Diese Arbeit befasst sich mit der Herstellung monoklonaler Antikörper gegen Gliotoxin und eine Charakterisierung der Eigenschaften dieser Antikörper sowie ihrer Fab-Fragmente im ELISA sowie in Zellkulturen. Insgesamt konnten fünf monoklonale Antikörper generiert werden, die spezifisch für das Mykotoxin Gliotoxin waren. / This work focuses at the production of Gliotoxin specific monoclonal antibodies and their characterization as well as their fab fragments in ELISA and cell culture. All in all, five specific antibodies could be isolated.

The occurrence of mould in food and animal feed is a severe problem due to the secondary metabolites, called mycotoxins, which can possess toxic activity. Aspergillus fumigatus is a common fungus found in improperly stored animal feed and the abundance of spores of the fungus is frequently spread into the air. Gliotoxin has been identified as one of the most toxic second metabolites produced by A. fumigatus. Although A. fumigatus is known to produce mycotoxins that induce neurological syndromes, the neurotoxic properties of gliotoxin have not previously been studied. In this thesis a neurotoxic activity of gliotoxin was demonstrated by using differentiated human neuroblastoma SH-SY5Y cells as a surrogate for the nervous system. The major findings were as follows: i. Gliotoxin is highly toxic to SH-SY5Y cells and there is a correlation between the toxicity and the cellular redox status. ii. Gliotoxin reduces the number of neurites, but does not affect the cell bodies morphologically, at non-cytotoxic concentrations. This indicates that the toxin may induce peripheral axonopathy in vivo. iii. The intracellular free Ca2+ concentration is increased after exposure to gliotoxin, an effect that is the most ubiquitous feature of neuronal cell death. Simultaneously, calpains and caspases, proteases known to be involved in neuronal death and axonal degeneration, are activated. iv. The observed irreversible neurite degenerative effects of gliotoxin are mainly dependent on caspase activation, whereas calpains are involved in the gliotoxin-induced cytotoxicity. v. Gliotoxin induces a decreased rate of protein synthesis at non-cytotoxic concentration, which may contribute to the degeneration of neurites. vi. We did also succeed in developing an in vitro method for determination of toxic activity in animal feed. This study was done in collaboration with National Veterinary Institute (SVA) in Uppsala, and the method is today established and in use at Department of Animal Feed, SVA.

This thesis describes the cultivation and extraction of filamentous fungi isolated from extreme environments in the search for new antibiotic compounds. Filamentous fungi are a rich source of medicines including antibiotics, and it is believed that many currently unknown fungal species and bioactive fungal metabolites remain to be discovered. Aspergillus fumigatus and Penicillium nalgiovense strains were isolated from an antibiotic-contaminated riverbed near Hyderabad, India, and soil taken from a penguin’s nest on Paulete Island, Antarctica, respectively. It was anticipated that the extreme conditions within these environments would exert unusual selective pressures on their filamentous fungi, possibly causing the secretion of new bioactive compounds. The cultivation, extraction and analysis of metabolites from the A. fumigatus strain resulted in the isolation of the antimicrobial substance gliotoxin. Subsequent investigations revealed that this strain’s secretion of gliotoxin was increased by as much as 65 % when it was cultivated in the presence of pathogen-associated molecular patterns. These results indicate the existence of a fungal receptor/signaling system for detecting nearby bacteria. The scope for using gliotoxin and the related metabolite bis(methyl)gliotoxin as biomarker metabolites for diagnosing the lethal pulmonary condition invasive aspergillosis was also investigated. Bronchoalveolar lavage fluid from 42 patients with and without possible invasive aspergillosis was extracted and analyzed. The results obtained suggest that gliotoxin and bis(methyl)gliotoxin are not suitable markers for diagnosing invasive aspergillosis. Studies on the P. nalgiovense strain from Antarctica resulted in the isolation of the antifungal agent amphotericin B. The secretion of this compound increased when P. nalgiovense was cultured on a potato-dextrose agar enriched with coconut flakes rather than liquid RPMI 1640 medium. This was the first time amphotericin B was isolated from any organism other than the bacterium Streptomyces nodosus. The results presented in this thesis will be useful in the continuing search for novel bioactive compounds, the diagnosis of fungal infections, and as a source of insight into the interactions between microorganisms. Moreover, they show that even extensively studied fungal genera such as Aspergillus and Penicillium are not completely understood and may produce unexpected or previously unknown bioactive metabolites under appropriate conditions.

