The significance of fungal infections has been grossly underestimated. Only a few drugs are clinically available to treat life-threatening fungal infections, and resistance against these drugs is rising. Antifungal peptides (AFPs) are being actively explored as novel pharmaceuticals. PAF26 is a de novo designed hexapeptide possessing N-terminal cationic and C-terminal hydrophobic regions. Previously the roles of each of these motifs in the antifungal mode-of-action of PAF26 have indicated that it involves three stages: interaction with the plasma membrane, internalisation, and cell killing. The overall aim of my project was to obtain further insights into its mode-of-action and develop more active antifungal derivatives of PAF26. Three experimental fungal systems were used in this study: the model Neurospora crassa, the human pathogen Aspergillus fumigatus and the plant/human pathogen Fusarium oxysporum. The first objective of the study was to evaluate the impact of different fluorescent labels on the intracellular localisation and antifungal properties of PAF26. For this purpose a library of PAF26 labelled with 13 different fluorophores was synthesised. This library contained PAF26 conjugates of broad chemical and spectral diversity. These fluorescent PAF26 conjugates were analysed by live-cell imaging and tested for their antifungal activities. The different fluorescent labels were found to have significant impacts both on intracellular localisation and antifungal activities. TMR, carboxyfluorescein, NBD and DMN were found to be the best labels for live-cell imaging because they had the least influence on the intracellular localisation and antifungal activity of PAF26. The second objective was to identify target proteins of PAF26 in N. crassa cells. A large number of proteins were identified as binding to PAF26 from a protein pull-down and mass spectroscopy analysis using TMR- and fluorescein-labelled PAF26. One of these proteins was the highly abundant plasma membrane ATPase PMA-1. An in-silico analysis showed that PMA-1 is likely to be a major target protein of PAF26. The final objective was to develop novel antifungals based on PAF26 with improved activities and stability. Novel cyclic derivatives of PAF26 were designed in-silico against PMA-1. These peptides were synthesised and tested against N. crassa, A. fumigatus and F. oxysporum and were found to have higher activities (at the sub-micromolar level) and greater stability than the linear PAF26. Overall this study has provided novel mechanistic insights into the mode-of-action of PAF26 and discovered novel highly active antifungal peptides with clinical potential as therapeutics.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Michael Kanost / Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC₅₀ of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta. Biophysical analysis showed that diapausin-1 binds to the β-1,3 glucan component of the S. cerevisiae cell wall. A second insect peptide investigated in this project was M.sexta stress-response peptide 1(SRP1), an immune-related peptide upregulated under different stress conditions including immune-challenge. Preliminary results for NMR structure determination are presented. Most of the amino acid residue spin systems were assigned, and we determined the connectivities of many amino residues as a first step to solve the NMR structure. The circular dichroism spectrum of SRP1 indicates that the peptide lacks alpha-helical structure and may contain beta strands and turns.

Orientador: Saulo Santesso Garrido / Coorientador: Marlus Chorilli / Banca: Fernando Rogério Pavan / Banca: Eduardo Festozo Vicente / Resumo: Atualmente aproximadamente 75-88% das infecções fúngicas são causadas por espécies de Candida, que geram um custo de 1,7 bilhões de dólares para a saúde pública, somente nos EUA. Candida albicans é o principal microrganismo causador da candidíase bucal, uma infecção da mucosa oral do organismo humano. A patogenicidade dessa espécie está relacionada com a formação de biofilmes, que agem como um revestimento impermeável e protetor, e tornam o microrganismo resistente a medicamentos convencionais. Neste caso, os antifúngicos mais comumente utilizados, não apresentam ação eficaz. Por esse motivo, a busca por novas opções de tratamento e por novos medicamentos está em constante desenvolvimento, principalmente na área da biotecnologia. Um exemplo disso são os peptídeos antifúngicos da família das Histatinas, entre eles a Histatina-5. Trata-se de um peptídeo naturalmente encontrado na saliva humana, mas que sofre rápida degradação quando presente na cavidade bucal, que é seu local de ação. Diante disto, o objetivo deste trabalho é o desenvolvimento de lipossomas de diferentes composições lipídicas, na intenção de proteger o peptídeo e melhorar sua ação, preservando seu potencial antifúngico. Para isso foi sintetizado o peptídeo 0WHistatina-5, um análogo do peptídeo Histatina-5, que contém o aminoácido triptofano em sua sequência. Foi utilizado o método de síntese em fase sólida, seguido de clivagem e purificação, com confirmação da massa molecular utilizando HPLC e ESI-MS. Os liposs... