti centosettantacinque campioni di grano durante le stagioni agricole: 2013-2014, 2014-2015 e 2015-2016 in diverse regioni italiane. I tricoteceni (DON, NIV, tossine T2 e HT-2) e i livelli di ZEA sono stati monitorati attraverso l'uso di metodi analitici validati, per fornire una visione d’insieme della distribuzione italiana delle micotossine nel grano. Le specie di Fusarium isolate dai semi sono state identificate in base alle loro caratteristiche morfologiche. Nel Capitolo 2, lo sviluppo di una tecnologia innovativa per la bioremedation del mais contaminato dall'AfB1 e della sua bioconversione in mangime ad un alto apporto nutrizionale, è stato realizzato attraverso lo sfruttamento della capacità degradativa del Pleurotus eryngii. A tale scopo, è stata studiata l'attività degradativa dei confronti dell’AfB1 da parte di un estratto enzimatico grezzo ottenuto da un substrato esausto e la capacità del fungo commestibile P. eryngii di degradare l'AfB1 sia in vitro che in una coltivazione dei funghi su scala di laboratorio. Nel Capitolo 3, è stata valutata la capacità del micelio non vitale del P. eryngii (ITEM 13681) di assorbire l’AfB1. Sono stati valutati l'influenza di diversi parametri, quali: pH (5, 7), concentrazioni dell’AfB1 (da 50 a 1000 ng/mL), tempo (da 30 a 120 min), temperatura (25, 37 ° C), massa fungina (da 50 a 1000 mg), sulla capacità di assorbimento del micelio di P. eryngii. La stabilità del legame AfB1-bioassorbente e gli studi di desorbimento sono stati effettuati variando, rispettivamente, il pH a 7 e 3, per 24 ore di incubazione a temperatura ambiente e al buio. Nel capitolo 4, l'attività di degradazione di due laccasi ottenute da due funghi commestibili (P. eryngii e P. pulmonarius) nei confronti di AfB1, AfM1, FB1, ZEA e tossina T2, sono state valutate singolarmente, aggiungendo alla reazione mediatori naturali ed artificiali. L'effetto dei sistemi laccasi-mediatore (LMSs) è stato analizzato mediante cromatografia liquida ad elevata prestazione accoppiata ad uno specifico rivelatore, scelto in base alle caratteristiche chimiche di ciascuna tossina. Nel capitolo 5, l'obiettivo perseguito era quello di investigare l'azione dei sistemi laccase-mediatore (LMSS) verso più tossine, simultaneamente. A tal fine, sono stati eseguiti diversi test di degradazione, esaminando l'effetto dei diversi mediatori, come acetosyringone (AS), syringaldehyde (SA) e del mediatore sintetico 2,2,6,6-tetrametil-piperidinil-ossil (TEMPO), sull'attività della laccasi da Trametes versicolor (CE 1.10.3.2) verso l'acido fusarico (FA) e micotossine, come: DON, T2, FB1, AfB1, OTA e ZEA. Un metodo multi micotossina, è stato sviluppato per determinare simultaneamente queste sette tossine, mediante cromatografia liquida con spettrometria di massa in tandem (LC-MS/MS). Nel capitolo 6, l'attività di degradazione dell’enzima laccasi verso lo ZEA è stata ulteriormente investigata. I prodotti della biodegradazione sono stati monitorati mediante cromatografia liquida con spettrometria di massa ad elevata risoluzione (LC-HRMS). I dati sono stati elaborati dal software MassHunter Workstation (Qualitative Analysis Navigator e Workflow di analisi qualitativa, versione B.08.00), Mass Profile Professional (versione 14.08) e MassHunter Molecular Structure Correlator (versione B.08.00)) di Agilent Technologies, per consentirne l'identificazione. Nell'Annesso A, la produzione di Enniatine (A, A1, B e B1) e Beauvericina da varie specie di Fusarium è stata valutata mediante cromatografia liquida ad alte prestazioni accoppiata con array di fotodiodi e spettrometro di massa a singolo quadrupolo (UPLC-PDA-QDa).
Mycotoxins are secondary metabolites produced by certain filamentous fungi, which can contaminate crop plants or stored food and feed; among them the most important mycotoxigenic fungi involved in food contamination, belong to three genera: Aspergillus, Fusarium and Penicillium. More than 300 mycotoxins have been identified and these secondary metabolites can be harmful to human and animal health when ingested (Bennett and Klich, 2003). Main mycotoxins contaminant in food and feed are: aflatoxins, ochratoxins, fumonisins, trichothecenes and zearalenone. Aflatoxins represent the most important class of mycotoxins, commonly found in maize and other cereals, the main fungi responsible for their production are Aspergillus flavus and A. parasiticus (Shepard, 2008). Instead grains, coffee, cocoa, wine, beer, and foods from animal origin are often contaminated by ochratoxin A, that is mainly produced by A. ochraceus (Van der Merwe et al., 1965), A. carbonarius, Penicillium verrucosum and P. nordicum. Fusarium verticillioides and F. proliferatum produce fumonisins, which are often detected in maize and by-products (Dutton, 1996). Trichothecenes, which are often found in cereal grains, in particular in wheat and maize, are divided in four groups, the principal two groups are: type-A with T2 and HT-2 toxin, produced by F. langhsetiae and F. sporotrichioides (Van der Fels-Klerk and Stratakou, 2010); Type-B with deoxynivalenol (DON) and nivalenol (NIV) produced by F. graminearum and F. culmorum (Placinta et al., 1999; Turner, 2010). Moreover, DON contamination is frequently found in association with another mycotoxin produced by the same fungi, zearalenone (ZEA) (Logrieco et al., 2002a). These species are responsible for infections occurring both in the field and during postharvest storage, particularly when cereals are stored under inappropriate conditions (e.g. high temperatures and high humidity). A large variety of toxic effects in animals and humans has been observed due to the ingestion of food contaminated with mycotoxins, such as: immunosuppression, carcinogenic, genotoxic, teratogenic or mutagenic effects (Peraica et al., 1999; Richard, 2007). Mycotoxin contamination became a public health concern with serious economical and ethical implications. Since it is not completely possible to prevent the synthesis of mycotoxins, national and international authorities have adopted regulatory limits and guidelines to monitor mycotoxin levels in various food and feed products (EC 2006a and 2006b; Commission Recommendation 2013/165/UE). Different physical, chemical and biological methods have been recommended for detoxification of food and feed contaminated by mycotoxins. Nevertheless, only a few of them have been accepted for practical use. A lot of specialists think that the best approach for mycotoxin decontamination should be the biological degradation, giving the possibility to remove mycotoxins under mild conditions, without using harmful chemicals and without significant losses in nutritive value and palatability of detoxified food or feed. Depending on their mode of action, these feed additives may act either by binding mycotoxins to their surface (adsorption), or by degrading or transforming them into less toxic metabolites (biotransformation). The binder efficacy of these substances is based on the properties of both the binder and the mycotoxin. Biotransformation can be achieved by mycotoxin-degrading enzymes or by microorganisms (fungi and bacteria) producing such enzymes. Various inorganic adsorbents, aluminosilicate and activated carbons, have been tested and used as mycotoxins binders (MB). An interesting alternative to inorganic adsorbents for the detoxification of mycotoxins is the use of organic binders, such as, cell wall components of yeast, lactic acid bacteria, conidia of Aspergilli. These MB are used to feed animal diet in order to reduce the absorption of mycotoxins from the gastrointestinal tract and their distribution to blood and target organs, thus preventing or reducing mycotoxicosis in livestock. Recently, the use of such substances as technological feed additives has been officially allowed in the European Union (Commission Regulation 2015/786). Ligninolytic enzymes, such as laccase, from white-rot fungi, as Pleurotus spp. catalyzed the oxidation of a broad number of phenolic compounds and aromatic amines by using molecular oxygen as the electron acceptor, which is then reduced to water (Reinhammar and Malstrom, 1981). Adding the appropriate redox mediator to the reaction can extend the activity of the laccase enzymes to nonphenolic substrates, such as mycotoxins. This PhD thesis is organized into six chapters and one annex, where the following tasks are described. In Chapter 1, one hundred and seventy-five wheat samples were collected during the growing seasons: 2013-2014, 2014-2015 and 2015-2016 in different Italian regions. Trichothecenes (DON, NIV, HT-2 and T2 toxins) and ZEA levels were monitored through the use of validated analytical methods, to provide an overview of the Italian distribution of mycotoxins in wheat. The Fusarium species isolated from the kernels were identified, based on their morphological characteristics. In Chapter 2, the development of an innovative technology for the bioremediation of AfB1-contaminated maize and its bioconversion into high nutritional feed, was realized through the exploitation of the degradative capability of Pleurotus eryngii. For this purpose, the AfB1–degradative activity of a crude enzymatic extract from a spent substrate and the ability of the white-rot and edible fungus P. eryngii to degrade AfB1 both in vitro and in a laboratory-scale mushroom cultivation, were investigated. In Chapter 3, the power of ground not-viable mycelium of P. eryngii (ITEM 13681) to absorb AfB1, was assessed. The influence of different parameters: pH (5, 7), AfB1 concentrations (50 and 1000 ng/mL), time (30 and 120 min), temperature (25 and 37°C), fungal mass (50 and 1000 mg), on the absorption capability of the mycelium of P. eryngii. were evaluated. Binding stability of AfB1-biosorbent and desorption studies were carried out varying, respectively, the pH to 7 and 3, for 24 hours of incubation at room temperature in the dark. In Chapter 4, the degradation activity of two laccases from two edible fungi (P. eryngii and P. pulmonarius) towards AfB1, AfM1, FB1, ZEA and T2 toxin, were evaluated separately, adding to the reaction natural and artificial mediators. The effect of laccase-mediator systems (LMSs) were analyzed by liquid chromatography with specific detector, based on the chemical feature of each single toxin. In Chapter 5, the aim pursued was to investigate the action of LMSs toward multiple toxins. For this purpose, several degradation assays were performed, screening the effect of different mediators, as acetosyringone (AS), syringaldehyde (SA), and synthetic mediator as 2,2,6,6-tetramethyl-piperidinyloxyl (TEMPO), on the activity of laccase from Trametes versicolor (EC 1.10.3.2) towards fusaric acid (FA) and mycotoxins, such as: DON, T2, FB1, AfB1, OTA and ZEA. A multi mycotoxin method, was set up to simultaneously screen these seven toxins, by liquid chromatography/tandem mass spectrometry (LCMS/ MS). In Chapter 6, the biodegrading activity of laccases enzymes towards ZEA has been further investigated. The degradation products were monitored by liquid chromatography–high resolution mass spectrometry (LC-HRMS). Data were processed by MassHunter Workstation Software (Qualitative Analysis Navigator and Qualitative Analysis Workflow, version B.08.00), Mass Profile Professional (version 14.08) and MassHunter Molecular Structure Correlator (version B.08.00)) from Agilent Technologies, to allow their identification. In Annex A, the Enniatins (A, A1, B and B1) and Beauvericin production from various Fusarium spp. were measured by ultra-performance liquid chromatography coupled with photodiode array and single quadrupole mass spectrometer (UPLC-PDA-QDa).