Gliotoxin ist ein ubiquitär vorkommendes Mykotoxin und wird unter anderem von Aspergillus sp. gebildet. Das Gliotoxin ist toxisch und stellt für Mensch und Tier ein gesundheitliches Risiko dar. Über die Aufnahme von kontaminierten Futtermitteln (FM) kann es in tierische Produkte und anschließend in die Nahrungsmittelkette gelangen. Dem Mykotoxin werden starke immunmodulatorische Eigenschaften zugeschrieben. Gliotoxin gilt als eine sehr reaktive Verbindung, die in der Umwelt schnell zu bis(methylthio)Gliotoxin (bmGliotoxin) umgesetzt werden kann. Dieser Metabolit ist sowohl atoxisch als auch biologisch inaktiv und wird aufgrund seiner Stabilität als ein wichtiger und zuverlässiger Diagnostikmarker beschrieben. In der Mykotoxinanalytik werden Konzentrationsbestimmungen primär durch chromato-graphische Verfahren durchgeführt. Mit diesen Verfahren konnten bereits Gliotoxin-Konzentrationen in verschiedensten FM bestimmt werden. Im Gegensatz zu anderen prominenten Mykotoxinen gibt es für das Gliotoxin kein Nachweisverfahren für routinemäßige Kontrolluntersuchungen. Daher war das Ziel der Dissertationsarbeit die Entwicklung eines ELISA, mit dem Gliotoxin in FM einfach und schnell bestimmt werden kann. Es wurden zunächst geeignete Protein-Gliotoxin-Konjugate für die Anti-Gliotoxin-Immunisierung hergestellt. Kaninchen wurden nach einem festgelegten Protokoll mit diesen Hapten-Konjugaten immunisiert (Kurzzeit- bzw. Langzeitimmunisierung). Anschließend erfolgte die Titerbestimmung in den Antiseren und die Überprüfung der Paratophemmbarkeit durch freies Gliotoxin. Aus dem Antiserum mit der höchsten IgG-anti-Gliotoxin-Konzentration wurden die Antikörper antigenaffinitäts-chromatografisch aufgereinigt. Zusätzlich wurde für den kompetitiven ELISA ein geeignetes Gliotoxin-Peroxidase-Konjugat hergestellt. Alle Testkomponenten wurden vorab geprüft und danach der kompetitive Gliotoxin-ELISA validiert. Mit dem optimierten kompetitiven Testsystem wurden die Gliotoxin-Konzentrationen in Schimmelpilz-Kulturüberständen (Aspergillus sp.) gemessen. Anschließend erfolgte die Untersuchung von Futtermittelproben (Silagen) auf Gliotoxin. Nach der entsprechenden Probenaufbereitung der FM wurde das Gliotoxin im kompetitiven ELISA ebenfalls quantitativ bestimmt. Die hier gemessenen Gliotoxin-Konzentrationen wurden mit Ergebnissen aus parallel durchgeführten Untersuchungen mit der LC-MS/MS verglichen. Mit dem entwickelten kompetitiven ELISA sind quantitative Aussagen zu erhöhten Gliotoxin-Konzentrationen in verschiedenen Probenmaterialien möglich. Das Mykotoxin kann sowohl frei in seiner nativen Form, gebunden an Proteine oder metabolisiert als bis(methylthio)gliotoxin detektiert werden. Mit dem Testsystem kann die quantitative Produktion von Gliotoxin durch verschiedene Schimmelpilzarten beurteilt werden. Die untersuchten FM (Silageproben) konnten im entwickelten ELISA alle erfolgreich gemessen und beurteilt werden.:1 Einleitung 1 2 Literaturübersicht 2 2.1 Schimmelpilze 2 2.1.1 Definition und Zuordnung 2 2.1.2 Vorkommen in der Umwelt 4 2.1.3 Bedeutung und Nutzen 5 2.1.4 Schadwirkung für Mensch und Tier 7 2.2 Mykotoxine 9 2.3 Gliotoxin 10 2.3.1 Allgemeine Merkmale 10 2.3.2 Bis(methylthio)Gliotoxin 16 2.3.3 Ausgewählte Pathogenitätsmechanismen 17 2.3.3.1 Redox-Zirkel und ROS-Produktion 17 2.3.3.2 Verschiebung des TH1/ TH2-Gleichgewichtes 18 2.3.3.3 Inhibition des Transkriptionsfaktors NF-κB 19 2.3.3.4 Einleitung von Apoptose und Nekrose 20 2.3.3.5 Inhibition von Mastzellen 21 2.3.4 Nachweisverfahren 22 2.3.4.1 Chromatographie 22 2.3.4.2 Enzyme-linked Immunosorbent Assay (ELISA) 23 2.3.5 Quantitative Mengenbestimmungen in Proben 25 2.3.5.1 Klinisches Probenmaterial 25 2.3.5.2 Belastete Futtermittelproben 27 2.3.5.3 Belastete Lebensmittelproben 33 3 Material und Methoden 34 3.1 Gliotoxin, Chemikalien und ausgewählte Laborgeräte 34 3.2 Gliotoxin-Hapten für die Immunisierung 35 3.3 Immunisierung 37 3.4 Untersuchung der Anti-Gliotoxin-Antiseren 37 3.4.1 Protein-Gliotoxin-Konjugate für ELISA 37 3.4.2 ELISA für Antiserumuntersuchung 39 3.4.3 ELISA für die Bestimmung der IgG-Konzentrationen anti-Gliotoxin 40 3.5 Etablierung des kompetitiven Gliotoxin-ELISA 42 3.5.1 Isolierung der IgG-anti-Gliotoxin 42 3.5.2 Herstellung des Gliotoxin-HRP-Konjugates 43 3.5.3 Prüfung der Testkomponenten 44 3.5.4 Validierung des kompetitiven Gliotoxin-ELISA 45 3.6 Untersuchung von Kreuzreaktionen 45 