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently, approximately 75-88% of fungal infections are caused by Candida species, which generate a cost of $ 1.7 billion for public health in the US. Candida albicans is the main microorganism that causes oral candidiasis, an infection of the oral mucosa of the human organism. The pathogenicity of this species is related to the formation of biofilms, which act as an impermeable and protective coating, and make the microorganism resistant to conventional drugs. In this case, the most commonly used antifungals, have no effective action. For this reason, the search for new treatment options and new drugs is in constant development, especially in the biotechnology area. The antifungal peptides of the Histatin family, including Histatin-5, are an example. The Histatin-5 is a peptide naturally found in human saliva, but it undergoes rapid degradation when present in the oral cavity, which is its place of action. For this reason, this work aimed at developing liposomes of different lipid compositions, in order to protect the peptide, improve its action, and preserve its antifungal potential. For this, the peptide 0WHistatin-5, an analog of the peptide Histatin-5, was synthesized, which contains the amino acid tryptophan in its sequence. Therefore, the peptide was synthesized manually by the solid phase synthesis method, followed by cleavage and purification. The molecular weight was confirmed by using HPLC and ESI-MS. The liposomes were produced by the lipid film hydration method,... (Complete abstract click electronic access below) / Mestre

Submitted by Carolina Reis Zambom null (carolinarz@gmail.com) on 2018-03-20T17:16:52Z No. of bitstreams: 1 Dissertação Carol_versão correçãotodos_versãofinalpósdefesa-edit.pdf: 2874016 bytes, checksum: 09ff8b1558d91035a2e106afc33f8bd8 (MD5) / Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2018-03-23T17:38:22Z (GMT) No. of bitstreams: 1 zambom_cr_me_araiq_int.pdf: 2737311 bytes, checksum: 4eca302db8e9f1efe9d15f6049d7aa9a (MD5) / Made available in DSpace on 2018-03-23T17:38:22Z (GMT). No. of bitstreams: 1 zambom_cr_me_araiq_int.pdf: 2737311 bytes, checksum: 4eca302db8e9f1efe9d15f6049d7aa9a (MD5) Previous issue date: 2018-03-02 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Atualmente aproximadamente 75-88% das infecções fúngicas são causadas por espécies de Candida, que geram um custo de 1,7 bilhões de dólares para a saúde pública, somente nos EUA. Candida albicans é o principal microrganismo causador da candidíase bucal, uma infecção da mucosa oral do organismo humano. A patogenicidade dessa espécie está relacionada com a formação de biofilmes, que agem como um revestimento impermeável e protetor, e tornam o microrganismo resistente a medicamentos convencionais. Neste caso, os antifúngicos mais comumente utilizados, não apresentam ação eficaz. Por esse motivo, a busca por novas opções de tratamento e por novos medicamentos está em constante desenvolvimento, principalmente na área da biotecnologia. Um exemplo disso são os peptídeos antifúngicos da família das Histatinas, entre eles a Histatina-5. Trata-se de um peptídeo naturalmente encontrado na saliva humana, mas que sofre rápida degradação quando presente na cavidade bucal, que é seu local de ação. Diante disto, o objetivo deste trabalho é o desenvolvimento de lipossomas de diferentes composições lipídicas, na intenção de proteger o peptídeo e melhorar sua ação, preservando seu potencial antifúngico. Para isso foi sintetizado o peptídeo 0WHistatina-5, um análogo do peptídeo Histatina-5, que contém o aminoácido triptofano em sua sequência. Foi utilizado o método de síntese em fase sólida, seguido de clivagem e purificação, com confirmação da massa molecular utilizando HPLC e ESI-MS. Os lipossomas foram produzidos pelo método de hidratação do filme lipídico, em 3 composições lipídicas diferentes, denominadas de F1, F2 e F3. F1 possui somente DPPC e Chol em sua composição, F2 contém, além desses componentes, PEG e F3 contém DPPC, Chol e POPG. Os lipossomas foram submetidos a processo de extrusão e sonicação para padronização do tamanho das vesículas, e estudo da melhor técnica para sua produção. Os lipossomas foram caracterizados por espalhamento de luz dinâmico determinação da eficiência de encapsulação, cinética de liberação, estabilidade e avaliação da atividade antifúngica. O método de síntese do peptídeo 0WHistatina-5 foi adequado e o processo de purificação possibilitou a obtenção do peptídeo com alto grau de pureza. Os lipossomas obtidos por extrusão apresentaram tamanho médio na faixa de 100 nm, enquanto os lipossomas obtidos por sonicação apresentaram tamanho menor, na faixa de 90 nm. Os lipossomas contendo 0WHistatina-5, apresentaram