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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
111

Isolation and characterisation of antifungal compounds from medicinal plants that are active against selected fusarium species

Seepe, Hlabana Alfred January 2021 (has links)
Thesis (Ph.D. (Chemistry)) -- University of Limpopo, 2021 / Fusarium species are among pathogenic organisms responsible for massive yield and quality losses in crop production. They cause crop diseases in the field and during storage, and some species are capable of producing mycotoxins which contaminate products and threaten consumer s’ health. Conventional synthetic fungicides are available for the control of Fusarium pathogens, however, their applications have been restricted or discouraged due to their harmful effect on the environment, livestocks and human health. There are also reports about fungal-resistance to available fungicides. Moreover, the synthetic chemicals are not affordable to smallholder farmers and to some extent, they are not recommended for applications in organic farming. As an alternative to these fungicides, selected medicinal plant species were investigated as sources of natural chemicals or compounds with potential to be developed into plant-based fungicides to control Fusarium pathogens. This study aimed to identify antifungal extracts among the selected medicinal plant species which could be used to develop plant-based fungicides to control Fusarium diseases. It also focused on isolation and characterization of antifungal compounds from selected medicinal plant species. Thirteen medicinal plant species (Combretum erythrophyllum (Burch.) Sond , Melia azedarach L, Solanum mauritianum Scop, Nicotiana glauca Graham, Schotia brachypetala Sond, Lantana camara L, Combretum molle R. Br. ex G. Don, Quercus acutissima Carruth, Olea europaea L, Vangueria infausta Burch, Withania somnifera (L.) Dunal, Harpephyllum caffrum Bernh and Senna didymobotrya (Fresen.) H.S. Irwin & Barneby) were selected from literature based on their reported strong antimicrobial activity against human and/or animal pathogens. The leaves of these plant species were collected, shade-dried and extracted with water, petroleum ether, ethyl acetate and acetone. Extractant yield was recorded and each extract was evaluated for antifungal activity using a micro-dilution assay against nine Fusarium pathogens (Fusarium verticillioides, Fusarium proliferatum, Fusarium subglutinans, Fusarium graminearum, Fusarium solani, xxvii Fusarium oxysporum, Fusarium semitectum, Fusarium chlamydosporum and Fusarium equiseti). Similar solvent extracts from different plant species that demonstrated MIC value of less than 0.1 mg/ml against the same pathogen were combined and evaluated for antifungal activity. The interation effect of combined extracts was determined by calculating their fractional inhibitory concentration index (FICI) in order to determine their possible synergistic, additive, indifference or antagonistic antifungal activity against tested pathogens. Plant extracts demonstrating synergistic and or additive interaction were further evaluated in combination and individually for in vivo antifungal activity against maize seed Fusarium pathogens. At least, one of the extracts obtained from these medicinal plant species showed strong antifungal activity with minimum inhibitory concentration (MIC) of less than 0.1 mg/ml against at least one of the tested pathogens. Of the four solvent extracts evaluated, acetone and ethyl acetate extracts showed stronger antifungal activity compared to petroleum ether and water extracts. Of the nine pathogens tested, F. proliferatum was the most susceptible and was strongly inhibited (MIC < 0.1 mg/ml) by 41 plant extracts whilst F. equisite was found to be resistant with MIC < 0.1 mg/ml by only three plant extracts. In total, each pathogen was tested against 52 plant extracts. There were 17, 16 and 15 extracts from C. erythrophyllum, S. mauritianum and Q. acutissima, respectively, with MIC values less than 0.1 mg/ml. These species were the most active when tested individually. Schotia brachypetala was found to be the least active medicinal plant with only seven extracts demonstrating very strong activity (MIC < 0.1 mg/ml) against the tested pathogens. Minimum inhibitory dilution (MID) or total activity was also calculated and it was found that water and acetone extracts had the highest MID, followed by ethyl acetate extracts while petroleum ether extracts recorded the lowest. Of all plant extracts tested against the nine pathogens, 59 plant extracts demonstrated MID values of more than 1000 ml/g. Out of the 348 extract combinations evaluated, 116 and 87 extract combinations demonstrated synergistic and additive antifungal activity, respectively. The strongest activity xxviii recorded for the combined extracts resulted from synergistic interaction with MIC value of 0.001 mg/ml against F. proliferatum and F. verticilloides. Combined acetone extract of C. erythrophyllum and Q. acutissima was very active (95.75% inhibition) against F. verticilloides inoculated on maize seeds while individual preparation from M. azedarach acetone extract demonstrated 97.10% inhibition against F. proliferatum. The extracts showing good antifungal activity (≥ 50% inhibition) were further tested for phytotoxicity on maize seed germination and the lowest recorded seed germination was 86.25%, resulting from Q. acutissima ethyl acetate extract. Combined acetone extract of C. erythrophyllum and Q. acutissima did not significantly affect maize seedling growth when compared to negative control (water treatment). All plant extracts that showed strong activity (MIC < 0.1 mg/ml) when tested using micro-dilution assay were spotted on thin layer chromatography (TLC) bioautographic assay to establish and determine the number of active compounds or bands. The white spots observed on the chromatograms indicated the presence of antifungal compounds. Combretum erythrophyllum, W. somnifera and L. camara exhibited the presence of antifungal compounds against 7, 5 and 4 pathogens, respectively. Hence, these plant species were selected for isolation of antifungal compounds where open column chromatography and preparative TLC were used for compound purification. At least, three isolated fractions from the three plant species were found to be active (MIC values ranging from 0.0098 to 0.625 mg/ml) against more than five pathogens. The fractions were also found to contain different levels of phytochemicals such as glycosides, flavonoids, steroids, and terpernoids. The structures of isolated compounds or fractions were determined using nuclear magnetic resonance (NMR) and mass spectroscopic (MS) techniques. A mixture of apeginin (4′,5,7-trihydroxyflavone) and salvigenin (5-hydroxy-6,7,4'-trimethoxyflavone) isolated from the leaves of C. erythrophyllum showed strong antifungal activity (MIC values ranging from 0.01 mg/ml to 0.63 mg.ml) against 5 tested Fusarium pathogens. Also isolated from C. erythrophyllum was a derivative of maslinic acid and it has xxix shown antifungal activity with MIC values ranging from 0.08 mg/ml to 0.63 mg/ml against 6 tested pathogens. On the other hand, lantadene A (22- angeloyloxy-9-hydroxy-3-oxo-olean-12-en-28-oic acid), boswellic acid (11-keto-β-boswellic acid) and boswellic acid glycoside isolated from the leaves of Lantana camara showed good activity (MIC values ≤ 0.63 mg/ml) against one or more Fusarium pathogens. Withaferin A (4β,27-dihydroxy-1-oxo-5β,6β-epoxywitha-2-24-dienolide) glycoside isolated from the leaves of Withania somnifera showed antifungal activity with MIC value of 0.16 mg/ml against F. verticilloides. This study demonstrated potential applications of medicinal plant extracts as cheap, accessible and sustainable source of eco-friendly pesticides for fighting crop diseases in organic and smallholder farming. The extracts can be used as treatment agents to control maize seed spoilage during post-harvest storage. Additionally, characterised antifungals may serve as scaffold compounds during commercial synthesis of plant-based fungicides. / Agricultural Research Council (ARC) and National Research Foundation (NRF)
112

