Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-02-09T11:45:00Z No. of bitstreams: 2 Dissertação - Kamila Pereira da Silva - 2014.pdf: 1336089 bytes, checksum: 2658c99debc59b350392edc7239568d9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-20T10:55:13Z (GMT) No. of bitstreams: 2 Dissertação - Kamila Pereira da Silva - 2014.pdf: 1336089 bytes, checksum: 2658c99debc59b350392edc7239568d9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-20T10:55:13Z (GMT). No. of bitstreams: 2 Dissertação - Kamila Pereira da Silva - 2014.pdf: 1336089 bytes, checksum: 2658c99debc59b350392edc7239568d9 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-05-14 / Fungal infections caused by yeasts of Cryptococcus neoformans complex have increased considerably among immunosuppressed especially in patients infected with human immunodeficiency virus (HIV). Antifungal drugs available to treat these infections have a wide spectrum of action, but the high costs, side effects, besides the acquisition of resistance of fungi make their limited effectiveness. In these circumstances, the search for new drugs is needed. In the present study, we evaluated the antifungal activity of tamoxifen, bisabolol, indinavir, didanosine, UFMG, Labiocon 241, solasodine, Labiocon 237, Clonazepam, semicarbonado Benzaldehyde, Cardanol, AB 36 and three natural extracts of Psychotria spp, Sclerolobium paniculatum, Hymenaea courbaril and cell viability and mechanism of action of the compound showed antifungal activity of 20 isolates of Cryptococcus neoformans. To evaluate the antifungal activity , we used the in vitro susceptibility to yeast broth microdilution protocol M27 - A3 ( CLSI 2008) and to verify the mechanism of action, flow cytometry was performed with the cellular marker Propidium Iodide ( PI ) and cell viability assay with MTT salt. Among the compounds analyzed, only tamoxifen and olanzapine showed inhibitory action on fungal samples at concentrations 128-256 μg/mL. Analysis by flow cytometry was performed with tamoxifen and showed that the compound did not alter the cell membrane of Cryptococcus neoformans ATCC 28957 , however, the cell viability test showed that tamoxifen was able to inhibit the metabolism of the fungus in different concentrations ranging 4-1024 μg/mL. / As infecções fúngicas causadas por leveduras do complexo Cryptococcus neoformans tem sido reduzida entre os imunossuprimidos principalmente nos pacientes infectados pelo vírus da imunodeficiência humana (HIV). Os antifúngicos disponíveis para o tratamento destas infecções apresentam um amplo espectro de ação, porém os altos custos, efeitos colaterais, além da aquisição de resistência dos fungos tornam sua eficácia limitada. Nestas circunstâncias, a busca por novos fármacos é necessária. No presente trabalho, foi avaliada a atividade antifúngica de tamoxifeno, bisabolol, indinavir, didanosina, Olanzapina, UFMG, Labiocon 241, Solasodina, Labiocon 237, Clonazepam, Benzaldeído semicarbonado, Cardanol, AB 36 e três extratos naturais obtidos de Psychotria spp, Sclerolobium paniculatum, Hymenaea courbaril. A viabilidade celular e o mecanismo de ação do composto que apresentou ação antifúngica sobre 20 isolados de Cryptococcus neoformans formam determinados. A atividade antifúngica foi realizada utilizando-se o método microdiluição em caldo para leveduras. Para verificar do mecanismo de ação, foi realizada a citometria de fluxo com o marcador celular Iodeto de Propídio (PI), enquanto que o teste da viabilidade celular foi obtido usando-se o sal MTT. Dentre os compostos analisados, apenas o tamoxifeno e olanzapina apresentaram ação inibitória sobre as amostras fúngicas, em concentrações de 128 a 256 μg/mL. Pela análise por citometria de fluxo com o tamoxifeno não se verificou alteração da membrana celular de Cryptococcus neoformans ATCC 28957. Entretanto, o tamoxifeno inibiu o metabolismo do fungo em diferentes concentrações, variando de 4 a 1024 μg/mL verificado pelo teste da viabilidade celular.