Il fenomeno della contaminazione chimica dei sedimenti marini costieri è molto diffuso e rappresenta una grande preoccupazione per il benessere della biodiversità e dell’ecosistema. Il biorisanamento è una strategia ecocompatibile che sta ottenendo sempre più attenzione grazie al suo potenziale di ripulire sedimenti marini contaminati. In questa tesi di dottorato, prima di tutto, ho fornito una panoramica delle attuali conoscenze e prospettive sul biorisanamento di sedimenti marini, presenti in letteratura. Quindi ho valutato la diversità degli assemblaggi microbici in diversi sedimenti cronicamente contaminati delle aree di Bagnoli-Coroglio, Mar Piccolo di Taranto e Falconara Marittima (tutti inclusi nell'elenco dei Siti di Interesse Nazionale, SIN) e le loro relazioni con il livello e la tipologia degli inquinanti chimici. Ho testato l'efficienza delle strategie di biostimolazione nella degradazione degli Idrocarburi Policiclici Aromatici (IPA) in sedimenti a diverso livello di contaminazione sulla base di approcci di aggiunta di nutrienti inorganici e bioaumento utilizzando i singoli consorzi batterici o fungini o entrambi, precedentemente isolati e identificati; ho inoltre studiato i cambiamenti nella ripartizione dei metalli e nella diversità microbica dovuti ai biotrattamenti. I risultati qui presentati suggeriscono che i contaminanti chimici possono avere un ruolo importante nel modellare la diversità procariotica, potenzialmente selezionando taxa microbici tolleranti/resistenti. I sedimenti di Falconara Marittima ospitano taxa microbici con un'elevata capacità di biobonifica verso gli IPA. Questi taxa microbici, batteri e funghi, una volta isolati e cresciuti su terreni specifici, possono essere efficaci per il biorisanamento dei sedimenti di Bagnoli altamente contaminati da IPA. Nonostante i risultati riportati in questo studio non consentano di distinguere tra l'importanza relativa dei taxa microbici alloctoni e autoctoni sulla biodegradazione degli IPA, forniscono nuove conoscenze sulle interazioni batterico-fungine che si verificano durante il biorisanamento dei sedimenti marini altamente contaminati. Complessivamente, questi risultati suggeriscono che i biotrattamenti basati su consorzi batterici e/o fungini selezionati o su una combinazione di entrambi potrebbero costituire una strategia efficace per ridurre significativamente, in tempi relativamente brevi, la contaminazione da IPA dei sedimenti marini, portando possibilmente a opzioni di gestione alternative rispetto al dragaggio e allo smaltimento in discarica.
Chemical contamination of coastal marine sediments is a widespread phenomenon and represents a major concern for biodiversity and ecosystem health. Bioremediation is an environmental-friendly strategy gaining increasing attention for its potential to clean-up contaminated marine sediments. In this PhD thesis, first of all, I provided an overview of the current knowledge and perspectives on the bioremediation of marine sediments, based on literature review. Then I assessed the diversity of microbial assemblages in different chronically contaminated sediments of the Bagnoli-Coroglio, Mar Piccolo of Taranto and Falconara Marittima areas (all of them included in the list of Sites of National Remediation Interest) and their relationships with the level and typology of chemical pollutants. I tested the efficiency of biostimulation strategies based on inorganic nutrient addition and bioaugmentation approaches using selected bacterial or fungal consortia or both, previously isolated and identified, on PAH degradation in sediments displaying different contamination level and I investigated changes in metal partitioning and microbial diversity due to biotreatments. Results presented here suggest that chemical contaminants can have an important role in shaping prokaryotic diversity, potentially by selecting tolerant/resistant microbial taxa. Sediments of Falconara Marittima host microbial taxa with a high bioremediation capacity toward PAHs. These microbial taxa, including both bacteria and fungi, once isolated and growth on selected media, can be effective for the bioremediation of Bagnoli sediments highly contaminated with PAHs. Despite findings reported in this study do not allow disentangling the relative importance of the allochthonous vs. autochthonous microbial taxa on the biodegradation of PAHs, they provide new insights on bacterial-fungal interactions occurring during bioremediation of highly contaminated marine sediments. Overall, these results suggest that biotreatments based on selected bacterial and/or fungal consortia or a combination of both could be an effective strategy to significantly reduce in a relatively short time PAH contamination of marine sediments, possibly leading to alternative management options compared to dredging and landfill disposal.

