Systemic fungal infection is a life-threatening problem. Anti-fungal drugs are the most effective clinical strategy to cure such infections. However, most current anti-fungal drugs either have high toxicity or have a narrow spectrum of effect. Meanwhile, anti-fungal drugs are losing their clinical efficacy due to emerging drug resistance. To protect us from these deadly pathogenic fungi, scientists need to study new drug targets and to solve problems related to drug resistance. The cell wall is essential for fungal cell survival and is absent from animal cells, so it is a promising reservoir for screening safe and effective drug targets. Alpha-1,3-glucan is one of the major cell wall carbohydrates and is important for the virulence of several pathogenic fungi. In this thesis, molecular biology and microscopy techniques were used to investigate the function and the synthesis process of α-1,3-glucan in the model fungus A. nidulans. My results showed that α-1,3-glucan comprises about 15% of A. nidulans cell wall dry weight, but also that α-1,3-glucan does not have an important role in cell wall formation and cell morphology. Deletion of α-1,3-glucan only affects conidial adhesion and cell sensitivity to calcofluor white. In contast, elevated α-1,3-glucan content can cause severe phenotypic defects. To study the α-1,3-glucan synthesis process, I systematically characterized four proteins, including two α-1,3-glucan synthases (AgsA and AgsB) and two amylase-like proteins (AmyD and AmyG). Results showed AgsA and AgsB are both functional synthases. AgsB is the major synthase due to its constant expression. AgsA mainly functions in conidiation stages. AmyG is a cytoplasmic protein that is critical for α-1,3-glucan synthesis, likely being required for an earlier step in the synthesis process. In contrast to the other three proteins, AmyD has a repressive effect on α-1,3-glucan accumulation. These results shed light on therapeutic strategies that might be developed against α-1,3-glucan. I also developed a strategy to investigate drug resistance mutations. The tractability of A. nidulans and the power of next generation sequencing enabled an easy approach to isolate single mutation strains and to identify the causal mutations from a genome scale efficiently. I suggest this strategy has applications to study the drug resistance mechanisms of current anti-fungal drugs and even possibly future ones.

Philosophiae Doctor - PhD / The reproductive health of individuals is severely compromised by HIV infection, with candidiasis being the most prevalent oral complication in patients. Although not usually associated with severe morbidity, oropharyngeal candidiasis can be clinically significant, as it can interfere with the administration of medications and adequate nutritional intake, and may spread to the esophagus. Azole antifungal agents are commonly prescribed for the treatment and prophylaxis of candidal infections. However, the emergence of drug resistant strains and dose limiting toxic effects have complicated the treatment of candidiasis. Consequently, safe and effective and affordable medicine is required to combat this fungus. Commercial garlic (Allium sativum) has been used time since immemorial as a natural antibiotic, however very little is known about the antifungal properties of two indigenous South African species of garlic, namely Tulbaghia alliacea and Tulbaghia violacea, that are used as folk medicines for a variety of infections. This study compares the in vitro anti-candidal activity of Tulbaghia alliacea, Tulbaghia violacea and Allium sativum extracts. It was found that the greatest concentrations of inhibitory components were extracted by chloroform or water. The IC50 concentrations of Tulbaghia alliacea were between 0.007 - 0.038% (w/v). Assays using S. cerevisiae revealed that the T. alliacea extract was fungicidal, with a killing half-life of approximately 2 hours. This inhibitory effect of the T. alliacea extracts was observed via TLC, and may be due to an active compound called Marasmicin, that was identified using NMR. This investigation confirms that extracts of T.alliacea exhibit anti-infective activity against candida species in vitro. / South Africa