Made available in DSpace on 2015-04-22T22:14:14Z (GMT). No. of bitstreams: 1 Cristina Pessoa Veloso Freire.pdf: 2819799 bytes, checksum: 8919fceb66aefa886139385d2b54aae1 (MD5) Previous issue date: 2010-06-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / the species most frequently at least seven species, has been associated with diseases in humans: Candida albicans, Candida. tropicalis, Candida stellatoidea, Candida pseudotropicalis (now called C. Kefyr), Candida parapsilosis, Candida guilliermondii and Candida krusei. There is an increased occurrence of infections by Candida species, caused by population growth that immunosuppression with high predisposition to develop infections. Also, there was an increased use of antifungal agents, where some Candida showed a decline in sensitivity to these agents. Increasing numbers of fungal strains are becoming resistant to the drugs available. As a result, there is a constant search for new antifungal drugs more effective and safer than existing ones. The naphthoquinones are widely distributed in nature, with the known activities, the antibacterial, antifungal and antitumor. Thus the objective of this study was to evaluate the antifungal activity and cytotoxicity of naphthoquinones semisynthetic in Candida strains isolated from the oral cavity. The strains studied were C. albicans, C. krusei, C. parapsilosis, C. kefyr, C. tropicalis and C. dubliniensis. The susceptibility of semi-synthetic naphthoquinones was tested initially by using the simple diffusion from the hole in the culture medium (hole plate), to obtain a quantitative analysis, and then for a quantitative analysis technique was used in broth. Patterns known antifungals were used, for comparison of antifungal activity. The semi-synthetic naphthoquinone that showed the best antifungal activity in the hole plate technique was NORALFA, followed by IVS322 with average of inhibition zone against C. albicans 21,26 mm and 16,62 mm respectively, and quantitative analysis of antifungal activity, substances NORALFA and IVS322 stood out with MIC against C. albicans 0,125 μg/ml and 0.5 μg/ml. It was found that none of the substances tested at a concentration of 50 μg/ml caused hemolysis and fibroblasts tested remained viable at a concentration of 12,5 μg/ml. This result is favorable, since the hemolysis test is a measure of toxicity and a positive result would preclude the therapeutic use of these substances. Through this work we determine the potential antifungal and cytotoxic profile of the compounds tested. / A candidíase é uma doença infecciosa causada pelo fungo Candida, sendo a espécie albicans a mais freqüentes. Sete espécies pelo menos, tem sido associadas a doenças no homem: Candida albicans, Candida. tropicalis, Candida stellatoidea, Candida peseudotropicalis (hoje chamada de C. Kefyr), Candida parapsilosis, Candida guilliermondii e Candida krusei. Há um aumento da ocorrência das infecções por espécies de Candida, causada pelo crescimento da população com imunodepressão que grande predisposição ao desenvolvimento de infecções.Números cada vez maiores de cepas fúngicas estão se tornando resistentes aos fármacos disponíveis no mercado. Em virtude disso, existe uma constante procura de novos fármacos antifúngicos mais eficazes, e mais seguros que os já existentes. As naftoquinonas são amplamente distribuídas na natureza, tendo como atividades conhecidas, a atividade antibacteriana, antifúngica e antitumoral. Desta forma o objetivo deste estudo foi avaliar a atividade antifúngica e citotoxicidade de naftoquinonas semi-sintéticas em cepas de Candida isoladas da cavidade oral. As cepas estudadas foram de C. albicans, C. krusei, C. parapsilosis, C. kefyr, C. tropicalis e C. dubliniensis. A suscetibilidade das naftoquinonas semisintéticas foi testada inicialmente, através do teste difusão a partir de orifício no meio de cultura (hole plate). Posteriormente para uma analise quantitativa foi utilizada a técnica de microdiluição em caldo. Padrões antifúngicos conhecidos foram usados, para comparação da atividade antifúngica. A citotoxicidade foi avaliada em hemácias de camundongos através do teste de hemólise e através do teste de viabilidade celular em fibroblastos, utilizando o Alamar Blue. A naftoquinona semisintética que apresentou melhor atividade antifúngica na técnica hole plate foi a NORALFA, seguida da IVS322 com médias do halo de inibição frente a C. albicans de 21,26 e 16,62 mm respectivamente, e na análise quantitativa da atividade antifúngica, as substâncias NORALFA e IVS322 se destacaram com CIM frente a C. albicans 0, 125 μg/ml e 0,5 μg/ml. Verificou-se que nenhuma das substâncias testadas a uma concentração de 50 μL/ml provocou hemólise e os fibroblastos testados permaneceram viáveis na concentração de 12,5 μL/ml. Este resultado é favorável, uma vez que o teste de hemólise é um parâmetro de toxicidade e um resultado positivo poderia impossibilitar o uso terapêutico dessas substâncias. Com a realização deste trabalho conseguimos determinar o potencial antifúngico e o perfil citotóxico das substâncias testadas.

Made available in DSpace on 2016-04-28T20:15:36Z (GMT). No. of bitstreams: 1 Tatiana Xavier de Almeida.pdf: 937180 bytes, checksum: e0cda53e5c0c9829836d98da482b3737 (MD5) Previous issue date: 2010-02-09 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Fungal and mycotoxin contamination in food is one of the greatest problems around the world. Some natural products, such as essential oils, have been shown to be effective inhibitors of fungal growth and production of their toxins. In this study, a total of 40 vegetal extracts were tested for inhibition on Aspergillus section Flavi growth. These extracts were obtained from ten herbs cultivated in Brazil: basil (Ocimum basilicum), cinnamon (Cinnamomum zeylanicum), clove (Eugenia caryophyllata), cumin (Cuminum cyminum), marjoram (Origanum majorana), nutmeg (Myristica fragrans), oregano (Origanum vulgare), rosemary (Rosmarinus officinalis), spearmint (Mentha piperita) and sweet fennel (Pimpinella anisum). Preliminary in vitro tests using the agar diffusion method showed that essential oils were more effective in inhibiting the fungal growth when compared with ethanol and aqueous extracts. Essential oils obtained from oregano and rosemary, clove and cumin ethanolic extracts and the essential hexanic oil of oregano and cinnamon were selected for advanced tests. Differents concentrations of plant material (0, 50, 100, 150, 300 and 600 μg/g) were added to culture medium. A suspension of 103 conidia per inoculation