2015 - 2016
The research was devoted entirely to the study of funeral pyres dating from the second half of the 4th century BC and the beginning of II century BC, attested in nine different necropolis selected as a sample area - Verghina, Derveni, Thessaloniki, Aineia, Aghios Athanasios, Pydna, Methone, Lefkadia and Pella - and located along the Thermaic Gulf of Central Macedonia. The purpose of the investigation was to reconstruct the funerary rite of secondary deposition cremation, which in Macedonia is often performed by the sovereigns and the aristocratic class in the so-called "heroic" way described in the Homeric text of the Iliad. This funerary practice, in which pyre and burial do not coincide but constitute two distinct moments of a single complex funeral process, expresses behavior codes that are reflected in a series of clearly recognizable material signs in the archaeological excavation. The reconstruction has been attempted with the exclusive help of the archaeological data retrieved scattered in the bibliography so far published in modern Greek language, consisting of charred layers, outcome of funeral pyres, found rarely in situ, most frequently in a secondary deposition, accumulated around or above the corresponding burials. Interest has thus focused on the identification of this particular burial costume’s passage, the last ring with a strong ritual value, of a long chain that ends with the erection of the artificial mound. In single context, on the basis of the funeral or sacrificial nature of the investigated charred residue, a reconstructive hypothesis is proposed, of both the funeral pyre, which always goes beyond a simple pile of wood placed on the ground, and the sacrificial act - enagismòs - offered with fire in honor of the deceased, after his burial and erection of the mound. From the comparison of individual partial hypotheses, linked to a specific funerary context, facilitated by creating an elaborated ad hoc synoptic table, attempts were made to deduce considerations of a general nature which could give the idea of the entire ritual process’s carrying out, at least in its most macroscopic passages. The rearrangement of the data obtained from the edited bibliography enabled a comprehensive comparison of the charcoal layers, by listing the different aspects and variants, by highlighting the preferences regarding the location of the piles in relation to the grave, by distinguishing the pyres found in a primary deposition from those found in a secondary deposition, by considering the choices on the funeral setup, by analyzing the various classes of materials found inside them to argue recurrences and constants, linked to the rank, gender and age of the deceased. The archaeological data - the charred stratifications pertinent to funeral pyres - if identified in its distinctive features and interpreted in the correct manner, today renders likelihood to the so-called "homeric" or "heroic" funeral rite, so far considered simply a story produced by literary fiction. Such costume, made for and by royal or equestrian high rank personalities, is an expression of an aristocratic world with a purely warlike character; with the advent to the power of Philip II and then of Alexander the Great, we are witnessing the realization of monumental funeral pyres, the rediscovery and the voluntary imitation of the "homeric" funeral costume, practiced by the royalists and members of the Macedonian court in Aegae (modern Verghina) but also in the rest of the territorial area investigated, strongly marked by the presence of the Macedonians. [edited by author]
XXIX n.s.

Endocytosis is little understood in filamentous fungi. For some time it has been controversial as to whether endocytosis occurs in filamentous fungi. A comparative genomics analysis between Saccharomyces cerevisiae and 10 genomes of filamentous fungal species showed that filamentous fungi possess complex endocytic machineries. The use of the endocytic marker dye FM4-64, and various vesicle trafficking inhibitors revealed many similarities between endocytosis in the filamentous fungus Neurospora crassa, and endocytosis in budding yeast and mammalian cells. Actin polymerization was found to be crucial for endocytosis in N. crassa, and the microtubule cytoskeleton seemed to be necessary for long distance movement of putative early endosomes. Brefeldin A (BFA) blocked vesicular transport to the Spitzenkörper. Three putative endocytic proteins (WASP, clathrin light chain and Rab5) were labelled with fluorescent proteins in N. crassa. WASP-GFP was found to localise to motile, punctate structures in the plasma membrane just behind the hyphal apex in growing hyphae. This localisation changed to the hyphal apex when growth was temporarily arrested, indicating a possible role in endocytosis and polarized growth. Clathrin light chain-GFP was found to be concentrated in a region just behind the Spitzenkörper, which is consistent with there being a high concentration of clathrinmediated endocytosis in this region. Clathrin light chain-GFP also labelled putative Golgi and this labelling was found to be BFA sensitive, whereas BFA did not have a detectable effect on FM4-64 internalisation and organelle staining. GFP-Rab5 labelled putative early endosomes and decorated microtubules. Knock-outs of putative endocytic proteins in N. crassa, generated as part of the Neurospora genome consortium gene knock-out project, were analysed for defects in endocytosis. 14 out of 17 gene knock-outs were found to be ascospore lethal. The Rab5 knock-out was viable, but did not show a detectable effect on the endocytic internalisation of FM4-64 or its pattern of staining. However, it did exhibit a defect in sexual crossing.

This thesis discusses the issue of predicting of the effect of amino acid substitutions on protein funkcion, based on phylogenetic analysis method, inspired by tool MAPP. Significant number of genetic diseases is caused by nonsynonymous SNPs manifested as single point mutations on the protein level. The ability to identify deleterious substitutions could be useful for protein engineering to test whether the proposed mutations do not damage protein function same as for targeting disease causing harmful mutations. However the experimental validation is costly and the need of predictive computation methods has risen. This thesis describes desing and implementation of a new in silico predictor based on the principles of evolutionary analysis and dissimilarity between original and substituting amino acid physico-chemical properties. Developed algorithm was tested on four datasets with 74,192 mutations from 16,256 sequences in total. The predictor yields up to 72 % accuracy and in the comparison with the most existing tools, it is substantially less time consuming. In order to achieve the highest possible efficiency, the optimization process was focused on selection of the most suitable (a) third-party software for calculation of a multiple sequence alignment, (b) overall decision threshold and (c) a set of physico-chemical properties.

L'opinione pubblica percepisce fortemente il rischio derivante da sostanze chimiche sintetiche, ma ignora spesso quello derivante dalla presenza di sostanze tossiche naturali. Tra queste sostanze vengono classificate le micotossine; prodotti del metabolismo secondario di alcuni funghi. La maggior parte degli alimenti di origine vegetale e in particolare i cereali, possono andare incontro a contaminazione da parte di questi funghi, produttori di micotossine, in qualunque stadio. In particolare nel frumento le micotossine che si riscontrano più frequentemente sono l'ocratossina A (OTA) e il deossinivalenolo (DON), mentre nel mais, si riscontrano le Aflatossine e le fumonisine. Queste micotossine hanno effetti epatotossici, nefrotossici, mutageni e cancerogeni, è necessario perciò, un continuo monitoraggio della materia prima (frumento e mais), ma anche dei prodotti destinati all'alimentazione umana a base di questi cereali.
People experience deeply, the risk of synthetics chemicals substances, but often don't know the risk from naturals toxics substances. Between these substances, they are mycotoxins; fungi secondary metabolism's products. Most of vegetable food, as cereals, can suffer a mycotoxins contamination, for effect of these fungi, during any step of alimentary chain. In particular, in wheat , the mycotoxins more frequents are ochratoxin A end deossinivalenol, while in the maize can find aflatoxins and fumonisins. These mycotoxins can have hepatotoxics, nefrotoxics, mutagens and cancerogens effects. This study has monitored contamination's levels of raw material (wheat and maize), but also of foodstuff containing cereals.