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-27T14:45:53Z No. of bitstreams: 1 Arquivototal.pdf: 1693139 bytes, checksum: 05772ba5cdc4001f007fd50e10adb2b9 (MD5) / Made available in DSpace on 2018-04-27T14:45:54Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 1693139 bytes, checksum: 05772ba5cdc4001f007fd50e10adb2b9 (MD5) Previous issue date: 2017-04-18 / Seeds, rhizomes and tubers are welcomed with sources of bioactive molecules, which are related to health promotion, such as proteins and other components of plant sources. (Dioscorea cayennensis) is part of the Brazilian food habit, it is necessary to study the proteins present in this plant. The objective of this work was to isolate and characterize biochemically the dioscorin proteic fraction (DPF) present in the tubers of D. cayennensis, characterizing the presence of antinutritional compounds and antioxidant, antibacterial and antifungal biological activities. Isolation of DPF was performed with protein extraction in Tris-HCl 0.05M pH 8.3, followed by precipitation in Ammonium Sulfate (F45-75). The molecular weight of DPF was determined by SDS-PAGE and MALDI-ToF, characterized by the presence of a major component with a mass of approximately 28 kDa corresponding to dioscorin. When tested for the presence of phytochemicals and antinutritional compounds (lectins and trypsin inhibitors), the presence of these compounds was not detected in the DPF. The amino acid composition of the fraction was determined and the antioxidant activity estimated by the ABTS and DPPH methods. Glutamic acid, Aspartic acid and Arginine were the amino acids that showed higher levels in DPF, which explains the antioxidant activity, suggesting that the interaction of these amino acids seems to act on reactive molecules making them stable. Finally, in the evaluation of the antibacterial and antifungal activity of the total extract and DPF against different strains of microorganisms, an antibacterial potential was detected between 50-500 μg/mL of protein for inhibition of the growth of strain of L. monocytogenes. / Sementes, rizomas e tubérculos são geralmente boas fontes de moléculas bioativas, as quais estão relacionadas à promoção da saúde, as proteínas e outros compostos oriundos de fontes vegetais, vem ganhando relevância em pesquisas científicas. Considerando que o consumo do tubérculo de inhame (Dioscorea cayennensis) faz parte do hábito alimentar brasileiro, faz-se necessário o estudo das proteínas presentes neste vegetal. O objetivo deste trabalho foi isolar e caracterizar bioquimicamente a fração proteica dioscorina (FPD) presente nos tubérculos de D. cayennensis, detectando a presença de compostos antinutricionais e atividades biológicas antioxidante, antibacteriana e antifúngica. O isolamento do FPD foi realizado com a extração proteica em Tris-HCl 0,05M pH 8,3, seguido de precipitação em Sulfato de Amônio (F45-75). A massa molecular da FPD foi determinada por SDS-PAGE e MALDI-ToF. Caracterizado pela presença de um componente majoritário com massa de aproximadamente 28 kDa, correspondente a dioscorina. Quando submetida a testes de avaliação da presença de fitoquímicos e compostos antinutricionais (lectinas e inibidores de tripsina), não foi detectada na FPD a presença destes compostos avaliados. A composição aminoacídica da fração foi determinada e a atividade antioxidante estimada pelos métodos de ABTS e DPPH. Ácido glutâmico, Ácido Aspártico e Arginina foram os aminoácidos que apresentaram maiores teores na FPD, o que explica a atividade antioxidante verificada, sugerindo que a interação destes aminoácidos parece atuar em moléculas reativas tornando-as estáveis. Por fim, na avaliação da atividade antibacteriana e antifúngica do extrato total e FPD frente a diferentes cepas de micro-organismos, foi detectado potencial antibacteriano entre 50-500 μg/mL de proteína para inibição do crescimento da cepa L. monocytogenes.