were used. Plates were incubated for ten days while daily measurements of colony diameters were made to obtain growth rate (mm/day) and lag phase (h) of toxigenic strains. The comparison of selected essential oils effect on A. flavus and A. parasiticus showed better inhibition by incorporating oregano essential oil in the culture medium at 600 μg/g concentration. Nevertheless, the hexanic essential oil of oregano showed no statistical difference with the control group, a change occurred in the plant product due to the extraction method. Among the evaluated ethanol extracts, clove had the best result. Aspergillus flavus showed a slight reduction of growth (4.5 to 3.5 mm/day) when compared to control group, but a large increase in the latency period (37 h at a 600 μg/g concentration). Aspergillus parasiticus showed gradual decreases as increased concentrations were assayed, except for the 150 μg/g concentration, and a significant increase on lag phase at the highest concentrations tested (45 and 48 h, respectively). The data presented here are very important for the continued efforts of microbiological control in food, especially when it comes to control fungi. / A contamina??o de alimentos por fungos e micotoxinas constitui um grande problema mundial. Alguns produtos naturais, como por exemplo, ?leos essenciais, s?o demonstrados como potenciais inibidores do crescimento f?ngico, bem como da produ??o de suas toxinas. Neste estudo, um total de 40 extratos de plantas foi avaliado como inibidores do desenvolvimento f?ngico de cepas pertencentes ao g?nero Aspergillus se??o Flavi. Tais extratos foram obtidos a partir de dez ervas, utilizadas especialmente como condimentos e cultivadas no Brasil: alecrim (Rosmarinus officinalis), canela (Cinnamomum zeylanicum), cominho (Cuminum cyminum), cravo-da-?ndia (Eugenia caryophyllata), erva-doce (Pimpinella anisum), hortel? (Mentha piperita), manjeric?o (Ocimum basilicum), manjerona (Origanum majorana), noz moscada (Myristica fragrans) e or?gano (Origanum vulgare). Ensaios preliminares in vitro utilizando o m?todo de difus?o em agar demonstraram que os ?leos essenciais foram os mais efetivos na inibi??o do crescimento f?ngico quando comparados com extratos etan?licos e aquosos. Foram selecionados os ?leos essenciais de or?gano e alecrim, os extratos etan?licos de cravo-da-?ndia e cominho e, os ?leos essenciais hex?nicos de or?gano e canela para ensaios mais aprofundados. Diferentes concentra??es de extratos de plantas arom?ticas (0, 50, 100, 150, 300 e 600 μg/g) foram adicionadas ao meio de cultivo. Utilizou-se uma suspens?o de 103 con?dios por inocula??o. As placas foram incubadas por dez dias enquanto medi??es di?rias do di?metro da col?nia eram realizadas a fim de obter velocidade de crescimento (mm/dia) e fase de lat?ncia (h) das cepas tox?genas. Comparando os ?leos essenciais selecionados, tanto A. flavus quanto A. parasiticus apresentaram uma melhor inibi??o atrav?s da incorpora??o do ?leo essencial de or?gano no meio de cultivo a uma concentra??o de 600 μg/g. Apesar disso, o ?leo essencial hex?nico de or?gano n?o demonstrou qualquer diferen?a estat?stica em rela??o ao grupo controle, havendo ent?o altera??o em algum componente do extrato devido a forma de extra??o. Entre os extratos etan?licos avaliados, o cravo-da-?ndia obteve o melhor resultado. Aspergillus flavus demonstrou uma discreta diminui??o de crescimento (4,5 a 3,5 mm/dia), quando comparado ao grupo controle, mas um grande aumento em sua fase de lat?ncia (37 h a 600 μg/g). Aspergillus parasiticus apresentou uma diminui??o gradativa conforme ocorria o aumento das concentra??es, exceto pela concentra??o de 150 μg/g, e um aumento significativo da fase lag nas maiores concentra??es testadas (45 e 48 h respectivamente). Os dados apresentados aqui s?o de extrema relev?ncia para os constantes esfor?os de controle microbiol?gico em alimentos, principalmente em se tratando de controle f?ngico.

Made available in DSpace on 2016-04-28T20:17:28Z (GMT). No. of bitstreams: 1 2008 - Alexander Santos.pdf: 2028526 bytes, checksum: 5876d3f1d1f305df3af598f0bef1531d (MD5) Previous issue date: 2008-04-15 / This work was developed in the Department of Microbiology and Immunology of the Institute of Veterinary Rural Federal University of Rio de Janeiro in Serop?dica-RJ. We evaluated the antifungal activity plant Cymbopogon citratus (lemon-grass), Peumus boldus (boldo) and Shinus terebinthifolia (aroeira), inhibition of growth of species of the genus Aspergillus (A. flavus, A. niger, A. ochraceus, A. Parasiticus and A. carbonarius. Using the method of minimum inhibitory concentration in agar, with the technique of dilution plate (Pour-Plate), were held dilutions of different extracts, resulting in the final concentrations of 5%, 2.5% and 1.25%. Testing of commercial sensitivity to antifungal - ketoconazole were made in a final concentration of 1933.18 ? g /mL, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The results obtained in the experiments have shown that aqueous extracts of C. citratus and S. terebinthifolia, after 24 hours of incubation, were able to inhibit the growth of A. flavus and A. carbonarius, respectively. The other extracts being studied and tested in the concentrations were not able to inhibit the growth of mycelial species of the genus Aspergillus. / Este trabalho foi desenvolvido no Departamento de Microbiologia e Imunologia do Instituto de Veterin?ria da Universidade Federal Rural do Rio de Janeiro, em Serop?dica-RJ. Foram avaliadas a atividade antif?ngica das plantas Cymbopogon citratus (capim-lim?o), Peumus boldus (boldo) e Shinus terebinthifolia (aroeira), na inibi??o do crescimento de esp?cies do g?nero Aspergillus (A. flavus, A. niger, A. ochraceus, A. parasiticus e A. carbonarius). Utilizando o m?todo da concentra??o inibit?ria m?nima em ?gar, com a t?cnica de dilui??o em placa (Pour-Plate), foram realizadas dilui??es dos diferentes extratos, obtendo-se as concentra??es finais de 5%, 2,5% e 1,25%. Testes de sensibilidade ao antif?ngico comercial - cetoconazol foram realizados numa concentra??o final de 1933,18 ?g/mL, conforme recomendada pelo National Committee for Clinical Laboratory Standards (NCCLS). Os resultados obtidos nos experimentos demonstraram que os extratos aquosos de C. citratus e S. terebinthifolia, ap?s 24 horas de incuba??o, foram capazes de inibir o crescimento de A. flavus e A. carbonarius, respectivamente. Os demais extratos em estudo e nas concentra??es testadas, n?o foram capazes de inibir o crescimento micelial das esp?cies do g?nero Aspergillus.