L'opinione pubblica percepisce fortemente il rischio derivante da sostanze chimiche sintetiche, ma ignora spesso quello derivante dalla presenza di sostanze tossiche naturali. Tra queste sostanze vengono classificate le micotossine; prodotti del metabolismo secondario di alcuni funghi. La maggior parte degli alimenti di origine vegetale e in particolare i cereali, possono andare incontro a contaminazione da parte di questi funghi, produttori di micotossine, in qualunque stadio. In particolare nel frumento le micotossine che si riscontrano più frequentemente sono l'ocratossina A (OTA) e il deossinivalenolo (DON), mentre nel mais, si riscontrano le Aflatossine e le fumonisine. Queste micotossine hanno effetti epatotossici, nefrotossici, mutageni e cancerogeni, è necessario perciò, un continuo monitoraggio della materia prima (frumento e mais), ma anche dei prodotti destinati all'alimentazione umana a base di questi cereali.
People experience deeply, the risk of synthetics chemicals substances, but often don't know the risk from naturals toxics substances. Between these substances, they are mycotoxins; fungi secondary metabolism's products. Most of vegetable food, as cereals, can suffer a mycotoxins contamination, for effect of these fungi, during any step of alimentary chain. In particular, in wheat , the mycotoxins more frequents are ochratoxin A end deossinivalenol, while in the maize can find aflatoxins and fumonisins. These mycotoxins can have hepatotoxics, nefrotoxics, mutagens and cancerogens effects. This study has monitored contamination's levels of raw material (wheat and maize), but also of foodstuff containing cereals.

Forest fires have been the major stand-replacing/modifying disturbance in boreal forests. To adapt to fire disturbance, different strategies have evolved. This thesis focuses on wood fungi, and the effect of forest fire on this organism group. In many ways it is a study on adaptation to forest fire, in concurrence with adaptation to dry open habitats. In Paper I we study increased heat resistance in  mycelia from species prevalent in fire prone environments. Fungi were cultivated on fresh wood and exposed to different temperatures. Species prevalent in fire affected habitats had a much higher survival rate over all combinations of time and temperature compared to species associated with other environments. Based on this results the competitiveness was tested after temperature stress (paper II), three fire associated species, were tested against three non fire associated species. All fire associated species had a clear advantage after heat treatment, conquering a larger volume of wood than its competitor. In paper III we studied the effect of heat shock on decomposition rate, 18 species was tested. Species were cultivated and monitored for CO2 accumulation for 8 weeks and then heat shocked. All species including non fire associated species seemed to up-regulate decomposition after heat shock, this response was more pronounced in fire associated species. To look at the possible effect of forest fire on population structure (Paper IV), we developed 29 SNP/INDELs for Phlebiopsis. gigantea. We amplified the marker containing fragments in 132 individuals of P. gigantea in 6 populations, 3 which were found in areas affected by forest fire and 3 in unaffected areas. We found no genetic structure in accordance to forest fire. However we detected geographic structure, which stands in contrast to earlier studies. This might be due to the method, using SNP´s and number of individuals in the study. Finally we collected cross-sections of decayed logs to evaluate the number of fungal species domains that are likely to be hit when drilling a saw-dust sample in a log. We used these estimates to simulate how many species that will be found by a certain number of samples. We found that in 99% of the

Endophytic fungi inhabit the living tissue of a host plant for at least a portion of their life cycle. While some researchers have shown that various endophytic fungi participate in litter decomposition, we do not know whether such fungi are actually saprotrophic, meaning that they can obtain energy from litter. Therefore, I determined if endophytic fungi are saprotrophs using leaf litter as the energy source. All 49 tested isolates were found to be saprotrophic. To compare the saprotrophic capacities of fungi from different habitats, which produce different types of litter, a universal litter proxy needs to be used. I hypothesized that pure cellulose would be an adequate proxy for litter for in vitro studies because of its abundance in litter. This was tested in the first study. Saprotrophic capacity on pure cellulose was not highly correlated with that on leaf litter. I conclude, therefore, that cellulose may not be a good proxy for leaf litter. Some endophytic fungi are biotrophs, presumably acquiring energy from photosynthate produced by the host plant. This suggests that the level of exposure to sunlight by the plant should influence the competitive ability of such fungi. If saprotrophic endophytic fungi do exist, they ought to be less competitive against biotrophic endophytic fungi in leaves receiving full sunlight than in shaded leaves. I, therefore, hypothesized that the frequency of saprotrophy will be influenced by the level of sun exposure of the leaf from which the fungi were isolated. This was tested in the second study. Moreover, because closely related organisms ought to be more similar to each other than more distantly related organisms, I also hypothesized that saprotrophic capacity has a strong phylogenetic component, which was also tested in the second study. Unexpectedly, isolate identity within genus accounted for far more variability in saprotrophic capacity than genus identity, and sun exposure did not have a significant effect on saprotrophy. These results suggest that saprotrophic capacity may not be highly consequential in the ecology of these organisms.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The aim of this dissertation is to present a simpli cation of the proof of the following result obtained rst by Happel in [3]: If A is a nite-dimensional algebra over a eld algebraically closed K, then there is a triangulated, full and faithful functor of triangulated categories H : Db(modA) ! modA^, where A^ is the repetitive algebra obtained from A, which is also dense if A is of nite global dimension. We begin with a succinct presentation of the categorical language, approaching in general terms on the localization of categories, triangulated categories and their localizations, and nally derived categories, which are localized and triangulated categories. We also introduce the stable category of modules of a repetitive algebra A^. In the last chapter, we demonstrate the main result with the help of a result found in [8], in addition to the previously mentioned concepts.
O objetivo dessa disserta c~ao e trazer uma simpli ca c~ao da demonstra c~ao do seguinte resultado obtido primeiramente por Happel [3]: Se A e uma K- algebra de dimens~ao nita, ent~ao existe um funtor pleno, el e triangulado H : Db(modA) ! modA^, onde A^ e a a lgebra repetitiva obtida de A, que e tamb em denso se A e de dimensa~o global nita. Iniciamos com uma apresenta c~ao sucinta da linguagem categ orica, abordando de maneira geral sobre localiza c~ao de categorias, categorias trianguladas e suas localiza c~oes, e nalmente categorias derivadas, que s~ao categorias localizadas e trianguladas. Tamb em introduzimos a categoria est avel de m odulos da algebra repetitiva de A. No ultimo cap tulo, demonstramos o resultado principal com o aux lio de um resultado encontrado em [8], al em dos conceitos citados anteriormente.
São Cristóvão, SE

The Rosetta Stone is one of the most important stone fragments in history. It is the most popular single object in London’s British Museum, has been the object of scholarly research and has had much written about it. Indeed, any account of the history of translation will at least mention the Rosetta Stone. Today, the name “Rosetta” is used metaphorically in the context of translation, foreign-language learning, and even space exploration. In the light of this, one would assume that all sources are in agreement on the facts but, surprisingly, this is not the case. This article shows that sources disagree even in the most obvious aspects, such as the material, colour, condition of the stone and, in particular, with respect to its discovery. Based on an excursion to Alexandria, Rashíd and the – in all likelihood – real discovery site in the Nile delta, this article provides facts and casts some doubt on the reliability of internet sources.