3.7 Untersuchung von Pilzkulturen auf Gliotoxin 45 3.8 Untersuchung von Futtermitteln auf Gliotoxin 47 3.8.1 Untersuchung von dotiertem Futtermittel 47 3.8.2 Futtermitteluntersuchung mit dem Gliotoxin-ELISA 48 3.8.3 Futtermitteluntersuchung mit der LC-MS/MS 49 3.9 Datenauswertung 49 4 Ergebnisse 50 4.1 Hapten für die Immunisierung 50 4.2 Untersuchung der Anti-Gliotoxin-Antiseren 50 4.2.1 Titerbestimmung 50 4.2.2 Bestimmung der Hemmungsrate durch freies Gliotoxin 51 4.2.3 IgG-anti-Gliotoxin-Konzentration in den Antiseren 53 4.3 Untersuchung der Antiseren 55 4.3.1 Isolierung des IgG-anti-Gliotoxin 55 4.3.2 Voruntersuchungen für die Etablierung des kompetitiven Gliotoxin-ELISA 57 4.3.3 Validierung des kompetitiven Gliotoxin-ELISA 60 4.4 Untersuchung von Kreuzreaktionen 64 4.5 Untersuchung von Pilzkulturen auf Gliotoxin 65 4.6 Untersuchung von Futtermitteln auf Gliotoxin 68 4.6.1 Untersuchung mit Gliotoxin dotierter Futtermittel 68 4.6.2 Futtermitteluntersuchung mit Gliotoxin-ELISA und LC-MS/MS 71 4.7 Auswertung und Bewertung 73 5 Diskussion 78 5.1 Bewertung der Gliotoxin-Antigen Entwicklung 78 5.2 Bewertung der Antiseren nach Immunisierung (28d- und 91d-Protokoll) 79 5.3 Etablierung des kompetitiven ELISA 83 5.4 Bewertung der Pilze und der Kulturüberstände 85 5.5 Bewertung der Futtermittel 88 6 Zusammenfassung 96 7 Summary 98 8 Literaturverzeichnis 100 9 Anhang 116

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, <em>Anguilla rostrata</em>. <br> <br> RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders. <br><br> HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues. <br><br> Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked <em>Mx</em> induction, which is the first demonstration of PKR mediating Mx expression. <br><br> The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication. <br><br> The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, <em>Anguilla rostrata</em>. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis. <br><br> This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

Subterranean termites encounter numerous kinds of fungi during foraging and feeding activities. Nearly nine decades of research have exposed only a small fraction of the termitefungal interactions that exist in nature. The first portion of research presented here focused on how feeding behaviors of Reticulitermes virginicus (Banks) were affected by the fungus Trichoderma virens (Miller, Giddens & Foster) von Arx. Tests were performed with 'P' (GLT+) and 'Q'(GLT-) strains of T. virens. Both strains were applied to filter paper and wood disks cut from southern yellow pine and Sentricon® monitoring devices. The first bioassay assessed the termites' feeding responses to fungal extracts removed from liquid media on days 2 through 7, and again on day 15. Only the GLT+ extracts from days 6 and 7 inhibited termite feeding significantly from the controls (16% and 54% less area loss, respectively). Response to wood covered by live T. virens mycelia was tested in the second bioassay. No significant differences in termite consumption were seen between fungal strains, but both substantially reduced the area loss due to termite feeding of treated wood by an average of 35%. A vacuum impregnation system was used to inoculate wood disks with fungal homogenate in the third bioassay. The wood treated with either GLT+ or GLT- homogenates did not have significant differences in area loss due to termite feeding. Overall, these results reiterated the plasticity that exists with termitefungal relationships. The second research topic addressed the applicability of labrum-based identification techniques to Reticulitermes Holmgren in Texas. Soldier labral morphology of four species, R. flavipes (Kollar), R. hageni (Banks), R. tibialis Banks, and R. virginicus (Banks), was evaluated as a character to separate species. Length and width measurements of five soldier labra were taken from each of the eight collection sites. These results were then judged against molecular analysis of the mtDNA 16S rRNA gene. Findings showed that labral shape was an unreliable diagnostic characteristic when comparing all species. A combination of length and length-towidth ratio successfully segregated all four Reticulitermes species. Comparison of a morphology-based dendogram to the phylogenetic analysis revealed a shared pattern between phenotypic and genotypic variations.

