Les phénylpropanoïdes (PPs) constituent l'une des familles les plus abondantes des métabolites secondaires dans le règne végétal. Ils protègent les plantes contre les stress biotiques et abiotiques. De nos jours, les études portent sur l'utilisation des PPs comme alternatifs aux agents antimicrobiens, antioxydants et anti-inflammatoires de synthèse pour leur incorporation dans la formulation des produits alimentaires et pharmaceutiques. Cependant, l'utilisation de PPs est généralement limitée en raison de leur faible solubilité, stabilité et volatilité. L'objectif de notre travail a été d'encapsuler sept PPs dans des molécules cages, les cyclodextrines (CDs), en vue de développer des systèmes naturels et éco-compatibles ayant des applications potentielles dans les domaines alimentaire et pharmaceutique. Trois axes ont été abordés. Le premier axe a porté sur la préparation et la caractérisation des complexes d'inclusion CD/PP en solution et à l'état solide. Les techniques d'"headspace" couplé à la chromatographie en phase gazeuse (HS-CG), spectroscopie UV-visible, ¹H RMN, (2D) ROESY RMN, FTIR, DSC et de la modélisation moléculaire ont été utilisées comme outils pour la caractérisation des complexes obtenus. Des études de phase de solubilité ont été également réalisées. Le deuxième axe a porté sur l'évaluation de l'effet des CDs sur la photostabilité et la vitesse de libéralisation des PPs. Le dernier axe a été orienté vers l'étude des activités anti-radicalaire, antibactérienne et antifongique des complexes d'inclusion CD/PP. Les résultats montrent que les CDs sont capables d'encapsuler les PPs étudiés, réduire leur volatilité, augmenter leur solubilité et photostabilité ainsi que de générer des systèmes de libération prolongée. De plus, l'encapsulation conserve les propriétés antioxydante, antibactérienne et antifongique des PPS. Les résultats de cette étude suggèrent que les complexes d'inclusion des PPs avec les CDs peuvent être considérés comme outils prometteurs pour l'optimisation des formulations alimentaires et pharmaceutiques. / Phenylpropanoids (PPs) are one of the largest families of plants secondary metabolites. They protect plants against biotic and abiotic stresses. Nowadays, extensive research has been dedicated to PPs aiming their use as natural alternatives to synthetic antimicrobial, antioxidant and anti-inflammatory agents in food and pharmaceutical formulations. However, PPs suffer from a low water solubility, high volatility, high light and thermal sensitivity that limit their further use. This current study aimed to encapsulate seven PPs in host cage molecules, cyclodextrins (CDs), in order to develop natural and biocompatible formulation that may find applications in food and pharmaceutical fields. It focused on three main research axes. The first part dealt with the preparation and the characterization of CD/PP inclusion complexes both in solution and in solid state. Characterizations were performed with Static-Headspace-Gas Chromatography (SH-GC), UV-Visible, ¹H NMR, (2D) ROESY NMR, FTIR, DSC and molecular modeling. These investigations were complemented with phase solubility studies. The second part was devoted to the evaluation of the effect of CDs on the PPs photostability and controlled release. The last part aimed to evaluate the CD/PP inclusion complexes as radical scavengers, antibacterial and antifungal agents. Results showed that CDs could successfully encapsulate PPs, reduce their volatility, enhance their solubility and photostability and generate controlled release system. In addition, encapsulation maintained the antioxydant, antibacterial and antifungal properties of PPs. Thus, the CD/PP inclusion complexes could be considered as a promising tool for formulation optimization.