This thesis is focused influence of external electromagnetic fields to function of speed sensors. In this work is created summary of individual principles speed sensors and possible sources electromagnetic fields.

In questa tesi analizzerò la tradizione sarda dei canti funebri, gli attitos, concentrandomi in particolar modo sulle capacità poetiche delle donne dedite a questo canto, le attitadoras. Partendo dai ricordi delle persone, mostrerò i particolari significati che questa pratica era in grado di creare e i contesti sociali e culturali capaci di attivare il canto. Nell'analizzare quello che in sardo si chiama "su donu", ovvero il dono poetico, la capacità innata di verseggiare, approfondirò tematiche come l'improvvisazione e l'eventuale iter formativo che le attitadoras affrontano per essere in grado di eseguire un bel canto. Ulteriore elemento di indagine, è l'aspetto del "saper piangere", affrontato anche da de Martino, così come il "saper ridere". Una disamina verterà anche sullo stato attuale di questa tradizione, ovvero su quanto si è conservato e quanto si è mutato o addirittura scomparso.

Endophytes are mutualistic fungi living in green tissue of all plants examined so far.Some of these fungi can produce compounds that are beneficial to the host plant, and it isalso known that some pathogenic fungi live parts of their lives as endophytes. Endophyticinteractions have been well characterized in various grasses, but much is unknown abouttheir interactions with trees. One reason for this is that the fungal biodiversity is muchlarger among endophytes in trees than in grasses, another is that screening for endophytestakes a lot of work. The goal of this thesis work was to develop a polymerase chainreaction (PCR) based method that is simple, fast and reliable for detection of endophytesin aspens. Eleven primer pairs were designed, each pair specific for one fungus. Afteroptimization and evaluation four of the primer pairs were found to be both specific andsensitive, and could detect fungus in DNA preparations from leaf samples.

Four aspects of horizontal genetic transfer during heterokaryon formation were examined in the asexual pathogen Fusarium oxysporum f.sp. cubense (Foe): 1) variability based on method of heterokaryon formation 2) differences in nuclear and mitochondrial inheritance 3) the occurrence of recombination without nuclear fusion 4) the occurrence of horizontal genetic transfer between distantly related isolates. The use of non- pathogenic strains of Fusarium oxysporum as biocontrol agents warrants a closer examination at the reproductive life cycle of this fungus, particularly if drag resistance or pathogenicity genes can be transmitted horizontally. Experiments were divided into three phases. Phase I looked at heterokaryon formation by hyphal anastomosis and protoplast fusion. Phase II was a time course of heterokaryon formation to look at patterns of nuclear and mitochondrial inheritance. Phase III examined the genetic relatedness of the different vegetative compatibility groups using a multilocus analysis approach. Heterokaryon formation was evident within and between vegetative compatibility groups. Observation of non-parental genotypes after heterokaryon formation confirmed that, although a rare event, horizontal genetic transfer occurred during heterokaryon formation. Uniparental mitochondria inheritance was observed in heterokaryons formed either by hyphal anastomosis or protoplast fusion. Drag resistance was expressed during heterokaryon formation, even across greater genetic distances than those distances imposed by vegetative compatibility. Phytogenies inferred from different molecular markers were incongruent at a significant level, challenging the clonal origins of Foe. Mating type genes were identified in this asexual pathogen Polymorphisms were detected within a Vegetative Compatibility Group (VCG) suggesting non-clonal inheritance and/or sexual recombination in Foe. This research was funded in part by a NIH-NIGMS (National Institutes of Health-National Institute of General Medical Sciences) Grant through the MBRS (Minority Biomedical Research Support), the Department of Biological Sciences and the Tropical Biology Program at FIU.

The interaction between several species of arbuscular mycorrhizal fungi, micropropagated strawberry plants and Phytophthora fragariae, the pathogen that causes red stele disease of strawberry plants, was investigated. The optimum temperature for germination of zoospore cysts of P. fragariae in vitro was found to be 15°C, and growth of the emerging germ tube was significantly orientated towards the strawberry root tip. Cyst germination was reduced in the presence of a mycorrhizal strawberry root. Elsanta was more susceptible to P. fragariae than the cultivar Rhapsody. A low level of colonisation of Elsanta with the arbuscular mycorrhizal fungi Glomus mosseae, Glomus intraradices or Glomus fistulosum resulted in a significantly greater amount of total phosphorus in plant shoots compared to non-mycorrhizal plants, although further increases in the percentage of root colonisation by the fungi had no effect on the plants. The presence of these mycorrhizal fungi had no effect on disease due to subsequent inoculation of the plants by P. fragariae. Increasing colonisation of Elsanta by Scutellospora nodosa was correlated with a significant increase in plant size and additional phosphorus uptake. However, these same plants exhibited greater levels of disease due to the following inoculation with P. fragariae. A low level of root colonisation of Elsanta by Acaulospora scrobiculata caused significant increases in plant size and phosphorus uptake up to a threshold level of root colonisation beyond which further increases had no affect on the plant. The results are discussed in relation to the utilisation of specific strains of arbuscular mycorrhizal fungi as inoculants of micropropagated strawberry plants of particular cultivars with the potential to increase plant growth and reduce the level of disease due to soil-borne plant pathogens.

Two field studies were conducted to assess the potential benefit of arbuscular mycorrhizal (AM) inoculation of elite strawberry plants on plant multiplication, and fruit yield, under typical nursery conditions, in particular soils classified as excessively rich in P. To study plant productivity, five commercially in vitro propagated elite strawberry cultivars ('Chambly', 'Glooscap', 'Joliette', 'Kent', and 'Sweet Charlie') were not inoculated with AM fungi or were inoculated with either a single species (Glomus intraradices), or a mixture of species (G. intraradices, Glomus mosseae, and Glomus etunicatum). AM inoculation was found to impact strawberry plant productivity in a soil with excessive P levels. The AM fungi introduced into the field by inoculated mother plants established a mycelial network in the soil through colonization of the daughter plant roots, however, persistence of colonization was determined to below (<12% in inoculated plant roots). In soils excessively rich in P, individual crop inoculation may be the only option for management of the symbiosis, as the host and non-host rotation crops, planted prior to strawberry production, had no effect on plant productivity or soil mycorrhizal potential.
To study the impact of AM inoculation on fruit production, three commercially grown strawberry cultivars (Glooscap, Joliette, and Kent) were not inoculated with AM fungi or were inoculated with either G. intraradices or G. mosseae. AM fungi impacted the fruit yield, with all inoculated cultivars producing more fruit than noninoculated cultivars during the first harvest year. The percentage of root colonization could not be used to explain the differences in total fruit yield during the first harvest year, or the increase in total fruit yield the second harvest year.
We wished to examine the effects of various P treatments on C metabolism within the intraradical mycelia (IRM) of the fungus. Specific primers were developed for the Glomus intraradices glucose-6-phosphate dehydrogenase (G6PDH) gene. Real-time quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) was used to measure the gene expression of the G. intrarardices G6PDH gene in response to external P conditions of colonized transformed carrot roots. The results showed a significant down-regulation of G6PDH in the IRM of G. intraradices when cultures were grown in a high P (350 muM P) medium compared to those grown in the low P (35 muM P) medium. The down-regulation may suggest a reduction in the C flow from the host to the fungus. There was no effect on G6PDH expression following a two-hour incubation with additional P applications (No P, low P and high P).