<p>This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996).</p>

<p>This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996).</p>

Magister Scientiae - MSc / This investigation focused on Rhoicissus tomentosa, belonging to the family, Vitaceae in an attempt to assess the phytochemistry of this plant which is widely used by traditional healers in South Africa to ensure the safe delivery during pregnancy and childbirth (Hutchings et al., 1996). / South Africa

Neem cake is a product of the cold pressing from the neem kernels to obtain neem oil. Bio-active substances from neem cake extracted solutions were evaluated for their potential to control the root knot nematodes and other pests of plants. In this study different concentrations of the solution extracted from neem cake was tested against the second stage juveniles of the plant parasitic nematode Meloidogyne spp. and four phytopathogenic fungi: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytopthora capsici. Toxicity of neem cake extractions is represented by the EC50 value for the second-stage juvenile (J2) of Meloidogyne spp. and the four phytopathogenic fungi via Probit analysis. A 5% dilution of the solvent extracting from neem cake already caused 100% larval mortality after 24 hours exposure. Undiluted neem cake extraction effectively inhibited the growth of the four phytopathogenic fungi. The EC50 value of neem cake on J2-larvae of Meloidogyne nematode and on the fungi Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici was 0.51, 0.74, 0.30, 0.51 and 4.33%, respectively. / Bánh dầu neem là sản phẩm của quá trình ép nhân hạt neem để lấy dầu. Các hoạt chất sinh học từ dịch chiết bánh dầu neem đã được đánh giá có tiềm năng lớn trong phòng trừ tuyến trùng nốt sưng và các loài dịch hại khác của nhiều loại cây trồng. Trong nghiên cứu này các nồng độ dịch chiết khác nhau của bánh dầu neem đã được thử nghiệm khả năng diệt tuyến trùng (ấu trùng tuổi 2 thuộc giống Meloidogyne spp.) và ức chế 4 loài nấm bệnh như: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. và Phytopthora capsici. Độc tính của dịch chiết bánh dầu neem được biểu diễn bởi giá trị EC50 đối với ấu trùng tuổi 2 của tuyến trùng Meloidogyne spp. và các loài nấm bệnh thông qua phân tích Probit. Dịch chiết bánh dầu neem ở nồng độ 5% đã làm chết 100% cá thể IJ2 của Meloidogyne spp sau 24 giờ phơi nhiễm. Dịch nguyên chất bánh dầu neem ức chế cả 4 loài nấm bệnh. Giá trị EC50 của bánh dầu neem lên ấu trùng tuổi 2 của Meloidogyne spp và các loài nấm bệnh Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici tương ứng là 0.51, 0.74, 0.30, 0.51 và 4.33%.

La vectorisation des médicaments est une approche très prometteuse tant sur le plan médical qu’économique pour la livraison des substances actives ayant une faible biodisponibilité. Dans ce contexte, les polymères en étoile et les dendrimères, macromolécules symétriques et branchées, semblent être les solutions de vectorisation les plus attrayantes. En effet, ces structures peuvent combiner efficacement une stabilité élevée dans les milieux biologiques à une capacité d’encapsulation des principes actifs. Grâce à leur architecture bien définie, ils permettent d’atteindre un très haut niveau de reproductibilité de résultats, tout en évitant le problème de polydispersité. Bien que des nombreuses structures dendritiques aient été proposées ces dernières années, il est cependant à noter que la conception de nouveaux nanovecteurs dendritiques efficaces est toujours d’actualité. Ceci s’explique par des nombreuses raisons telles que celles liées à la biocompatibilité, l’efficacité d’encapsulation des agents thérapeutiques, ainsi que par des raisons économiques. Dans ce projet, de nouvelles macromolécules branchées biocompatibles ont été conçues, synthétisées et évaluées. Pour augmenter leur efficacité en tant qu’agents d’encapsulations des principes actifs hydrophobes, les structures de ces macromolécules incluent un coeur central hydrophobe à base de porphyrine, décanediol ou trioléine modifié et, également, une couche externe hydrophile à base d’acide succinique et de polyéthylène glycol. Le choix des éléments structuraux de futures dendrimères a été basé sur les données de biocompatibilité, les résultats de nos travaux de synthèse préliminaires, ainsi que les résultats de simulation in silico réalisée par une méthode de mécanique moléculaire. Ces travaux ont permis de choisir des composés les plus prometteurs pour former efficacement et d’une manière bien contrôlable des macromolécules polyesters. Ils ont