Peptídeos antimicrobianos (PAMs) fazem parte de um dos mecanismos da imunidade inata contra infecções. A microplusina é um PAM de 10.204 Da, isolado da hemolinfa livre de células e dos ovos do carrapato Rhipicephalus (Boophilus) microplus. É um PAM aniônico em pH fisiológico, possui seis resíduos de cisteína, com formação de três pontes dissulfeto, além de sete resíduos de histidina concentrados principalmente na sua porção C-terminal. O presente trabalho teve como objetivo investigar o mecanismo de ação antimicrobiana da microplusina. A microplusina recombinante é ativa contra várias bactérias Gram-positivas e fungos, porém não apresenta atividade contra bactérias Gram-negativas. Para avaliar o seu mecanismo de ação, foram utilizados dois modelos: a bactéria Micrococcus luteus e o fungo Cryptococcus neoformans. A microplusina é bacteriostática contra M. luteus e apresenta localização intracelular na bactéria. Além disso, observamos que a microplusina liga cobre e que a adição deste metal ao meio de cultivo reduz sua atividade antibacteriana. Bactérias M. luteus pré-incubadas com microplusina retomam o seu crescimento quando cobre é adicionado ao meio. Estes dados indicam que a atividade da microplusina está relacionada à sua habilidade de depletar cobre do meio extra ou intracelular, sugerindo um efeito nutricional para o peptídeo. A microplusina apresenta estrutura terciária com cinco a-hélices e sua ligação ao cobre não induz mudanças conformacionais. Observou-se que as histidinas 1, 2 e 74 da microplusina podem estar envolvidas na formação de um sítio de ligação ao cobre. Quanto à C. neoformans, verificou-se que a microplusina inibe a melanização do fungo, um fator de virulência catalisado pela lacase, uma enzima cobre-dependente. Entretanto, a microplusina não afeta a atividade da lacase, nem sua expressão gênica. O peptídeo também não inibe a auto-polimerização de substratos fenólicos que levam à melanização. Sendo assim, mais estudos são necessários a fim de avaliar o mecanismo pelo qual a microplusina inibe a melanização. Adicionalmente, a microplusina afeta a viabilidade do fungo e reduz o tamanho de sua cápsula, outro importante fator de virulência. As atividades da microplusina sobre C. neoformans sugerem o seu potencial terapêutico. Experimentos in vivo com modelo murino, mostraram que a microplusina reduz o processo inflamatório e a viabilidade de C. neoformans nos pulmões, indicando que em condições otimizadas, o peptídeo pode atuar no controle de infecções. / Antimicrobial peptides (AMPs) take part of innate immune mechanisms against infections. Microplusin is a 10,204 Da AMP, isolated from cell-free hemolymph and eggs of the tick Rhipicephalus (Boophilus) microplus. It is an anionic AMP at physiological pH, with six cysteine residues forming three disulfide bridges and seven histidine residues clustered mainly at the carboxy end portion. The goal of the present work was investigate the antimicrobial action mechanism of microplusin. Recombinant microplusin is active against Gram-positive bacteria and fungi, however, no activity is detected for Gram-negative bacteria. Two models were used to evaluate the action mechanism of microplusin: the bacteria Micrococcus luteus and the yeast Cryptococcus neoformans. Microplusin is bacteriostatic against M. luteus and its localization is intracellular for these bacteria. Moreover, microplusin binds copper and the addition of this metal into the medium reduces its antibacterial activity. M. luteus bacteria pre-treated with microplusin recover its growth when copper is added. These data indicate that microplusin activity is related to its ability to deplete copper present in the extracellular or intracellular environment, suggesting a nutritional effect. Microplusin presents a tertiary structure with five a-helix and the copper binding does not induce conformation changes. In addition, it was observed that histidines 1, 2 and 74 from microplusin may be involved in the formation of a copper binding site. About C. neoformans, it was verified microplusin inhibits its melanization, a virulence factor catalyzed by laccase, a copper dependent enzyme. However, microplusin does affect neither laccase activity nor its gene expression. The melanization caused by auto-polymerazation of phenolic substrates, is also not inhibited by microplusin. Hence, additional studies are required to evaluate the mechanism by which microplusin inhibits melanization. In addition, microplusin also affects the fungi viability and reduces the capsule size, another important virulence factor.The microplusin activities against C. neoformans suggest its therapeutic potential. In vivo experiments with murine model showed that microplusin reduces the inflammation and the viability of C. neoformans in the lungs, indicating that, in optimized conditions, the peptide may act in the infection control.