Interactions are all around us, and as humans we may use words and gestures to communicate our intentions. At the micro level of fungi, communications are replaced by chemical signals and structure. These interactions fall into three distinctive categories: synergistic, where organisms help each other, as is the case with ectomycorrhizal fungi and tree roots, deadlock, or combat, where organisms fight for or defend a resource. When it comes to fungi-tree interactions, the fungi group of basidiomycetes fall into the latter category. At the onset of fungal infection, a living tree defends itself by producing resinous substances such as terpenes. These compounds are frequently found in hydrodistilled turpentine, which makes turpentine a prime source of antifungal compounds. A D-optimal design of fractionated turpentine together with gas chromatography (GC) coupled to a mass spectrometer was employed to find the most biologically active constituent of turpentine. Growth rate of Coniophora puteana was used to assess the efficacy of the mixed fractions. The partial least squares projection model had an excellent predictive power (R2 = 0.988, Q2 = 0.825) and validity. A putative sesquiterpene was identified as the most active compound for inhibiting fungal growth. The model was corroborated by an external validation assay employing preparative GC. After the death of a tree, fungi are no longer hindered by secondary metabolites from the tree. Instead, other interspecies interactions and intraspecies interactions, such as fungi-fungi interactions, occur. We found that when the white-rot fungus Heterobasidion parviporum and brown-rot fungus Gloeophyllum sepiarium interact with each other, amino acids are used to a higher extent. Amino acids may be used to produce antifungal compounds to hinder the other species from growing. Lysine in particular was utilized to a greater extent during interaction. Glutamine was the only amino acid that increased in concentration. Glutamine might be exuded or converted by enzymes from already existing glutamic acid. Dry weights suggest that the fungi were in a deadlock and that nutrient limitation might be a determining factor. It seemed that H. parviporum was favoured by a decrease in pH while the opposite pattern may be true for G. sepiarium.

A study on cuticle-degrading enzymes (CDE) of three hyphomycete entomopathogens has produced information on enzyme types, levels, characteristics, mode of action, regulation, sequence of production, cellular localisation and production during host penetration. This is the first critical work on CDE of any entomopathogen. Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as sole carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular and bound enzymes corresponding to the major components of insect cuticle e.g. 3 endo-proteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase and N-acetylglucosaminidase. Considerable variations occurred in levels of production between spp. and even within a sp., but endo-proteases were exceptional in being produced in large amounts by all the isolates. CDE were produced rapidly and sequentially in culture. The first activities to appear (< 24 h) were those of the proteolytic complex, chitinases were always produced substantially later. Properties of CDE were investigated in terms of pH and tem- erature optima, substrate specificity, molecular weight, iso-electric point, mechanism of substrate degradation and the effect of specific inhibitors. Studies with culture filtrates and purified CDE revealed that substrates in intact cuticles are amenable to degradation but the prior action of protease is necessary for significant degradation of the chitin. Staining of chitin by a fluorescent lectin (FITC-WGA) and calcofluor only in cuticles from which protein has been removed (by protease or KOH) also suggests initial masking of chitin. This and determination of amino acid composition of peptides solubilised by endo-protease revealed the potential of CDE in studying the physicochemical structure of insect cuticles. The apparently localised action of CDE during host penetration may result from molecular sieving, binding to fungal walls or binding to cuticle. The first possibility is lessened by the small size of the endo-enzymes (<34 K daltons) which could allow diffusion via the various canals which traverse cuticle. However, cuticle effectively binds (ionically) CDE, and also activities of several CDE remain partly bound in various ways to hyphae and conidia (by ionic binding to walls, by disulphide bonds, and on or within membrane structures). The involvement of proteolytic enzymes in infection was suggested by their presence in conidia, penetration structures, and infected cuticle (detected histochemically and following extraction from cuticles). Also the constitutive production of endo- and exo-proteases lends weight to their possible significance in parasitism as synthesis will be subject only to catabolite repression. Chitinase is induced by N-acetylglucosamine and was not detected in infected cuticle. Possible mechanisms and significance of enzymic degradation of cuticle during infection are discussed, particularly in comparison to host penetration by phytopathogenic fungi.

Single beam and holographic optical tweezer micromanipulation have been explored. The single beam system used was a simple, compact, easy-to-use, safe and robust optical tweezer setup mounted on a standard research grade light microscope. It was specifically designed to produce high quality images and to be used with brightfield, phase contrast, differential interference contrast and fluorescence optics. The holographic optical tweezer system used involved the creation of multiple traps and complex patterns of light, and was employed with a range of laser wavelengths. The various optical tweezer systems were used in a wide range of applications to trap and micromanipulate whole fungal cells, organelles within cells, and synthetic beads. The experimental studies demonstrated how optical tweezers can be used to: unambiguously determine whether hyphae are actively homing towards each other; move the Spitzenkörper and change the pattern of hyphal morphogenesis; create ‘pseudowalls’ of light to control hyphal growth over extended distances; make piconewton force measurements; investigate the tethering of organelles within cells; mechanically stimulate hyphal tips; produce stable, fixed arrays of cells; and, deliver chemicals to localized regions of hyphae. A significant finding was that germ tubes seem to generate significantly lower growth forces than leading vegetative hyphae. An assessment of the photodamage caused to spores showed that ungerminated spores could be trapped for short periods of time without damage, but could not germinate whilst being continuously trapped. However once germinated, spores continually trapped for 25 min exhibited no deleterious effects with regard to conidial anastomosis tube growth, homing or fusion.