Antifungal activities of metergoline, purpurin and baicalein on Candida species. / CUHK electronic theses & dissertations collection

January 2010 (has links)
Baicalein is known to be a potent antifungal agent and induces programmed cell death in Candida albicans. In the present study, we found that baicalein also inhibited the growth of C. krusei isolates. The minimal inhibitory concentrations of baicalein against eight C. krusei isolates were 1.35--2.70 microg/ml. One-hour exposure to baicalein elicited a consistent and moderate post-antifungal effect on the C. krusei isolates. Further flow cytometric study demonstrated a depolarization of mitochondrial membrane potential. However, both the levels of reactive oxygen species and DNA fragmentation were not significantly changed after baicalein treatment in C. krusei. It can be concluded that the antifungal activity of baicalein was mitochondria-dependent in both C. krusei and C. albicans, but the antifungal mechanism was different. Reactive oxygen species may not play a direct role and baicalein does not initiate programmed cell death or apoptosis in C. krusei. The structure-activity relationship study showed that the three hydroxyl groups in baicalein were essential for its antifungal potency. / Candidiasis has become a serious infection with very high mortality and morbidity in the world if not providing effective treatments. However, due to clinical limitation and resistance of the current antifungal agents, there is an urgent need to search for novel antifungals. In this study, after screening a compound library (n=400) for antifungal activity, three members (metergoline, purpurin and baicalein) were chosen for further study. Their antifungal characteristics and the antifungal mechanisms were investigated. / Metergoline, a serotonin receptor antagonist, was found to have potent antifungal activity against the intrinsically fluconazole-resistant human fungal pathogen Candida krusei. The minimal inhibitory concentration and minimal fungicidal concentration of metergoline against C. krusei were 4 microg/ml and 8 microg/ml respectively. Metergoline induced post-antifungal effect. Significant synergism was found in combination of metergoline with amphotericin B by a checkerboard assay, which may be due to the perturbation of cell permeability and increase in the intracellular accumulation of antifungal agents. Metergoline also inhibited extracellular phospholipase production in C. krusei. To gain insights into the mechanisms, intracellular changes that accompany apoptosis were examined by flow cytometry and spectrophotometry. The results showed an increase in the level of reactive oxygen species, depolarization of mitochondrial membrane potential, phosphatidylserine externalization, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling in the metergoline-treated C. krusei . Taken together, we conclude that metergoline may promote apoptosis in C. krusei through reactive oxygen species production and perturbation in mitochondrial homeostasis, implying its antifungal potential to treat candidiasis. / The antifungal activity of purpurin, a natural red anthraquinone pigment in madder root (Rubia tinctorum L.), was evaluated against Candida isolates by a broth microdilution assay. The minimal inhibitory concentrations of purpurin against Candida species isolates were 1.28--5.12 microg/ml. Mechanistic studies indicated that purpurin inhibited energy-dependent efflux pumps of Candida isolates. Furthermore, purpurin demonstrated a depolarization of mitochondrial membrane potential, suggesting a possible linkage of the antifungal mechanism of purpurin to Candida apoptosis. / Kang, Kai. / Adviser: Fong Wing Ping. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 98-123). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
113