Neste estudo, o extrato bruto obtido da cultura do fungo E. nigrum foi extraído com hexano (HEX), diclorometano (DCM) e acetato de etila (AE). Após fracionamento por HPLC, a atividade antifúngica das frações foi avaliada pelo ensaio de concentração inibitória mínima (CIM) contra Candida albicans (CA), Trychophyton rubrum (TR), Cryptococcus neoformans (CN) e Aspergillus fumigatus (AF). O extrato de AE não foi efetivo, e os extratos HEX e DCM inibiram os patógenos na faixa de 31,25 a 250 µg/mL. Das frações hexânicas, apenas HEX-F9 apresentou atividade antifúngica, com CIM entre 31,25 e 250 mg/mL, exceto sobre AF. As frações DCM-F3, F5, F6, F9 e F10 apresentaram atividade antifúngica com CIM de 31,25 a 500 mg/mL contra todos os patógenos testados. A fração DCM-F9 foi efetiva com CIM entre 62,5 e 250 mg/mL contra CA, TR e CN. As frações DCM-F9 e DCM-F11 estão em fase final de purificação para posterior caracterização físico-química. / The aim of this study was to purify the crude extract produced from E. nigrum and characterize antifungal isolated molecules. E. nigrum was cultivated and the culture supernatant extracted with hexane (HEX), dichloromethane (DCM) and ethyl acetate (AE). Fractions were obtained by HPLC. The antifungal activity were evaluated by minimal inhibitory concentration (CIM) against C. albicans (CA), T. rubrum (TR), C. neoformans (CN) and A. fumigatus (AF). The HEX extract inhibited all pathogens with CIM from 31,25 to 62,5 mg/mL while DCM from 62,5 to 250 mg/mL. From HEX fractions only HEX-F9 showed antigungal activity with CIM from 31,25 to 250 mg/mL, except AF. The DCM-F3, F5, F6, F9 and F10 fractions showed antifungal activity, mainly DCM-F6 (CIM from 31,25 to 62,5 mg/mL against all pathogens) and DCM-F9 (CIM from 62,5 to 250 mg/mL against CA, TR and CN). DCM-F6 was not produced anymore by the fungus in subsequent cultures. DCM-F9 LC/MS, CG/MS and RMN results suggested impure fraction, making difficult to determine the main compound. New approaches are being considered.

Os óleos essenciais de plantas representam uma fonte de potenciais princípios ativos que podem ser utilizados como defensivos agrícolas para combate de insetos, bactérias e fungos. Vários estudos científicos relatam o potencial fungitóxico de óleos essenciais inclusive de óleos provenientes de algumas espécies de Eucalyptus. O presente trabalho teve como objetivo avaliar o pontencial dos óleos provenientes de diferentes espécies de Eucalyptus, sobre a microflora natural do grão de milho em pós-colheita. Utilizou-se para isto três diferentes avaliações: a) a avaliação inicial que consistiu em avaliar o potencial antifúngico dos óleos de dez espécies de Eucalyptus sobre grãos de milho plaqueados sobre papel de filtro umedecido e considerando a microflora natural dos grãos; b) a avaliação da simulação de armazenamento dos grãos com apenas os óleos que se mostraram com maior potencial antifúngico na avaliação inicial e com misturas deles. Essa avaliação simula as condições dos grãos armazenados considerando a microflora natural e c) a avaliação em meio de cultura utilizando o meio Maize Meal Extract Agar e os fungos produtores de aflatoxinas, Aspergillus flavus e A. parasiticus. Em todas as avaliações foram avaliados os modos de exposição aos voláteis e por contato com os óleos. Os óleos de E. grandis, E. citriodora, o híbrido E. grandis x E. urophylla e o E. staigeriana, que apresentaram melhor eficiência antifúngica, na avaliação inicial, sobre a microflora natural dos grãos de milho da amostra utilizada, foram analisados quanto a sua composição por cromatografia gasosa acoplada à espectrometria de massas e utilizados nas demais avaliações. Na avaliação de armazenamento para o modo de ação por contato, a mistura E. grandis + E. staigeriana obteve o melhor resultado e no modo de ação por exposição aos voláteis, o melhor óleo foi o E. citriodora. Comparando os dois modos de ação, o por contato teve o melhor potencial antifúngico. Na avaliação em meio de cultura no modo de ação por contato, a mistura E. staigeriana + (E. grandis x E. urophylla) obteve melhor potencial antifúngico para ambos os fungos e para o modo de ação por exposição aos voláteis, o melhor potencial antifúngico foi com o óleo de E. staigeriana. Ambos os modos