aumento em seu tamanho médio, indicando que o peptídeo está contido no compartimento interno aquoso dos lipossomas. A eficiência de encapsulação foi maior para os lipossomas obtidos por sonicação, sendo de 34,5% para a formulação F1, que contém DPPC e Chol em sua composição. A composição lipídica dos lipossomas está relacionada com a sua eficiência de encapsulação, e a presença de colesterol dificultou a encapsulação do peptídeo. A estabilidade das formulações também está relacionada com sua composição, sendo a formulação F3, obtida por sonicação e com POPG em sua formulação, a que apresentou melhor estabilidade quando armazenada por 60 dias a temperatura de 4°C. As formulações desenvolvidas apresentaram capacidade de liberar o peptídeo pelo tempo total de 96 horas, com o primeiro pico de liberação após 5 horas, e novo aumento do conteúdo liberado após 30 horas. O ensaio antimicrobiano foi realizado utilizando C. albicans ATCC 10231, e demostrou que no tempo de 4 horas a inibição para F1 foi de 66,5%. Assim, este trabalho demonstrou que a utilização de sistemas nanoestruturados é de grande importância para viabilizar a aplicação do peptídeo Histatina-5 em estudos terapêuticos, e em estudos in vivo no futuro. / Currently, approximately 75-88% of fungal infections are caused by Candida species, which generate a cost of $ 1.7 billion for public health in the US. Candida albicans is the main microorganism that causes oral candidiasis, an infection of the oral mucosa of the human organism. The pathogenicity of this species is related to the formation of biofilms, which act as an impermeable and protective coating, and make the microorganism resistant to conventional drugs. In this case, the most commonly used antifungals, have no effective action. For this reason, the search for new treatment options and new drugs is in constant development, especially in the biotechnology area. The antifungal peptides of the Histatin family, including Histatin-5, are an example. The Histatin-5 is a peptide naturally found in human saliva, but it undergoes rapid degradation when present in the oral cavity, which is its place of action. For this reason, this work aimed at developing liposomes of different lipid compositions, in order to protect the peptide, improve its action, and preserve its antifungal potential. For this, the peptide 0WHistatin-5, an analog of the peptide Histatin-5, was synthesized, which contains the amino acid tryptophan in its sequence. Therefore, the peptide was synthesized manually by the solid phase synthesis method, followed by cleavage and purification. The molecular weight was confirmed by using HPLC and ESI-MS. The liposomes were produced by the lipid film hydration method, with three different lipid compositions, named F1, F2 and F3. F1 has only DPPC and Chol in its composition, F2 contains, in addition to these components, PEG and F3 contains DPPC, Chol and POPG. The liposomes were submitted to extrusion and sonication processes, in order to standardize their vesicle size, and determine the best technique for their production. The dynamic light scattering technique was used to characterize the liposomes, which were tested for encapsulation efficiency, release kinetics, stability and evaluation of the antifungal activity. The synthesis method of the Histatin-5 was adequate and the purification process allowed to obtain the peptide in high purity. The liposomes obtained by extrusion presented average size in the range of 100 nm, while the liposomes obtained by sonication presented a smaller size, in the range of 90 nm. Liposomes loaded with Histatin-5 showed an increase in their mean size, which indicated that the peptide was contained in the intern aqueous compartment of the liposomes. The encapsulation efficiency was higher for the liposomes obtained by sonication. The F1 formulation presented an encapsulation efficiency of 34.5%, which contains DPPC and Chol in its composition. The lipid composition of the liposomes was related to their encapsulation efficiency, and the presence of cholesterol hindered the encapsulation of the peptide. The stability of the formulations was also related to their compositions. The F3 formulation obtained by sonication and containing POPG presented a higher stability when stored for 60 days at 4°C. The formulations showed the ability to release the peptide in the total period of 96 hours. The first release peak was observed after 5 hours, and a further increase of the released content was verified after 30 hours. The antimicrobial assay was performed using C. albicans ATCC 10231. It demonstrated that the inhibition for F1 was 66.5% after four hours of incubation, and it was maintained at 30% until the end of the experiment. Thus, this work conclusions showed that the use of nanostructured systems is of great importance to enable the application of Histatin-5 in therapeutic studies, and in vivo studies in the future. / 132393/20166