<p>Cilj istraživanja doktorske disertacije bio je da se ispita pojedinačni i sinergistički uticaj<br />ekstrakata kima (Carum carvi L.), bosiljka (Ocimum basilicum L.), origana (Origanum<br />vulgare L.) i etarskih ulja crnog (Allium cepa L. kultivar Kupusinski jabučar) i belog luka<br />(Allium sativum L. kultivar Bosut) na rast plesni izolovanih iz prehrambenih proizvoda, kao i<br />njihov uticaj na biosintezu mikotoksina.<br />Ukupan broj plesni u uzorcima salata od povrća spremnih za konzumiranje kretao se od<br />10,0 do 5,5&acute;102 cfu/g, u uzorcima poslastičarskih proizvoda do 6,1&acute;102 cfu/g i u<br />proizvodima od mesa do 60,0 cfu/g. Najveći broj plesni izolovan je na DG18 podlozi<br />(1,53&acute;102 cfu/g), a najmanji na MY50G (42,0 cfu/g). U ukupnoj mikopopulaciji svih ispitivanih<br />uzoraka dominirale su vrste rodova Penicillium (39,07%), Cladosporium (23,40%) i<br />Aspergillus (20,42%). Vrste iz rodova Alternaria, Fusarium i Eurotium su bile zastupljene sa<br />5,85%, 4,97% i 2,76%. Dominantne vrste u ukupnoj mikopopulaciji bile su C.<br />cladosporioides (21,63%), A. niger (16,0%) i P. aurantiogriseum (11,81%).<br />Dominirali su potencijalni producenti ohratoksina A (31,89%), proizvo&ntilde;ači fumonizina<br />(4,74%), moniliformina (1,43%) i sterigmatocistina (1,54%). Izolati A. versicolor su<br />biosintetisali sterigmatocistin u koncentracijama od 56,3 i 109,2 ng/mL. Ostale potencijalne<br />toksin-produkujuće vrste nisu pokazale sposobnost produkcije mikotoksina.</p><p>Mikotoksikolo&scaron;kim ispitivanjem hrane u dva uzorka salata spremnih za konzumiranje<br />(kupus beli rezani i FIT salata) utvr&ntilde;en je sadržaj sterigmatocistina u koncentracijama od 3,5<br />i 5,5 mg/kg.<br />Kao glavna komponenta u ekstraktu kima odre&ntilde;en je karvon (43,98%), u ekstraktu bosiljka<br />estragol (metil kavikol) (86,72%), a u ekstraktu origana karvakrol (34,20%) i karvon (18,05%).<br />Najveći deo etarskog ulja crnog luka činili su: dimetil-trisulfid, metil-propil-trisulfid, dimetiltetrasulfid,<br />dietil-1,2,4-tritiolan, metil-(1-propenil)-trisulfid, metil-(1-propenil)-disulfid. Dialildisulfid,<br />dialil-trisulfid, metil-alil-trisulfid i metil-alil-disulfid su glavne komponente koje su<br />odre&ntilde;ene u etarskom ulju belog luka.<br />Koncentracija od 0,35 mL/100 mL ekstrakta kima je bila fungicidna (MFC) prema C.<br />cladosporioides, dok je 0,70 mL/100 mL potpuno inhibirala rast A. carbonarius, A. wentii, E.<br />nidulans, Eurotium spp., C. cladosporioides, P. glabrum, P. brevicompactum, F.<br />subglutinans i F. verticillioides. Na rast P. chrysogenum i P. aurantiogriseum ista<br />koncentracija bila je inhibitorna (MIC). Najslabije delovanje ovaj ekstrakt ispoljio prema A.<br />niger, A. versicolor, F. oxysporum i F. proliferatum.<br />Primena ekstrakta bosiljka u koncentraciji od 0,70 mL/100 mL pokazala je fungicidno<br />delovanje na C. cladosporioides. Koncentracija od 1,50 mL/100 mL potpuno je inhibirala<br />rast A. wentii, A. versicolor, E. nidulans, E. herbariorum, E. chevalieri, E. rubrum, P.<br />chrysogenum i Fusarim spp. Ekstrakt bosiljka je najslabije delovao prema A. niger, A.<br />carbonarius, P. aurantiogriseum, E. amstelodami, P. glabrum i P. brevicompactum.<br />Ekstrakt origana je pokazao najslabije ihibitorno delovanje na rast ispitivanih plesni.<br />Primena ekstrakta u koncentraciji od 1,50 mL/100 mL je bila fungicidna prema E. rubrum.<br />Koncentracija od 2,50 mL/100 mL je pokazala fungicidno delovanje na E. rubrum, E.<br />herbariorum, A. wentii, C. cladosporioides i P. aurantiogriseum, a inhibitorno prema E.<br />nidulans, E. chevalieri, E. amstelodami, P. glabrum i P. brevicompactum. Ovaj ekstrakt je<br />najslabije delovao na A. niger, A. carbonarius, F. proliferatum, F. subglutinans i P.<br />chrysogenum.<br />Etarsko ulje crnog luka pokazalo je signifikantno jače antifungalno delovanje na ispitivane<br />plesni u odnosu na etarsko ulje belog luka. Koncentracija od 14,0 mL/100 mL ulja belog luka<br />fungicidno je delovala prema E. rubrum, E. chevalieri i C. cladosporioides, dok je ulje crnog<br />luka na ovoj koncentraciji pokazalo fungicidni efekat i na E. herbariorum i E. amstelodami.<br />Za ostale plesni fungicidna koncentracija iznosila je 28,0 mL/100 mL, osim za A. niger i P.<br />aurantiogriseum.<br />Neke od ispitivanih sme&scaron;a bosiljka i kima, bosiljka i origana, origana i kima i etarskih ulja<br />lukova pokazale su sinergističko delovanje na inhibiciju rasta A. wentii, E. herbariorum, F.<br />verticilllioides i P. aurantiogriseum sa FICindex od 0,63 do 0,97.<br />Začinski ekstrakti i etarska ulja lukova su pored ograničavanja rasta kolonija plesni<br />uzrokovali i promene u makro i mikromorfologiji.<br />Potpuna inhibicija biosinteze sterigmatocistina i rasta A. versicolor postignuta je pri<br />koncentraciji od 0,20 mL/100 mL ekstrakta kima i origana u periodu od 21 dana. Na ovoj<br />koncentraciji ekstrakt bosiljka je inhibirao biosintezu sterigmatocistina za 88,73% i rast<br />plesni za 52,56%. Sme&scaron;a koja je sadržavala 75% ekstrakta kima i 25% ekstrakta bosiljka<br />potpuno je inhibirala rast plesni i biosintezu sterigmatocistina u YES bujonu tokom 21 dana<br />inkubiranja.<br />Pojedinačne koncentracije etarskih ulja crnog i belog luka od 5,0 i 10,0 mL/100 mL i u sme&scaron;i<br />sa 1,50 mL/100 mL etarskog ulja crnog luka i 0,50 mL/100 mL etarskog ulja belog luka bile su<br />potrebne za potpunu inhibiciju rasta A. versicolor i biosintezu sterigmatocistina.