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene, as an antifungal and biocontrol agent

Carstens, Maryke,1976- 04 1900 (has links)
Thesis (MScAgric) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can colonise various other organisms, including humans, plants and animals. The effect of this is usually detrimental, not only to agricultural crops and livestock, but also to human well-being. The extensive farming of crops and livestock requires persistent control of fungal populations, commonly through the use of chemical fungicides. However, the exclusive use of fungicides is no longer a sustainable practice, as a result of serious problems, such as increasing fungicide resistance in pathogen strains, the high costs of fungicides, as well as concern about the environment. The search by producers and scientists for alternative control measures is an ongoing process. The fungal cell wall consists of polysaccharides that not only playa role in protection of the fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic linkages, is one of the major components of the fungal cell wall, where it plays an important role in the apical growth of the vegetative hyphae. Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of many plant species by a pathogen, the production of a specific group of proteins, designated pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and crucial part of the plant's natural defence response. Chitinases have increasingly been targeted to upregulate plants' endogenous disease resistance mechanisms through transgenic overexpression in a variety of hosts. Several species of fungi, including various Trichoderma spp., are potent biocontrol agents of plant pathogenic fungi and insects. The antagonistic activities of these biological control agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes, such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to naturally occurring biocontrol agents. Through the process of genetic transformation, other fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial substances more effectively in order to yield potent biocontrol agents. Various types of chitinases have been applied in the production of fungal resistant plants and some research has been done on the application of chitinases, from a variety of microorganisms, as biological control agents. In contrast, very little is known about the antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2 gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit fungal growth. The first objective of this study involved the high level expression and optimisation of the secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators were included in the study and the secretion of the chitinase gene was evaluated by testing three different secretion signals. The secretion signals included: the native CTS1-2 secretion signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1 (PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to achieve high-level expression. The results obtained from the analysis of the recombinant yeasts showed that the PGK1 promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently secrete the overexpressed chitinase was analysed and it was found that the non-native secretion signals delivered significantly more protein to the extracellular environment. It was thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that of the native secretion signal. The antifungal activities of the recombinant chitinases produced by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to a significant reduction in hyphal development, caused by extreme structural damage to the hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2 promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be detected with any of these strains. The reasons for this still remain unclear. The second objective of this study was to assess the potential of the yeast chitinase gene to upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2 gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the intra- and the extracellular environment. The results obtained showed that the transgenic tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene expression as well as the production of a biologically active yeast chitinase enzyme. The F, progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection; some of the lines showed disease resistance of 65 and 70%. The plants expressing an extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected relating to the effect of the chitinase on the plant surface with regards to disease resistance to fungal pathogens. In conclusion, the combined set of results from both the yeast and plant overexpression studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2 chitinase could be instrumental in the development of a new generation of yeast strains with improved antifungal capabilities. This enzyme could also play an important role in genetic transformation technologies aimed at enhanced disease resistance. / AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie, hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende proses. Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in 'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc) residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel. Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR) proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere. Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige bio-antagoniste. Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1) sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om hoë-vlak uitdrukking te dryf. Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer, en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2 sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm. Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op hede onbekend. Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is. Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die plant se oppervlak met betrekking tot siekteweerstand teen swampatogene. Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.
114

Biofilmes mistos de Mycobacterium tuberculosis e Paracoccidioides brasiliensis e ação de chalcona livre e carreada no sistema nanoemulsionado /