Fungi and Oomycetes represent the two most important groups of eukaryotic plant pathogens. Besides chemical and physical control of these pathogens, biological control is an approach enjoying increasingly more focus. One of the biological agents increasingly employed in biological control of plant pathogenic fungi is ironically the fungus Trichoderma, more specifically Trichoderma harzianum. Besides control of the fungal plant pathogens, another interesting aspect observed when plants are treated with Trichoderma harzianum are effects such as complete and even stand of plants, faster seed germination, increases in plant height and overall enhanced plant growth. Though there have been various studies on this effect, almost no research has yet been conducted to elucidate the mechanism by which these effects occur. In particular, effects such as faster seed germination suggest that Trichoderma harzianum produces a metabolite that may mimic the plant growth hormone gibberellic acid. Through an evaluation of the various metabolites produced by Trichoderma harzianum; gliotoxin seemed structurally most similar to gibberellic acid. To verify that gliotoxin can indeed serve as an analogue for gibberellic acid and elicit similar physiological responses in plants, a two–pronged approach was followed. Firstly, molecular similarity evaluation through common pharmacophore evaluation was conducted, followed by docking simulations into the recently discovered receptor for gibberellic acid. Common pharmacophore evaluation between gibberellic acid and gliotoxin showed successful alignment of gliotoxin into the gibberellic acid based pharmacophore space. Furthermore, docking simulations further strengthened this by the similarity in docking scores calculated and the similar poses of the ligands (gliotoxin and gibberellic acid) in the receptor space. However, similarity in pharmacophore alignment and docking simulation results only suggest that gliotoxin should be able to occupy the receptor space, but it is not a guarantee that similar physiological responses will be elicited. In the second part of the project, the ability of gliotoxin to elicit similar physiological responses in plants to gibberellic acid was investigated. For this, a–amylase induction; plant emergence and height; and chlorophyll fluorescence were compared for both gliotoxin and gibberellic acid treatments. In terms of a–amylase induction, gliotoxin was able to induce production of the enzyme as visualised by starch–containing native gel electrophoresis (zymograms). Gliotoxin induced the strongest response at a 10–6 M dilution which is typically the range expected for hormones in biological systems in de–embryonated seeds of Phaseolus vulgaris. Gibberellic acid was able to induce the strongest response at a 10–7 M dilution. In essence, similar physiological responses were observed. In terms of plant emergence and plant height, treatment with gliotoxin or gibberellic acid resulted in plant emergence a day earlier than the untreated control. However, even though there were slight differences in plant height favouring the gliotoxin or gibberellic acid treated plants, the differences were not statistically significant. Thus, in this regard similar responses were again observed for both gliotoxin and gibberellic acid treatments. In the final evaluation the effect of gliotoxin and gibberellic acid treatments on the chlorophyll fluorescence of mature plants was investigated. Overall, both gliotoxin and gibberellic acid elicited beneficial effects on plant vitality, expressed through PI(Abs) with the gliotoxin treatment performing better than the equivalent gibberellic acid treatment. Overall, the physiological tests demonstrated that gliotoxin can indeed elicit similar positive physiological responses to gibberellic acid in Phaseolus vulgaris. Furthermore the test used in this project can serve as a standard evaluation bench for screening for gibberellic acid analogues on a laboratory scale before larger scale field trials are considered. / Thesis (Ph.D. (Microbiology))--North-West University, Potchefstroom Campus, 2012.