<br />Dodatak sme&scaron;e ekstrakata kima i bosiljka (0,35 mL/100 mL ekstrakta kima + 0,70 mL/100 mL<br />ekstrakta bosiljka) u svež kupus rezanac uticao je na smanjenje inicijalne kontaminacije<br />plesnima za 93,9%, uz pojavu intenzivnijeg, ali prihvatljivog mirisa i neznatne promene boje.</p><p>Definisani matematički model za komparaciju uticaja ekstrakata i etarskih ulja na rast plesni<br />može se primenjivati u formiranju matrica inhibicije i optimizaciji vremena i koncentracije<br />antifungalnih agenasa.<br />Dobijena saznanja o antifungalnom delovanju ekstrakata začina i etarskih ulja lukova mogu<br />biti značajna u pobolj&scaron;anju antifungalne za&scaron;tite namirnica, smanjenju biosinteze<br />mikotoksina i ukupnim smanjenju &scaron;teta izazvanih delovanjem plesni.</p> / <p>The aim of this PhD thesis was to study the individual and synergistic effects of extracts of<br />caraway (Carum carvi L.), basil (Ocimum basilicum L.), oregano (Origanum vulgare L.) and the<br />essential oils of onion (Allium cepa L. cultivar Kupusinski jabučar) and garlic (Allium sativum<br />L. cultivar Bosut) on the growth of moulds isolated from food products. The study also<br />focused on the impact of extracts and the essential oils on mycotoxins biosynthesis.<br />The total number of moulds detected in samples of vegetable salads &ldquo;ready for use&rdquo; ranged<br />from 10.0 to 5.5&acute;102 cfu/g. In cake and pastries, as well as, meat samples, the number<br />reached 6.1&acute;102 cfu/g and 60.0 cfu/g, respectively. The highest number of mould colonies was<br />isolated in DG18 medium (1.53 &acute; 102cfu/g) and the lowest in MY50G medium (42.0 cfu/g). The<br />species of the genera Penicillium (39.07%), Cladosporium (23.40%) and Aspergillus (20.42%)<br />prevailed in the entire mycopopulation of all tested samples. Species of the genera Alternaria,<br />Fusarium and Eurotium were represented with 5.85%, 4.97% and 2.76%, respectively, while C.<br />cladosporioides (21.63%), A. niger (16.0%) and P. aurantiogriseum (11.81%) were the most<br />dominant species in the entire mycopopulation.<br />Potential producers of ochratoxin A (31.89%) accounted for the largest share of the isolated<br />mycopopulation. The share of producers of fumonisin, moniliformin and sterigmatocystin<br />amounted to 4.74%, 1.43% and 1.54%, respectively. Sterigmatocystin was biosynthesised in</p><p>the concentration of 56.3 ng/mL and 109.2 ng/mL by both isolates of A. versicolor, while other<br />potential toxin producers did not show the ability of mycotoxin production.<br />Mycotoxicological investigation showed the sterigmatocystin content in two samples of<br />vegetable salads &quot;ready for use&quot; (shredded white cabbage and FIT salad - carrot, lettuce and<br />red chicory) in concentrations of 3.5 mg/kg and 5.5 mg/kg, respectively.<br />The major component in the extract of caraway was carvon with a share of 43.98%. The basil<br />extract contained estragol (methyl cavicol) in the highest percentage (86.72%), while<br />carvacrol (34.20%) and carvon (18.05%) were major components of the oregano extract. As for<br />the essential oil of onion, dimethyl trisulphide, methyl propyl trisulphide, dimethyl tetrasulfid,<br />diethyl-1, 2, 4-tritiolan, methyl-(1-propenyl)-trisulphide, and methyl-(1-propenyl) &ndash; disulfide<br />constituted the largest share. The major components isolated in garlic essential oil were<br />diallyl disulfide, diallyl-trisulphide, allyl methyl trisulphide and allyl methyl disulfide.<br />The concentration of the caraway extract of 0.35 mL/100mL exhibited fungicidal effect (MFC)<br />on C. cladosporioides, while the concentration of 0.70 mL/100mL completely inhibited the<br />growth of A. carbonarius, A. wentii, E. nidulans, Eurotium spp., C. cladosporioides, P.<br />glabrum, P. brevicopmactum, F. subglutinans and F. verticillioides. The same concentration<br />showed the inhibitory effect (MIC) on the growth of P. chrysogenum and P. aurantiogriseum.<br />The poorest effect of the caraway extract was expressed on the growth of A. niger, A.<br />versicolor, F. oxysporum and F. proliferatum.<br />The basil extract application in the concentration of 0.70 mL/100mL showed fungicidal effects<br />(MFC) on the growth of C. cladosporioides. The concentration of 1.50 mL/100mL completely<br />inhibited (MFC) the growth of A. wentii, A. versicolor, E. nidulans, E. herbariorum, E.<br />chevalierii, E. rubrum, P. chrysogenum and Fusarim spp. The poorest effect of the basil<br />extract was exhibited on A. niger, A. carbonarius, P. aurantiogriseum, E. amstelodami, P.<br />glabrum and P. brevicompactum.<br />The oregano extract showed the weakest growth inhibition influence on all of the tested<br />moulds. The application of this extract in the concentration of 1.50 mL/100mL was fungicidal<br />(MFC) to E. rubrum. The concentration of 2.50 mL/100mL showed fungicidal effects (MFC) on<br />the growth of E. rubrum, E. herbariorum, A. wentii, C. cladosporioides and P. aurantiogriseum<br />and inhibitory effects (MIC) on E. nidulans, E. chevalieri, E. amstelodami, P. glabrum and P.<br />brevicompactum. The weakest effect of this extract was expressed on the growth of A. niger,<br />A. carbonarius, F. proliferatum, F. subglutinans and P. chrysogenum.<br />Onion essential oil showed a significantly stronger antifungal effect on the tested moulds in<br />comparison to garlic essential oil. While the concentration of 14.0 mL/100mL of garlic oil had a<br />fungicidal effect on E. rubrum, E. chevalieri and C. cladosporioides, the same concentration<br />of onion oil was also fungicidal to E. herbariorum and E. amstelodami. With an exception of A.