Alarcón, Kaila Petronila Medina. January 2018 (has links)
Orientador: Ana Marisa Fusco Almeida / Coorientador: Fernando Rogério Pavan / Banca: Katiany Rizzieri Caleffi Ferracioli / Banca: Luís Otávio Regasini / Banca: Maria José Soares Mendes Giannini / Banca: Marlus Chorilli / Resumo: As infecções pulmonares são um problema global e estão classificadas em terceiro lugar entre as principais causas de morte. Entre as infecções pulmonares crônicas estão a tuberculose (TB) e a paracoccidioidomicose (PCM). A TB é uma doença cujo principal agente etiológico é o Mycobacterium tuberculosis e a PCM tem como agente etiológico o complexo Paracoccidioides spp. As duas doenças podem ocorrer na forma de coinfecção, com incidência aproximada de 2,8 a 5,5% dos casos nos países da América do Sul e 10% no Brasil. Ambos os microrganismos já foram descritos na forma de biofilme monoespécie e o tratamento de ambas as doenças tem merecido atenção, devido a casos de resistência e as poucas opções terapêuticas. Diante do exposto, este trabalho visa mostrar, pela primeira vez, a capacidade de M. tuberculosis e P. brasiliensis se associarem por meio do crescimento na forma de um biofilme misto in vitro, a fim de elaborar um novo protótipo de 3'chalcona carreada numa nanoemulsão com atividade para as formas planctônica e de biofilme. Para isso, inicialmente foi padronizada e caracterizada a formação dos biofilmes monoespécie da linhagem ATCC M. tuberculosis (H37Rv), da linhagem clínica (Rv40) e da linhagem de P. brasiliensis (Pb18) e dos biofilmes mistos das associações de H37Rv+Pb18 e Rv40+ Pb18 (nesta ordem de crescimento), ambas as associações mostraram um aparente sinergismo entre elas. Na formação do biofilme misto inverso denominado assim pela ordem de associação, onde primeir... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pulmonary infections are a global problem and rank third among the leading causes of death. Among the chronic lung infections are tuberculosis (TB) and paracoccidioidomycosis (PCM). The TB is a disease whose main etiologic agent is Mycobacterium tuberculosis and PCM has the etiological agent Paracoccidioides spp. The two diseases may occur in the form of coinfection, with an approximate incidence of 2.8 to 5.5% of the cases in the countries of South America and 10% in Brazil. Both microorganisms have already been described in the form of monospecies biofilm and the treatment of both diseases has deserved attention, due to cases of resistance and the few therapeutic options. In view of the above, this research aims to show, for the first time, the ability of M. tuberculosis and P. brasiliensis to associate by means of growth in the form of a mixed biofilm in vitro, in order to elaborate a new prototype in a nanoemulsion with activity for planktonic and biofilm forms. For this purpose, it was initially standardized and characterized the formation of monospecies biofilms of the ATCC strain M. tuberculosis (H37Rv), the clinical lineage (Rv40) and the P. brasiliensis (Pb18) lineage and the mixed biofilms of the associations of H37Rv + Pb18 and Rv40 + Pb18 (in this order of growth), both associations showed an apparent synergism between them. In order to establish the mixed biofilm (Pb18 + H37Rv), it was first proposed the association of P. brasiliensis (Pb18), followed by ATCC M. tuberculosis (H37Rv) with order of inverse growth, in this association a apparent competition between the microorganisms was observed. All biofilms, monospecies and mixed, were consolidated from 30 to 45 days... (Complete abstract click electronic access below) / Doutor
115

Infections in patients with hematological malignancies : etiology, trends and management /

Cherif, Honar, January 2005 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.
116

Study of antimicrobial activity and mechanism of zinc oxide nanoparticles against foodborne pathogens

Liu, Yang, Li, Men`gshi. January 2009 (has links)
The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 23, 2010). Thesis advisor: Dr. Mengshi Lin. Includes bibliographical references.
117

Estudo da atividade antifúngica da associação do óleo essencial de Cymbopogon Winterianus Jowitt (Citronela) com antifúngicos sintéticos sobre espécies de Aspergillus

Barbosa, Danilo Batista Martins 26 August 2010 (has links)
Made available in DSpace on 2015-05-14T12:55:58Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 527726 bytes, checksum: 9d9d888b1040d17c42f2b3e23c69fcd7 (MD5) Previous issue date: 2010-08-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This research aimed to study the antifungal properties of essential oil of Cymbopogon winterianus Jowitt associated with synthetic antifungals against strains of Aspergillus. The parameters used for this purpose were based on the determination of Minimum Inhibitory Concentration, Fractional Inhibitory Concentration Index (method of association - Checkerboard) as well as in microbial death curve. Amphotericin B and fluconazole antifungal synthetic were used as positive controls, and is also used in combination with tests of the natural product. All tests were performed in triplicate. The present study allowed further our understanding about the therapeutic effects of essential oil of C. winterianus, when used in isolation or drug related alluded to earlier. In addition, we attempted to find viable alternatives to the treatment of Aspergillus infections, currently based on the prolonged use of allopathic drugs, where the observed side effects are a limiting factor to its full success. / Esta pesquisa se propôs a estudar as propriedades antifúngicas do óleo essencial de Cymbopogon winterianus Jowitt associado a antifúngicos sintéticos contra cepas de Aspergillus. Os parâmetros utilizados para esse fim se basearam na determinação da Concentração Inibitória Mínima, Índice de Concentração Inibitória Fracionada (método de associação Checkerboard), bem como na curva de morte microbiana. A anfotericina B e o fluconazol, antifúngicos sintéticos de uso consagrado, foram empregados como controles positivos, sendo também utilizados nos testes de combinação com o produto natural. Todos os ensaios foram realizados em triplicata. A realização desse estudo possibilitou aprofundar os conhecimentos acerca dos efeitos terapêuticos do óleo essencial de C. winterianus, quando usado de forma isolada ou se relacionado às drogas aludidas anteriormente. Além disso, buscou-se encontrar alternativas viáveis ao tratamento das infecções por Aspergillus, atualmente baseados no uso prolongado de drogas alopáticas, onde os efeitos colaterais observados representam um fator limitante para seu pleno sucesso.
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Susceptibilidade de Candida albicans resistente a fluconazol ao efeito fotodinâmica e inibidores dos sistemas de efluxo /