de ação foram estatisticamente iguais nesta avaliação para maioria dos tratamentos. Observou-se que os tratamentos apresentaram uma ação diferenciada sobre o crescimento e a produção de aflatoxinas que foi dependente do óleo, do modo de ação e da espécie aflatoxigênica avaliada. / The essential oils of plants are a potential source of active ingredients that can be used as pesticides to combat insects, bacteria and fungi. Several studies have been reported in the literature on the fungitoxic potential of essential oils, including oils from some species of Eucalyptus. This study aimed to evaluate the potential of essential oils from different species of Eucalyptus on the natural microflora of post-harvest corn. Three different assessments were used for this study: a) the initial assessment was used to grade ten Eucalyptus oils relating to its antifungal potential. In this assessment the effect of oil was checked on corn grains that were placed on moistered filter paper, considering the natural microflora of the grains, b) the second assessment was carried out just on higher antifungal potential oils observed in the initial evaluation and their binary blends. This evaluation simulates the conditions of grain storage considering the natural microflora of grain and c) the thrid assessment was carried out using basal medium (Maize Meal Extract Agar) and aflatoxigenic species, Aspergillus flavus and A. parasiticus. In all assessments, two assays were evaluated, headspace volatile exposure assay and contact assay. The oils of E. grandis, E. citriodora, the hybrid of E. grandis x E. urophylla and E. staigeriana which had the best antifungal efficiency in the initial evaluation on the natural microflora of corn were analyzed by gas chromatography with mass spectrometry for their composition. In the evaluation of grain storage for the contact assay, the mixture of E. grandis + E. staigeriana obtained the best result and for the headspace volatile exposure assay the best oil was E. citriodora. Comparing the two assays, contact and headspace volatile exposures, the contact assay had the best antifungal potential. In the evaluation using the basal medium in the contact assay, the mixture of E. staigeriana + (E. grandis x E. urophylla) achieved the best antifungal potential for both fungal species, and in the haedspace volatile exposure assay, the best antifungal potential was achieved with the oil of E. staigeriana. Both assays of this assessment were statistically identical to most treatments. It was observed that the treatments had a different influence over the fungal growth and aflatoxin production. And these depended on the oil, the assays and the aflatoxigenic species.

O Brasil tem uma posição de destaque mundial na produção de frutas e alguns grãos, os quais são comercializados in natura ou na forma de produtos processados. O processamento têm várias vantagens, porém gera grandes quantidades de resíduos, os quais têm sido aproveitados para a alimentação animal e geração de energia. Entretanto, muito tem sido relatado sobre o potencial bioativo desses materiais, dentre eles a ação fungitóxica de alguns de seus compostos sobre fungos fitopatogênicos. Assim, o objetivo desse trabalho foi avaliar o potencial de extratos de resíduos agroindustriais de abacate, uva, café, manga, abacaxi, maracujá e caju na inibição do crescimento micelial in vitro dos fungos Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani e Phomopsis sp., bem como a composição química do resíduo mais promissor. Os resíduos foram liofilizados, moídos e submetidos a extração com etanol 80% (etanol:água; 8:2, v/v) em banho de ultrassom (extrato denominado bruto ou EB). O EB também foi tratado com a resina Amberlite XAD-2, visando à eliminação de açúcares e interferentes, dando assim origem ao extrato semi-purificado (EP). Os extratos que apresentaram alto rendimento foram avaliados quanto à atividade antifúngica in vitro e teor de compostos fenólicos totais. A amostra que apresentou os melhores índices de inibição foi selecionada para dar continuidade ao estudo, sendo a mesma fracionada em coluna de gel Sephadex LH-20. Os extratos e as frações ativas, detectadas pelo ensaio de bioautografia, foram analisadas quanto a composição química pelas técnicas