<br />niger and P. aurantiogriseum, the concentration that showed a fungicidal effect on the<br />remaining moulds equalled 28.0 mL/100mL.<br />Some of the tested mixtures of basil with caraway, basil with oregano, oregano with caraway,<br />and essential oils of onion and garlic, showed a synergistic effect on the growth inhibition of<br />A. wentii, E. herbariorum, F. verticilllioides and P. aurantiogriseum with the FIC index ranging<br />from 0.63 to 0.97.<br />Apart from the inhibitory effect on the mould colony growth, the spices extracts and the<br />essential oils of onion and garlic also caused changes in the macro- and micro- morphology<br />of the moulds.<br />Complete inhibition of the growth of A. versicolor and sterigmatocystin biosynthesis was<br />achieved at a concentration of 0.20 mL/100mL of the extract of caraway and oregano in the<br />period of 21 days. At this concentration the basil extract delayed the sterigmatocystin<br />biosynthesis by 88.73% while the mould growth was inhibited by 52.56%. Mixtures containing</p><p>75% of the caraway extract and 25% of the basil extract completely inhibited the mould<br />growth and sterigmatocystin biosynthesis in YES broth during 21 days of incubation.<br />The concentrations of 5.0 m L/100mL (onion essential oil) and 10.0 m L/100mL (garlic essential<br />oil) applied in a mixture containing 1.50 mL/100mL of onion and 0.50 mL/100mL of garlic<br />essential oil were necessary for a complete inhibition of the growth of A. versicolor and<br />sterigmatocystin biosynthesis.<br />The addition of the mixture of caraway and basil extracts (0.35 mL/100mL of caraway + 0.70<br />mL/100mL of basil) to fresh shredded cabbage influenced the reduction of initial mould<br />contamination by 93.9%. This was accompanied by the occurrence of acceptable more<br />intense flavour and slight discoloration.<br />The defined mathematical model for comparing the effects of extracts and essential oils on<br />the growth of moulds can be applied in establishing inhibition matrices and optimisation of<br />the time and the concentration of antifungal agents.<br />The obtained results on the antifungal effects of the spices extracts and onion and garlic<br />essential oils can be beneficial for improving the antifungal protection of food and reducing<br />the mycotoxin biosynthesis as well as the overall damage caused by the action of moulds.</p>

Candida albicans représente la première cause d’infection fongique chez l’Homme. Chez les patients immunodéficients, ce pathogène est extrêmement virulent et le nombre de décès suite à une infection systémique par Candida atteint 200 000 chaque année dans le monde. Quatre classes de médicaments antifongiques existent mais les traitements ont un coût élevé et des problèmes récurrents de résistance. Il y a donc un besoin urgent de trouver de nouvelles options thérapeutiques.Notre projet explore une nouvelle cible potentielle de la famille des protéines BET (Bromo and extra-terminal) pour traiter les infections fongiques. Nous étudions la protéine BET de levure, bromodomain factor 1 (Bdf1), impliquée dans la régulation de la transcription. Afin de caractériser son rôle fonctionnel, j’ai combiné différentes approches en biochimie, protéomique et transcriptomique. J’ai démontré que Bdf1 et ses deux bromodomaines, Bd1 et Bd2, sont essentiels pour la croissance et la survie de C. albicans.La seconde partie de ma thèse s'inscrit dans un projet de recherche d'inhibiteurs des bromodomaines de Bdf1 chez C. albicans, inspiré par la découverte de composés anti-cancéreux ciblant les bromodomaines mammifères. Grâce à un criblage haut débit, nous avons identifié des inhibiteurs sélectifs des bromodomaines de Bdf1. Ils agissent comme des molécules antifongiques et inhibent la croissance de C. albicans.Ces découvertes indiquent que les bromodomaines de Bdf1 sont une nouvelle cible pour le développement de nouveaux traitements antifongiques. Leurs inhibitions pourraient représenter une stratégie thérapeutique innovante pour traiter les patients infectés par C. albicans. / C. albicans is the most prevalent human fungal pathogen. In immunocompromised patients, this pathogen is highly virulent and the number of deaths because of systemic infections by C. albicans reaches 200.000 per year worldwide. The limited number, high cost and toxicity of currently available antifungal drugs indicate that new therapeutic agents against C. albicans are urgently needed.We propose to investigate a novel target of BET (Bromo and extra-terminal) proteins family to treat fungal infections. We are interested in the yeast BET protein named bromodomain factor 1 (Bdf1), involved in transcription regulation. To characterize its functional role, I combined several approaches in biochemistry, proteomic and transcriptomic. I discovered that Bdf1 and its two bromodomains Bd1 and Bd2 are essentials for the growth of C. albicans.The second part of my thesis is involved in a research project that aims to identify Bdf1 bromodomains inhibitors in C. albicans, inspired by recently discovered anti-cancer compounds that target mammal bromodomains. High-throughput chemical screens have identified selective Bdf1 bromodomain inhibitors. They act as antifungal compounds and inhibit the growth of C. albicans.Altogether, these discoveries indicate that Bdf1 bromodomains are a valid antifungal target. Hopefully, their inhibition represents a new and innovative therapeutic strategy to treat patients infected with C. albicans