Vega Chacón, Yuliana del Pilar January 2018 (has links)
Orientador: Ewerton Garcia de Oliveira Mima / Resumo: Um dos principais mecanismos de resistência microbiana são os sistemas de efluxo, que transportam medicamentos antimicrobiano para fora da célula. A eficácia de alguns agentes de inibição dos sistemas de efluxo tem sido reportada para reverter a resistência microbiana e também para potencializar as terapias antimicrobianas. Além disso, métodos alternativos aos agentes antimicrobianos convencionais têm sido investigados, como a Terapia Fotodinâmica antimicrobiana (aPDT). O objetivo desse estudo foi avaliar in vitro o efeito da aPDT e de dois inibidores de sistemas de efluxo microbiano (curcumina e verapamil) na resistência à inativação de C. albicans. Foram utilizadas duas cepas de C. albicans, uma susceptível (CaS) e outra resistente (CaR) a fluconazol. Os parâmetros de inativação fúngica foram determinados submetendo-se culturas planctônicas de ambas as cepas à curcumina, ao verapamil, ao fluconazol e também à aPDT (mediada pela curcumina 40 μM (14,73 μg/mL) e luz de LED azul de ≅455 nm a 5,28 J/cm2). As duas cepas foram cultivadas e tratadas associando-se os agentes de inibição do efluxo ao fluconazol em concentrações não letais. Os dados de UFC/mL foram analisados pelos testes paramétricos t de Student, ANOVA/Welch e post-hoc de Games-Howell e pelo teste não paramétrico de Mann-Whitney (α=0,05; n=12). Os resultados demostraram que aPDT promoveu uma redução significativa (p<0,001) de 4,5 e 4,42 log10 para CaS e CaR, respectivamente. Para CaS, os valores de Concentrações Ini... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: One of the major mechanisms of microbial resistance is the efflux systems, or efflux pumps, present in the plasma membrane of microorganisms that carry an antimicrobial drug out of the cell. The efficacy of some inhibitors of efflux systems has been reported to reverse microbial resistance, including C. albicans, and also to potentiate antimicrobial therapies. In addition, alternative methods to conventional antimicrobial agents have been investigated, such as antimicrobial Photodynamic Therapy (aPDT). The objective of this study was to evaluate in vitro the effect of aPDT and two inhibitors of microbial efflux systems (curcumina and verapamil) on the resistance to inactivation of C. albicans. For this, two strains of C. albicans, one susceptible (CaS) and another resistant (CaR) to fluconazol were used. Fungal inactivation parameters were determined by subjecting planktonic cultures of both strains to curcumina, verapamil, fluconazol, and also aPDT (mediated by curcumin at 40 μM (14.73 μg/mL) and blue LED light of ≅455 nm and 5.28 J/cm2). These strains were then cultured and treated associating one of the efflux inhibitors with fluconazole using non-lethal concentrations. For the statistical analysis, the normality and the homogeneity of variances were evaluated by the Shapiro-Wilk and Levene tests, respectively. Data were analyzed by Student's t-tests, Welch-corrected ANOVA and Games-Howell post-hoc and Mann-Whitney non-parametric test (α = 0.05) (n = 12). aPDT promoted a s... (Complete abstract click electronic access below) / Mestre
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Modelagem farmacocinética-farmacodinâmica do antifúngico voriconazol