de HPLC e GC/MS. Na análise de fenólicos totais os maiores teores encontrados foram para a casca de abacate, enquanto que para o ensaio de inibição de crescimento micelial in vitro o melhor resultado foi para a semente de abacate, tanto para o EB quanto para o EP. Desta maneira, a semente de abacate foi selecionada para as etapas posteriores. Para o fracionamento em gel Sephadex LH-20 foi eleito o EP para F. pallidoroseum enquanto que para os demais fungos foi eleito o EB. No fracionamento do EP obtiveram-se 13 frações, sendo que as frações 3 e 4 foram ativas, enquanto que no do EB obtiveram-se nove, sendo as frações 3, 4 e 5 ativas. Pela técnica de HPLC foram detectados em comum nas frações 3, 4 e 5 do EB dois compostos majoritários, e nas frações 3 e 4 do EP, sete compostos, os quais não puderam ser identificados pelos padrões comerciais disponíveis. Já pela técnica de GC/MS foi possível a identificação de quatro compostos em comum nas frações ativas do EB e de 11 nas frações do EP. Dentre os compostos presentes nas frações ativas, foram identificados ácidos graxos, os quais têm sido reconhecidos por apresentarem ação antifúngica. Assim, pode-se concluir que os resíduos agroindustriais estudados são fontes de compostos com atividade antifúngica, podendo assim ser uma alternativa para o controle de fungos fitopatogênicos à cultura da soja / Brazil holds an important position in the world production of fruit and some grains, which are commercialized in natura or in processed products. The processing has several advantages, but generates large amounts of by-products which have been used for animal feed and power generation. However, bioactive potential of these materials have been reported, including the fungitoxic effect. The goal of this study was to evaluate the potential of agro-industrial by-products extracts of avocado, grape, coffee, mango, pineapple, passion fruit and cashew to inhibit in vitro mycelial growth of fungi Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani and Phomopsis sp. as well as the chemical composition of the most promising by-products. The by-products were freeze-dried, milled and the extraction was done with ethanol 80% (ethanol:water, 8:2, v/v) in an ultrasound bath (called crude extract or CE). The CE was also treated with Amberlite XAD-2 to eliminate sugars and interfering compounds so that the semi-purified extract (PE) could be obtained. Extracts with high yield were evaluated to in vitro antifungal activity and phenolic compounds content. Samples with the best inhibition rates were selected to continue this study, and it was fractioned on gel Sephadex LH-20. The extracts and the active fractions, detected by bioautography, were analyzed by HPLC and GC/MS. The highest phenolic content was found in the avocado peel, meanwhile for the mycelial growth inhibition in vitro, the best result was found in the avocado seeds for both extracts (CE and PE). Thus, avocado seed was selected for subsequent steps. For fractionation on Sephadex LH-20 gel, PE was chosen for F. pallidoroseum and CE for the other fungi. For PE, 13 fractions were obtained in which fractions 3 and 4 were active. For CE, 9 fractions were obtained and the actives were 3, 4 and 5. In CE, HPLC technique detected two major compounds in common in fractions 3, 4 and 5. In PE, seven major compounds were detected in fractions 3 and 4, which could not be identified by commercial available standards. By the GC/MS technique was possible to identify four compounds in common in the active CE fractions and 11 in PE. Among the compounds presented in the active fractions, fatty acids were identified. It has been reported that antifungal action has been found in these compounds. Therefore, the studied agroindustrial by-products are sources of compounds with antifungal activity and they can be used as an alternative to control phytopathogenic fungi on soybean