Dissertation (PhD)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that activated cytoplasmic or peroxisomal acetyl-groups are made available for mitochondrial energy production when the cells utilise non-fermentable carbon sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups are incorporated into each cycle, which releases a C4 intermediate. This intermediate is then transported to the mitochondria where it can enter the tricarboxylic acid cycle. The second pathway is the carnitine shuttle. Activated acetyl-groups react with carnitine to form acetylcarnitine, which is then transported to the mitochondria where the acetyl group is transferred. In this study it was shown that the deletion of the glyoxylate cycle specific citrate synthase, encoded by CIT2, results in a strain that is dependent on carnitine for growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in carnitine-dependent metabolic activities were generated. Complementation of the mutants with a genomic library resulted in the identification of four genes involved in the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase, encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent strain. The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable carbon sources suggested that S. cerevisiae does not biosynthesise carnitine. Measurements using electrospray mass spectrometry confirmed this hypothesis. As a result an investigation was initiated into carnitine biosynthesis in order to genetically engineer a S. cerevisiae strain that could endogenously biosynthesise carnitine. The filamentous fungus, Neurospora crassa, was one of the first organisms used in the seventies to identify the precursor and intermediates of carnitine biosynthesis. However, it was only about twenty years later that the first genes encoding these enzymes where characterised. Carnitine biosynthesis is a four-step process, which starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine is cleaved to trimethylamino-butyraldehyde by the hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH). The N. crassa TMLH homologue was identified in the genome database based on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing, which then revealed that the gene indeed had seven introns. Functional expression of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent Scii? strain on non-fermentable carbon sources when y-butyrobetaine was present. This is the first report of an endogenously carnitine biosynthesising strain of S. cerevisiae. The cloning of the remaining two biosynthesis genes presents particular challenges. To date, the HTMLA has not been characterised on the molecular level making the homology-based identification of this protein in N. crassa impossible. Although the TMABA-DH has been characterised molecularly, the protein sequence is conserved for its function as a dehydrogenase and not conserved for its function in carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is probably due to the fact that the enzyme is involved in other metabolic processes. The use of N. crassa carnitine biosynthesis mutants would probably be one way in which to overcome these obstacles. The !1cit2 mutant proved useful in studying carnitine related metabolism. We therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels. The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase (PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The catalytic subunits phosphorylate a variety of regulators and enzymes involved in metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt? mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of wild-type cells grown on glycerol. However, the enhancement of proliferation was much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde response may play a role. Overexpression of RAS2 resulted in elevated levels of intracellular citrate and citrate synthase activity. It therefore appears that the suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation of the respiratory capacity and possible leakage of citrate and/or citrate synthase from the mitochondria. The phenotype of RAS2 overexpression contrasts with the hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However, both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the RAS2val19dependent activation is more severe. Finally, this study implicated the Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed. Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable carbon sources, a notion that is supported by the literature.
AFRIKAANSE OPSOMMING: In die gis Saccharomyces cerevtstee, is daar twee metaboliese weë waarmee geaktiveerde asetielgroepe na die mitochondrium vervoer kan word wanneer die sel op nie-fermenteerbare koolstofbronne groei. Die een weg is die glioksilaatsiklus, waar die geaktiveerde asetielgroepe geïnkorporeer word in die siklus en dan vrygestel word as Ca-intermediêre. Hierdie intermediêre word dan na die mitochondrium vervoer waar dit in die trikarboksielsuursiklus geïnkorporeer word. Die ander weg is die karnitiensiklus, waar geaktiveerde asetielgroepe met karnitien reageer om asetielkarnitien te vorm wat dan na die mitochondrium vervoer word waar dit die asetielgroep weer vrygestel. Hierdie studie het getoon dat die delesie van die glioksilaatsiklus spesifieke sitraatsintetase, gekodeer deur CIT2, die gisras afhanklik maak van karnitien vir groei op nie-fermenteerbare koolstofbronne. Deur gebruik te maak van 'n ócit2 gisras, kon mutante, wat geaffekteer is in karnitien-verwante metaboliese aktiwiteite, gegenereer word. Komplementering van die mutante met 'n genomiese biblioteek het gelei tot die identifisering van vier gene betrokke by die karnitiensiklus. Hierdie gene sluit in: (i) die mitochondriale en die peroksisomale karnitienasetieltransferase, gekodeer deur CAT2; (ii) die buite-mitochondriale karnitienasetieltransferase, gekodeer deur YAT1; (iii) die mitochondriale karnitientranslokase, gekodeer deur CRC1; en (iv) 'n nuutgeïdentifiseerde karnitienasetieltransferase, gekodeer deur YAT2. Daar benewens, is ook gewys dat al drie karnitienasetieltransferases noodsaaklik is in 'n karriltienafhanklike gisras. Die afhanklikheid van eksogene karnitien van die ócit2 gisras, wanneer dit gegroei word op nie-fermenteerbare koolstofbronne, was aanduidend dat S. cerevisiae nie karnitien kan biosintetiseer nie. Metings deur middel van elektronsproeimassaspektrometrie het hierdie veronderstelling bevestig. Gevolglik is 'n ondersoek deur ons geïnisieer in die veld van karnitienbiosintese om 'n S. cerevisiae gisras geneties te manipuleer om karnitien sodoende endogenies te biosintetiseer. Die filamentagtige fungus, Neurospora crassa, was een van die eerste organismes wat in die sewentiger jare gebruik is om die voorloper en intermediêre van karnitienbiosintese te identifiseer. Dit was egter eers sowat twintig jaar later dat die eerste gene wat vir hierdie ensieme kodeer, gekarakteriseer is. Karnitienbiosintese is 'n vierstap-proses wat met trirnetlellisten as voorloper begin. Trimetiellisien word omgeskakel na hidroksi-trimetiellisien deur die ensiem trimetiellisienhidroksilase (TMLH). Hidroksietrimetlelllsien word dan gesplits om trimetielaminobuteraldehied te vorm deur die werking van die hidroksitrimetiellisienaldolase (HTMLA) met die gevolglike vrystelling van glisien. Trimetielaminobuteraldehied word dan na trimetielaminobuteraat (y-butirobeteïen) deur trimetielaminobuteraldehied dehidrogenase (TMABA-DH) gedehidrogeneer. In die laaste stap word y-butirobeteïen deur middel van die y-butirobeteïen hidroksilase (BBH) na L-karnitien omgeskakel. Op grond van die proteïenvolgordehomologie in die genoomdatabasis tussen die menslike TMLH en N. crassa se TMLH is laasgenoemde geïdentifiseer. As gevolg van die groot getal introns wat vir hierdie geen voorspel is, is die cDNA-weergawe daarvan gekloneer en aan volgordebepaling onderwerp. Dit het getoon dat die geen inderdaad sewe introns bevat. Funksionele uitdrukking van die geen in S. cerevisiae en ensiematiese analise het getoon dat die geen vir 'n TMLH kodeer en is gevolglik cbs-1 genoem; dit staan vir "karnitien biosintese geen no. 1tt. Meeste van die kinetiese parameters was ook soortgelyk aan die van die menslike TMLH-ensiem. Hierna is 'n genomiese kopie van N. crassa se BBH-homoloog gekloneer en funksioneel in S. cerevisiae uitgedruk. Biochemiese analise het getoon dat die uitgedrukte BBH-ensiem L-karnitien vanaf y-butirobeteïen kan biosintetiseer en die geen is cbs-2 genoem. Daar benewens kon die geen die groeidefek van die karnitien-afhanklike tlcit2-gisras ophef wanneer dit op nie-fermenteerbare koolstofbronne in die teenwoordigheid van y-butirobeteïen aangekweek is. Hierdie is die eerste verslag oor 'n endogeniese karnitien-biosintetiserende ras van S. cerevisiae. Die klonering van die oorblywende twee karnitienbiosintetiserende gene het sekere uitdagings. Tot op datum, is die HTMLA nog nie tot op genetiese vlak gekarakteriseer nie, wat dan die homologie-gebaseerde identifikasie van hierdie proteïen in N. crassa onmoontlik maak. Alhoewel die TMABA-DH geneties gekarakteriseer is, is die proteïenvolgorde ten opsigte van sy funksie as 'n dehidrogenase gekonserveer, maar nie vir sy funksie in karnitienbiosintese soos in die geval van TMLH en BBH nie. Die rede hiervoor is moontlik omdat die ensiem ook in ander metaboliese prosesse betrokke is. Die gebruik van N. crassa karnitienmutante sal moontlik een manier wees om hierdie probleme te oorkom. Die tlcit2-mutant het handig te pas gekom vir die bestudering van karnitienverwante metabolisme. Dus is daar vir onderdrukkers van die tlcit2-mutant gesoek wat gelei het tot die klonering van die RAS2-geen. In S. cere visiae , kodeer twee gene vir Ras-proteïene, RAS1 en RAS2. GTP-gebonde Ras-proteïene aktiveer adenilaatsiklase, Cyr1 p, wat verhoogde intrasellulêre cAMP-vlakke tot gevolg het. Die cAMP bind aan die regulatoriese subeenheid van die cAMP-proteïenkinase (PKA), Bcy1 p, en daardeur word die katalitiese subeenhede, Tpk1 p, Tpk2p en Tpk3p, vrygestel. Die katalitiese subeenheid fosforileer 'n verskeidenheid van reguleerders en ensieme betrokke by metabolisme. Ooruitdrukking van RAS2 het die groeidefek van die tlcit2-mutant op gliserolonderdruk. Oor die algemeen, verbeter die ooruitdrukking van RAS2 die proliferasie van die wildetipe op gliserol bevattende media. Alhoewel, die verbetering van proliferasie was baie meer opmerklik in die tlcit2-gisras. In hierdie verband, speel die gedegenereerde response dalk 'n rol. Ooruitdrukking van RAS2 het verhoogde intrasellulêre vlakke van sitraat- en sitraatsintetase-aktiwiteit tot gevolg gehad. Dit wou dus voorkom asof die onderdrukking van die ócit2-groeidefek deur RAS2 se ooruitdrukking die gevolg was van algemene opreguiering van respiratoriese kapasiteit en die lekkasie van sitraat en/of sitraatsintetase uit die mitochondria. Die fenotipe van RAS2 ooruitdrukking kontrasteer die hiperaktiewe RAS2va / 19 alleel, wat 'n groeidefek op gliserol media veroorsaak. Alhoewel beide RAS2-00ruitdrukking en RAS2va / 19 die cAMP/PKA-weg aktiveer, is gevind dat die RAS2va/19-afhanklike aktivering strenger is. Ten slotte, die cAMP/PKA-weg is in die proliferasie effek op gliserol media geïmpliseer deur te wys dat in 'n Mpk1-gisras, die groeieffek geblokkeer is. Alhoewel, die verbeterde proliferasie is steeds waargeneem in die Mpk2-en Mpk3-gisrasse toe die RAS2-geen ooruitgedruk is. Dus, dit wil voorkom asof Tpk1 p 'n belangrike rol in die groei van gisselle op nie-fermenteerbare koolstofbronne speel; 'n veronderstelling wat deur die literatuur ondersteun word.