Araújo, Bibiana Verlindo de January 2008 (has links)
Objetivos: O objetivo deste trabalho foi o desenvolvimento de um modelo farmacocinético/farmacodinâmico (PK/PD) para descrever o efeito antifúngico voriconazol (VRC) contra espécies de Candida. Método: Para alcançar este objetivo as seguintes etapas foram realizadas: i) foi adaptado e padronizado modelo de candidíase disseminada em ratos Wistar imunocompetentes e imunocomprometidos com Candida sp.; ii) foram validados métodos analíticos de LC-MS/MS e LC-UV para o doseamento do VRC em amostras de plasma e microdialisado de tecido; iii) foram estabelecidas as condições para microdiálise do VRC e as taxas de recuperação in vitro, por perda e ganho, e em tecido renal in vivo, por retrodiálise, foram determinadas; iv) foi avaliada a PK não-linear do VRC após administração i.v. bolus das doses de 2,5, 5 e 10 mg/kg e a biodisponibilidade oral foi determinada em roedores; v) a penetração renal do VRC após administração oral das doses de 40 e 60 mg/kg foi determinada em ratos Wistar sadios e infectados com C. albicans ou C. krusei; e (vi) o perfil fungistático do VRC contra C. albicans e C. krusei foi determinado utilizando modelo de infecção experimental in vitro onde foram simuladas as concentrações livres renais do VRC esperadas em humanos após administração oral e i.v. de diferentes posologias. Os dados de cinética e dinâmica obtidos foram modelados com equação de Emax modificada, com auxílio do Scientist®. Resultados e Conclusões: i) O modelo de candidíase disseminada foi adaptado com sucesso para ratos Wistar. C. albicans apresentou maior virulência com Log UFC/g de tecido renal de 5,51 ± 0,56 e 7,29 ± 0,26, após 2 e 7 dias de infecção em animais imunocompetentes, respectivamente. Em animais imunocomprometidos a contagem foi de 6,43 ± 0,59 Log UFC/g após 2 dias de infecção, com morte de todo o grupo dentro de 4 dias. As espécies não-albicans (C. krusei e C. glabrata) apresentaram um perfil de infecção semelhante em animais imunocompetentes (Log UFC/g = 2,98 ± 0,27 para C. krusei e 2,48 ± 0,46 para C. glabrata). Entretanto, nos animais imunocomprometidos, C. krusei promoveu morte de todo o grupo em até 7 dias, enquanto C. glabrata causou apenas um aumento no grau de infecção (Log UFC/g = 6,98 ± 0,48). ii) Os métodos analíticos por LC-UV e LCMS/ MS para quantificação do VRC foram validados. As curvas de calibração foram lineares na faixa de 50 a 2500 ng/mL (r > 0,98) para ambos os métodos. Os ensaios de precisão intra e inter-dia foram > 94,9 e 95,8 %, para microdialisado por HPLC-UV e > 87,5 e 92,3 % para LC-MS/MS em plasma, respectivamente. A exatidão foi > 89,1 % para HPLC-UV e > 88,4 % para LC-MS/MS. iii) A avaliação do VRC por microdiálise mostrou que a recuperação é concentração independente (0,1–2,0 μg/mL). O VRC entretanto, devido a sua moderada lipofilia, liga-se às tubulações do sistema de microdiálise, gerando diferenças entre a recuperação determinada pelo método de perda (retrodiálise) e de ganho (diálise) in vitro, as quais puderam ser corrigidas após o cálculo do coeficiente de ligação do fármaco ao sistema. A recuperação in vivo após correção da ligação ao sistema foi de 24,5 ± 2,8 % iv) A análise dos perfis de plasmáticos do VRC obtidos em ratos Wistar após administração oral mostrou comportamento não-linear, compatível com saturação de eliminação. A avaliação compartimental dos perfis i.v. de diferentes doses, utilizando modelo de três compartimentos com eliminação de Michaelis-Menten, permitiu a determinação da constante de Michaelis (KM) de 0,58 μg/mL e da velocidade máxima da eliminação (VM) de 2,63 μg/h, em média. A modelagem simultânea dos dados plasmáticos (40 mg/kg) e i.v. (10 mg/kg) permitiu a determinação da biodisponibilidade oral do VRC em ratos, que foi de 82,8%. v) A fração de penetração renal do VRC, determinada por microdiálise em ratos sadios e infectados, foi de 0,34 ± 0,01, similar a fração livre do fármaco no plasma (0,34), indicando que as concentrações livres renais de VRC são semelhantes às concentrações livres plasmáticas e que as mesmas não se modificam devido a infecções causadas por Candida sp. vi) Os parâmetros da modelagem PK/PD do efeito do VRC contra espécies de Candida em modelo de infecção experimental in vitro obtidos foram: CE50 de 2,96 μg/mL e Kmax = 0,26 h-1 para C. albicans e CE50 de 3,47 μg/mL e Kmax = 0,51 h-1 para C. krusei. Houve diferença estatística apenas no Kmax para as duas espécies (α = 0,05) indicando uma maior suscetibilidade da C. krusei ao VRC. O modelo PK/PD de Emax modificado utilizado foi capaz de descrever adequadamente os perfis de inibição do crescimento de Candida sp em função do tempo, para todos os regimes terapêuticos do VRC avaliados, podendo ser usado para otimização da terapia com esse fármaco. / Objectives: The aim of this work was