aussi permis d’évaluer au préalable la capacité de futurs dendrimères de capter une molécule médicamenteuse (itraconazole). Durant cette étape, plusieurs nouveaux composés intermédiaires ont été obtenus. L’optimisation des conditions menant à des rendements réactionnels élevés a été réalisée. En se basant sur les travaux préliminaires, l’assemblage de nouveaux dendrimères de première et de deuxième génération a été effectué, en utilisant les approches de synthèse divergente et convergente. La structure de nouveaux composés a été prouvée par les techniques RMN du proton et du carbone 13C, spectroscopie FTIR, UV-Vis, analyse élémentaire, spectrométrie de masse et GPC. La biocompatibilité de produits a été évaluée par les tests de cytotoxicité avec le MTT sur les macrophages murins RAW-262.7. La capacité d’encapsuler les principes actifs hydrophobes a été étudiée par les tests avec l’itraconazole, un antifongique puissant mais peu biodisponible. La taille de nanoparticules formées dans les solutions aqueuses a été mesurée par la technique DLS. Ces mesures ont montré que toutes les structures dendritiques ont tendance à former des micelles, ce qui exclue leurs applications en tant que nanocapsules unimoléculaires. L’activité antifongique des formulations d’itraconazole encapsulé avec les dendrimères a été étudiée sur une espèce d’un champignon pathogène Candida albicans. Ces tests ont permis de conclure que pour assurer l’efficacité du traitement, un meilleur contrôle sur le relargage du principe actif était nécessaire. / The drug molecule vectorization is a very promising approach in terms of both medical and economical factors for the delivery of active substances with low bioavailability. In this context, the star polymers and dendrimers, symmetrical and branched macromolecules, seem to be more attractive solutions. Indeed, these structures can effectively combine a high stability in biological media and the ability to encapsulate active ingredients. Thanks to the well-defined architecture, they can achieve a high level of reproducibility of results, while avoiding the problem of polydispersity. In recent years, many dendritic structures have been proposed; however, the design of new effective dendritic nanocarriers is still relevant. This is due to many reasons such as related to biocompatibility, encapsulation efficiency of therapeutic agents, as well as economic reasons. In this project, new branched biocompatible macromolecules were designed, synthesized and evaluated. To increase their effectiveness as encapsulation agents for hydrophobic active principles, the structures of the proposed macromolecules include a hydrophobic central core on the basis of porphyrin, decanediol or modified triolein, and also a hydrophilic outer layer based on succinic acid and polyethylene glycol. The choice of structural elements of future dendrimers was based on the data on their biocompatibility and the results of our preliminary synthesis works, as well as the in silico simulations performed by using the method of molecular mechanics. The preliminary studies allowed for selecting the most promising compounds to effectively form polyesters macromolecules in well controlled manner, as well as to assess in advance the ability of future dendrimers to capture a drug molecule (itraconazole). During this phase, several new intermediates were obtained. The optimization of reaction conditions leading to high yields was performed. Based on the preliminary work, the assembly of new dendrimers of first and second generations was performed, by using the divergent and convergent synthesis approaches. The structures of new compounds were characterized by proton and 13C carbon NMR, FTIR, UV-Vis, elemental analysis, mass spectrometry, and GPC techniques. The biocompatibility of products was evaluated by cytotoxicity tests with MTT on murine RAW 262.7 macrophages. The ability to encapsulate hydrophobic active principles was studied by testing with itraconazole, an antifungal agent with low bioavalability. The size of nanoparticles formed in aqueous solutions was measured by the DLS technique. These measurements showed that all dendritic structures tend to form micelles, which excludes their application as unimolecular nanocapsules. The antifungal activity of itraconazole formulations with dendrimers was studied in a kind of a pathogenic fungus Candida albicans. These tests lead to the conclusion that to ensure the effectiveness of treatment, more control over the release of the active ingredient has been needed.

