Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Bacterial resistance against antibiotic treatments is a global concern and resistance to almost every known antibiotic has already been reported. There is thus a significant need for the development of novel antimicrobial drugs. In addition to probiotic traits, certain bacteria have the ability to produce antimicrobial peptides, referred to as bacteriocins. Lantibiotics, a group of small ribosomally synthesized bacteriocins, recently gained interest for their application in the medical field. Lantibiotics have a very specific structure, including lanthionine rings, that stabilise the peptides. Due to their small size and specific action, these peptides reach specific sites of infection without affecting the composition of the host’s natural microbiota. As with any therapeutic agent, antimicrobial peptides are also prone to in vivo degradation, binding, clearance via immune action and development of bacterial resistance. Nisin F, a class Ia lantibiotic produced by Lactococcus lactis subsp. lactis F10, has already shown activity against the well-known pathogens Stapylococcus aureus, Listeria monocytogenes and various antibiotic resistant strains. The aim of this study was to assess the antimicrobial activity of nisin F against systemic S. aureus infections in mice and possible immune responses elicited by the peptide. A single administration of nisin F to the peritoneal cavity protected mice from S. aureus infection for at least 15 min. After continuous administration, the peptide showed no significant antimicrobial activity against S. aureus. The peptide did, however, convey some degree of protection to infected mice by stimulating a pro-inflammatory action through lymphocyte protection. When administered to uninfected mice, nisin F had an immune boosting effect via interleukin (IL)-6 and IL-10 without being detrimental to the host. The ex vivo effects of nisin F was compared to nisin A, a natural nisin variant, and Nisaplin®, a commercially purified form of nisin A. None of the three peptides inhibited the functional capacity of leukocytes in terms of 1L-1β en IL-6 production, not even in the presence of an external stimulus (lipopolysaccharides from Escherichia coli). Cytotoxicity was detected in response to high dosages of nisin F. Serum inhibited the antimicrobial effect of nisin F and nisin A, but Nisaplin® remained unaffected. Nisin F was applied against systemic infection for the first time and the immunological effect of the peptide was investigated. Nisin F partially protected mice against S. aureus infections through immunomodulatory effects. This study provided valuable knowledge on the in vivo application of nisin F. With further optimization of nisin F preparation and application systems, the peptide might be more effective against in vivo infections. / AFRIKAANSE OPSOMMING: Bakteriële weerstand teen antibiotika wek wêreldwyd kommer en weerstand teen amper elke bekende antibiotikum is reeds aangemeld. Daar is dus 'n groot behoefte vir die ontwikkeling van nuwe antimikrobiese middels. Bykomend tot probiotiese eienskappe, het sekere bakterieë die vermoë om antimikrobiese peptiede, bekend as bakteriosiene, te produseer. ‘n Groep klein ribosomaal-gesintetiseerde bakteriosiene, lantibitiotika, is onlangs vir mediese toepassing oorweeg. Lantibiotika beskik oor 'n baie spesifieke struktuur, insluitend lantionien ringstrukture, wat die peptied stabiliseer. Weens hul klein grootte en spesifieke aksie is hierdie peptiede daartoe in staat om spesifieke areas van infeksie te bereik sonder om die gasheer se natuurlike mikrobepopulasie te beïnvloed. Soos met enige terapeutiese middel, is bakteriosiene ook geneig tot in vivo afbreking, binding, klaring via die immuunsisteem en ontwikkeling van bakteriële weerstand. Nisien F, 'n klas Ia lantibiotikum, deur Lactococcus lactis subsp. lactis F10 geproduseer, het reeds aktiwiteit teen die bekende patogene Stapylococcus aureus, Listeria monocytogenes en verskeie antibiotika-weerstandige stamme getoon. Die doel van hierdie studie was om die antimikrobiese aktiwiteit van nisien F teen sistemiese S. aureus infeksies in muise te bepaal, asook die moontlike immuunreaksies wat die peptied mag veroorsaak. 'n Enkele toediening van nisien F het muise vir ten minste 15 min teen S. aureus beskerm. Na deurlopende administrasie het die peptied geen beduidende antimikrobiese aktiwiteit teen S. aureus getoon nie. Die peptied het egter 'n mate van beskerming aan geinfekteerde muise verleen deur ‘n pro-inflammatoriese aksie te inisieer deur limfosiet beskerming. Met toediening aan gesonde diere, het nisien F 'n immuunversterkende effek teweeg gebring via interleukin (IL)-6 en IL-10 vlakke, sonder nadelige uitwerking op die gasheer. Die ex vivo effek van nisien F is ook vergelyk met nisien A, 'n natuurlike variant van nisien, asook Nisaplin®, 'n kommersieël-gesuiwerde vorm van nisien A. Nie een van die drie peptide het leukosiete se funksionele kapasiteit in terme van 1L-1β en IL-6 produksie inhibeer nie, selfs nie in die teenwoordigheid van ‘n eksterne stimulus (lipopolisakkariede van Escherichia coli) nie. Seltoksisiteit is na blootstelling aan hoë dosisse van nisien F waargeneem. Serum het die antimikrobiese effek van beide nisien F en nisien A geïnhibeer, terwyl die werking van Nisaplin® nie beïnvloed is nie. Nisien F is vir die eerste keer teen sistemiese infeksies ingespan en die immunologiese impak van die peptied is ondersoek. Nisien F het gedeeltelike beskerming aan muise met S. aureus infeksies verleen deur die immuunsisteem te versterk. Die resultate het ‘n waardevolle bydrae gelewer tot die in vivo toediening van nisien F. Met verdere optimisering van nisien F voorbereiding en toedieningsisteme, mag die peptied moontlik meer effektief teen in vivo infeksies aangewend word. / The National Research Foundation (NRF) of South Africa for financial support and funding of the research