the development of a pharmacokineticpharmacodynamic model (PK/PD) to describe the fungistatic effect of voriconazole (VRC) against Candida species. Method: To reach this objective, the following steps were done: i) a disseminated candidiasis model to immunocompetent and immunocompromised Wistar rats with Candida sp was adapted and standardized; ii) analytical methods of LC-MS/MS and LC-UV for measurement of VRC in plasma and microdialysate tissue samples were validated; iii) microdialysis conditions of VRC and the recoveries rate in vitro, by loss and gain, in renal tissue in vivo, by retrodialysis, were determined; iv) the non-linear PK of VRC after i.v. bolus administration of 2.5, 5 e 10 mg/kg doses were evaluated and the oral bioavailability in rodents was estimated; v) tissue penetration of VRC after oral administration of 40 and 60 mg/kg was determined in healthy and infected by C. albicans or C. krusei Wistar male rats; vi) the fungistatic profile of VRC against C. albicans and C. krusei was determined using a experimental infection model in vitro, where the free renal concentrations of VRC expected in humans after oral and iv administration of different dosing regimens were simulated. The kinetic and dynamic data obtained were modeled using an Emax modified model, with aid of Scientist®. Results and Conclusions: i) The disseminated candidiasis model was successfully adapted to Wistar rats. C. albicans showing high virulence with Log CFU/g of renal tissue of 5.51 ± 0.56 and 7.29 ± 0.26, after 2 and 7 days of infection in immunocompetent animals, respectively. In immunocompromised animals, the counting was 6.43 ± 0.59 Log CFU/g after 2 days of infection, with whole group death within 4 days. Non-albicans especies (C. krusei e C. glabrata) showed a similar infection profile in immunocompetent and immunocompromised animals (Log CFU/g = 2.98 ± 0.27 to C. krusei e 2.48 ± 0.46 to C. glabrata). However, in immunocompromised animals, C. krusei causes death in the whole group up to 7 days, instead, C. glabrata causes only a low increase in the infection degree (Log CFU/g = 6.98 ± 0.48). ii) The analytical methods of HPLC-UV and LC-MS/MS to VRC quantification were validated. Linearity was between 50 - 2500 range ng/mL (r > 0.98) for both methods. The intra and inter-day precision assays were > 94.9 e 95.8 %, for microdialysate using LC-UV and > 87.5 e 92.3 % using LCxx MS/MS for plasma, respectively. The accuracy was > 89.1 % for HPLC-UV and > 88.4 % for LC-MS/MS. iii) The evaluation of VRC by microdialysis showed that recovery is concentration independent (0.1–2 μg/mL). VRC, however, due to its moderate lipophilic characteristic, binds to the microdialysis system tubing’s, generating differences between recoveries determined by loss (retrodialysis) and gain (dialysis) in vitro methods, which could be corrected after determination of drug’s binding coefficient to the system. The in vivo recovery determined after correction of system binding was 24.5 ± 2.8 %. iv) VRC plasma profiles analysis obtained from Wistar rats after oral administration showed a nonlinear behavior, compatible with saturable elimination. The compartmental evaluation of i.v. profiles in different doses, employing the a compartment model with Michaelis-Menten elimination, allowed to determine the Michaelis-Menten constant (KM) of 0.58 μg/mL and the maximum velocity (VM) of 2.63 μg/h, in average. The simultaneous modeling of oral (40 mg/kg) and iv (10 mg/kg) plasma data allowed the determination of the oral bioavailability of VRC in rats, equal to 82.8%. v) The VRC renal penetration fraction, determined by microdialysis in healthy and infected rats, was 0.34 ± 0.01, similar to the free unbound fraction in plasma (0.34), showing that VRC free renal concentration levels are similar to the unbound plasma concentrations and that did not change due the infection associated to Candida sp. vi) The parameters of PK-PD modeling of VRC effect against Candida species in the in vitro experimental infection model obtained were: EC50 de 2.97 μg/mL and Kmax = 0.203 h−1 to C. albicans and EC50 of 3.47 μg/mL and Kmax = 0.51 h−1 to C. krusei. There is a statistical difference only in Kmax value for the two species (α = 0.05), showing a higher susceptibility of C. krusei to VRC. The PK/PD Emax modified model employed was able to describe adequately the growth inhibition profiles of Candida sp in function of time, for all VRC dosing regimens evaluated, and can be used for therapy optimization with this drug.
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Avaliação da formação de biofilme de fungos emergentes e sua susceptibilidade a antifúngicos na forma livre e nanoencapsulada / Assessment of biofilm formation of emerging fungi and their susceptibility to antifungal agents in free form and in nanocapsules