The growing neutropenic patient population provides an ideal target for opportunistic fungal infections. Several effective antifungal drugs are toxic at high doses and contraindicated for long-term treatment. Recombinant human granulocyte colony stimulating factor (rhG-CSF) has been shown to increase neutrophilic numbers and functions, thus providing enhanced host defense. Improved efficacy by using rhG-CSF in conjunction with various antifungal agents was the primary focus of these studies. Use of rhG-CSF in a murine model of vaginal candidiasis did not reduce vaginal colony counts, or improve vaginal histophathology scores. Administration of rhG-CSF in a murine model of pulmonary aspergillosis improved survival, clinical signs, and gross pathology and histophathology scores of the lungs, and increased weight gain. The rhG-CSF was not shown to be an effective therapeutic treatment in this model of vaginal candidiasis. The rhG-CSF was, however, an effective prophylactic treatment in this model of pulmonary aspergillosis.

Introdução: Nas últimas décadas, as infeções fúngicas invasivas por leveduras tornou-se um importante problema de saúde pública, dado sua incidência crescente relacionada ao aumento da população suscetível. O reconhecimento destes patógenos em aspectos como, distribuicão ambiental e caraterísticas fenotípicas, são pilares essenciais para sua vigilância e controle. Objetivo: Descrever a frequência dos agentes de criptococose e outras leveduras com potencial patogênico e comparar essa frequência em excretas de aves silvestres em três municípios do estado de São Paulo, com vistas a melhor conhecimento da distribuição desses agentes no ambiente, além de determinar o perfil de suscetibilidade in vitro a antifúngicos de uso clínico. Método: No período de 2 anos, aves silvestres foram identificadas em áreas de circulação de 3 municípios de São Paulo (Praia Grande, Santos e Rio Claro) e submetidas à coleta de excretas para isolamento de leveduras com potencial patogênico. Análise microscópica e macroscópica para classificação presuntiva de gênero foram realizados em todas as colônias de leveduras obtidas das amostras de excretas. A suscetibilidade dos isolados de leveduras aos antífúngicos: fluconazol, voriconazol e anfotericina B foi determinada segundo método de referência europeu (AFST-EUCAST). Análise de dados: Foi utilizada a regressão de Poisson com a opção robusta para estimar razões de prevalência e identificar variáveis associadas com os principais isolados identificados, com opção de cluster para agrupar os isolados por excreta. Foi avaliado o nível de concordância entre os dois métodos de identificação (fenotípico e MALDI-TOF), utilizando o coeficiente Kappa. Adicionalmente, foi estimada a correlação entre os MIC´s dos fármacos estudados no total de espécies identificadas, utilizando o coeficiente de correlação de Spearman. Resultados: Das 294 excretas coletadas, 42,2 por cento continham leveduras, incluindo espécies de Candida 62 por cento , seguido por Rhodotorula 16,4 por cento , Cryptococcus 10,4 por cento , Trichosporon 6,6 por cento e Pichia 2,7 por cento . Muitas espécies, verificadas em alta frequência, tem forte potêncial de causar infecção invasiva, como: C. parapsilosis stricto sensu, C. tropicalis, Clavispora lusitaniae, C. krusei, C. orthopsilosis, C. glabrata, C. laurenti, C. albicans, C. metapsilosis, C. nivariensis e Meyerozyma guilliermondii. A resistência ao fluconazol, voriconazol e anfotericina B ocorreu nesses isolados, sendo documentada uma forte correlação entre a susceptibilidade, principalmente entre os azois (fluconazol e voriconazol), no entanto, a correlação mesmo sendo menor também foi significativa entre esses fármacos e a anfotericina. De 13 espécies de aves silvestres dispersoras de leveduras, as de maior frequência foram: Sula leucogaster 26,2 por cento , Turdus leucomelas 17 por cento , Larus dominicanus 15 por cento , Thalasseus maximus 11,2 por cento , Thalasseus acuflavidus 5,4 por cento , Tangara sayaca 4,4 por cento , Turdus amaurochalinus 3,7 por cento , Sterna hirundinacea e Pitangus sulphuratus 2,7 por cento . Os gêneros identificados apresentaram associações entre local, estação do ano e espécies de aves. Conclusões: Dentre as principais espécies de aves estudadas, 3 eram de hábitos migratórios (Thalasseus maximus, Thalasseus acuflavidus e Sterna hirundinacea) o que permite inferir dispersão interamericana de leveduras patogênicas. Diversas espécies resistentes a antifúngicos foram descritas, pela primeira vez, em excretas de aves silvestres conferindo a este estudo o valor de contribuir para o conhecimento da epidemiologia das infecções fúngicas por leveduras. / Background: In recent decades, invasive yeast fungal infections have become an important public health problem, due to their increasing incidence related to the increase in the susceptible population. The recognition of these pathogens in aspects such as environmental distribution and phenotypic characteristics are essential pillars for their surveillance and control. Objective: To describe the frequency of cryptococcosis agents and other yeasts with pathogenic potential and to compare this frequency in excreta of wild birds in three municipalities of the state of São Paulo, with a view to a better knowledge of the distribution of these agents in the environment, in addition to determining the