Made available in DSpace on 2014-06-11T19:27:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-12Bitstream added on 2014-06-13T19:35:39Z : No. of bitstreams: 1 boldrin_pk_me_arafcf.pdf: 821120 bytes, checksum: 24da3e2629cace4da1696a044c5c3fdd (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A família Fabaceae tem ocorrência em toda faixa tropical e em regiões temperadas, sendo uma família que apresenta grande potencial econômico. Machaerium, um dos gêneros pertencente a esta família, tem sido utilizado para diversos fins, tais como a recomposição de áreas degradadas ou uso medicinal para o tratamento de diarréia, cólicas menstruais, tosse e aftas bucais. Já o gênero Crotalaria, tem sido empregado na agricultura como adubo verde no combate à erosão e também despertado interesse para a indústria farmacêutica, já que os componentes produzidos por plantas deste gênero podem atuar como antimicrobiano, antimalárico, anti-inflamatório, anti-hepatotóxico e até mesmo no tratamento contra o câncer. As plantas medicinais têm sido cada vez mais empregadas na recuperação da saúde, sendo nas últimas décadas, muitos estudos direcionados na identificação de produtos naturais com propriedades terapêuticas. Apesar da notável utilização das plantas no tratamento das mais diversas patologias, a pesquisa científica tem mostrado que alguns componentes presentes nestas plantas podem apresentar efeitos nocivos, como a mutagenicidade, o que pode restringir seu uso. Assim, é indispensável a realização de ensaios que possam garantir o uso seguro desses compostos. Diante disso, o teste de Ames foi empregado no presente trabalho para análise do potencial mutagênico de Machaerium hirtum e Crotalaria pallida, já que consiste num ensaio de triagem para avaliação da atividade mutagênica, identificando mutágenos entre substâncias puras, em misturas e em amostras ambientais. Tanto o extrato de folhas, quanto o de caule de M. hirtum não se mostraram mutagênicos em nenhuma das concentrações testadas, o que é interessante, visto a grande utilização popular e propriedades farmacológicas já descritas... / The Fabaceae family has occurred throughout the tropical and temperate regions, been a family that has great economic potential. Machaerium, one of the genera belonging to this Family, has been used for various purposes since the restoration of degraded areas, to treat diarrhea, menstrual cramps, coughing, and thrush. The genus Crotalaria has been used in agriculture as fertilizer to prevent erosion and also aroused interest in the pharmaceutical industry, since the components produced by these plants can act as antimicrobial, antimalarial, anti-inflammatory, anti-hepatotoxic and even in cancer treatment. Medicinal plants have been increasingly employed in health recovery, and in recent decades, many studies have focused on the identification of natural products with therapeutic properties. Despite the remarkable use of plants in the treatment of several pathologies, scientific research has shown that some components present in these plants may have adverse effects as mutagenicity, which may restrict their use. Thus, assays that ensure the use of these compounds are extremely important. Therefore, Ames test was employed in this study to analyze the mutagenic potential of Crotalaria pallida and Machaerium hirtum because this assay is a screening test for evaluation of mutagenic activity, identifying mutagens between pure substances and mixtures and in environmental samples. Both leave and stem extracts from M. hirtum were not mutagenic in any of the tested concentrations, which is interesting because the wide use and pharmacological properties from this plant. In the extracts of leaves and seeds from C. pallida, positive results were obtained in the Ames test, which make unsafe their use. Knowing that beyond the mutagenic activity, activation of the enzyme cyclooxygenase-2 (COX-2) is also involved... (Complete abstract click electronic access below)

Orientador: Eliana Aparecida Varanda / Banca: Denise Crispim Tavares / Banca: Flávia Aparecida Resende / Resumo: A família Fabaceae tem ocorrência em toda faixa tropical e em regiões temperadas, sendo uma família que apresenta grande potencial econômico. Machaerium, um dos gêneros pertencente a esta família, tem sido utilizado para diversos fins, tais como a recomposição de áreas degradadas ou uso medicinal para o tratamento de diarréia, cólicas menstruais, tosse e aftas bucais. Já o gênero Crotalaria, tem sido empregado na agricultura como adubo verde no combate à erosão e também despertado interesse para a indústria farmacêutica, já que os componentes produzidos por plantas deste gênero podem atuar como antimicrobiano, antimalárico, anti-inflamatório, anti-hepatotóxico e até mesmo no tratamento contra o câncer. As plantas medicinais têm sido cada vez mais empregadas na recuperação da saúde, sendo nas últimas décadas, muitos estudos direcionados na identificação de produtos naturais com propriedades terapêuticas. Apesar da notável utilização das plantas no tratamento das mais diversas patologias, a pesquisa científica tem mostrado que alguns componentes presentes nestas plantas podem apresentar efeitos nocivos, como a