Jesus, Roberta Stefanello de January 2013 (has links)
Nos últimos anos, várias espécies de fungos, até então conhecidos como saprófitas do ambiente, têm emergido como importantes patógenos na prática clínica, associados muitas vezes com a resistência aos antimicrobianos disponíveis comercialmente. Dessa forma, o conhecimento de aspectos relacionados à patogenicidade desses micro-organismos, como a formação de biofilme, assim como o perfil de susceptibilidade aos antifúngicos e a novas alternativas terapêuticas faz-se necessário. Uma importante tecnologia neste contexto é a utilização de sistemas nanoestruturados a partir de polímeros biodegradáveis para a veiculação de fármacos. Este trabalho visa verificar em isolados fúngicos emergentes a expressão fenotípica de biofilme através do teste em microplaca de poliestireno e o perfil de susceptibilidade frente a antifúngicos na forma livre e incorporados em sistemas nanoestruturados, através da técnica de microdiluição em caldo. Foram selecionados para o estudo 82 isolados fúngicos potencialmente patogênicos, sendo 26 oriundos do ambiente e 56 procedentes de espécimes clínicos. Quanto à capacidade de formação de biofilme verificou-se que 68 (82,9%) isolados produziram biofilme pelo teste da microplaca, sendo 38 classificados como fortes, 17 como moderados e 13 como fracos produtores. Além disso, observou-se que os isolados clínicos apresentaram maior capacidade de formação de biofilme do que os isolados ambientais. Não houve correlação entre a produção de biofilme e a susceptibilidade aos antifúngicos testados nas células planctônicas. De um modo geral, os fungos foram sensíveis à maioria dos antifúngicos testados neste trabalho. Entretanto, o fluconazol apresentou baixa atividade (CIM≥64μg/mL) para 45% dos isolados avaliados. Com o intuito de investigar o efeito de cetoconazol e de fluconazol associados a nanoestruturas sobre os diferentes fungos apresentados no estudo, também foram desenvolvidas nanocápsulas de poli-ɛ-caprolactona contendo estes fármacos. Análises físico-químicas das nanoestruturas revelaram tamanho médio de partícula variando de 206 nm para as nanocápsulas de cetoconazol (CN) e de 211 nm para as nanocápsulas de fluconazol (FN). Ambas as formulações apresentaram características homogêneas e demonstraram estruturas monodispersas. A eficiência de encapsulação do cetoconazol e do fluconazol nas formulações foi de 86,35 e de 80,5%, respectivamente. Os diversos gêneros investigados parecem se comportar de maneira diferente quando expostos às nanoestruturas dos dois fármacos em estudo, sugerindo que as espécies de Candida poderiam apresentar uma maior susceptibilidade in vitro frente a estas nanoestruturas quando comparadas à forma livre dos antifúngicos em questão. Os resultados obtidos demonstram a potencialidade destas formulações para a veiculação de cetoconazol e fluconazol. Como perspectivas, sugere-se a realização de estudos adicionais para evidenciar o efeito in vitro das nanopartículas sobre os diferentes fungos de importância clínica e ambiental. / In recent years, several fungal species known as environmental saprophytes have emerged as important pathogens in clinical practice, often associated with antimicrobial resistance commercially available. Thus, the knowledge of aspects related to these pathogenic micro-organisms, such as biofilm formation, as well as the profile of susceptibility to antifungal agents and new therapeutic options is necessary. An important technology in this context is the use of nanostructured materials from biodegradable polymers for the placement of drugs. This work aims to examine the emerging fungal isolates in the phenotypic expression of biofilm through the test on polystyrene microplate and the susceptibility profile to antifungals front in the free trade and in the nanostructured systems by the broth microdilution method. In this study, 82 potentially pathogenic fungal isolates were selected, being the 26 from environment originating and the 56 from the clinical specimens. Regarding the ability of biofilm formation was found that 68 (82,9%) isolates produced biofilm by the microplate assay. The isolates were classified as 38 strong, 17 moderate and 13 weak biofilm producers. Moreover, it was noted that clinical isolates showed greater capability for the biofilm formation than the environmental isolates. There was no correlation between the biofilm production and the susceptibility to antifungal agents tested in planktonic cells. In general, the fungi were susceptible of the majority of the antifungal agents tested in this study. However, the fluconazole showed low activity (MIC≥64μg/mL) for 45% of the isolates. In order to investigate the effect of the ketoconazole and the fluconazole associated with nanostructures against different fungi contained in the study, polymeric nanocapsules have also been developed containing these drugs. Physicochemical analyzes showed an average size of nanostructures ranging from 206 nm for the nanocapsules of ketoconazole (CN), and 211 nm for the fluconazole (FN). The formulations showed homogeneous characteristics and demonstrated monodisperse structures. The encapsulation efficiency of ketoconazole and fluconazole in the formulations was 86,35 and 80,5%, respectively. The diverse genera investigated appear to behave differently when exposed to the nanostructures of the two drugs have being studied, suggesting that Candida species could exhibit a greater sensitivity to these nanostructures compared to the free form of antifungal concerned. The results demonstrate the potential of the formulations for the delivery of fluconazole and ketoconazole. As perspectives, we suggest further studies to demonstrate the in vitro effect of the nanoparticles against different fungi from the clinical and the environmental importance.

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