La vectorisation des médicaments est une approche très prometteuse tant sur le plan médical qu’économique pour la livraison des substances actives ayant une faible biodisponibilité. Dans ce contexte, les polymères en étoile et les dendrimères, macromolécules symétriques et branchées, semblent être les solutions de vectorisation les plus attrayantes. En effet, ces structures peuvent combiner efficacement une stabilité élevée dans les milieux biologiques à une capacité d’encapsulation des principes actifs. Grâce à leur architecture bien définie, ils permettent d’atteindre un très haut niveau de reproductibilité de résultats, tout en évitant le problème de polydispersité. Bien que des nombreuses structures dendritiques aient été proposées ces dernières années, il est cependant à noter que la conception de nouveaux nanovecteurs dendritiques efficaces est toujours d’actualité. Ceci s’explique par des nombreuses raisons telles que celles liées à la biocompatibilité, l’efficacité d’encapsulation des agents thérapeutiques, ainsi que par des raisons économiques. Dans ce projet, de nouvelles macromolécules branchées biocompatibles ont été conçues, synthétisées et évaluées. Pour augmenter leur efficacité en tant qu’agents d’encapsulations des principes actifs hydrophobes, les structures de ces macromolécules incluent un coeur central hydrophobe à base de porphyrine, décanediol ou trioléine modifié et, également, une couche externe hydrophile à base d’acide succinique et de polyéthylène glycol. Le choix des éléments structuraux de futures dendrimères a été basé sur les données de biocompatibilité, les résultats de nos travaux de synthèse préliminaires, ainsi que les résultats de simulation in silico réalisée par une méthode de mécanique moléculaire. Ces travaux ont permis de choisir des composés les plus prometteurs pour former efficacement et d’une manière bien contrôlable des macromolécules polyesters. Ils ont aussi permis d’évaluer au préalable la capacité de futurs dendrimères de capter une molécule médicamenteuse (itraconazole). Durant cette étape, plusieurs nouveaux composés intermédiaires ont été obtenus. L’optimisation des conditions menant à des rendements réactionnels élevés a été réalisée. En se basant sur les travaux préliminaires, l’assemblage de nouveaux dendrimères de première et de deuxième génération a été effectué, en utilisant les approches de synthèse divergente et convergente. La structure de nouveaux composés a été prouvée par les techniques RMN du proton et du carbone 13C, spectroscopie FTIR, UV-Vis, analyse élémentaire, spectrométrie de masse et GPC. La biocompatibilité de produits a été évaluée par les tests de cytotoxicité avec le MTT sur les macrophages murins RAW-262.7. La capacité d’encapsuler les principes actifs hydrophobes a été étudiée par les tests avec l’itraconazole, un antifongique puissant mais peu biodisponible. La taille de nanoparticules formées dans les solutions aqueuses a été mesurée par la technique DLS. Ces mesures ont montré que toutes les structures dendritiques ont tendance à former des micelles, ce qui exclue leurs applications en tant que nanocapsules unimoléculaires. L’activité antifongique des formulations d’itraconazole encapsulé avec les dendrimères a été étudiée sur une espèce d’un champignon pathogène Candida albicans. Ces tests ont permis de conclure que pour assurer l’efficacité du traitement, un meilleur contrôle sur le relargage du principe actif était nécessaire. / The drug molecule vectorization is a very promising approach in terms of both medical and economical factors for the delivery of active substances with low bioavailability. In this context, the star polymers and dendrimers, symmetrical and branched macromolecules, seem to be more attractive solutions. Indeed, these structures can effectively combine a high stability in biological media and the ability to encapsulate active ingredients. Thanks to the well-defined architecture, they can achieve a high level of reproducibility of results, while avoiding the problem of polydispersity. In recent years, many dendritic structures have been proposed; however, the design of new effective dendritic nanocarriers is still relevant. This is due to many reasons such as related to biocompatibility, encapsulation efficiency of therapeutic agents, as well as economic