Stipitatic acid 12 is an aromatic seven-membered ring produced by Talaromyces stipitatus (previously known as Penicillium stipitatus). The identification of this secondary metabolite changed the concept of aromaticity and paved the way for the structural elucidation of many non-benzenoid aromatic natural products called tropolones. Stipitatic acid has undergone one of the most detailed isotopic labelling studies to understand how nature could form such an aromatic seven-membered ring under biological conditions. The incorporation of labelled acetate units into the carbon skeleton of stipitatic acid pointed to a polyketide origin for this fungal tropolonoid metabolite. Further labelling study using radioactive molecules of 3-methylocinaldehyde 29 suggested an oxidative ring expansion rearrangement for the formation of the tropolone seven-membered ring.

Sexual reproduction is an important process amongst eukaryotic organisms, with one function being to maintain genetic variation. The idea that complex eukaryotic species can persist for millions of years in the absence of sex defies fundamental evolutionary dogma, yet a group of organisms known as ancient asexuals were thought to have evolved clonally under deep evolutionary time. Prominent among these are the arbuscular mycorrhizal fungi (AMF), which are obligate plant symbionts that colonize the root cells of plants and extend their hyphae into the soil assisting the plant in acquiring key nutrients. Unlike most eukaryotes, AMF cells are multinucleate with thousands of nuclei moving through a continuous cytoplasm. Genomic analyses have identified a putative mating-type (MAT) locus within the nuclear genomes of model AMF Rhizophagus irregularis, a region that in other fungi dictates the process of sexual reproduction. Additional findings demonstrated that AMF strains carry one of two nuclear organizations. They can be either homokaryotic (AMF homokaryons), where all nuclei within the cytoplasm are virtually identical, or heterokaryotic (AMF dikaryons), where two MAT-locus variants co-exist within the cytoplasm. Despite a lack of observable traits indicative of sex, this homo/heterokaryotic dichotomy is reminiscent of the nuclear organization of sexual fungi. My research aims to build on these findings to investigate the actual role of the MAT-locus in driving AMF reproduction. To address this, I build my thesis into three main chapters. The first chapter reviews our current understanding of AMF genetics and what drives genome evolution in these organisms. The second chapter establishes a relatively easy, inexpensive, and reproducible approach to genotype known MAT variants of R. irregularis in natural and experimental conditions. The last chapter uses experimental crossings between strains to assess cytoplasmic compatibility and nuclear exchange. I demonstrate that dikaryotic spore progenies can be formed after co-culturing two distinct AMF homokaryotic strains. Further analyses of various genomic regions also reveal possible recombination in homokaryotic spore progenies from co-cultures. Overall, this research provides new experimental insights into the origin of genetic diversity in AMF. These findings open avenues to produce genetically new AMF strains in the lab using conventional crossing procedures and provide a glimpse of the mechanisms that generate AMF genetic diversity in the field.

Endophytic fungi from Cassia fistula or golden shower, a well known medicinal plant in Thailand and Asia, were isolated from trees growing in three geographical separate sites. These locations were Kanchanaburi, Nakhon Ratchasima and Bangkok and were selected to allow comparisons between their endophytic assemblages and to evaluate these data in relation to differences in plant diversity and density and local environment. Kanchanaburi which was the site closest to a natural forest situation provided the highest number of isolates with Bangkok, where the trees were isolated individuals, having the least. Members of the Xylariaceae proved to be common and frequent isolates especially species of Xylaria and Daldinia but Nemania and Hypoxylon were also obtained. Phomopsis was also well represented and clearly was dominant at the Kanchanaburi site. Species of Fusarium, Colletotrichum, Penicillium, Nigrospora, Coprinus and Psathyrella were also identified but were occasional isolates. Differences in endophytic assemblages between samples obtained early in the rainy season (July, 2001) with those sampled towards the end of the rainy season (December, 2001) were found to occur in the Nakhon Ratchasima samples with over twice as many isolates obtained from the December samples. This is likely to be a reflection on the longer exposure period to the potential inoculum of these leaves. A total of 956 endophytic isolates were obtained from the three sites with samples from Kanchanaburi (December 2000) and Bangkok and Nakhon Ratchasima in July 2001 with a further samples from Nakhon Ratchasima in December, 2001. Isolations were also made from different anatomical regions of the leaf, leaf lamina, midrib and veins. There were no appreciable differences in either the number of isolates obtained or an association between leaf area and specific fungal species. Identification of many xylariaceous endophytic isolates is well known to be problematic since Xylaria species rarely produce their anamorphic form in culture and virtually no members of the Xylariaceae develop their teleomorph in culture. Therefore molecular techniques were used to compare DNA sequences of the ITS region from a selection of endophyes with sequences obtained from teleomorphic material, or cultures derived from teleomorphs of identified and authenticated Xylariaceae. Comparisons were also made with data held in GenBank. This enabled the identity of a number of taxa to be made although more sequences from Xylaria species are required for future investigations. A number of non-xylariaceous taxa were also named as a result of DNA sequence comparisons. Secondary metabolites from the xylariaceae were also investigated and their metabolite profiles used to support identifications. The metabolite profiles proved to be a useful tool to confirm doubtful endophytic isolates when their DNA sequences could not place them with certainty in a right group. Together with extracts from other endophytic species, their inhibitory effects on bacteria and fungi were tested. Cassia endophytes were found to show low antimicrobial activity. However, they may later be shown to have other activities when when tested e. g. anti-malarial, anti-cancer and anti-HIV.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第10274号
農博第1346号
新制||農||869(附属図書館)
学位論文||H15||N3795(農学部図書室)
UT51-2003-H695
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 熊谷 英彦, 教授 井上 國世, 教授 江崎 信芳
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