profile of in vitro susceptibility to antifungals for clinical use. Method: During two years, wild birds were identified in circulation areas of three municipalities of São Paulo (Praia Grande, Santos and Rio Claro) and were screened for yeasts with pathogenic potential. Microscopic and macroscopic analysis for presumptive genus classification were performed in all yeast colonies obtained from excreta samples. Susceptibility of yeast isolates to antifungals: fluconazole, amphotericin B and voriconazole was determined according to the European reference method (AFST-EUCAST). Data analysis: Poisson regression was used with the robust option to estimate prevalence ratios and to identify variables associated with the main isolates identified, with option of cluster to group the isolates by excreta. The level of agreement between the two identification methods (phenotype and MALDI-TOF) was evaluated using the Kappa coefficient. Additionally, the correlation was estimated between MICs of the drugs studied in total of species identified, using Spearman\'s correlation coefficients. Results: Of the 294 excreta collected, half contained yeasts, including Candida species (62 per cent ), followed by Rhodotorula (16.4 per cent ), Cryptococcus (10.4 per cent ), Trichosporon (6.3 per cent ) and Pichia (2.7 per cent ). Many species, verified at high frequency, have a strong potential to cause invasive infection, such as: C. parapsilosis stricto sensu, C. tropicalis, Clavispora lusitaniae, C. krusei, C. orthopsilosis, C. glabrata, C. laurentii, C. albicans, C. metapsilosis, C. nivariensis and Meyerozyma guilliermondii. Resistance to fluconazole, voriconazole and amphotericin B occurred in these isolates and a strong correlation was reported between susceptibility, mainly between azole (fluconazole and voriconazole), however, the correlation, even though it was lower, was also significant between these drugs and amphotericin. From 13 species of wild birds dispersing yeasts, the ones with the highest frequency were: Sula leucogaster 26,2 per cent , Turdus leucomelas 17 per cent , Larus dominicanus 15 per cent , Thalasseus maximus 11,2 per cent , Thalasseus acuflavidus 5,4 per cent , Tangara sayaca 4,4 per cent , Turdus amaurochalinus 3,7 per cent , Sterna hirundinacea e Pitangus sulphuratus 2,7 per cent . The identified genera presented associations between site, season of the year and species of birds. Conclusion: Among the main species of birds studied, 3 were of migratory habits (Thalasseus maximus, Thalasseus acuflavidus and Sterna hirundinacea), which allows inferring the inter - American dispersion of pathogenic yeasts. Several species resistant to antifungal were described for the first time in excreta of wild birds, conferring to this study the value of contributing to the knowledge of the epidemiology of fungal infections by yeasts.

Encapsulation is a novel technique that involves the entrapment of materials such as cells, enzymes or chemicals within a semi-permeable matrix and is being explored as a drug delivery system. This project investigated the encapsulation of Streptomyces nodosus in alginate to assess whether this organism can produce the antifungal drug amphotericin B from within the matrix. New methods were developed to immobilise S. nodosus mycelia and spores in alginate capsules, assess bacterial viability and detect ng mL–1 quantities of amphotericin B in culture fluids. When capsules were cultured and cell proliferation was encouraged, organisms formed protrusions on the surface of the capsules. Differentiated branched hyphae that never progressed to sporogenic hyphae were observed on the surface of these structures. Viability was maintained for up to 30 days and low levels of amphotericin B were produced. The emergence of a co-existing free-dwelling population was also observed. Culturing immobilised organisms using conditioned media from an amphotericin deficient S. nodosus strain, augmented the development of the free-dwelling population resulting in the detection of amphotericin B in the culture fluid and full differentiation to sporogenic hyphae. This is the first report of sporulation of S. nodosus in liquid environments and demonstrates that immobilised S. nodosus can produce antibiotics. The sporulation of free-dwelling organisms was also induced using conditioned media and manipulation of quorum size, indicating a solid surface is not required for sporulation. Conditioned media from other Streptomyces spp. induced variable responses including sporulation, pigment formation and antibiotic production, possibly demonstrating communication between species and/or alteration in nutritional status. This new model for the life cycle of S. nodosus will permit the study of developmental pathways, antibiotic production, microbial community structure and inter-species and intra-species signalling. / Doctor of Philosophy (PhD)

Bibliography: leaves 87-96. This thesis investigates non-proteinaceous low molecular weight flavonoid and alkaloid compounds in Scurulla oortiana (Korth.) Danser grown on Camellia sinens. Three flavonols are identified in S. oortiana (Korth.) Danser growing on different hosts. The identification and characterisation of these flavonoids are carried out using various chromatographic and spectrometric procedures. Two purine alkaloids are isolated from and identified in S. oortiana (Korth.) Danser parasitizing tea plant, C. Sinensis. The antifungal activity of the phenolic compounds isolated from mistletoe parasitizing tea plant is examined.