reasons. In this project, new branched biocompatible macromolecules were designed, synthesized and evaluated. To increase their effectiveness as encapsulation agents for hydrophobic active principles, the structures of the proposed macromolecules include a hydrophobic central core on the basis of porphyrin, decanediol or modified triolein, and also a hydrophilic outer layer based on succinic acid and polyethylene glycol. The choice of structural elements of future dendrimers was based on the data on their biocompatibility and the results of our preliminary synthesis works, as well as the in silico simulations performed by using the method of molecular mechanics. The preliminary studies allowed for selecting the most promising compounds to effectively form polyesters macromolecules in well controlled manner, as well as to assess in advance the ability of future dendrimers to capture a drug molecule (itraconazole). During this phase, several new intermediates were obtained. The optimization of reaction conditions leading to high yields was performed. Based on the preliminary work, the assembly of new dendrimers of first and second generations was performed, by using the divergent and convergent synthesis approaches. The structures of new compounds were characterized by proton and 13C carbon NMR, FTIR, UV-Vis, elemental analysis, mass spectrometry, and GPC techniques. The biocompatibility of products was evaluated by cytotoxicity tests with MTT on murine RAW 262.7 macrophages. The ability to encapsulate hydrophobic active principles was studied by testing with itraconazole, an antifungal agent with low bioavalability. The size of nanoparticles formed in aqueous solutions was measured by the DLS technique. These measurements showed that all dendritic structures tend to form micelles, which excludes their application as unimolecular nanocapsules. The antifungal activity of itraconazole formulations with dendrimers was studied in a kind of a pathogenic fungus Candida albicans. These tests lead to the conclusion that to ensure the effectiveness of treatment, more control over the release of the active ingredient has been needed.

Neem cake is a product of the cold pressing from the neem kernels to obtain neem oil. Bio-active substances from neem cake extracted solutions were evaluated for their potential to control the root knot nematodes and other pests of plants. In this study different concentrations of the solution extracted from neem cake was tested against the second stage juveniles of the plant parasitic nematode Meloidogyne spp. and four phytopathogenic fungi: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytopthora capsici. Toxicity of neem cake extractions is represented by the EC50 value for the second-stage juvenile (J2) of Meloidogyne spp. and the four phytopathogenic fungi via Probit analysis. A 5% dilution of the solvent extracting from neem cake already caused 100% larval mortality after 24 hours exposure. Undiluted neem cake extraction effectively inhibited the growth of the four phytopathogenic fungi. The EC50 value of neem cake on J2-larvae of Meloidogyne nematode and on the fungi Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici was 0.51, 0.74, 0.30, 0.51 and 4.33%, respectively. / Bánh dầu neem là sản phẩm của quá trình ép nhân hạt neem để lấy dầu. Các hoạt chất sinh học từ dịch chiết bánh dầu neem đã được đánh giá có tiềm năng lớn trong phòng trừ tuyến trùng nốt sưng và các loài dịch hại khác của nhiều loại cây trồng. Trong nghiên cứu này các nồng độ dịch chiết khác nhau của bánh dầu neem đã được thử nghiệm khả năng diệt tuyến trùng (ấu trùng tuổi 2 thuộc giống Meloidogyne spp.) và ức chế 4 loài nấm bệnh như: Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. và Phytopthora capsici. Độc tính của dịch chiết bánh dầu neem được biểu diễn bởi giá trị EC50 đối với ấu trùng tuổi 2 của tuyến trùng Meloidogyne spp. và các loài nấm bệnh thông qua phân tích Probit. Dịch chiết bánh dầu neem ở nồng độ 5% đã làm chết 100% cá thể IJ2 của Meloidogyne spp sau 24 giờ phơi nhiễm. Dịch nguyên chất bánh dầu neem ức chế cả 4 loài nấm bệnh. Giá trị EC50 của bánh dầu neem lên ấu trùng tuổi 2 của Meloidogyne spp và các loài nấm bệnh Rhizoctonia solani, Sclerotium rolfsii, Collectotrichum spp. and Phytophthora capsici tương ứng là 0.51, 0.74, 0.30, 0.51 và 4.33%.