Fungal infections are one of the leading causes of death in humans, causing infections that range from mild superficial infections to severe, and life-threatening invasive infections that affect the bloodstream and vital organs. Invasive fungal infections have a high mortality rate, leading to approximately 1.5 million deaths annually. The most common pathogens responsible for these infections are Candida, Cryptococcus, and Aspergillus. Currently, treatment options for invasive fungal infections are limited to three main classes of antifungal drugs: azoles, polyenes, and echinocandins. The emergence of new fungal species, such as Candida auris, which displays high resistance and mortality rates (30-60%), has further complicated treatment efforts. Thus, there is a critical need for new therapeutic strategies to combat these life-threatening pathogens. C. auris isolates have demonstrated significant resistance, especially to azoles (fluconazole) and polyenes (amphotericin B, AmB). To address this, we screened approximately 2,600 FDA-approved drugs and clinical compounds to identify agents capable of inhibiting C. auris growth and enhancing or restoring the antifungal activity of existing antifungals. This screening revealed that HIV protease inhibitors, such as lopinavir, atazanavir, saquinavir and ritonavir, significantly enhanced the antifungal activity of azoles (fluconazole, voriconazole, itraconazole, and posaconazole) and polyene (AmB). Mechanistic studies showed that the HIV protease inhibitors inhibited the fungal efflux pump, and interfered with glucose utilization, leading to reduced ATP levels in C. auris. Moreover, HIV protease inhibitors, in combination with AmB, were able to inhibit the virulence factors of Candida species. Furthermore, HIV protease inhibitors, in combination with itraconazole or posaconazole, resulted in a significant reduction of the C. auris burden in mice kidneys. Two additional drugs, darapladib and rilapladib, phospholipase A2 inhibitors, were identified as potent inhibitors of C. auris. Darapladib and rilapladib demonstrated superior killing kinetics compared to itraconazole. Interestingly, C. auris did not develop any detectable resistance to both drugs at sub-inhibitory concentration over 16-passages. Mechanistic studies revealed that darapladib increased the plasma membrane permeability and caused DNA leakage, likely due to a direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. We further validated the broad-spectrum, fungicidal, and potent activity of darapladib in combination with AmB, showing significant synergy against multidrug-resistant fungal pathogens, including C. auris, Aspergillus fumigatus and mucormycosis-associated species. Additionally, darapladib demonstrated a superior safety profile compared to AmB, exhibiting lower affinity for cholesterol, and no toxicity in combination with AmB to kidney cells. Finally, rilapladib (at 2× MIC) inhibited the C. auris burden by 1.5 Log10, whereas darapladib (at 2× MIC) achieved complete eradication of the C. auris burden in an in vivo C. elegans model. / Doctor of Philosophy / Fungi are a group of living organisms that are neither plants nor animals. While Some fungi are beneficial, others can cause serious diseases in humans. Candida auris is a human fungal pathogen that causes serious infections with mortality rate up to 60%. It has become a growing threat to global public health. This pathogen is particularly concerning due to its high virulence, resistance to multiple antifungal drugs, and ability to rapidly spread, often leading to hospital outbreaks. In our current research, we have identified several compounds that either inhibit the growth of C. auris and/or enhance the efficacy of existing antifungals, such as azoles and amphotericin B. We have identified HIV protease inhibitors (lopinavir, atazanavir, saquinavir, and ritonavir) that were able to enhance the antifungal activities of both azoles (fluconazole, itraconazole, voriconazole, and posaconazole) and polyene (amphotericin B) against multidrug-resistant C. auris. The combination of HIV protease inhibitors with amphotericin B inhibited the virulence factors of Candida species, including biofilm and hyphae formation. Two drugs darapladib and rilapladib were found to not only inhibit the growth of C. auris alone but also enhance the antifungal activity of amphotericin B against all medically important fungal pathogens tested. Additionally, we have explored the mechanisms by which these compounds act on the pathogen. Finally, we have confirmed the effectiveness of these drugs in live models, including mice and worms.