mutagenicidade, o que pode restringir seu uso. Assim, é indispensável a realização de ensaios que possam garantir o uso seguro desses compostos. Diante disso, o teste de Ames foi empregado no presente trabalho para análise do potencial mutagênico de Machaerium hirtum e Crotalaria pallida, já que consiste num ensaio de triagem para avaliação da atividade mutagênica, identificando mutágenos entre substâncias puras, em misturas e em amostras ambientais. Tanto o extrato de folhas, quanto o de caule de M. hirtum não se mostraram mutagênicos em nenhuma das concentrações testadas, o que é interessante, visto a grande utilização popular e propriedades farmacológicas já descritas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Fabaceae family has occurred throughout the tropical and temperate regions, been a family that has great economic potential. Machaerium, one of the genera belonging to this Family, has been used for various purposes since the restoration of degraded areas, to treat diarrhea, menstrual cramps, coughing, and thrush. The genus Crotalaria has been used in agriculture as fertilizer to prevent erosion and also aroused interest in the pharmaceutical industry, since the components produced by these plants can act as antimicrobial, antimalarial, anti-inflammatory, anti-hepatotoxic and even in cancer treatment. Medicinal plants have been increasingly employed in health recovery, and in recent decades, many studies have focused on the identification of natural products with therapeutic properties. Despite the remarkable use of plants in the treatment of several pathologies, scientific research has shown that some components present in these plants may have adverse effects as mutagenicity, which may restrict their use. Thus, assays that ensure the use of these compounds are extremely important. Therefore, Ames test was employed in this study to analyze the mutagenic potential of Crotalaria pallida and Machaerium hirtum because this assay is a screening test for evaluation of mutagenic activity, identifying mutagens between pure substances and mixtures and in environmental samples. Both leave and stem extracts from M. hirtum were not mutagenic in any of the tested concentrations, which is interesting because the wide use and pharmacological properties from this plant. In the extracts of leaves and seeds from C. pallida, positive results were obtained in the Ames test, which make unsafe their use. Knowing that beyond the mutagenic activity, activation of the enzyme cyclooxygenase-2 (COX-2) is also involved... (Complete abstract click electronic access below) / Mestre

Les iminosucres, analogues de sucres dans lesquels l'oxygène endocyclique a été remplacé par un atome d'azote, constitue une classe importante de mimes de sucres. Aujourd'hui, leurs applications se limitent au domaine biologique car ces composés ont montré un potentiel thérapeutique prometteur. Il serait intéressant d'élargir le domaine d'application de ces iminosucres, et la combinaison de ces structures présentant un azote endocyclique pourrait conduire à de nouveaux macrocycles inédits présentant des propriétés de chélation innovantes. Pour cela, l'introduction d'une chaîne alkyle en position pseudoanomérique donne accès à une nouvelle classe de composés, les iminosucres C-glycosides dont la fonctionnalisation en positions C-5 et C-1 est nécessaire.La première partie de ce travail se focalise sur le développement d'une voie de synthèse rapide et efficace d'iminosucres C-glycosides à six et sept chaînons au moyen d'une réaction tandem de Staudinger aza-Wittig. Pour accéder à ces composés de choix, notre stratégie se base sur des réactions de fonctionnalisation diastéréosélectives et stéréocontrollées. La seconde partie de cette thèse a été consacrée à la synthèse d'iminosucres aza-couronnes, dont les structures constituent un nouveau type de récepteurs moléculaires. L'étude des propriétés de chélation de ces nouveaux macrocycles des cations métalliques a montré des premiers résultats prometteurs et encourageants grâce à des analyses par RMN et par fluorimétrie. / Iminosugars, sugar analogs in which the endocyclic oxygen has been replaced by nitrogen, constitute a major class of sugar mimetics. Their application has been limited to the biological field so far as these compounds have shown promising therapeutic properties[1]. Interestingly, their structural analogy with sugars combined with the presence of an endocyclic nitrogen atom could deliver innovative macrocycles that could display chelation properties as well as catalytic potential when bound to metals and associated as duplex or higher multiplicity scaffolds. For this purpose, efficient introduction of an alkyl chain at the pseudoanomeric position of the iminosugar to yield an iminosugar C-glycoside[2] displaying two arms at C-5 and C-1 position is necessary. The first part of this work focused on the development of an efficient and convergent synthesis of seven and six membered iminosugars C-glycosides using a highly diastereoselective tandem Staudinger-Aza-Wittig reaction is presented. To access to these new compounds, our strategy is based on a highly diastereoselective and stereocontrolled functionalization. The second part of this work is based on the use of these structures to build up unprecedented iminosugar-aza-crowns, a new type of molecular receptors, using the strategy way developed in the first part. These news sweet aza-crowns displaying with various linkages showed promising results through their preliminary chelation properties by NMR and fluorimetric techniques.