Fungi and Oomycetes represent the two most important groups of eukaryotic plant pathogens. Besides chemical and physical control of these pathogens, biological control is an approach enjoying increasingly more focus. One of the biological agents increasingly employed in biological control of plant pathogenic fungi is ironically the fungus Trichoderma, more specifically Trichoderma harzianum. Besides control of the fungal plant pathogens, another interesting aspect observed when plants are treated with Trichoderma harzianum are effects such as complete and even stand of plants, faster seed germination, increases in plant height and overall enhanced plant growth. Though there have been various studies on this effect, almost no research has yet been conducted to elucidate the mechanism by which these effects occur. In particular, effects such as faster seed germination suggest that Trichoderma harzianum produces a metabolite that may mimic the plant growth hormone gibberellic acid. Through an evaluation of the various metabolites produced by Trichoderma harzianum; gliotoxin seemed structurally most similar to gibberellic acid. To verify that gliotoxin can indeed serve as an analogue for gibberellic acid and elicit similar physiological responses in plants, a two–pronged approach was followed. Firstly, molecular similarity evaluation through common pharmacophore evaluation was conducted, followed by docking simulations into the recently discovered receptor for gibberellic acid. Common pharmacophore evaluation between gibberellic acid and gliotoxin showed successful alignment of gliotoxin into the gibberellic acid based pharmacophore space. Furthermore, docking simulations further strengthened this by the similarity in docking scores calculated and the similar poses of the ligands (gliotoxin and gibberellic acid) in the receptor space. However, similarity in pharmacophore alignment and docking simulation results only suggest that gliotoxin should be able to occupy the receptor space, but it is not a guarantee that similar physiological responses will be elicited. In the second part of the project, the ability of gliotoxin to elicit similar physiological responses in plants to gibberellic acid was investigated. For this, a–amylase induction; plant emergence and height; and chlorophyll fluorescence were compared for both gliotoxin and gibberellic acid treatments. In terms of a–amylase induction, gliotoxin was able to induce production of the enzyme as visualised by starch–containing native gel electrophoresis (zymograms). Gliotoxin induced the strongest response at a 10–6 M dilution which is typically the range expected for hormones in biological systems in de–embryonated seeds of Phaseolus vulgaris. Gibberellic acid was able to induce the strongest response at a 10–7 M dilution. In essence, similar physiological responses were observed. In terms of plant emergence and plant height, treatment with gliotoxin or gibberellic acid resulted in plant emergence a day earlier than the untreated control. However, even though there were slight differences in plant height favouring the gliotoxin or gibberellic acid treated plants, the differences were not statistically significant. Thus, in this regard similar responses were again observed for both gliotoxin and gibberellic acid treatments. In the final evaluation the effect of gliotoxin and gibberellic acid treatments on the chlorophyll fluorescence of mature plants was investigated. Overall, both gliotoxin and gibberellic acid elicited beneficial effects on plant vitality, expressed through PI(Abs) with the gliotoxin treatment performing better than the equivalent gibberellic acid treatment. Overall, the physiological tests demonstrated that gliotoxin can indeed elicit similar positive physiological responses to gibberellic acid in Phaseolus vulgaris. Furthermore the test used in this project can serve as a standard evaluation bench for screening for gibberellic acid analogues on a laboratory scale before larger scale field trials are considered. / Thesis (Ph.D. (Microbiology))--North-West University, Potchefstroom Campus, 2012.

SCH-64874 (5) is a fungal metabolite that inhibits the epidermal growth factor receptor (EGFR), a high-profile oncology target, with an IC50 of 1.0µg/mL. It is of particular interest because it is unlikely to inhibit the protein’s intramolecular kinase domain (as typical chemical EGFR inhibitors do), and may act instead by obstructing the receptor’s ligand binding and/or dimerisation processes. / In this work, the epipolythiodiketopiperazine family of natural products is reviewed, leading to a discussion of the probable biosynthetic pathways by which these complex molecules are produced in nature. A laboratory synthesis based on this proposed biosynthesis was subsequently proposed and undertaken. / The oxidation of aromatic systems was investigated, which led to the synthesis, for the first time, of complex functionalised arene oxides such as 178. The regioselective epoxidation of 178 was accessed by derivatisation as the Diels-Alder adduct 180. Subsequent epoxidation and manipulation led to the amino alcohol 195b, possessing the exo-epoxide endo-alcohol stereochemistry shown. / This stereochemical assignment was based on detailed NMR analysis of the product, and also on AM1 semi-empirical molecular modelling and Ab initio molecular orbital calculations, which were used to evaluate the relative stabilities of the cyclisation products.