No / A library of 53 benzimidazole derivatives, with substituents at positions 1, 2 and 5, were synthesized and screened against a series of reference strains of bacteria and fungi of medical relevance. The SAR analyses of the most promising results showed that the antimicrobial activity of the compounds depended on the substituents attached to the bicyclic heterocycle. In particular, some compounds displayed antibacterial activity against two methicillin-resistant Staphylococcus aureus (MRSA) strains with minimum inhibitory concentrations (MICs) comparable to the widely-used drug ciprofloxacin. The compounds have some common features; three possess 5-halo substituents; two are derivatives of (S)-2-ethanaminebenzimidazole; and the others are derivatives of one 2-(chloromethyl)-1H-benzo[d]imidazole and (1H-benzo[d]imidazol-2-yl)methanethiol. The results from the antifungal screening were also very interesting: 23 compounds exhibited potent fungicidal activity against the selected fungal strains. They displayed equivalent or greater potency in their MIC values than amphotericin B. The 5-halobenzimidazole derivatives could be considered promising broad-spectrum antimicrobial candidates that deserve further study for potential therapeutic applications.

La sicurezza e la qualità degli alimenti sono tutt’ora un problema critico per i paesi in via di sviluppo. Le diete a basso contenuto di acido folico, per esempio, possono causare gravi problemi di salute, soprattutto nei bambini. Gravi disturbi legati al tubo neurale (DTN) nei neonati possono derivare infatti da madri che hanno insufficiente apporto di acido folico (400-600 g / giorno) durante il periodo di gravidanza. Inoltre, se non adeguatamente protetti o trattati, I prodotti alimentari possono essere vettori di funghi e batteri patogeni rappresentando una fonte potenziale di malattie per l’uomo e una perdita economica per le industrie agro-alimentari. Nella seguente tesi si è quindi quindi studiato il ruolo di batteri lattici selezionati (LAB) in grado di aumentare il valore nutrizionale del latte attraverso la produzione di acido folico durante il processo di fermentazione. Inoltre, ci si è concentrati sul loro uso come "bio-conservanti" contro funghi e batteri, attraverso la sintesi di composti antimicrobici (batteriocine) in grado di inibire la crescita di funghi filamentosi e/o batteri patogeni. / The safety and quality of food are still a critical issue in developing countries. Diets with a low content of folic acid, for example, may cause serious health problems, especially in children. Severe disorders related to neural tube (NTD) in infants may arise from mothers having inadequate intakes of folic acid (400-600 g/dia) during the mother pregnancy period. Moreover foods, when not properly protected or treated, can be vectors of pathogenic fungi and bacteria thereby representing a potential source of human diseases and an economical loss for the food industry. In the following thesis we have therefore investigated the role of selected lactic acid bacteria (LAB) in increasing the nutritional value of milk through the production of folic acid during the fermentation process. In addition, we focused on their use as “bio-preservatives” against fungal and bacterial spoilage, through the synthesis of antimicrobial compounds (bacteriocins) able to inhibit the growth of filamentous fungi and /or pathogenic bacteria.

Thesis (MScAgric)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. Although integration of the MJ-EXG1 expression cassette was confirmed, no mRNA levels could be detected with northern blot or RT-PCR analysis of the MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse.

Rhododendron tomentosum is an evergreen shrub with a high content of secondary metabolites, particularly essential oils with antimicrobial effects. Diversity of endophytic fungi in this species and their possible adaptation to growth in the essential oil environment is not much explored. Therefore, the first aim of this thesis was to reveal the diversity of endophytic fungi colonising leaves of R. tomentosum on seven localities in the Czech Republic and one in Estonia. I isolated and determined (using comparison of ITS1 and ITS2 rDNA with the sequences from GenBank and morphological signs) 37 species of endophytic fungi. Among them the ubiquitous species colonising the most of the plants as endophytes were dominant. The second aim of my thesis was to explore whether the essential oil from R. tomentosum influences its endophytic fungi. The hypothesis that the strains obtained from R. tomentosum would be adapted to growth in the environment of the essential oil was postulated. I supposed that they would grow better on mediums with different concentrations of these chemical compounds added, in comparison with strains of the same species obtained from different substrates. Within four of seven species tested, the strains obtained from R. tomentosum grew better, but also on the medium without the...